131 stefano bertuzzi ceo of asm diversity/inclusion in microbiology and science policy perspective“ copyright 2018, fine focus. all rights reserved. 132• fine focus, vol. 4(1) the challenge and the power of diversity a few years ago, i read an illuminating book by social psychologist claude steele, with the intriguing title of whistling vivaldi: how stereotype affect us and what we can do1 . the book presents overwhelming evidence from solid scholarly behavioral research on the implicit biases that reside inside all of us. steele is african-american and the title of his book derives from a story told him by brent staples, an african-american friend who had been a graduate student at the university of chicago. staples observed that while walking at night through the streets of chicago’s hyde park neighborhood wearing a hoodie, oncoming pedestrians would cross the road so as not to be on the same sidewalk. to them, there was something threatening about a young black man in casual clothing at night in a sketchy neighborhood. however, when staples— the same young black man, in the same outfit, at the same time, and in the same neighborhood— tried cheerfully whistling the “spring” tune from vivaldi’s “four seasons,” passersby were much friendlier. they would say hello, and wouldn’t cross the road. the abundant evidence provided in steele’s book which spans enormous breath of situations and stereotypes, is solidly datadriven, based on the results from many controlled behavioral experiments that testify to these widespread biases. steele’s point is not merely that negative biases exist—he says there are noxious biases about virtually every group in american life—young, old, male, female, northerners, southerners, geeks, jocks, or californians— but often the real damage is internal for the person on the receiving end. self-worth and personal-expectations fall. the problem is especially serious when someone is made to feel out-of-place and under suspicion like an inner city minority student on a college campus that seems populated only by privileged whites. a dynamic is set up, says steele, where these individuals systematically underperform when compared to their already demonstrated potential. this is a real and difficult problem to address, particularly in science education. by not fostering a diversified and multicultural environment, we lose talent because individuals cannot express or perform as well as they could. the power of diversity is that it creates better groups, better schools, and ultimately a better society. stereotypes are deeply rooted in our society. the challenge is to recognize them and to act. if you think that you are immune to bias, think again, your immune system may be working hard to fight it, but the antigen is certainly around. try taking the harvard implicit association test (iat) [link to website https://implicit.harvard.edu/ implicit/demo/], it takes about five minutes. iat is a very solid instrument literally taken by hundreds of thousands of people; it is a very robust and validated instrument. it is illuminating to see what kind of biases may be creeping into your daily life. diversity produces benefits, although it can be hard to look beyond the snap biases that steele shows so clearly envelop our daily lives. we know on one level that people from different identity groups or cultures bring different tools, ways of thinking and of solving problems to society. and yet rather than leveraging those differences for the collective benefit, we often allow them to impede progress and innovation. certainly leveraging diversity is not an easy job. the political scientist robert putnam presents data indicating that the level of civic engagement and general trust decreases 133 when communities become more diverse. the problem is compounded; not only do people trust other groups less, they trust people within their own group less. however, other researchers, most notably the mathematician, economist, “decision scientist,” scott page, author of the wonderful book the difference2 builds on organizational theorist i.d. steiner’s work providing us with an interesting conceptual framework. when we are tasked to do something as a group, we need to analyze the nature of the assignment. is it a disjunctive task where only one person needs to succeed in the group for the whole group to succeed or is it a conjunctive task, when everyone’s contribution is critical? a group tackling a complex math problem is an example of a disjunctive task. the more diverse the thinking, the easier, it is for the team to solve the problem. football, though, is a conjunctive task. the offensive linemen must complete their task of protecting the quarterback or the quarterback cannot succeed in his assignment. if a lineman fails to do his job, the quarterback gets sacked. so, the more we can deal with disjunctive tasks, the easier it will be to leverage diversity and differences. the problem in bioscience is that tasks are rarely either disjunctive or conjunctive, but a mix of the two. the trick is to transform conjunctive tasks into disjunctive tasks. the best example is possibly crowdsourcing and the greatest current example is wikipedia where distributed problem solving can be a powerful source of innovation. so, in this framework, where decentralized problemsolving and innovation go hand-in-hand, diversity deserves some credit. we have to think of sharing ideas—and sharing our scientific community—not as protecting privileged information or advantages but as building a collective scientific enterprise, a lever large enough to lift the world, as archimedes said. 13 rona robinson-hill ph.d assistant professor of biology and science education ball state university faculty perspective perspective“ faculty perspective • copyright 2018, fine focus. all rights reserved. 14• fine focus, vol. 4(1) serving as the only african-american in the biology department at ball state university (bsu) and a consultant on the fine focus – catalyzing stem opportunities for undergraduates immersive project with university of detroit-mercy (udm) i am tasked with addressing: 1) what does diversity mean today in stem?; 2) how have scientists changed how diversity is being defined?; 3) what is the importance of society vs. science vs. individual? webster’s dictionary defines diversity as, “the condition of having or being composed of differing elements; variety; especially, the inclusion of different types of people (such as people of different races or cultures) in a group or organization with the goal of programs intended to promote diversity in schools.” informed by this definition, then the goal for stem should be to make sure it is inclusive of all cultures and genders, so that each group is afforded the same benefits that come out of the research that a commitment to diverse stem produces. an analogy to describe what true diversity should look like can be depicted with a jigsaw puzzle. the border of the puzzle represents the typical scientist (white males); and the object revealed in the completed puzzle represents the other members of the population, such as white females, males and females from the african diaspora and males and females from other cultures. diversity in stem too often our classrooms in stem and other disciplines lack inclusive practices with both a focus and a value for diversity. conversation and collaborations like the fine focus and udm project dedicated to promoting social justice play a critical role in improving diversity in education generally and stem specifically. we (myself and two colleagues) discovered the importance of this diversity when the bsu students trained and immersed the udm students in the blind review process that a manuscript goes through to be considered for publication in a journal. this experience revealed an array of comments for the article. two groups evaluated the article with diversity in comments due to the diversity in backgrounds and experiences each student brought with them. the udm students were selected because of their ethnic diversity and their variations in research in stem, so their comments did not match the comments of the predominately white students from bsu, who only shared diversity in their socio-economic status. for example, the first day after our first session working with the udm students, two students approached me and said, “why did your students say they never thought about our ideas?” i replied, “they did not because their experiences are totally different from yours.” for me, these excerpts reaffirmed the importance of diversity in stem. 15 faculty perspective • scientists and diversity from my point of view many scientists, particularly at bsu, believe they have diverse academic environments. currently several white females have been extended opportunities to participate in research and conversations, not previously accessible to women. the inclusion of female scientists has paved the way for some research that might not have otherwise been addressed by men. however, the voices and perspectives of cultural groups that have been historically marginalized in areas like stem are still absent. the udm program is addressing this issue through their rebuild detroit initiative. to train and immerse the udm students in the blind review process that a manuscript goes through to be considered for publication in a journal fortifies each student to perform research on issues that plague their community and/or their personal lives. because these student’s rationale is, “if i do not do the research, who will?” this is true diversity in action that will impact the future ills of our world. the impact of this program hopefully will change the look of stem eventually, because the partnership between the bsu and udm students are conscientiously making an effort to make sure their diverse student body has access to authentic research experiences that effect diverse populations. stem in society if science is not addressing the ills of the diverse society then we are not preparing the individuals in society to be scientifically literate. research from the washington university human genome center in st. louis, mo has identified the one million base pairs (bps) that create the human being regardless of your ethnicity. according to this research they discovered that it is less than 10,000 bps distinguish humans from one another. however, these ten thousand bps are crucial when it comes to science. if science only focuses on one group of people in their research or just one gender, white males, then the ills of individuals from other groups will not be addressed. we cannot assume a “one-size fits all” approach when it comes to science and until we diversify our research, science research we will never solve the problems that are destroying our world on a daily basis. as a science educator, i am committed to shaping the mindset of my students in my courses by raising their awareness on how we have to cause a paradigm shift in education. the educational system for historically underserved and marginalized students is currently ineffective, even though their schools are filled with highly qualified predominately white female teachers that are ill prepared to work with the population of students they serve. this flawed system has produced a prison pipeline, 16• fine focus, vol. 4(1) students that are hopeless and teachers that are leaving their classrooms after only ten weeks, due to fear. conversely, the programs i have designed train the teachers how to teach science using an inquiry approach to underserved and marginalized students and also train the teachers love and respect their students. in return these teachers receive love and respect from their students that can do science and love doing it with qualitative and quantitative evidence. the biggest attribute of this program is the diminishment of the fears that the bsu students held at the beginning of the semester, and the confidence and competency they gained in teaching science, making them change agents in the schools and communities they will inhabit. copyright 2018, fine focus all rights reserved faculty perspective“rebuildetroit and fine focus collaboration to foster sucessful stem education opportunities stephanie conant, ph.d associate professor & chair, department of biology, university of detroit mercy 155 156 • fine focus, vol. 4 (2) overview of the nih rebuildetroit program the building infrastructure leading to diversity (rebuildetroit) nih grant was awarded in 2014 as a consortium-wide effort to expand and develop avenues for students from underrepresented and socio-economically disadvantaged backgrounds to experience an undergraduate education designed to expose them to biomedical research and career opportunities in stem fields. the consortium consists of faculty and staff from the university of detroit mercy (udm) and wayne state university, with the overarching goal to provide collaborative academic, pedagogical, and research-based opportunities for students that are accepted as rebuildetroit scholars. rebuildetroit is one of only 10 nih-funded build awards in the country and has been an exciting and transforming opportunity for both students and faculty across the consortium. the focus and timing of this grant support aligns nicely with the re-envisioning and revitalization of detroit as a hub for urban invention and opportunity. benefits of the nih rebuildetroit program at udm and consortium institutions the components of this nih funded program for students and faculty at all institutions are numerous and diverse but include a pre-college summer enrichment program, cross-institutional collaborations, curricular re-design, and faculty-mentored research opportunities for rebuildetroit scholars as soon as the summer after their first year. in addition, udm students are provided stipend and tuition support and intensive mentoring to help them navigate the academic and personal struggles that may come as they progress through their undergraduate education. educational opportunities and research based pedagogy is available to scholars interested in one of three areas of research: biology, chemistry, and health disparities. rebuildetroit scholars begin their first year with courses designed to provide hands-on authentic research experiences in the classroom that give them the technical and theoretical skills to approach learning and data collection with critical thinking, persistence, and confidence. students are matched to research mentors in their summer after their first year and begin a collaborative relationship with a research mentor where they are able to further develop the skills they have learned in their research based courses and begin their transformation into productive and prolific research scientists. intensive mentoring, cohort building, leadership roles, and exposure to opportunities for these students in the different areas of science support academic and personal goals as well as open students’ eyes to the wealth of career possibilities this unique undergraduate educational experience can lead to. all of this on top of a traditional degree-granting curriculum in their academic department of choice means that the rebuildetroit scholars are immersed in a unique and supportive environment to guide them through the trials and tribulations of the college experience all the while giving them a great opportunity for experience and growth as future research scientists. over the last four years udm and its rebuildetroit scholars have learned a tremendous amount. exposure to the process of how to choose a mentor that fits your learning style and goals, how to think like a scientist, and how to identify as a contributing member of a research team are new ideas and objectives for rebuildetroit scholars. these skills are a model to ultimately build and sustain diversity in biomedical and health research professions in detroit and around the country. faculty have been provided mentoring training and experiences, pedagogical training for course-based undergraduate research experiences (cures), as well as professional training and guidance for advising and working through unique issues that students from diverse and disadvantaged groups may bring to their undergraduate education. these types of personal and professional development opportunities disseminate throughout the student body at faculty perspective • 157 our university and make for stronger and more effective faculty to serve our students. students in the rebuildetroit program at udm have met with leading scientists and researchers in many fields of study from around the world. they have traveled to and presented at scientific conferences in michigan, california, georgia, louisiana, and washington dc, just to name a few. these travel and presentation opportunities are few and far between in a traditional undergraduate education so all academic institutions participating in rebuildetroit are excited and humbled by the opportunity to expose undergraduate researchers to as many avenues of growth as possible. participation in the rebuildetroit nih program has therefore expanded student horizons and their view of possibility. udm rebuildetroit/fine focus collaboration when we at udm were approached by dr. john mckillip, faculty mentor and managing editor of ball state’s fine focus publication, to collaborate on a project designed to explore and comment on diversity and its role in undergraduate educational opportunities in stem fields, along with providing an opportunity for udm rebuildetroit scholars to gain experience in the process of double-blind peer-review and assembling a scientific publication we jumped at the opportunity. initially the collaboration was most attractive as an avenue to expose young scientist in training at udm to another aspect of being successful in biomedical research, peer-review and publishing of scientific manuscripts. scholars have participated in generation and analysis of data, and even presentation of data in a conference setting however they had not delved into the process of getting that data published in a peer-reviewed publication; a hallmark of biomedical research success. ten of our highly motivated rebuildetroit scholars, ball state student editors, staff, and faculty from both udm and ball state met four times over the course of the fall 2017 semester for intensive manuscript review sessions and student interview opportunities. the fine focus team of students interviewed and recorded personal accounts of success and struggle that our udm scholars have experienced in their pursuit of a career in the biomedical sciences. these candid and wellthese candid and well-structured interviews drew out the most honest and humbling accounts of persistence, self-awareness, and tenacity from our udm scholars and opened even our eyes here at udm to the potential that our students possess. what we found when our two schools provided these opportunities for student interactions was a deep reflection from students from both universities as to what has led them to scientific fields of study, what challenges they have faced, and how they have overcome these challenges and walked away with a better understanding of themselves and the strength it takes to reach the goals they have set for themselves both personally and professionally. student perspectives published in the spring 2018 issue (finefocus. org) label this collaboration as “insightful”, “enjoyable”, and “fullfilliing”. these descriptions, i believe, hold true for faculty and staff involved as well. as a faculty member advising and guiding rebuildetroit scholars through academic programs and research opportunities i am thoroughly impressed and proud of the mature and open attitude that all students demonstrated throughout this collaboration. students, faculty, and staff from both universities were able to share meals, stories, goals, and a lot of laughs over the course of these campus visits leading to a bond and respect that we at udm hope will continue for years to come. we at udm believe that opportunities such as this collaboration with the fine focus team and others like it show that the mission of the rebuildetroit grant and collaborative opportunities such as this one are vital to the future of science education and providing quality professionals in all areas of stem. copyright 2016, fine focus all rights reserved perspective“undergraduate perspectives from the fine focus student team, fall term 2015, university of akureyri, iceland as an undergraduate science student, i feel like i have the constant urge to expand my knowledge in every possible way. with fine focus i’ve reached an unknown area of science, the other side. when referring to the other side, i’m talking about the process of how things are handled after completing the actual experiments; that is, submitting a paper, based on those experiments and up to the point where it is accepted or rejected. having said that, i’ve gotten the opportunity to understand the amount of work spent by each scientist out there performing experiments that leads to the expression of their thoughts and discoveries in articles, which then go through the exhausting review process. after being a part of fine focus, i feel like i’ve realized how much mutual respect is among people from each of these sides, where the scientist depend on, and trust the reviewers to judge their work with a positive perspective. at the same time, reviewers depend on the scientists to do their work in such a manner that their free time is not poorly spent. with that new realization, i also feel like my future work as a scientist, or a reviewer, will be better spent since i now know that genuine people out there are doing the same things. i also really believe that these people do their job with good intentions, to make science accessible. fine focus has not only expanded my knowledge on and respect for people in journalism but it has also opened an expression frame for undergraduate students to publish their work, which otherwise might be lost in the chaotic field of science. thank you fine focus, and last but not least, thank you john mckillip. i didn’t know much about the fine focus journal until i started the class, although i knew it was a journal for undergraduate students. i decided to take the fine focus class because i figured, since i plan on submitting papers in the future, i was able to understand the process of journal submissions and principles of how to write a good paper. the fine focus class has taught me a lot! i was not aware of the process of journal systems, when journals receive manuscripts or papers until it gets submitted, resubmitted or rejected, i had questions like; “how can i submit a paper?”, “who decides whether my paper gets submitted or rejected?” and “am i allowed to resubmit?”. this class has allowed me to be a part of this birta líf fjölnisdóttir biotechnology pálína haraldsdóttir biotechnology 66 • fine focus, vol. 2 (1) this course has opened up for me the importance of cooperative working and international communication in a world of rapid change in science. the launching of this many-sided fine focus project is an intriguing initiative. it is a healthy and meaningful way of schooling undergraduates in scientific writing, reading and reviewing a paper. you are reminded of the importance of keeping up with the latest published material. the course is probably a reflection of how things work out in the field, a combination of teamwork and taking on different tasks with feedback from other scientific milieus. when selecting three persons from an external worldwide editorial board, i came to realize, being a student of fisheries science, the various subdisciplines of microbiology and how they can relate to other expertise, even outside of biology. as a product-based course it challenges our integrity with being held responsible as lead editors for one paper throughout the whole scenario from submission to decisionmaking, publishing and promotion. you have to work professionally from start and have your research and writing support each other along the process. this is good training in how to summarise the content of a paper. peer reviewing taught me the importance of clarity. it all revolves around the hypothesis when structuring a research paper. you adjust to the language and it is vital for your career to learn from start the rules of ethics, to handle citations and sigríður kristinsdóttir fisheries science ““[fine focus] is a healthy and meaningful way of schooling undergraduates in scientific writing, reading and reviewing a paper whole process, from receiving a manuscript or paper until the final decision has been made. this takes a lot of time and hard work for editors, reviewers and everybody involved. i think each and every person in this class has improved the fine focus journal in many ways, by spreading our knowledge, opinions and suggestions. i highly recommend students to take the fine focus class, it is fun, instructive and improves your writing in so many ways. in addition, i think the fine focus journal has great potential and will become a platform for many undergraduate students who are taking their first step into the world of scientific publication. perspective • 67 references with respect. it also puts focus on originality and how good work adds to the body of knowledge. i am more familiar with how to rate a paper for publishing, the scope of journals, their policies, rate of rejections, regulations and checkboxes. rejection of a paper is something scientists will have to learn to live with, and we had to rationalize our final decision on student papers as for what to improve. the situation of this student group is probably distinct from others in this field of study, with exchange students and people of different ages, some with previous work experience. my special contribution to the journal was updating the website. despite my presence through skype, with a few burls, being a distance student, i felt amazing as part of the group. john is definitely the right person to run the complicated tasks of this course and keeping up the spirit. fine focus is a great opportunity for me to get to know a real experience with marketing plans and raising funds. the big advantage is the fact that everything is real and not a training of hypothetical situations. fine focus is an unique opportunity for improving my soft skills, too. i used my psychological and journalistic knowledge for creating a website called first giving for potential donors, and for improvement of our existing marketing plan. it was also helpful to share information in the classes; we have made many useful decisions, thanks to brainstorming. as i am not a microbiology student, i could not develop the ideas regarding this particular field, but i appreciated interesting discussion about this topic as well as proofing the manuscripts, with interesting microbiological information. it has opened the doors to the new world. also, i liked taking photos for the first giving website in a lab because students showed us their tools in examining microbes. i liked that the lessons were open discussion and a friendly atmosphere. i hope our effort, when we tried to improve the marketing and funding issue, will provide background and better conditions for student’s work and manuscripts. i really like the fact that fine focus is a real journal where i can practice my knowledge. as it is not only theoretical schema, everything that i did was discussed and i could see the changes regarding fine focus during this semester. i learned something from marketing and from the system of getting donations, as well as the steps which are necessary for success in this area. i find interesting the communication with administrators of the first giving site, too. finally, we could see our result and we can wait what new our effort will bring to fine focus. i am glad i could be a part of the fine focus team. hopefully we did something worthwhile! kristýna tronečková journalism/psychology 68 • fine focus, vol. 2 (1) my involvement in the fine focus course has been rewarding and fun. before i signed up for this course, i didn’t know much what i was going into and i was not sure if this course would benefit me and if it would be useful to my study. but really soon i changed my mind. it was really fun to be a participant and learn how the real peer-reviewed journal is produced from start to finish. the fine focus course has also helped me a lot to step considerably outside my comfort zone. i was trained in critical thinking, i improved a lot reading english scientific articles and my english vocabulary has increased considerably. i think without a doubt that this course will be useful to me in many ways after my graduation. in my opinion, a subject like this should be taught in almost all courses that revolve largely about writing and publish articles and it would be a good preparation for students when they start working. i hope that my contribution will help so we will publish a good and wellmade peer-reviewed journal, by helping review articles and other important projects within this course. there was also a large part of the course devoted to marketing and presentation of the journal, and it was very informative, though i was not directly involved in that part. i hope this course will thrive and grow with each period and i look forward to watching this project develop for the better. i am a third-year international business student, and being part of a group of student editors of fine focus has been an engaging way to relate my theoretical studies to real life, as well as to learn more about the world of microbiology, which used to be like an undiscovered land for me before. through fine focus, my interest towards working in multinational teams, challenging myself and gaining experience from multiple fields, such as microbiology, has increased notably. during fine focus autumn 2015, i was mainly responsible for improving the marketing plan to increase the awareness of fine focus, especially in europe, in order to increase the visibility of the journal and thereby the number of submitted undergraduate papers. i enjoyed the most the process of innovative thinking for bringing in fresh ideas for improving the marketing of fine focus, knowing that out of which eventually the most creative ideas would be selected and applied to the actual marketing plan after discussing as a group with the rest of the student editors. fine focus has not only been a truly sigþóra brynja kristjánsdóttir biotechnology saara-maria kallio international business perspective • 69 interesting course completed as a part of my international studies, but also it will be part of my life again in the near future. as a marketing student, i got offered an opportunity to represent fine focus at the british conference of undergraduate research (bcur) in march 2016 in the uk. through this exciting opportunity i will get to develop my skills in a real world context and gain further practical experience on international marketing. i have found it truly fascinating to participate in running the development of an international journal and being part of the growth of fine focus. attending the conference in manchester will not only be a great opportunity for networking but also it will be one step closer to reaching our goals in expanding fine focus to europe. taking part in fine focus has made me better at representing scientific work, both according to structure and content. being good at representing my work will save time both for me as a writer and others that will read my work in the future. another important thing that i have learned is to make my material interesting from the beginning and let it stay that way throughout. keeping material interesting is essential to the readers so they don’t loose interest and give up on reading and perhaps miss the good part of the paper. i have also improved my skills in recognising a good paper, not only a well-written one but also one that involves good scientific practises. recognising a good paper is critical when searching for good information on given topic. learning how the peer review process works in detail, from submission to publication, is a valuable lesson and will make me more confident when publishing in the future. participating in this course also gave me a glance of both marketing and graphics, two fields that i don’t know much about but are highly connected to the scientific world. for example, it’s useful to know how to promote my work after it has been published. overall, participating in fine focus has given me valuable insight into the scientific world of representing and publishing data, an insight that, unfortunately, many undergraduates don’t get. helga helgadóttir biotechnology ““ fine focus has given me valuable insight into the scientific world of representing and publishing data, an insight that, unfortunately, many undergraduates don’t get. 70 • fine focus, vol. 2 (1) what if the next antibiotic is in your backyard? post/biotics is a platform that facilitates finding new antibiotics by integrating citizen scientists in the process of identifying natural substances with antimicrobial properties. opportunity most known antibiotics are developed from natural extracts, implying a potential new antibiotic could be anywhere. post/biotics distributes empirical research to the public by creating a platform and the according tools for participating in this process. the pop-up lab allows anyone to discover substances with antimicrobial values by testing locally available plants, vegetables, fruits, fungi, mushrooms & soil. with the help of the corresponding app, the results are stored on an online platform to create a library of antimicrobials. the value of this “microbial fingerprint” of our environment is significantly important for developing new drugs. by partnering with universities & pharmaceutical companies, the massive costs for finding new drugs can be diminished when giving access to this platform. impact the coming cost of antimicrobial resistance is 10 million deaths per annum by 2050. this is estimated to cost about 3% of the global gdp. antibiotic resistance is growing faster than pharmaceutical companies are finding new ones. corporate social responsibility of a pharma company can largely benefit from an educational initiative that promotes growth of young minds in stem careers in research. method users test their samples like a crushed bark of a tree in their backyard with the toolkit that contains e.coli bacteria. if the tested tree bark has antimicrobial properties, it will be recognized by the platform and the user is encouraged to submit the original platform. using gamification, users are incentivized to test different samples, geographical areas, and to use their peers for verifying their findings. 2015 © copyright post-biotics. all rights reserved. vidhi mehta theresa schachner www.post-biotics.com find more about post/biotics on bbc, wired, fastcompany & sciencealert vision: post/biotics is a vision for revolutionizing drug discovery and raising awareness for the problem of antibiotic resistance. frequency of antibiotic residues in a central wisconsin dairy morgan l. beilke and jeffery d. fritz* department of biological sciences university of wisconsin – marshfield/wood county marshfield, wi usa copyright 2016, fine focus all rights reserved manuscript received 27 july 2015; accepted 2 september 2015 16 • fine focus, vol. 2 (1) antibiotics, used to maintain healthy dairy animals, persist in milk produced by treated animals for several days after therapy. antibiotic residues, if present in the milk supply, negatively impact suitability for processing and consumption. this is an area of concern in the dairy industry and for the general public. this study explores the incidence of antibiotic residues in milk provided to a central wisconsin dairy. random, unidentified samples were obtained over a four month period from producers located within a 100-mile radius of a dairy located in granton, wisconsin. all samples were tested within one week of their receipt, and maintained at refrigerated temperatures at all times prior to testing. following testing, the origin of the samples was revealed. samples were derived from two milk populations: those intended for human consumption (bulk milk), and those from cows under surveillance following antibiotic treatment (milk removed from the commercial supply). all bulk milk samples tested negative for over 55 different antibiotic residues, while all samples from the surveillance population tested positive for antibiotic residues. our results are consistent with those observed nationally, and suggest that dairy producers recognize the ongoing concern of antibiotic contamination in the milk supply and are taking steps to prevent antibiotic contamination in milk. our findings also suggest the public should have minimal concern with respect to antibiotic residues in the commercial milk supply. abstract corresponding author *jeffery d. fritz jeffery.fritz@uwc.edu keywords • antibiotics • drug residues • dairy foods • food safety • bacillus stearothermophilus var. calidolactis antibiotic use on dairy farms is imperative to treating ill cows. use of antibiotics is most common in the treatment of mastitis, a painful inflammation of the cow’s udder (8). there are approximately 40 drugs approved for use in lactating dairy cows (14). once an ill cow is treated, the antibiotics may be retained in the cow body for several days. this makes it important for producers to test regularly for drug residues in treated cows. generally, all antibiotics should be excreted from milk within 72 hours of treatment (7). however, if not used properly, antibiotics can be retained in the cow for longer periods. the food and drug administration surveys indicate that improper use of drugs in the introduction applied/environmental • 17 milk samples were obtained from lynn dairy inc. (located in granton, wisconsin), between march 09th, 2015 – june 07th, 2015. at the time of collection, samples were identified through a six-digit number with no indication of milk supplier or type of milk. milk suppliers for these samples spanned a 100-mile radius from the dairy plant where milk samples were processed. samples were received in batches of 25 and each batch was tested within a week of receipt. prior to testing, samples were stored in a refrigerator below 6°c. materials and methods control of mastitis is the major source of antibiotic residues found in milk supply (10). if present, antibiotic drug residues in our milk supply pose a public health problem. specifically, the consumption of milk contaminated with antibiotics can detrimentally influence our ability to treat human infections, because infectious agents adapt to the continual exposure of drugs (12). further, for those individuals who are already sensitized to antibiotics, overuse of these antibiotics in dairy cows could result in allergies if these products make their way to our local grocery stores. for example, penicillin is the most frequent human drug allergy and is common affecting approximately 10% of the population with anaphylaxis in 0.01% and fatal reactions in 0.0015% of cases (1). most concern with antibiotic persistence is placed on the development and spread of virulent and resistant bacteria within the dairy animal. these antimicrobialresistant pathogens may emerge in the food production chain, and can be transmitted to humans, and cause infection. salyers and shoemaker outline the potential concern of the ability of bacteria to exchange resistance genes through their exposure to the intestinal tracts of consumers, as well as their potential to interact with any pathogenic bacteria in passage through the human colon (13). human exposure to resistant bacteria from non-human usage of antibiotics can create new, more severe, infections that would not have otherwise occurred, and cause an increased frequency of treatment failures (5). because the foodborne route is a major transmission pathway for bacteria and resistance genes (5), as well as antibiotic residues themselves, understanding farming practices relating to managing and treating ill cows becomes relevant to our everyday lives, especially for those purchasing commercial milk and milk products. consequently, educating the public on milk sanitation practices and the safety of our nation’s milk supply is an important area of research. our investigation was aimed at exploring the possible public health issue of antibiotic persistence in milk products through the collection and testing of raw milk samples collected from a dairy in granton, wi. current national findings place the frequency of antibiotic persistence in the commercial milk supply below 0.015% (14-16), and suggest that the public, at present, should have minimal concern regarding antibiotic residues when consuming commercial milk products. we hypothesize that the commercial milk supply contains very little (and likely undetectable levels) antibiotic residues. 18 • fine focus, vol. 2 (1) a total of 268 samples were tested for antibiotic residues using dsm’s delvotest sp nt, a simple and reliable test that detects over 55 different types of antibiotics including beta lactams, sulphonamides, aminoglycosides, quinolones, macrolides and tetracyclines (4). the delvotest incubator allows for 10 ampoules to be tested at a time. in this study, 9 milk samples and 1 control sample of penicillin were tested each time. separate pipettes were used to add 0.1 ml of each milk sample into the designated ampoules. each ampoule was marked with the six-digit sample number provided at time of collection for sample identification. individual pipettes were dipped 1 cm into the sample which was then transferred onto the agar medium in the designated ampoule. the incubation temperature was set at 64 degrees celsius and testing took approximately 3 hours (2). the test was read through analysis of the solid and buffered agar medium containing a ph indicator and a test organism, bacillus stearothermophilus var. calidolactis (3). a purple/blue reading implies the reduced growth of the test organism, and therefore a positive test for antibiotics in the raw milk. a green/yellow test implies the growth of the test organism, and therefore a negative test result for antibiotics in the sample. following testing, milk sample numbers were identified as originating from bulk milk samples or milk from animals under surveillance for antibiotic residues. the bulk milk samples total 264 of the 268 samples tested negative for antibiotic residues. the remaining four samples were obtained from animals under surveillance for antibiotic residues and all four of these samples tested positive for antibiotic residues (table 1). results table 1. antibiotic residue analysis of milk samples in this study source of sample total samples number positive percent positive disposition per pounds bulk milk pickup tanker 3,147,302 429 0.014% 17,754,000 pasteurized fluid milk and milk products 37,707 0 0.000% 0 producer 445,223 266 0.060% 240,000 other 49,953 8 0.016% 99,000 applied/environmental • 19 bulk tank samples all 264 samples originating from bulk milk samples were those that were deemed fit for bulk tank inclusion by the dairy producer, and thus added to the truckload. however, prior to acceptance, dairy processors will test the truckload once it arrives at the processing establishment as a precautionary measure. if the bulk tank is negative for antibiotic residues, it is presumed that the tank is antibiotic free and safe to use. if a bulk tank is positive for antibiotic residues, the truckload will be dumped. additionally, individual bulk, raw milk samples from the truckload will be tested in order to determine the cause of contamination. in our study, all samples deemed fit for bulk tank inclusion were completely antibiotic free. non-bulk tank samples from animals under surveillance next, it is important to note that the four positive test results observed in our study were not considered “true positives” for antibiotics as these milk samples were not intended to be included into the bulk tank by the dairy producer. specifically, milk producers may send individual milk samples from treated cows to milk processors for antibiotic testing. this precautionary routine is used to see if the drug still persists within the treated cow’s system (6). as it is unlawful to place unfit milk on the market for human consumption, this primary screening method is integral in determining whether the milk is fit for inclusion in the bulk tank (6). importantly, these samples are relevant to our data as it provides evidence that milk producers are exercising safe milk sanitation practices by monitoring treated cows and separating their milk from the central milk supply. conclusions our findings are consistent with surveys from the food and drug administration’s annual report on national milk drug residues. the national survey under the fda has been conducted since 1994, and illustrated the findings of bulk milk antibiotic testing over the course of 20 years (16). these surveys show a peak in 1996 with 0.1% of bulk milk samples testing positive for antibiotic residues to its current low of 0.014% in 2014 (16). the most recent fda survey was conducted from october 1, 2013 to september 30, 2014 (16). a total of 4,008,662 tests were conducted, consisting of nine different groups of individual drugs or different drug families. a total of 3,680,185 samples were collected and analyzed for animal drug residues. of those samples, only 703 were positive for a drug residue. the majority of samples (3,147,302) were samples from bulk milk pickup tankers of raw milk. only 429 samples, or 0.014%, tested positive for a drug residue. importantly, no antibiotic residues were found in samples that came from pasteurized fluid milk, or samples considered “retail-ready” (16). this would suggest that for our study, over 7000 bulk milk samples from our dairy would need to be surveyed before a single positive test would be likely. the national milk drug residue database is a voluntary industry reporting program that identifies the following: the extent of national testing activities, the analytical methods used, the kind and extent of the animal drug residue identified, and the amount of contaminated milk (16). mandatory reporting of drug residues in milk is required under the pasteurized milk ordinance; a strict set of requirements discussion 20 • fine focus, vol. 2 (1) for milk production, transportation, pasteurization, equipment sanitation, and labeling (15). dairy farmers and processors work closely with the food and drug administration, the u.s. department of agriculture (usda), and state regulatory agencies to promote the highest safety standards for milk (9). as such, it is not surprising that we found no bulk milk samples testing positive for antibiotic residues in our study given its sample size and these control measures. reducing the use of antibiotics minimizes the likelihood of antibiotic resistant bacteria to develop, as well as drug residues to exist in the milk supply. maintaining antibiotic free milk begins with the efforts of dairy producers in implementing effective milk sanitation practices. further, it is essential that producers and milk processors build trusting partnerships that allow for effective and economically sound business relationships. preliminary screening methods for drug residues at milk processing establishments, previously discussed in this study, are critical in the continued prevention of contaminating truckloads of milk. in conclusion, our study is in agreement with national surveys, which have demonstrated that these efforts are being maintained, as our results, along with results on a national level, show extremely low incidences of antibiotic persistence in milk. ultimately, these findings should provide reassurance that milk products will continue to be safe for consumption, and that efforts in reducing antibiotic contamination are ongoing and well implemented by the industry. acknowledgments the authors are grateful to lynn dairy for donating milk samples and to nelson-jameson for technical assistance. this work was funded by an undergraduate research grant from the university of wisconsin colleges, madison, wisconsin. references 1. adelman, d. 1991. penicillin allergy. western j. med., 154:456. 2. dsm. 2011. instruction for use: delvotest sp nt. netherlands: dsm food specialties. 3. dsm. 2011. product sheet: delvotest sp nt. netherlands: dsm food specialties. 4. dsm. 2012. technical bulletin: delvotest sp nt. netherlands: dsm food specialties. 5. fao, oie and who. 2004. expert workshop on non-human antimicrobial usage and antimicrobial resistance: management options. oslo: world health organization geneva. 6. food standards agency. 2009. information and guidance on the testing of milk for antibiotic residues (pp. 5-7). london: food standard agency. 7. fox, p. 2000. fundamentals of cheese science. gaithersburg, md: aspen pub. 8. gunther, a. 2010. dairy cattle antibiotic residue review | animal welfare approved. animalwelfareapproved.org. 9. international dairy foods association. 2015. overview of fda drug residue sampling survey confirming safety of milk supply. applied/environmental • 21 10. jones, g.m. 1999. on farm tests for drug residues in milk, virginia cooperative extensions, knowledge for the common wealth, virginia polytechnic and state university, u.s.a 11. kandc-sbcc.com. 11. lederberg, j. 1997. infectious disease as an evolutionary paradigm. emerg. infect. dis., 3: 417-423. 12. salyers, a., and shoemaker, n. 2006. reservoirs of antibiotic resistance genes. animal biotechnology, 17(2), 137-146. 13. u.s. food and drug administration. 1996. milk monitoring with antimicrobial drug screening tests. washington, dc: center for veterinary medicine. 14. u.s. food and drug administration. 2007. grade “a” pasteurized milk ordinance (2007 revision). silver spring: u.s. department of health and human services. 15. u.s. food and drug administration. 2015. national milk drug residue data base fiscal year 2014 annual report (pp. 1-3). lighthouse point: glh, incorporated. 16. 2006. k&c national milk drug residue database. recovery and enumeration of staphylococcus aureus by the selective agar overlay method jill bange*, emily brumfield, and alysha l ellison department of biology, ball state university muncie, in usa copyright 2016, fine focus all rights reserved manuscript received 2 july 2015; accepted 3 august 2015 52 • fine focus, vol. 2 (1) staphylococcus aureus is an example of a commensal bacterium responsible for emesis, acute diarrheal syndrome, and sepsis. s. aureus often must be isolated from patient samples in a clinical setting or from food samples during food processing in an industrial setting, although these bacterial cells may be injured by the human immune system or by food processing measures. therefore, injured cells may not be fully recovered on media selective for s. aureus and enumeration (e.g., cfu/ml) may not reflect the true concentration of the original sample. the objective of this study was to determine whether the selective agar overlay method of recovery is more sensitive, selective, and time-effective for enumeration of artificially injured s. aureus cultures when compared to more traditional techniques. the selective agar overlay method involves pour plating s. aureus in non-selective medium, allowing the sample to incubate for a four hour recovery period, and then overlaying selective medium over the non-selective medium. artificial injury of s. aureus cells was accomplished by treatment with carvacrol, an extract from oil of oregano. our results indicated that carvacrol-injured s. aureus cells were recovered by the selective agar overlay at the same concentration as recovery on non-selective media, and at a significantly higher concentration than recovery on selective media. this method allows for more rapid and accurate diagnoses, and may be more cost-effective due to the reduction or elimination of false negative results. abstract corresponding author *jill bange jillsbange@gmail.com keywords • staphylococcus aureus • overlay • sublethally injured • carvacrol staphylococcus aureus is a gram-positive, ubiquitous, coccoid bacterium, which is a prevalent organism in the food and clinical microbiology arenas (1,10). this commensal organism is an opportunist and an emerging pathogen for both nosocomial and community-acquired infections (6). staphylococcus aureus enterotoxins, exotoxins, and hemolysin are responsible for emesis, acute diarrheal syndrome, and sepsis in humans (6,10). in addition, methicillinresistant and vancomycin-resistant strains are troublesome because they are difficult to treat effectively (14). introduction pathogens and antimicrobial factors • 53 a variety of media can be used to grow s. aureus, using both non-selective and selective techniques. however, the human immune response as well as food processing techniques may injure s. aureus, limiting growth and detectability in lab cultures, particularly if bacterial cells are not allowed to repair. using a non-selective medium for isolating a pure bacterial culture is not warranted, as other species may grow (7). employing a selective medium alone may be an improvement, but unfortunately, cells may not repair well enough to grow visible colonies on the medium (7), and may result in a false negative for presence of bacteria in the original sample. to solve this problem, enrichment of bacterial samples in broth is needed before plating onto selective media. however, enrichment in broth will not allow for accurate calculation of the original sample’s bacterial cell concentration because only cells enriched in agar form countable colony forming units (cfu); each cfu represents one cell from the original sample. therefore, the selective agar overlay method allows isolation of a pure strain and recovery of injured cells as well as calculation of the bacterial cell concentration from the original sample (13). unfortunately, methodology on the use of mannitol salt agar (msa; hardy diagnostics, santa maria, ca) as a differential and selective medium with clinical relevance (3) in the selective agar overlay method of enumerating s. aureus has not been documented. others have found that gram-positive agar (gpa) was the most effective selective medium component of the overlay method for s. aureus (10). however, gpa is selective for all gram-positive bacteria, not just s. aureus. furthermore, the selective agar overlay method has been studied for s. aureus in the context of food microbiology but not for clinical microbiology (13). several stressors have the potential to sublethally injure s. aureus cells prior to recovery by the selective agar overlay method. carvacrol, an extract from oil of oregano, increases the bacterial cell membrane permeability to potassium, which alters potassium and hydrogen concentration gradients. this disrupts atp synthesis and leads to a decreased pool of atp available within the cell (15). carvacrol has the potential to be incorporated into the clinical arena in conjunction with antibiotics and, therefore, has been chosen as a clinically relevant stressor for this study. ultraviolet (uv) radiation injures bacterial cells by damaging dna. uv light initiates a reaction between thymine bases in dna to create a thymine dimer; two thymine bases are bonded together, which is not a normal base pairing. this mutation is stable and difficult for the cell to repair; therefore, the cell cannot transcribe its dna normally and a loss of function occurs (11). uv light is used as a disinfecting measure in clinical settings and is, therefore, considered a clinically relevant stressor. hydrogen peroxide acts at the bacterial cell membrane as an oxidizer, which would alter the activity of certain bacterial membrane proteins (9). hydrogen peroxide produced by immune cells can be used to combat bacterial infection; therefore, hydrogen peroxide is relevant in the clinical arena. by stressing s. aureus cultures with carvacrol, uv light, and hydrogen peroxide, a stressor which is most useful for testing the selective agar overlay method can be chosen. in order to be effective for this assay, the selected stressor needed to sublethally injure cells and therefore produce significantly lower cell counts on non-selective media than counts for non-stressed cells. this stressor also needed to allow for consistent 54 • fine focus, vol. 2 (1) recovery of enough injured cells in order to conduct reliable statistical analyses. the goal of this study was to develop a time-efficient selective agar overlay method of enumerating s. aureus which is sensitive and selective in the clinical arena. the objectives of this study were: 1) to show the selective agar overlay method is significantly more sensitive to enumerate s. aureus than plating directly onto selective media, such as msa, and 2) to show the selective agar overlay method is equally as sensitive, but more selective than plating s. aureus onto non-selective tryptic soy agar (tsa; teknova, hollister, ca). we hypothesize that if selective overlay plating (tsa-msa) allows for recovery of injured s. aureus and selects for s. aureus, then selective overlay cfu/ml will be significantly higher than direct selective plating (msa-msa) cfu/ ml alone and comparable to direct nonselective plating (tsa-tsa) cfu/ml. bacteria staphylococcus aureus strain atcc baa977 was obtained from microbiologics (st. cloud, mn). cultures were incubated in tryptic soy broth (tsb; alpha biosciences, baltimore, md) for 24h at 37°c. optical density (od) was measured as 0.46 using a beckman coulter du 530 spectrophotometer at 550 nm (beckman coulter, brea, ca). all laboratory work was performed at ball state university. bacterial injury staphylococcus aureus cells from tsb cultures were serially diluted 1:100 in sterile saline before undergoing stress treatments. three stressors were tested: carvacrol (safc supply solutions, st. louis, mo), uv light, and 3% hydrogen peroxide (vwr international, llc, radnor, pa). our intent was to find a stressor that caused injury, but did not completely kill all cells. carvacrol was added to the 10-2 diluted s. aureus cells at a volume of 100 ul, and these cells incubated for 5 min at room temperature. serial dilutions up to 10-6 cfu/ml were then performed on the carvacrol-injured cells to determine efficacy. injury by uv light was achieved by pouring cells from the 10-2 dilution to a layer of 5 mm thick into two separate, sterile petri dishes. this allowed for injury by uv light for either 60 s or 90 s at room temperature, with agitation at every 30 s. three percent hydrogen peroxide was added to the 10-2 diluted s. aureus cells at a volume of 5 ml, and these cells incubated for 5 min at room temperature. serial dilutions up to 10-6 cfu/ ml were then performed on the hydrogen peroxide-injured cells. stressor analysis carvacrol-injured, uv-injured (60s and 90s), hydrogen peroxide-injured, and 10-6 serially diluted non-injured (control) s. aureus cells from tsb cultures were pour plated with non-selective media (tsa) in accordance with methods used by leboffe and pierce (8). plates were incubated 4 h at 37°c, and then overlaid with 20 ml of tsa. plates were then incubated for 24 h at 37°c. each of the five treatment groups was inoculated in triplicate. colonies were counted and converted to cfu/ml. materials and methods pathogens and antimicrobial factors • 55 media analysis three base layer-top layer test media combinations were used in this study: non-selective (tsa-tsa), selective (msamsa), and selective overlay (tsa-msa). non-selective plates and selective overlay plates used tsa as the base layer medium for pour plating, while selective plates used msa as the base layer (fig. 1). non-injured s. aureus cells from tsb cultures were serially diluted to 10-6 cfu/ml and pour plated with the base layer of each test media (nonselective, selective, and overlay) and labeled. carvacrol-injured s. aureus cells were pour plated at a 10-6 dilution with the base layer of each test media (non-selective, selective, and overlay). all plates were incubated 4 h at 37°c. plates were then overlaid with a top layer of 20 ml of either tsa (nonselective plates) or msa (selective overlay and selective plates). plates were then incubated at 37°c for an additional 24 h after which colonies were counted and converted to cfu/ml. each test media group for both non-injured and carvacrol-injured treatments was inoculated in triplicate. statistics two sets of statistical analysis were run. first, recovered s. aureus cfu/ml (response variable) were compared by a general linear model to determine how they were impacted by the four stressors and control (predictor variables). once the most appropriate stressor was identified (i.e., carvacrol), s. aureus was subjected to the stressor and grown using non-selective, selective, and selective overlay media (predictor variables). second, comparison of s. aureus cfu/ml (response variable) was done using a general linear model and a tukey’s honest significant difference test (minitab 17, state college, pa). significance was set at p = 0.05. bacteria from a non-injured or injured treatment were pipetted into an empty, sterile petri dish the first layer of media was poured and all plates incubated at 37°c for 4h. this allowed for recovery of injured cells. 3x for each treatment tsa-tsa tsa-msa msa-msa the second layer of media was poured. all plates incubated at 37°c for 24h. figure 1. schematic of the methods used for recovery of staphylococcus aureus cells by nonselective (tsa-tsa), selective (msa-msa), and selective overlay (tsa-msa) plating techniques. 56 • fine focus, vol. 2 (1) concentration of recovered staphylococcus aureus cells on non-selective media (tsatsa) varied depending upon the stressor type used (glm, f = 445.81, n = 15, p < 0.001). hydrogen peroxide-injured cell recovery was not statistically different from non-injured recovery, but was higher than carvacrol and uv-injured cell recovery. carvacrol-injured were recovered at significantly lower concentrations than non-injured cells, but were recovered at significantly higher concentrations than uvinjured cells. uv-injured cells for both 60 and 90 seconds of exposure were recovered at significantly lower concentrations than all other treatment groups (fig. 2). differences were shown in the recovery of carvacrol-injured s. aureus cells based on the media type. cells were recovered from selective media at significantly lower concentrations than non-selective media and selective overlay (glm; f = 11.90, n = 9, p = 0.008). carvacrol-injured s. aureus cell recovery from selective overlay was not significantly different than recovery from non-selective media (fig. 3). results c on ce nt ra tio n (10 7 c fu /m l) non-injured hydrogen peroxide carvacrol uv, 60 sec uv, 90 sec 30 25 20 15 10 5 0 a a b c c figure 2. staphylococcus aureus density (cfu/ml) recovered on non-selective media (tsatsa) for five stressor treatments: non-injured, carvacrol-injured, hydrogen peroxide-injured, uv-injured for 60 sec, and uv-injured for 90 sec. bars are 2 standard errors from the mean. letters indicate significant difference as indicated by tukey’s honest significant difference test. we hypothesized that using a selective agar overlay method would result in more accurate enumeration of viable cell counts of s. aureus compared to direct plating onto a selective agar. as hypothesized, the recovery of injured cells via the selective agar overlay method was comparable to recovery on nonselective media, but greater than recovery on selective media. we intended to choose a stressor which would result in significantly less recovery of s. aureus cells on non-selective media than non-injured cells, and would allow for high enough recovery to ensure reliable colony counts. hydrogen peroxide and uv light were not chosen as stressors for assessing the selective agar overlay because they did not meet this requirement. hydrogen peroxide treatment allowed for recovery of enough cells to perform statistical analyses, but did not stress cells significantly from control, which is a necessity for ensuring the presence of sublethally injured s. aureus. uv-injured cells were recovered at a significantly lower concentration than non-injured cells, but viable cell counts from cells treated with this stressor were too low (fewer than 100 colonies per plate) to ensure consistency upon replication of the experiment. carvacrol significantly stressed s. aureus cells compared to control, but not discussion c on ce nt ra tio n (10 7 c fu /m l) tsa-tsa tsa-msa msa-msa 17 16 15 14 13 12 11 10 9 a a b figure 3. carvacrol-injured staphylococcus aureus concentration (cfu/ml) recovered on non-selective media (tsa-tsa), selective overlay (tsa-msa), and selective media (msa-msa). bars are 2 standard errors from the mean. letters indicate significant difference as indicated by tukey’s honest significant difference test. pathogens and antimicrobial factors • 57 acknowledgments to the extreme degree as seen in the uv treatments. in recent studies, carvacrol has been used to cease the synthesis of proteins that are important in bacteria such as escherichia coli (2). therefore, the selection of carvacrol to sublethally injure s. aureus was ideal for determining the effectiveness of the selective agar overlay method. the selective agar overlay assay was effective because recovered carvacrolinjured viable cell counts on the selective agar overlay were not statistically different from non-selective counts. in addition, the selective agar overlay counts were statistically higher than selective media cell counts. this indicates that the four hour recovery period in the selective overlay method promotes sensitivity. our findings and others (12) support the conclusion that the selective agar overlay method does allow for sensitive recovery of injured cells in the same manner that tsa would, but the overlay method does not compromise the selectivity of msa for s. aureus. this was concluded because the recovered cfu/ ml for the overlay method are comparable to non-selective cfu/ml but higher than selective cfu/ml. the selective agar overlay method is a relevant detection method for the clinical microbiology arena. this assay is more sensitive than direct selective plating, but maintains the selective and differential nature of selective media. these traits together minimize the potential for false negative diagnoses of s. aureus infection and allow for effective enumeration of s. aureus in samples with multiple bacterial species. the selective agar overlay assay is also time-efficient, which is necessary to expedite detection of s. aureus cells in patients and to prevent long-term damage resulting from illness. this may reduce financial burdens that long-term hospital stays can place on patients and families. the selective agar overlay method has advantages compared to current clinical methods of s. aureus isolation, which are direct plating on selective media containing antibiotics and multiplex polymerase chain reaction (pcr; 4,5). as shown in our results, recovery of s. aureus on selective media results in lower enumeration compared to the original sample. multiplex pcr is both rapid and sensitive, but it is extremely expensive and is a difficult technique to execute. the selective agar overlay method is inexpensive and can be performed correctly with minimal training. future studies may use alternative methods of stressing cells, including higher concentrations of hydrogen peroxide or less exposure time to uv. additional in vitro and in vivo studies, using clinical samples and/or animal models, are required before application of this assay to a clinical setting. we thank dr. thomas lauer for guidance and support in manuscript writing and editing. we also thank dr. john mckillip for assisting with experimental design and for supplying materials. in addition, we acknowledge the ball state university department of biology for funding the project. 58 • fine focus, vol. 2 (1) references 1. bennett, s.d., walsh, k.a., and gould, h.l. 2013. foodborne disease outbreaks caused by bacillus cereus, clostridium perfringens, and staphylococcus aureus– united states, 1998-2008. clinical infectious diseases 57:425-433. 2. burt, s.a., zee, r., koets, a.p., graaff, a.m., knapen, f., gaastra, w., haagsman, h.p., and veldhuizen, e.j.a. 2007. carvacrol induces heat shock protein 60 and inhibits synthesis of flagellin in escherichia coli o157:h7. appl. environ. microbiol. 73:4484-4490. 3. campos, g.b., souza, s.g., lobão, t.n., da silva, d.c.c., sou, d.s., oliveira, p.s., santos, v.m., amorim, a.t., farias, s.t., cruz, m.p., yatsuda, r. and marques, l.m. 2012. isolation, molecular characteristics and disinfection of methicillin-resistant staphylococcus aureus from icu units in brazil. new microbiologica 35:183-190. 4. fang, h., & hedin, g. 2003. rapid screening and identification of methicillin-resistant staphylococcus aureus from clinical samples by selective-broth and real-time pcr assay. j. clin. microbiol. 41:2894-2899. 5. francois, p., pittet, d., bento, m., pepey, b., vaudaux, p., lew, d., & schrenzel, j. 2003. rapid detection of methicillin-resistant staphylococcus aureus directly from sterile or nonsterile clinical samples by a new molecular assay. j. clin. microbiol. 41: 254-260. 6. kadariya, j., smith, t.c., and thapaliya, d. 2014. staphylococcus aureus and staphylococcal food-borne disease: an ongoing challenge to public health. biomed res. int. 2014, 1-9. 7. kang, d.h., and siragusa, g.r. 1999. agar underlay method for recovery of sublethally heat-injured bacteria. appl. environ. microbiol. 65:5334-5337. 8. leboffe, m.j. and pierce, b.e. 2015. microbiology: laboratory theory and application. morton publishing company, edgewood, co. 9. linley, e., denyer, s.p., mcdonnell, g., simons, c., and maillard, j.y. 2012. use of hydrogen peroxide as a biocide: new consideration of its mechanisms of biocidal action. j. antimicrob. chemother. 67:1589-1596. 10. sandel, m.k., wu, y.-f.g., and mckillip, j.l. 2003. detection and recovery of sublethally-injured enterotoxigenic staphylococcus aureus. j. appl. microbiol. 94:90-94. 11. sinha, r.p., and hader, d.p. 2002. uv-induced dna damage and repair: a review. photochem. photobiol. sci. 1:225-236. 12. speck, m.l., ray, b., and read, r.b. 1975. repair and enumeration of injured coliforms by a plating procedure. appl. microbiol. 29:549-550. 13. smith, a.r., ellison, a.l., robinson, a.l., drake, m., mcdowell, s.a., mitchell, j.k., gerard, p.d., heckler, r.a., and mckillip, j.l. 2013. enumeration of sublethally injured escherichia coli o157:h7 atcc 43895 and escherichia coli strain b-41560 using selective agar overlays versus commercial methods. j. food prot. 76:674-679. 14. tokajian, s.t., khalil, p., jabbour, d., rizk, m., farah, m.j., hashwa, f.a., and araj, g.f. 2010. molecular characterization of staphylococcus aureus in lebanon. epidemiol. infect. 138:707-712. 15. ultee, a., kets, e.p.w., and smid, e.j. 1999. mechanisms of action of carvacrol on the foodborne pathogen bacillus cereus. appl. environ. microbiol. 65:4606-4610. pathogens and antimicrobial factors • 59 characterization of indigenous bacterial communities in crude-oil impacted sites at obagi town, onelga, rivers state, nigeria. chioma blaise chikere*, chinyere augusta ajuzieogu, and michael chukwugoziem miller department of microbiology, faculty of science, university of port harcourt, rivers state, nigeria copyright 2016, fine focus all rights reserved manuscript received 26 may, 2015; accepted 9 september, 2015 hydrocarbon utilizers are expected to be indigenous in crude-oil polluted environments. the isolation and characterization of hydrocarbon utilizers is often a key strategy in bioremediation of hydrocarbon-polluted environments. in this study, crude-oil polluted soil samples from obagi town, onelga, rivers state were enumerated and characterized for putative hydrocarbon utilizing bacterial populations. biochemical characterization identified five bacterial species representative of five genera: bacillus, pseudomonas, acinetobacter, micrococcus and staphylococcus. amongst the genera of bacteria isolated, bacillus had the highest frequency of occurrence (40%). the mean count of total heterotrophic bacteria was 1.7 x 107 cfu/g, while hydrocarbon utilizing bacteria (hub) count mean density was 1.0 x 107 cfu/g for the three soil samples. statistical analyses revealed no significant difference at p>0.05 between total heterotrophic bacterial (thb) and hydrocarbon utilizing bacterial (hub) counts, suggesting that most of the bacteria present in the sampled sites were hydrocarbon utilizers. findings from this study suggest the presence of indigenous putative hydrocarbon utilizing bacteria in the crude-oil polluted soil of obagi town. hence, a promising potential exists for future bioremediation studies on the site. abstract corresponding author *chioma blaise chikere chioma.chikere@uniport.edu.ng keywords • hydrocarbon-utilizers • indigenous • bacteria • crude-oil • bioremediation one of the major environmental problems today is hydrocarbon pollution by the petrochemical industry (1), and widespread release of aromatic hydrocarbons through spillages and leakage from underground tanks and steamers, causing extensive contamination of surface soils, ground water, seas and oceans (2). mechanical and chemical methods for remediation of hydrocarbon polluted environments are often expensive, technologically complex and lack public acceptance (3). biodegradation by microorganisms is fundamental in the removal of hydrocarbons and xenobiotic substances (4). irrespective of the wealth of research relating to microbial degradation of hydrocarbons, knowledge pertaining to which organisms are the key players in hydrocarbon degradation in the environment is limited (5). introduction 8 • fine focus, vol. 2 (1) description of sample sites the study site, obagi town, is a mangrove environment whose center lies at a latitude of 5.25114 and longitude of 6.61298. surface soil from the three sample sites in obagi town were collected to enumerate and characterize bacterial isolates that have the potential for utilizing hydrocarbons. the samples were placed in sterile polyethylene bags and transported to the laboratory for analysis. enumeration of total heterotrophs and hydrocarbon utilizing bacteria one gram (1g) each of the soil samples were serially diluted (10-1 to 10-6) in 9ml normal saline. aliquots (0.1ml) from dilutions of 104, 10-5 and 10-6 of soil samples were plated in duplicate on sterile plate count agar (merck, germany) and incubated at 37oc for 24 hours for total culturable heterotrophic bacteria counts. for hydrocarbon utilizing bacterial counts, enumeration was performed as described by hamamura et al. (15) where appropriate dilutions of soil sample suspensions were plated on busnell-haas agar (sigmaaldrich, usa), and hydrocarbons were supplied through the vapour phase to putative hydrocarbon utilizers by placing sterile filter papers impregnated with 5ml okono crude oil on the lids of the inverted plates and incubated for 7 days at 37oc. purification and identification of putative hydrocarbon utilizing bacterial isolates discrete colonies of different putative hydrocarbon utilizing bacteria (hub) were randomly picked using a sterile wire loop and subcultured for purification by streaking on nutrient agar plates and incubated at 37oc for 24 hours. individual bacterial colonies were presumptively identified using morphological and biochemical tests as described in bergy’s materials and methods in order to combat this challenge, it is pertinent to first assess the hydrocarbon degrading potential of the microorganisms before any bioremediation intervention rather than just focus on the removal of individual hydrocarbon compounds via mechanical and chemical methods for remediation (6). this approach will provide new insights for improving the management of such environments. it has been observed that low molecular weight hydrocarbons like anthracene and naphthalene are usually readily degraded by bacteria in soil and under laboratory conditions (7). other studies have also shown that petroleum hydrocarbons can be degraded by microorganisms such as bacteria, fungi, yeast, and microalgae. hydrocarbon degrading bacteria and fungi are widely distributed in marine, freshwater and soil habitats (16). typical bacterial groups already known for their capacity to degrade hydrocarbons include pseudomonas, marinobacter, alcanivorax, microbulbifer, sphingomonas, micrococcus, cellulomonas, dietzia and gordonia groups (8). the aim of this study was to evaluate the microbial heterogeneity of crude-oil polluted soils at obagi town, onelga, rivers state to be able to predict their inherent potential for hydrocarbon utilization. applied/environmental • 9 manual for determinative bacteriology (gram stain, motility test, catalase test, oxidase test, citrate utilization test, indole test, hydrogen sulphide test, urease test, triple sugar iron test, methyl red, and voges-proskauer test)(17). statistical analysis data obtained from the study were subjected to statistical analysis using t-test and one way analysis of variance (anova) at 0.05 confidence level (p<0.05). total heterotrophic bacterial counts for each soil sample were (1.9×107cfu/g, 2.0×106cfu/g, and 3.0×107cfu/g) (fig. 1) and hydrocarbon utilizing bacterial counts were (2.1×106cfu/g, 1.4×106cfu/g and 2.7×107cfu/g) respectively (fig. 2). we observed that there was a significant difference between thb and hub in soil sample a (p=0.001), which suggests that the hydrocarbon utilizers (hub) present in soil sample a are not a majority proportion of the bacterial community (thb). however, in samples b and c (i.e, thb and hub in sample b, and thb and hub in sample c) there was no significant difference observed (p=0.084 and 0.441, respectively), which suggests that most of the culturable bacterial population (thb) have become putative hydrocarbon utilizers (hubs). a total of 28 bacterial species were isolated as thb, while 19 bacterial species were isolated on mineral salt medium (bushnell haas agar) and identified morphologically and via biochemical tests. results figure 1. log cfu/g of total heterotrophic bacteria of the various oil impacted soil samples *= significant difference exists at p=0.05 soil sample soil sample * * * * th b (l og 10 c fu /g ) h u b (l og 10 c fu /g ) figure 2. log cfu/g of hydrocarbon utilizing bacteria of the various oil impacted soil samples *= significant difference exists at p=0.05 8.0 7.0 6.0 5.0 4.0 3.0 2.0 1.0 0.0 8.0 7.0 6.0 5.0 4.0 3.0 2.0 1.0 0.0 a b c a b c 10 • fine focus, vol. 2 (1) table 1: characterization of bacterial isolates. table 2: frequency of occurrence of hydrocarbon utilizing bacteria genera isolated from oil impacted soil samples. isolates gram reaction tentative identity b2sa1 r klebsiella sp. b2sa2 + r corynebacterium sp. b2sa3 + c micrococcus sp. b2sa4 r enterobacter sp. b2sa5 r flavobacterium sp. b2sa6 r azotobacter sp. b4sa1 r escherichia coli b6sa1 + c proteus sp. b2sb1 + c staphylococcus sp. b2sb2 r proteus sp. b6sb1 r serratia sp. b8sb1 r pseudomonas sp. b8sc1 r pseudomonas sp. 1a1 + r bacillus sp. 1a2 + r bacillus sp. 2a1 + c staphylococcus sp. 2a2 c acinetobacter sp. 2a3 + c micrococcus sp. 1b1 c acinetobacter sp. 1b2 r pseudomonas sp. 2b1 + c micrococcus sp. 2b2 r pseudomonas sp. 2b3 + r bacillus sp. 1c1 + r bacillus sp. 1c2 + c staphylococcus sp. 1c3 + r bacillus sp. 2c1 + r bacillus sp. 2c2 r pseudomonas sp. genus frequency of occurrence (%) bacillus spp. 31.6 pseudomonas spp. 26.3 acinetobacter spp. 10.5 micrococcus spp. 15.8 staphylococcus spp. 15.8 legends: r: rod; c: cocci; +: positive; -: negative the characteristics of the total hydrocarbon bacteria are presented in table 1. the isolates were gram positive and negative rods and cocci, and were tentatively identified to be representatives of the genera staphylococcus, bacillus, micrococcus, pseudomonas acinetobacter, klebsiella, and enterobacter. nineteen putative hub species were isolated with the dominance (frequency of occurrence) of representatives related to the genera bacillus and pseudomonas (table 2). bacillus spp. was observed to be the most isolated bacterial genera from the soil samples. discussion the study site, obagi town, is a mangrove environment challenged by crude oil pollution. in this study, a culture dependent technique was used to isolate and characterize a putative hydrocarbon utilizing bacterial population indigenous in crude-oil impacted soil. culture dependent techniques have been used by several researchers to isolate bacteria involved in petroleum hydrocarbon utilization (2,3,6,7,10,13,21,22-26). applied/environmental • 11 results showed that the mean values of total culturable heterotrophic and hydrocarbon utilizing bacterial counts from each soil sample (soil a, b and c) were all moderateto-high. these high counts may be attributed to the presence of organic matter content (nutrients) and favorable ecological factors that underpin the survival of these bacterial species. consequently, it indicates a viable population with the potential to initiate and maintain hydrocarbon degradation. a similar finding was made by abu and dike (1) and chikere and ekwuabu (11). results revealed that most of the microorganisms present in the various sample sites were hydrocarbon degraders. a similar observation was reported by chikere and ekwuabu (11). furthermore, a one-way analysis of variance (anova) showed that significant difference existed in the thb (log10 cfu/g) counts in soil samples a, b and c with p-values of <0.0001 and hub (log10 cfu/g) counts in soil samples a, b and c with p-values of <0.0001. it was also observed that hub counts in soil c were significantly higher compared to soil a and b. this is likely due to the hydrocarbon concentration in soil c. soil c may have been chronically impacted by hydrocarbons compared to the other soils and the indigenous microbes as part of a survival strategy, and may have adapted to degrading/utilizing these hydrocarbons as carbon sources, thus increasing their counts in the area of concern. when environmental changes occur as a result of the presence of a pollutant, there will be the development of new metabolic pathways and acquisition of new functions by mutations. this increases diversity with one population by the formation of a new niche (hydrocarbon utilizers) such that under in vitro isolation processes, microorganisms with beneficial mutations have growth advantages in comparison to unadapted organisms (24). a total of 28 bacterial isolates were obtained as thb while a total of 19 bacterial isolates were confirmed on mineral salt medium (bushnell haas agar) with crude oil as the carbon source. these bacterial species were gram positive and mostly gram negative representative of the gammaproteobacteria group. this correlates with earlier studies (11,20). the putative hydrocarbon utilizing bacterial isolates were observed to be representative of the genera bacillus, staphylococcus, acinetobacter, micrococcus, and pseudomonas. these genera of bacteria have been reported as hydrocarbon utilizing bacteria by several researchers (9,12,18,27,28), and this suggests that these species are key players in biodegradation of hydrocarbons in these sites. bacillus spp. was recorded to be the dominant bacterial genus from the soil samples having 6 out of 15 isolates, followed closely by pseudomonas spp. having 5 as shown in table 1. this corroborates results observed by chikere et al. and kadali et al. (9,19). bacillus spp. have been reported to be more tolerant to high concentrations of hydrocarbons in soils due to their resistant endospores (14,19,29). the capability of pseudomonas species to grow and degrade different hydrocarbon content has also been reported. they have been isolated from worldwide polluted sites (5). 12 • fine focus, vol. 2 (1) references 1. abu, g.o. and dike, p.o. 2007. a study of natural attenuation processes involved in a microcosm model of crude oil-impacted wetland sediment in the niger delta. bioresource technol. 99:4761-4767. 2. adoki, a. and orugbani, t. 2007. removal crude petroleum hydrocarbons by heterotrophic bacteria in soils amended with nitrogenous fertilizer plant effluents. afric. j. biotechnol. 6:1529-1535. 3. ayotamuno, m.j., kogbara, r.b., ogaji, s.o.t. and pobert, s.d. 2006. bioremediation of a crude oil polluted agricultural soil at port harcourt, nigeria. appl. energy. 83:1249-1257. 4. bayoumi, r. a. and abul-hamd, a. t. 2010. optimization of bacterial biodegradation of toluene and phenol under different nutritional and environmental conditions. j. appl. sci. res. 68: 10861095. 5. brito, e. m. s., guyoneaud, r., goni-urriza, m., duran, r. 2006. characterization of hydrocarbonoclastic bacteria communities from mangrove sediments in guamabra bay, brazil. res. microbiol. 1578: 752762. 6. chaillan, f., bury, e., phantavong, y. h., grimont, p., saliot, a. and oudot, j. 2004. identification and biodegradation potential of tropical aerobic hydrocarbon-degrading microorganisms. res. microbiol. 1557: 587-595. 7. chikere, b.o. and chijoke-osuji, c. 2006. microbial diversity and physicochemical properties of a crude oil polluted soil. nig. j. microbiol. 20:1039-1046. 8. chikere, c.b. 2013. application of molecular microbiology techniques in bioremediation of hydrocarbons and other pollutants. brit. biotechnol. j. 3: 90-115. 9. chikere, c.b., okpokwasili, g.c. and chikere, b.o. 2009a. bacterial diversity in a tropical crude oilpolluted soil undergoing bioremediation. afric. j. biotech. 8:2535-2540. 10. chikere, c.b., surridge, k., okpokwasili, g.c. and cloete, t.e. 2011. dynamics of indigenous bacterial communities associated with crude oil degradation in soil microcosms during nutrient-enhanced bioremediation. waste manag. res. 303:225-236. 11. chikere, c. b. and ekwuabu, c. b. 2014. culturedependent characterization of hydrocarbon utilizing bacteria in selected crude oil-impacted sites in bodo, ogoniland, nigeria. afric. j. environ. sci. technol. 86: 401-406. 12. ebrahimi, m., sarikkhani, m.r. and fallah, r. 2010. assessment of biodegradation efficiency of some isolated bacteria from oil contaminated sites in solid and liquid media containing oil compounds. intl. res. j. appl. basic sci. 31:138-147. mangroves are intertidal ecosystems along coastlines of tropical and subtropical regions, thus they are prone and subjected to urban and industrial effluent discharges and accidental oil spills. results from this study revealed the utilization of hydrocarbons by the indigenous bacterial community in crude-oil polluted mangrove soil of obagi town, evidenced by appreciable total heterotrophic bacterial and hydrocarbon utilizing bacterial counts from a mineral salt medium (bushnell haas) containing crude-oil as the only carbon source. the isolated bacterial species such as bacillus and pseudomonas amongst others have also been isolated and implicated as hydrocarbon utilizers in a number of studies. this study therefore provides a database of the indigenous hydrocarbon utilizing bacterial community present in obagi town for application in bioremediation studies, although these data are based on culturable microbial populations present in the sampled locations. hence, molecular methods that characterize functional genes would be a more powerful approach to study hydrocarbon utilizers in a mixed microbial community (8,30,31). conclusion applied/environmental • 13 13. ebuehi, o.a.t., abibo, i.b.,shekwolo, p.d., sigismund, k.i., adoki, a. and okoro, i.c. 2005. remediation of crude oil contaminated soil by enhanced natural attenuation technique. j. appl. sci. environ. manag. 9:103-105. 14. ghazali, f.m., rahman, r.n.z., salleh, a.b. and basri, m. 2004. biodegradation of hydrocarbons in soil by microbial consortium. intl. biodet. biodeg. 54:61-67. 15. hamamura, n., olson, s. h d., ward, m. and inskeep, w. p. 2006. microbial population dynamics associated with crude oil biodegradation in diverse soils. appl. environ. microbiol. 72: 6316-6324. 16. head, i.m., jones, m.d. and roling, w.f.m. 2006. marine microorganisms make a meal of oil. natl. rev. microbiol. 4:173-182. 17. holt, j.g., krieg, n.r., sneath, p.h.a., stanley, j.t. and williams, s.t. 1994. bergey’s manual of determinative bacteriology. 9th ed. williams and wilkins company, baltimore, usa. pp. 71-561. 18. ibiene, a. a., orji, f. a., ezidi, c. o. and ngwobia, c. l. 2011. bioremediation of hydrocarbon contaminated soil in the niger-delta using spent mushroom compost and other organic wastes. nig. j. agric. food environ. 73: 1-7. 19. kadali, k.k., simon, k.l., skuza, p.p., moore, r.b. and ball, a.s. 2012. a complementary approach to identifying and assessing the remediation potential of hydrocarbonclastic bacteria. j. microbiol. meth. 88:348-355. 20. kaplan, c.w. and kitts, c.l. 2004. bacterial succession in a petroleum land treatment unit. appl. environ. microbiol. 70: 1777-1786. 21. maila, m.p., randima, p., dronen, k. and cloete, t.e. 2006. soil microbial communities: influence of geographic location and hydrocarbon pollutants. j. soil biol. biochem. 38:303-310. 22. nweke, c.o. and okpokwasili, g.c. 2004. effects of bioremediation on treatments on the bacterial populations of soil at different depths. nig. j. microbiol. 18:368-372. 23. odokuma, l.o. and dickson, a.a. 2003. bioremediation of a crude oil-polluted tropical rain forest soil. global j. environ. sci. 2:29-40. 24. papke, r.t. and ward, d.m. 2004. the importance of physical isolation to microbial diversification. fems microbiol. ecol. 48:293-303. 25. peng, r-h., xiong, a-s., xue, y., fu, x-y., gao, f., zhao, w., tian, y-s. and yao, q-h. 2008. microbial biodegradation of polyaromatic hydrocarbons. fems microbiol. rev. 326: 1574-6976. 26. rojas-avelizapa, n.g., roldan-carrillo, t., zegarramartinez, h., munoz-colunga, a.m. and fernandezlinares, l.c. 2007. a field trial for an ex-situ bioremediation of a drilling mud-polluted site. chemosphere 66:1595-1600. 27. said, b. o., goni-urriza, m. s., el-bour, m., aissa, p. and duran, r. 2008. characterization of aerobic polycyclic aromatic hydrocarbon degrading bacteria from bizerte lagoon sediments, tunisia. j. appl. microbiol. 107: 987-997. 28. sarma, a. and sarma, h. 2010. enhanced biodegradation by some microbial isolates supplemented with heavy metals. int. j. bot. 6:441-448. 29. singh, c. and lin, j. 2010. bioaugumentation efficiency of diesel degradation by bacillus pumilus jl8 and acinetobacter calcoaceiticus lt1 in contaminated soils. afric. j. biotech. 9:6881-6888. 30. vidali, m. 2001. bioremediation an overview. pure appl. chem. 737: 11631172. 31. zhu, x., venosa, a.d., suidan, m.t. and lee, k. 2001. guidelines for the bioremediation of marine shorelines and freshwater wetlands, report under a contract with u.s. environmental protection agency. 1-116 14 • fine focus, vol. 2 (1) relationships between algal biomass and diversity with stream size and adjacent land use rachel a. habegger and jordan m. marshall* department of biology, indiana universitypurdue university fort wayne, fort wayne, indiana copyright 2014, fine focus all rights reserved manuscript received 15 september, 2014; accepted 30 october, 2014 land use adjacent to waterways, such as development or agriculture, alters hydrological patterns leading to increases in runoff and nutrient input. forests and wetlands, as natural land cover types, reduce water movement and allow infiltration into soil. we measured algal biomass and diversity in order to quantify the influence neighboring land cover types have on streams in northeastern indiana. in the study area, cultivated crops were the dominant land cover type, with open development and deciduous forest following. emergent wetland area had the greatest influence on algal biomass, with increases in wetland area decreasing biomass. however, open development, low intensity development, grassland, shrub, and forested wetlands added to increases in biomass. conversely, forested wetlands reduced algal richness, while open development and pastures increased richness. because open development (i.e. dominated by turf grass, lawns, parks, golf courses) was the second most common land cover type and positively influenced both algal biomass and richness, management of those properties will likely have direct impact on nutrient flow into streams. additionally, adding functional wetlands dominated by emergent herbaceous plants will directly impact future algal biomass. abstract corresponding author * jordan m. marshall marshalj@ipfw.edu keywords • algae • eutrophication • nutrient flow • chlorophyll • runoff introduction land use and cover types can directly influence water infiltration and runoff patterns within a watershed (22). decreases in natural vegetation often results in increases in surface runoff (15). additionally, the complexity of layers within the vegetation cover can influence infiltration of water into soil (26). developed land cover types have decreased in tree cover in the u.s., while increasing in impervious surfaces (18). both changes in tree and impervious land cover have direct impacts on runoff volume and soluble nutrient content (2). assessing the anthropogenic changes to land cover types may be important in identifying potential water quality issues (20). in addition to decreases in infiltration and increases in runoff, there is a resultant increase in sedimentation and nutrient content associated with anthropogenic land cover changes (i.e. simplified vegetative structure, impermeable surfaces) (7,15,17,23). degradation of freshwater 22 • fine focus, vol. 1 has been clearly linked to increased nitrogen and phosphorous fertilization (24). increases in human population densities have resulted in increases in nitrate and suspended solids within streams (1). while agricultural fertilization may add to eutrophication, urban development imparts major influence on biologically available phosphorus (4). increased nutrient content within streams leads to human and environmental health issues including increases in disease vector populations and toxic algal blooms (11,13,16). in the midwest region of the united states, agricultural land dominates the land cover types, with cultivated crops and pasture lands making up 80-90% of land cover (6). in indiana major changes in land cover between 2001 and 2006 were focused around the state’s larger urban areas (i.e. indianapolis, fort wayne, evansville) (8). while there is minimal change in land cover types in indiana, those persistent land cover types adjacent to streams may have sustained and continual influence on water quality. the objectives of this study were to 1) quantify algal diversity and biomass within streams in northeastern indiana, 2) compare the influence of stream channel size and land use on algal communities, and 3) test the hypothesis that neighboring land use types influence algal diversity and biomass. materials and methods we selected thirteen sites in adams, huntington, and wells counties, indiana, based on size and access from public property (i.e. road crossings; fig. 1). each stream was measured at the sampling location from bank to bank. six small streams (< 8m channel width), four medium streams (≥ 8 and < 20m width) and three large streams (≥ 20m width) were selected (fig. 1). monthly from may to august 2013, we collected 40 ml of water from the center of the channel for each stream. samples were immediately separated into 20 ml containers, each for chlorophyll a biomass and diversity assessments. we used methods described by epa (5) for chlorophyll a extraction. briefly, the 20 ml samples were centrifuged (9000 rpm, 20 minutes), aqueous layer decanted, and 10 ml of acetone was added and mixed with the pellet on a vortex. the acetone solution was stored for 24 hours at 5°c, centrifuged (7000 rpm, 5 minutes), and aqueous layer decanted for spectrophotometry. using the equation presented by epa (5), we calculated μg/l of chlorophyll a in the streams. for diversity, we identified algal morphospecies as a rapid assessment tool within three random microscope views per sample (100 μl per slide, 100x magnification). morphospecies were defined based on shape (i.e. spherical, filamentous), motility (i.e. non-motile, flagellated), and colonial status (unicellular, colonial). we used morphospecies richness (counts of different morphospecies) as a measure for algal diversity. land use adjacent to the stream were assessed using 2011 national land cover data (10). in arcmap (version 10.1, esri inc., redlands, ca), land use were categorized in a 0.5 km buffer around the stream channel 2 km upstream from the sample location (including all tributaries within the 2 km buffer). the area of land within the assessment buffers for each land use type were calculated. additionally, the number of tributaries within the assessment buffer were counted for each stream. stream size classes were compared using oneway analysis of variance (anova) for total land area within assessment buffers and numapplied/environmental • 23 ber of tributaries, as well as repeated measures anova for algal richness and biomass. tukey hsd was used as a post-hoc multiple comparison test. pearson correlations were used to identify relationships between algal richness, algal biomass, and number of tributaries. multiple regression with reverse variable selection was used to identify relationships between stream size and land use types, with biomass and morphospecies richness. all analyses were conducted in r (version 3.1.1, the r foundation for statistical computing, vienna, austria). results large streams had significantly more tributaries within the 2 km upstream from the sampling location than small streams (f2,10 = 8.19, p = 0.008). however, the total area within the assessment buffers were not significantly different between the size classes (f2,10 = 1.06, p = 0.382). stream size classes were not significantly different in chlorophyll a biomass (f2,6 = 1.59, p = 0.279, table 1) using repeated measures over the months. however, stream sizes were significantly different in monthly algal richness, with large and medium streams having greater numbers of morphospecies than small streams (f2,6 = 13.48, p = 0.006, table 1). algal biomass and richness were not significantly correlated (r = 0.34, p = 0.259). mean algal biomass was significantly correlated with number of tributaries upstream (r = 0.61, p = 0.026), while richness was not correlated with tributaries (r = 0.36, p = 0.223). using different models defining the relationship between algal biomass and total phosphorus (12,25), estimated mean phosphorus ranged from 0.003 (± 0.002) to 0.057 (± 0.018) mg/l. we encountered thirteen of the 20 land cover types in the land assessment buffers (table 2). cultivated crop agriculture dominated the land cover types accounting for 77.2% of the area within the study. open development and deciduous forest lands were distant second and third in area, accounting for 6.7% and 6.5%, respectively. land cover types provided significant multiple regression models for both algal richness and biomass fig. 1. sampling locations within adams, huntington, and wells counties, indiana. squares indicate small stream sampling locations (<8 m channel width), triangles indicate medium streams (>8 and <20 m width), and circles indicate large streams (>20 m width). 24 • fine focus, vol. 1 table 1. algal biomass measured in μg/l chlorophyll a (se) and species richness (se) per size class over four sampling periods. land use cover type changes can greatly influence runoff and fertilization of local streams (2,15,18,22,26). the majority of changes occur as shifting from natural land cover with complex vegetative layers to simplified layers (17,20,23). within the study area in northeastern indiana, little land cover has changed recently; agriculture has dominated. dominance of both agricultural and developed land, both anthropogenic simplifications of the natural land cover, greatly alter stream nitrogen and phosphorus contents (1,7,24). stream size classes were designated as arbitrary categories based on channel width at the sampling location. as evidenced by the significant correlation between algal biomass and number of tributaries two km upstream from the sampling point, a categorical index based on tributaries may have been a more appropriate size category. the land cover/tributary interaction on the landscape can have substantial impact downstream (19). however, since our buffer production began with a 500 m buffer around all streams and then used a two km buffer, the area of land that was potentially draining into the sample stream channel didn’t differ between size classes. the tributary influence would have been included in the arbitrary size class categorization (large streams with more tributaries than small). while nitrogen and phosphorous are imdiscussion (table 2). for algal richness, open developed land and pasture had positive influence, while woody wetlands had negative influence. for algal biomass, open and low developed land, grass, shrubs, and woody wetlands had a positive influence. emergent wetlands had the strongest influence and negatively impacted algal biomass. applied/environmental • 25 ta bl e 2. l an d us e ty pe s oc cu rr in g w ith in s tr ea m b uf fe rs (0 .5 k m x 2 k m u ps tr ea m ) w ith m ea n nu m be r of tr ib ut ar ie s (s e) a nd m ea n ha ar ea (s e) fo r sm al l ( < 8 m ), m ed iu m (≥ 8 m , < 2 0 m ), an d la rg e (≥ 2 0 m ) w id th s tr ea m s. ta bl e 3. m ul tip le r eg re ss io n eq ua tio ns fo r pr ed ic tin g al ga l r ic hn es s an d bi om as s w ith a dj ac en t l an d co ve r ty pe a re a w ith in a 0 .5 k m w id e bu ff er 2 k m u ps tr ea m f ro m s am pl in g lo ca tio n. 26 • fine focus, vol. 1 portant nutrients in driving algal population size, phosphorus is considered the more important of the two in freshwater systems (3). because of this, we wanted to use published models for predicting algal biomass to inversely estimate stream phosphorus content. due to differences in systems and model techniques, we estimated a very broad range of phosphorus (0.003-0.057 mg/l) (12,25). even with this broad range, the estimated phosphorus content of the streams were well below the benchmark values set by the indiana state department of agriculture of 0.3 mg/l (9). while there are limitations to interpreting this conversion of algal biomass to estimated total phosphorus, it does indicate that the streams we sampled were well below the state’s benchmark value. in our study area, combined agriculture (pasture and cultivated crops; 80% of area) and combined development (open, low, medium, and high; 10% of area) were the clearly dominant land cover types. however, agriculture was not included in either algal richness or biomass models. likely this is due to the ubiquitous nature of agriculture as a dominant land cover within the buffers for every stream. changes in open developed land for algal richness, as well as both open and low intensity for algal biomass, significantly added positively to the multiple regression models. while phosphorus is often intentionally added to agricultural fields, total biologically available phosphorus during baseline flow rates in agricultural streams appears similar to urban streams (1,4,24). increasing urban development dramatically increases coverage of impermeable surfaces, decreasing water infiltration, and increases total biologically available phosphorus in streams (2,4,17,20). the influence of open and low intensity land use in the two models fits with an expectation of a potential increase in phosphorus with increasing area. the effect of wetlands on algal biomass and richness may be explained through absorption of nutrient-rich runoff from the dominating agricultural land use. however, emergent herbaceous wetlands have limited phosphorus retention capacity, while forested wetlands may serve as phosphorus sinks (14,21). our results may indicate interactions between natural systems: stream channel and natural wetlands. the two strongest influences within both models were negative associations with woody and emergent wetlands. both are common wetland types, when wetlands occur, within the study region. within our buffers, wetlands only accounted for 1.7% of land area. even though they were not a dominant land type, wetlands play a substantial and dramatic role in the algal richness and biomass responses within the study streams. our results highlight the importance of responsible land management near waterways to maintain high water quality. as part of the indiana nutrient reduction strategy (9), there is a focus on urban residential fertilizer application in addition to the typical focuses on agriculture and sewer management. open development land use (i.e. dominated by turf grass, lawns, parks, golf courses) has strong positive influence on algal richness and biomass, which could have future water quality implications if land uses change with increases in urban and suburbanization within the region. applied/environmental • 27 1. ahearn, d. s., sheibley, r. w., dahlgren, r. a., anderson, m., johnson, j., & tate, k.w. 2005. land use and land cover influence on water quality in the last free-flowing river draining the western sierra nevada, california. j. hydrol. 313:234-247. 2. barbec, e., schulte, s., & richards, p. l. 2002. impervious surface and water quality: a review of current literature and its implications for watershed planning. j. plan. lit. 16:499-514. 3. correll, d. l. 1998. the role of phosphorus in the eutrophication of receiving waters: a review. j. environ. qual. 27:261-266. 4. ellison, m. e., & brett, m. t. 2006. particulate phosphorus bioavailability as a function of stream flow and land cover. water res. 40:1258-1268. 5. epa. 1991. ess method 150.1: chlorophyllspectrophotometric. retrieved from http://www.epa. gov/grtlakes/lmmb/methods/methd150.pdf 6. fuelling, r. r. 2014. diversity and distribution of plant communities related to forest fragment size, shape, age, and structure. thesis. fort wayne, in: indiana university-purdue university fort wayne. 7. houser, j., & richardson, w. 2010. nitrogen and phosphorus in the upper mississippi river: transport, processing, and effects on the river ecosystem. hydrobiologia 640:71-88. 8. indianamap. n.d. land cover change, 2001-2006 (nlcd 2006). retrieved from http://maps.indiana. edu/previewmaps/environment/land_cover_ change_2001_2006.html 9. indiana state department of agriculture. n.d. indiana nutrient reduction strategy: a framework to reduce nutrients entering indiana’s waters. retrieved from http://www.in.gov/isda/files/indiana_nutrient_ reduction_strategy_(2).pdf 10. jin, s., yang, l., danielson, p., homer, c., fry, j., & xian, g. 2013. a comprehensive change detection method for updating the national land cover database to circa 2011. remote sens. environ. 132:159-175. 11. johnson, p. t. j., townsend, a. r., cleveland, c.c., gilbert, p. m., howarth, r. w., mckenzie, v. j., rejmankova, e., & ward, m. h. 2010. linking environmental nutrient enrichment and disease emergence in humans and wildlife. ecol. appl. 20:16-29. 12. jones, j. r., & bachmann, r. w. 1976. prediction of phosphorus and chlorophyll levels in lakes. j. water poll. control fed. 48:2176-2182. 13. kneitel, j. m., & lessin, c. l. 2010. ecosystem-phase interactions: aquatic eutrophication decreases terrestrial plant diversity in california vernal pools. oecologia 163:461-469. 14. masscheleyn, p. h., pardue, j. h., delaune, r. d., patrick, w. h. 1992. phosphorous release and assimilatory capacity of two lower mississippi valley freshwater wetland soils. j. amer. water resource assoc. 28:763-773. 15. mohammad, a. g., & adam, m. a. 2010. the impact of vegetative cover type on runoff and soil erosion under different land uses. catena 81:97-103. 16. morris, j. g. 1999. harmful algal blooms: an emerging public health problem with possible links to human stress on the environment. ann. rev. energy environ. 24:367-390. 17. mouri, g., takizawa, s., & oki, t. 2011. spatial and temporal variation in nutrient parameters in stream water in a rural-urban catchment, shikoku, japan: effects of land cover and human impact. j. environ. manag. 92:1837-1848. 18. nowak, d. j., & greenfield, e. j. 2012. tree and impervious cover change in u.s. cities. urb. fores. urb. green. 11:21-30. 19. oliver, a. a., dahlgren, r. a., & deas, m. l. 2014. the upside-down river: reservoirs, algal blooms, and tributaries affect temporal and spatial patterns in nitrogen and phosphorus in the klamath river, usa. j. hydrol. 519:164-176. 20. pauleit, s., & duhme, f. 2000. assessing the environmental performance of land cover types for urban planning. landscape urb. plan. 52:1-20. 21. richardson, c. j. 1985. mechanisms controlling phosphorous retention capacity in freshwater wetlands. science 228:1424-1427. 22. sriwongsitanon, n., & taesombat, w. 2011. effects of land cover on runoff coefficient. j. hydrol. 410:226-238. 23. sutherland, a. b., meyer, j. l., & gardiner, e. p. 2002. effects of land cover on sediment regime and fish assemblage structure in four southern appalachian streams. freshwater biol. 47:1791-1805. 24. u.s. geological survey. 1999. the quality of our nation’s waters: nutrients and pesticides. reston, va: usgs circular 1225. 25. van nieuwenhuyse, e. e., & jones, j. r. 1996. phosphorus-chlorophyll relationship in temperate streams and its variation with stream catchment area. can. j. fish. aquat. sci. 53: 99-105. 26. zimmermann, b., elsenbeer, h., & de moraes, j. m. 2006. the influence of land-use changes on soil hydraulic properties: implications for runoff generation. for. ecol. manag. 222: 9-38. references 28 • fine focus, vol. 1 book review: lab manual – “microbiology– the laboratory experience” (steve keating) w.w. norton & company, isbn 978-0-393-92364-3 autumn trice undergraduate student biology ball state university copyright 2017, fine focus. all rights reserved. book review• 193192 • fine focus, vol. 3 (2) • fine focus, vol. 3 (2) in microbiology: the laboratory experience, author steve keating takes lab manuals where they have not gone before. he states his purpose, “… to write a microbiology lab manual as a means of speaking directly to students to increase their interest and understanding of the subject and to decrease the number of mistakes.” through his dedication to providing students with a manual that is interesting, simple yet informative, engaging, and inclusive, keating presents the most impressive lab manual i have seen as an undergraduate student. the bulk of the manual is made up of 7 units: (1) the fundamentals, (2) staining microbes, (3) effects of the environment on microbial growth, (4) identifying medically significant bacteria, (5) applied food and water microbiology, (6) microbial ecology, and (7) viruses; each of these sections an umbrella for the various other important subsections. each section provides a significant amount of relevant background information for the corresponding laboratory experiment. unit 1: the fundamentals is accurately titled as it covers laboratory procedures which will be utilized in each of the subsequent units (the microscope, working with and storing pure cultures, quantitative plate counts). unit 2: staining microbes covers each of the familiar microbe staining topics including general staining and cell morphology, the gram stain, acid-fast stain, and endospore stain. however, this manual goes so far as to take the idea of the normal one-day staining lab and separates it into individual staining labs, making the information for each more detailed and easily digestible. unit 3: effects of the environment on microbial growth is one of the more expansive units (along with unit 4). the unit covers environmental factors including: media components, bacterial enzymes, temperature, pressure, ph, oxygen, uv, disinfectants, antiseptics, and antibiotic sensitivity. it also provides techniques for calculating the minimum inhibitory concentration (mic). unit 4: identifying medically significant bacteria includes techniques for isolation, pcr, and identification of both known microbes (streptococcus spp., enterococcus spp., staphylococcus spp., micrococcus spp.) and unknown bacteria. it also provides elaborate detail on the enterobacteriaceae family with corresponding identification techniques. units 5, 6, and 7 though short, continue to do an adequate job of providing contextual information that is thorough and accompanying it with procedures that give students realworld laboratory experience. unit 5: applied food and water microbiology deals with milk microbiology, water, meat, and wine microbe analysis. unit 6: microbial ecology cover topics such as the nitrogen cycle, winogradsky columns (something that i have never encountered in my microbiology experience as an undergrad student), and antibiotic producers in soil. the final unit, viruses, focuses primarily on understanding bacteriophages and how to type them. aside from providing sufficient background content and real-world lab experience, keating begins each laboratory procedure with clearly outlined learning objectives and ends the lab with conceptual post-lab questions. learning objectives allow students to get an idea of the key concepts and techniques they will be learning about/performing, while the post-lab questions facilitate conceptual understanding and real-world application of each procedure. what separates this manual from others i’ve come across is that keating refuses to cut corners. he makes sure that the manual is an object which can stand on its own, providing sufficient content in all areas, no matter where your instructor may lack. it is obvious that keating has students in mind when noting the overall clarity of the lab manual. as a 4-year science major, i still find myself getting lost in some scientific jargon. however, with this manual, i did not once experience that lost feeling. i could better understand the information and techniques presented, and i was able to reflect on them through the post-lab questions, without feeling as though the questions were set out to confuse me. one of my favorite things about keating’s manual is his willingness to be racially inclusive with his figures. in lab 8: the endospore stain, keating uses a visual diagram of the steps involved in the staining method. within this figure, the individual performing the stain is shown with dark skin. in all my years of schooling i have never seen a diagram or figure with a skin tone like mine. even with it just being a figure, it erased all doubt of my abilities as a scientist from my mind. this small bit of representation is something that should be recognized and acknowledged by all that review this work. i strongly applaud keating for his forward and inclusive thinking. all images and figures included in the manual are clear, visually appealing, and easy to interpret/ follow. the quality of many images and figures in this manual can be compared to those found in a standard textbook, something i find extremely impressive for a lab manual. once again, keating goes the extra mile to provide a manual that is engaging, inclusive and studentfriendly. in conclusion, keating’s manual goes above and beyond what is expected from a laboratory manual. he provides substantial information for each laboratory procedure in a way that is easy to understand and does not feel overwhelming. the organization of each laboratory is clear and structured. he also includes visual content that is stimulating and lively. he provides information about himself in the preface of the manual which sheds light on his dedication and authenticity as an author, making the manual a greater object of appreciation. i would recommend this for any microbiology professor to adopt into their curriculum. the views expressed in this book review do not necessarily reflect the opinions of fine focus. language figures conclusion context book review • 195194 67applied & environmental microbiology • waterfowl influence on fecal indicator bacteria in central florida freshwater lakes rachel schomaker and brittany gasper* department of biology, florida southern college, lakeland, fl usa copyright 2018, fine focus all rights reserved manuscript received 30 october 2017; accepted 23 february 2018 68 • fine focus, vol. 4(1) bacteria are important natural components of virtually every environment, including water systems. while many are beneficial to the ecosystem in which they are found, some can be indicators of pathogens that can endanger human health. fecal coliform bacteria such as escherichia coli are bacterial indicators that can originate from many of the same sources as pathogenic bacteria and serve as a sign that pathogens may be present. these bacterial counts can be influenced by many different well-studied environmental factors, including ph, temperature, and nutrient availability. in addition to these factors, mammalian and waterfowl presence can influence coliform abundance. while this area of research has been examined before, conflicting conclusions have been reached as to whether or not waterfowl abundance positively correlates with coliform bacteria abundance. levels of e. coli as well as enterococcus, a genus of noncoliform bacterial organisms that are also found in high concentrations in feces, were measured by membrane filtration of water samples collected from six freshwater lakes around lakeland, fl and were isolated from fresh fecal samples that were simultaneously collected from waterfowl species present at the lakes. results suggest a correlation between the abundance of e. coli and the presence of waterfowl. abstract corresponding author *brittany gasper bgasper@flsouthern.edu keywords • waterfowl • fecal indicator • freshwater • e. coli • enterococcus introduction bacteria are natural components of virtually every environment and many play important roles in the ecosystems of which they are found. for example, the human microbiome is imperative for metabolic processes that occur in the human digestive tract and is responsible for variations in metabolic phenotypes in humans (21). human health would drastically change if these bacteria did not exist as human symbionts (16). further, bacteria play a great part in the natural world. nitrogen fixation and nitrification, for instance, are processes by which bacteria fix atmospheric nitrogen from the air and convert it into a usable form for other organisms. both of these 69applied & environmental microbiology • processes are parts of the nitrogen cycle, which is a key process for almost all living organisms (17). in freshwater and marine systems, bacterial roles vary from primary producers to decomposers to pawns in the carbon cycle (14). additionally, bacteria can produce oxygen (5, 12, 31), recycle many primary elements of living systems (6, 28, 29), and detoxify systems (3, 11), among other processes. while bacteria occupy many important and beneficial niches across ecosystems, the presence of certain types of bacteria can indicate environmental contamination that can raise concerns for public health, such as coliform bacteria in aquatic environments (20, 33). coliform bacteria can be naturally occurring in the environment; however, they can also be found within the digestive tracts and therefore from there the feces of warm-blooded animals, such as mammals and birds (1, 23). because coliform bacteria can be found in the digestive tracts and from there the feces of these organisms, their presence in aquatic habitats can be indicative of fecal contamination. this is a human health concern because many pathogens survive under similar conditions as these fecal coliforms. thus, high levels of coliform bacteria may indicate high levels of human pathogens. the hypothesis that the presence of fecal coliforms in water suggests the presence of harmful bacteria was first introduced in 1892 and is still used as a basis for water quality standards today (15, 33). standards for measuring fecal coliform levels in water can vary, but the most commonly measured bacterial indicators can be split into three groups: total coliforms (tc), fecal coliforms (fc), and enterococcus organisms (ec). however, because some coliform bacteria are naturally occurring in the environment, tc counts can be inaccurate and may not be the best indicators of fecal contamination (26). escherichia coli counts, which is a subset of the fc count, and the enterococcus ec counts are becoming the most preferred methods, primarily because e. coli strains and enterococcus spp. have not yet been shown to be naturally occurring, while some other fecal coliforms can have questionable origins (26). many factors can impact coliform abundance in aquatic environments, including ph, nutrient availability, temperature, and anthropogenic pollution, as well as mammalian and avian abundances (8, 22, 30, 36). waterfowl excrete a large amount of fecal matter, and their feces have commonly been found to harbor pathogens (4, 9, 13, 25). microorganisms that are excreted in the feces of ducks have been linked to human health hazards such as salmonella, pathogenic bacteria that reside in the digestive tracts of animals and can lead to gastrointestinal disruptions in humans (32). moreover, a parasite known as echinostoma revolotum harbored in waterfowl feces has been linked to a condition known as ”swimmers’ itch”, an infection that appears as a skin rash, but is actually an allergic reaction to the parasite (24). ecologically, increases in duck fecal concentrations have affected shellfish beds in the past and have been shown to influence algal blooms, which can have multiple negative impacts on aquatic systems (7). the question asking how waterfowl abundance and fecal coliform bacteria abundance are related is not a new one. a previous study conducted by standridge et 70 • fine focus, vol. 4(1) materials and methods al., suggested that fecal coliform counts were high during their study time frame due to increased duck abundances and increased waterfowl fecal matter (32). however, in a similar study by brierley et al. there was no direct correlation between waterfowl abundance and fecal coliform counts observed (7). this pattern of conflicting results can be seen across many other similar studies, leaving the question as to whether or not waterfowl abundance plays a significant sampling methods five lakes in lakeland, florida were sampled: lake hollingsworth, lake morton, lake mirror, lake belulah, and lake hunter. these lakes were selected based on their close proximity and locations surrounding florida southern college. at each lake, water was collected from ten different widely distributed and randomly selected accessible sites on the same day using sterilized beakers. the gps coordinates, ph, and temperature of each sample site was measured and recorded, as well as plant abundance, anthropogenic pollution, and human trafficking that may influence water quality and therefore bacterial abundance. acreage for each lake was determined from the polk county water atlas (http://www.polk.wateratlas.usf.edu/). the waterfowl present at each lake was quantified and identified using the waterfowl identification guide from ducks unlimited (http://www.ducks.org/hunting/waterfowlid/). fresh fecal samples were collected from as many waterfowl as possible using sterile swabs and aseptic techniques. fecal samples role in fecal contamination levels unanswered (18). the goal of this study was to investigate this question and relationship by testing the hypothesis that the bacterial water quality of freshwater lakes in central florida will vary across a gradient of waterfowl abundance. from a big picture perspective, the study also addressed what varying water quality results could mean for overall ecosystem and human health. were collected from the ground, plated by continuous streaking on eosin methyl blue (emb) agar plates (remeltm) and m enterococcus agar plates (fisher scientific), and incubated overnight at 35°c. isolation and identification bacteria from water samples were filtered by membrane filtration using 0.2 µm membranes (pall corporation). for all enterococcus isolations, 100 ml of sampled water was vacuum filtered. for potential e. coli isolations, 10 ml of sampled water was filtered. all 0.2 µm filters used for isolating enterococcus bacteria were placed on enterococcus media plates, while all filters used for isolating e. coli were placed on emb plates. all of the plates were placed in a 35°celsius incubator for 24-48 hours. after incubation, the plates were removed and the colony forming units (cfu) count for each sample site was measured. cfu counts were determined based solely off of specific fecal coliform presence, meaning that for emb plate 71applied & environmental microbiology • isolates, only those bacteria that were observed by color change to be fecal coliforms (metallic green) were quantified, and for enterococcus plates, all of the bacteria were quantified. for each lake, colonies that appeared unique were isolated to obtain pure cultures of all isolated bacteria. isolated colonies from the emb plates were confirmed to be e. coli by 20e analytical profile index (api) strips (biomérieux). strips were inoculated with fresh overnight cultures following the manufacturer’s instructions and were incubated at 35°c for 16-24 hours. the profile index codes were obtained following the manufacturer’s guidelines and apiweb was used to determine identifications from the codes. all of the fecal coliform bacteria cultured from the fresh fecal samples were also isolated and quadrant streaked to obtain pure cultures for use in comparative experiments against the lake isolates. once pure cultures of all of the lake and fecal samples were obtained, antibiotic profiles were obtained for each pure culture bacterial isolate to determine if any lake isolates matched any strains of bacteria isolated from waterfowl at the same lake. to obtain antibiotic profiles, a single colony of each isolate was resuspended in a solution of 0.9% saline to an od600 value between 0.05 and 0.1. from this suspension, 100 µl was spread onto a mueller-hinton plate. antibiotic disks (carolina biological supply company) were dispensed onto the plate. the following disks were used: chloramphenicol (30 mcg), erythromycin (15 mcg), gentamycin (10 mcg), kanamycin (30 mcg), neomycin (30 mcg), novobiocin (30 mcg), penicillin g (10 units), streptomycin (10 mcg), and tetracycline (30 mcg). the plates were incubated at 35°c for 24 hours. the zones of inhibition for each antibiotic were measured, and isolates were classified as sensitive, intermediate, or resistant to each antibiotic using antibiotic disk diffusion interpretation guide (27). any isolates that showed a potential positive match was used for biochemical analysis and comparisons. the 20e analytical profile index (api) strips (biomérieux) were used to perform biochemical tests on the waterfowl fecal samples that were determined to be potential matches to the lake isolates based on identical results from the antibiotic susceptibility testing. the strips were inoculated from fresh cultures following the manufacturer’s guidelines and were incubated at 35°c for 16-24 hours. a known sample of e. coli k12 (atcc 10798) was cultured on a 20e api test strip as a positive control. the profile index codes were obtained following the manufacturer’s guidelines and apiweb was used to determine identifications from the codes. results the environmental protection agency (epa) defines the bacteriological water quality criteria for lake class waters,bodies of water that can be used for domestic, industrial, and agricultural water supply; stock watering; seafood rearing; wildlife habitat; ceremonial use; primary contact recreation; and commerce or navigational use, as water that should not exceed an enterococci density of 107 colony forming units (cfu) per 100 ml in any single water sample or water that should not exceed an enterococci density 72 • fine focus, vol. 4(1) geometric mean of 33 cfu/100 ml, where the mean was calculated with at least 5 samples over a period of 30 days (34). the e. coli standards for this same water class state that no single water sample should exceed 409 cfu/100 ml or that the geometric mean should not exceed 126 cfu/100 ml (34). a summary of the abundance of waterfowl relative to abundance of e. coli and enterococcus is shown in table 1. the waterfowl abundance was 2.7-fold greater at lake morton than the next most abundant site, lake mirror. abundance of e. coli was also the greatest at lake morton while abundance of enterococcus was greatest at lake mirror. lake hunter showed the lowest level of waterfowl abundance. the lowest levels of e. coli and enterococcus were found at lake belulah and lake hollingsworth, respectively. table 1 – average abundance of bacteria and waterfowl at each lake the table shows the average abundance of bacteria and the abundance of waterfowl at the five sampled lakes. abundance values for both e. coli and enterococcus represent the mean ± standard error of n=10 samples taken from unique locations on the same day around each lake. e. coli and enterococcus were isolated by membrane filtration. the number of samples exceeding the standards was determined by comparing the e. coli and enterococcus cfu counts from each individual sample to the epa standards of 409 cfu/100 ml and 107 cfu/100 ml for e. coli and enterococcus, respectively. 73applied & environmental microbiology • the abundance of e. coli from three lakes, lake hollingsworth, lake morton, and lake hunter exceeded the geometric mean standard of 126 cfu/100 ml, while 14 individual samples (three from lake hollingsworth, nine from lake morton, and two from lake hunter) exceeded the single sample standard of 409 cfu/100 ml. the abundance of enterococcus from one lake, lake mirror, exceeded the accepted epa geometric mean standard of 33 cfu/100 ml while the other four lakes fell within in the acceptable range. no single enterococcus sample exceeded 107 cfu/100 ml. figure 1 shows the abundance of e. coli (panel a) and enterococcus (panel b) vs. the waterfowl abundance for each lake. there is a strong positive correlation between e. coli and waterfowl abundance with a pearson’s correlation coefficient of 0.809. the abundance of enterococcus and waterfowl showed no real correlation with a coefficient of 0.067. in order to determine whether the presence of e. coli might affect levels of enterococcus and vice versa, their abundances were compared for each lake. the relationship between the average e. coli abundance and the average enterococcus abundance per lake is shown in figure 2. figure 1 – average bacteria abundance vs. waterfowl abundance per lake the figure shows the average abundance of e. coli (panel a) and enterococcus (panel b) vs. waterfowl abundance for each lake. average abundance values were calculated from membrane filtration of ten samples obtained from each lake. error bars represent the standard error of the mean. the trendline shows the best-fit linear line of regression. the pearson’s correlation coefficient for this data is 0.809 (e. coli) and 0.067 (enterococcus). a. b. figure 1 a. b. -200 0 200 400 600 800 1000 1200 0 50 100 150 200 250 300 350 400 450 500 av er ag e e. c ol i a bu nd an ce (c fu /1 00 m l) waterfowl abundance 0 5 10 15 20 25 30 35 40 45 50 0 50 100 150 200 250 300 350 400 450 500 av er ag e en te ro co cc us ab un da nc e (c fu /1 00 m l) waterfowl abundance 74 • fine focus, vol. 4(1) figure 2 – average enterococcus abundance per lake vs. average e. coli abundance per lake the figure shows the average abundance of enterococcus vs the average abundance of e. coli for each lake. average abundance values were calculated from membrane filtration of ten samples obtained from each lake. error bars represent the standard error of the mean. the trendline shows the best-fit linear line of regression. the pearson’s correlation coefficient for this data is -0.455. there is a negative correlation between the presences of these two bacterial varieties with a correlation coefficient of -0.455. since other factors besides waterfowl are believed to affect the abundance of fecal indicator bacteria the effects of ph and temperature on abundance of e. coli and enterococcus were examined. there is no apparent correlation between ph levels and either e. coli or enterococcus with correlation coefficients of -0.0121 and -0.221 (data not shown). neither e. coli nor enterococcus showed a correlation with temperature, producing coefficients of -0.104 and -0.0875, respectively (data not shown). the five lakes sampled in this study vary somewhat in size. to determine if there is any relationship between the size of the lake and the abundance of e. coli or enterococcus, the abundance of organisms from each lake was compared to the size of the lake. figure 3 shows the results of this comparison. the relationship between e. coli abundance and lake size resulted in a correlation coefficient of 0.191, suggesting a slight positive correlation may exist. however, the abundance of enterococcus and lake figure 2 0 5 10 15 20 25 30 35 40 45 50 0 200 400 600 800 1000 1200 av er ag e en te ro co cc us a bu nd an ce (c fu /1 00 m l) average e. coli abundance (cfu/100 ml) 75applied & environmental microbiology • figure 3 – average bacteria abundance per lake vs. lake size the figure shows the average abundance of e. coli (panel a) and enterococcus (panel b) as a function of lake size for each lake. average abundance values were calculated from membrane filtration of ten samples obtained from each lake. error bars represent the standard error of the mean. the trendline shows the best-fit linear line of regression. the pearson’s correlation coefficient for this data is 0.191 (e. coli) and -0.722 (enterococcus). size produced a strong negative correlation coefficient of -0.722. preliminary source tracking through the use of antibiotic susceptibility screening resulted in eight positive matches where the antibiotic susceptibility pattern of the e. coli sample from the lake matched the pattern from a fecal coliform sample isolated from waterfowl feces at the same lake (data not shown). after testing patterns of biochemical characterizations, one of these matches showed identical biochemical characterization using 20e api strips (data not shown). a. b. figure 3 a. b. 0 200 400 600 800 1000 1200 0 50 100 150 200 250 300 350 400 av er ag e e. c ol ia bu nd an ce (c fu /1 00 m l) lake size (acres) 0 5 10 15 20 25 30 35 40 45 50 0 50 100 150 200 250 300 350 400 av er ag e en te ro co cc us a bu nd an ce (c fu /1 00 m l) lake size (acres) 76 • fine focus, vol. 4(1) the question as to whether or not the abundance of waterfowl impacts the bacterial water quality of lakes is one that has been asked previously, leading to controversial results. this study aimed to provide insight into this topic in order to determine how the relationship between waterfowl and bacteria can impact the ecosystem as a whole and human health. results suggest that as the abundance of waterfowl present at a lake increases, the average e. coli abundance increases, with a positive correlation coefficient of 0.809, as shown in figure 1. while this coefficient is below the critical value for significance at the 95% confidence level, it is significant at the 90% confidence level and shows a strong positive trend worthy of future research with a larger sample size. this data lends support to the hypothesis that an increase in waterfowl abundance at a lake does increase the e. coli abundance and thus decreases the bacterial water quality and increases the potential human health risks. however, there was no definite correlation observed between waterfowl abundance and enterococcus abundance (figure 1), while there was a negative correlation (pearson’s correlation coefficient being -0.455) between the enterococci densities and e. coli densities at each lake (figure 2). while this correlation is not particularly strong, combined with the absence of a correlation between the waterfowl and the enterococcus spp., it suggests that there may be some type of competitive interaction occurring between the e. coli and enterococcus organisms and/ or the surrounding bacterial community that may be complicating the ecosystem dynamics observed in this study. a strong negative correlation (correlation coefficient of -0.722) was observed between lake size and enterococcus organisms (figure 3) while a very slight positive correlation was observed between e. coli and lake size (correlation coefficient 0.191). this further suggests there may be an interaction occurring between the two bacterial groups that is impacting their abundances. no relationship was found between ph and bacterial abundance, nor between temperature and bacterial abundance, so these factors do not appear to complicate the relationship. preliminary source tracking of fecal coliforms occurred in this study. a combination of antibiotic-susceptibility testing and 20e api strips was used to match samples isolated from water and fecal samples with the same patterns for both tests. one bacteria isolated from lake belulah was both positively identified as e. coli and matched the antibiotic susceptibility pattern and 20e api strip biochemical test results of an e. coli sample isolated from the feces of a white ibis at the same lake. sequencing of the 16s rrna and multiple other housekeeping genes would be necessary to confirm these two isolates are the same and identify the white ibis as the source of the fecal coliform isolated from the lake. while one positively source-tracked sample would not be considered significant, it would show that deposition of feces from waterfowl does contribute to the microbial community of discussion 77applied & environmental microbiology • freshwater lakes. it is important to note that not all bacteria were able to be tracked to a source for varying reasons. first, some of the bacteria did not survive in the lab until the end of the extensive study, and because of this, they could not be tracked to a source. additionally, some bacteria were not able to be matched back to an organism because the number of fecal samples gathered was limited. it was not possible to obtain fecal samples from every waterfowl, nor from every other potential source such as dogs, cats, livestock, and humans. future research will increase the number of samples collected and will examine the effects of plant abundance, anthropogenic pollution, and human traffic may have on waterfowl abundance. while the relationship between the bacteria and other factors at each lake was the primary focus of this study, the impacts that these relationships may have on the ecosystem and human health must be acknowledged. the bacterial water quality standards for lake class waters have been previously defined by the epa, and the results of this study were compared to those standards. only at one lake, lake mirror, was the average enterococcus density greater than the acceptable average, as displayed in table 1. moreover, none of the cfu counts for each individual site was greater than the acceptable value for enterococcus organisms. the e. coli densities, on the other hand, need to be addressed. the average e. coli abundance samples from three of the five lakes exceeded the epa geometric mean standards, and 14 of the 50 individual samples (28%) exceeded the individual sample standards. these potentially high levels of e. coli pose a threat to human health, given that such high levels of these coliforms may indicate high levels of pathogens. though a positive correlation between e. coli densities and waterfowl abundance was observed, it is possible that other factors may be contributing to these unusually high levels of bacteria, such as nutrient availability, competition between bacteria, vegetation presence and abundance, and anthropogenic pollution, such as the addition of fertilizer and oil run-off. in particular, if antibiotics have somehow entered these waters due to anthropogenic interactions and fecal contamination, any opportunistic bacteria or pathogens present in these waters may pose an even greater risk (10). a study conducted by costanzo, murby, and bates has shown that an increase in antibiotics in waterways can lead to bacterial resistance, contributing to the current worldwide antibiotic resistance crisis (10, 35). regardless of additional factors that may be influencing the bacterial abundance, these e. coli densities should not be ignored. these lakes are historically known to attract tourists and are often used for human recreational activities, such as kayaking, boating, and water skiing. on an ecological scale, bacteria occupy many niches, both harmful and beneficial. an increase in any number of microorganisms in an aquatic system may impact the bacterial ecosystem as a whole, changing the dynamic between organisms, as well as altering the biogeochemical cycles. for instance, increasing the nutrient availability in an aquatic ecosystem can lead to increased numbers of phytoplankton blooms, which can alter an ecosystem by depleting the oxygen and nutrients present in the water, 78 • fine focus, vol. 4(1) as well as decreasing the amount of sunlight to submerged vegetation, thus leading to a change in the entire ecosystem (2). increasing the bacterial abundance in an aquatic ecosystem may alter the entire dynamic, especially if these bacteria are introduced to the environment via contamination, thus essentially acting as invasive species, these introduced e. coli and enterococcus organisms must adapt and occupy niches that were previously occupied by the naturally occurring bacteria (19). overall, the bacterial water quality of the lakes sampled in this study was shown to be unacceptable based on the epa lake water standards when looking at e. coli densities. the ecosystem and 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antibiotic resistance crisis: part 1: causes and threats. j. formul. manag. 40:277–83. 36. wahyuni ea. 2015. the influence of ph characteristics on the occurance of coliform bacteria in madura strait. procedia environ. sci. 23:130–35. characterization of water-types and their influence on the antimicrobial properties of kombucha ferments against bacteria and yeast james k. lawton ii 1,2 and renu b. kumar 1* 1 minnesota state college and university (minneapolis community and technical college) 2 college of biological sciences, university of minnesota – twin cities, mn copyright 2016, fine focus all rights reserved manuscript received 15 april, 2015; accepted 1 november 2015 understanding the relationships between diet, gut microflora, and health is an increasingly important area of research. recent studies have demonstrated that kombucha tea provides variable antimicrobial activity against pathogenic microbes. in this study, we tested kombucha tea for antimicrobial activity against various gram-positive and gramnegative bacteria, as well as yeast, using an agar diffusion method. standard zone of inhibition assays were used to test the hypothesis that variance in antimicrobial activity against staphylococcus aureus, escherichia coli and candida albicans may be due to the varying levels of cations, like ca2+, found in different water-types (well water, artesian water, city water, type-ii water and distilled water). inductively coupled plasma-optical emission spectrometer (icp-oes) results indicated that high cationic (ca2+, mg2+, and na+) content water (well water) resulted in the largest zone of inhibition against s. aureus, with a 12.3% difference when compared to low cationic content water (typeii water). e. coli maintained a constant zone of inhibition regardless of water-type or batch-type, while c. albicans showed no zones of inhibition. inhibition is either through a synergistic relationship with the ph conditions, the other cations present (mg2+, na+, si, etc.) or a mix of both, as ph in the range of 4.5 3 is not enough to inhibit the growth of s. aureus. these results indicate that a direct relationship exists between cationic concentrations of water used to prepare kombucha, and antibacterial activity against s. aureus, due to the improved fermentation of the tea with high concentrations of cations. strong antimicrobial potential exists, particularly against s. aureus, which may be useful in determining novel approaches to synthesize antimicrobial drugs. further study is needed to assess other s. aureus strains, as well as to determine how this relationship translates to human microbiota interactions and their microbial metabolic profiles. abstract corresponding author *renu b. kumar renu.kumar@minneapolis.edu keywords • fermented tea • kombucha • antimicrobial • cation 40 • fine focus, vol. 2 (1) kombucha is a fermented tea produced with the help of a symbiotic culture of bacteria and yeast (scoby). the genus acetobacter is an aerobic, nitrogen-fixing bacteria which produces acetic acid, gluconic acid, and cellulose. saccharomyces is an aerobic to facultatively anaerobic, single celled yeast that produces ethanol and carbon dioxide. these two microorganisms have synergistic roles and are the most common components of scoby. the microbial composition of the scoby can vary and is dependent upon the origin of the culture (3). acetobacter produces cellulose, which is seen as a thin white film originating on the top of the tea as early as the first day of fermentation (1). this is when the bacteria and yeast cell mass accumulates to begin the synergetic fermentation process. the synergy of the yeasts cleaving the sugar and bacteria producing the acidic components is what establishes the unique cider-like carbonated characteristics of kombucha. saccharomyces strains cleave sucrose into glucose and fructose, with the former used to produce ethanol and carbon dioxide. acetobacter strains then oxidize ethanol to acetaldehyde and then into acetic acid (5). glucose also leads to the production of cellulose and gluconic acid. the acidic levels attained during fermentation (ph 3.0 2.5) result in unsuitable growing conditions for most microorganisms, reducing contamination (14). kombucha has been used as a beneficial health drink for several millennia. it has been suggested that kombucha aids in digestion, prevents microbial infections, can vitalize the physical body, increase the efficacy of the gastrointestinal tract, and is believed to enhance immunity (8). the popularity of kombucha in the united states has increased within the last 15 years, with annual reports showing kombucha sales expected to surpass $500m by the end of 2015 (10). this increase in popularity highlights the importance of the overall health implications of kombucha. kombucha has been identified as having antimicrobial properties against helicobacter pylori, salmonella typhimurium, staphylococcus aureus, agrobacterium tumefaciens, bacillus cereus, shigella sonnei, salmonella enteritidis and escherichia coli (8,12,15). no previous study has characterized water-types and identified its relationships with kombucha ferments relative to its antimicrobial activity. water-types five water types were selected on the basis of cationic concentration: 1.) well water taken from a private well located in prescott, wi usa; 2.) artesian water purchased from a local minnesota based company artesian fresh (leroy, mn usa); 3.) city water taken from a tap located in hennepin county, minneapolis, mn usa; 4.) type-ii water; 5.) distilled water from millipore water purification systems. water-types were analyzed for the major cations on a thermo scientific icap 6500 inductively coupled plasma-optical emission spectrometer (icp-oes) at the university of minnesota’s analytical geochemistry laboratory. triplicate measures were taken on each water sample, with the mean of each cation determined. high levels of ca2+, mg2+, and na+ were identified in well water and introduction materials and methods pathogens and antimicrobial factors • 41 artesian water, comparative to world health organization drinking water guidelines (7). silicon also appears with interesting values, but due to the higher levels of ca2+, mg2+, and na+ relative to si, and due to the biological role of these cations within cellular processes, this study focused on these cations, primarily ca2+. kombucha culture (scoby) kombucha cultures were purchased from anahata balance at organic-kombucha.com (buckley, mi usa). the primary strains used in these cultures were acetobacter xylinum and saccharomyces boulardii. the composition of the disks was specially constructed by anahata balance to fit the fermentation containers used, to prevent cross-contamination. each culture was then selected to ferment in only one water-type. a total of five cultures were assessed, one for each water-type in the experimental batches (as control batches did not have any cultures). preparation of tea ferments the recipe provided by anahata balance (fig. 1) was used to prepare solutions. kombucha was prepared by infusing 75.0g of organic sucrose (10% w/v) with 3.30g of hibiscus tea (0.44% w/v) into 750ml of boiling water in a 1l beaker, and allowed to steep for 20 min. organic sucrose and hibiscus tea were purchased from the wedge food co-op (minneapolis, mn usa). the contribution of the tea’s antimicrobial activity to kombucha ferments is shown to be insignificant (13) and as such, hibiscus tea was selected. five experimental batches (eb) and five control batches (cb) were prepared, each relative to the corresponding watertype. cbs were identical to the ebs except no kombucha culture was added (only water, sugar, and tea). after homogeneity of the sugar tea solutions in the ebs and cbs, the tea solutions were cooled to ambient temperature (20˚c) to allow for the addition of kombucha cultures to the ebs, in order to prevent harming the cultures. necessary ph sensor measurements were taken to ensure all ebs and cbs started the fermentation process at ph 4.5. ebs and cbs were then placed into a sterilized thermolyne type 4200 incubator at 30˚c for 8 – 9 days (ph dependent) until a ph of approximately 3.0 was reached in the ebs (as no change in ph would be noted in cbs). cbs ph was then adjusted to 3.0 with 1.0m acetic acid, which later acts as the ph controls. all ph determinations for the ebs and cbs were measured using a vernier ph sensor and vernier labquest palm pilot. ebs and cbs were covered with parafilm and then placed in a walk-in cooler for later experimentation. experimental test microorganisms two bacterial species were chosen to test against the antimicrobial activity of kombucha. one gram-positive bacteria: staphylococcus aureus atcc 27661 and a gram-negative bacteria: escherichia coli atcc 11303. both were provided by mctcs department of microbiology teaching stocks. s. aureus and e. coli were cultivated on nutrient agar (na) at 37˚c for 24h. a common human pathogenic yeast strain was chosen to test against the antimicrobial activity of kombucha. candida albicans sn76 was provided by dr. kirkpatrick at the university of minnesota. optical density determination optical density (od) determinations were measured with a vernier spectro visplus at 600nm, on a vernier labquest 42 • fine focus, vol. 2 (1) figure 1. kombucha mushroom tea culture starter recipe (anahatabalance.com) palm pilot. distilled water was used for calibration and for blank samples. a mcfarland standard of 0.5 was prepared using asm mcfarland standards sop 5.14.1 (2). bacterial and yeast colonies were transferred to 10ml of sterile distilled water and vortexed. after vortexing, od600 determinations were measured until each sample reached the target density at or around 0.20. each run included 2 blanks, 2 standards, 1 s. aureus, 1 e. coli, and 1 c. albicans. one run per zone of inhibition assay, three runs total, were performed. agar diffusion method the antimicrobial activity of kombucha was determined using an agar diffusion method. kombucha ebs and cbs were removed from the cold storage and allowed instructions: to make a 1 gallon batch of kombucha tea a. bring 1 gallon of distilled or filtered water to a boil. after the water starts to boil, slowly add 1 to 1 ½ cups of organic cane sugar. stir & cover solution, then simmer for another 10 min. or until sugar is completely dissolved. tea/sugar solution may be boiled in metal or glass container. it is suggested not to use nonorganic sugar, honey, or maple syrup, agave, etc. when brewing kombucha. alternative sugars may not provide the kombucha yeast cultures with the correct food source and is not recommended. b. remove tea solution from heat. add 6 tea bags (6 teaspoons) worth of green tea or black tea and allow solution to steep for 10 minutes. after 10 min. remove tea bags, cover, and allow solution to cool to room temperature. always keep tea solution securely covered with a tight weave cloth. c. transfer tea solution to a glass container for brewing the kombucha culture, never allow metal or ceramic to have long term contact with the culture. important note: add kombucha s.c.o.b.y. mushroom and starter liquid only after tea has cooled to room temperature. notes: make sure not to fill the brewing jar to full (it should be level with the straight side of the jar not up into the rounded edge. make sure that the smoother white side of mushroom faces upwards, it may float or sink, and this is acceptable. however, by gently placing the culture on top of the tea it should stay towards the top. if it does sink, the culture should start to rise to the surface as gases are forming in the fermenting tea. securely cover container with cloth (the culture needs to breath) and allow the inoculated tea solution to set undisturbed in a warm place out of direct sunlight. d. tea will be ready to drink after 5 to 14 days of fermentation, depending on temperature (around 75 -85 deg f is ideal) and amount of sugar added (more sugar = more time). if checking ph of a finished batch of tea, it should be around 3; a reading of 4 is to high and 2 too low. checking ph can greatly reduce the chance of contamination of the culture and is highly recommended. e. to make new batch of tea, make sure to save 10-15% of the old tea solution (as a starter to lower ph below 4.6) and one or two layers of the kombucha mushroom. when it is time for a new batch, just follow the directions above to start the process again. pathogens and antimicrobial factors • 43 cationic relationships results of the icp-oes water-type analysis are shown in table 1. high levels of ca2+ (104 ppm), mg2+ (56 ppm) and na+ (77 ppm) were observed in well water samples relative to the other water-type samples (fig. 2). of the 11 major cation concentrations determined in all five water-types, the three cations noted above (ca2+, mg2+, na+) demonstrated the largest variance throughout; in addition a gradient trend of ca2+ concentration levels was observed in the water-types and seemed to correlate with the zone of inhibition measurements in the s. aureus plates. due to ca2+ exhibiting the highest concentration levels, this study will focus on that cation. every metal cation has its own unique system to support prokaryotic and eukaryotic cellular regulation and growth requirements. of the most abundant cations required for these conditions (ca2+, mg2+, and na+), it is calcium that plays the universal role as a messenger, transmitting signals from the cell surface to the cytoplasm (6). calcium is also associated with the regulation of many types of cellular processes relative to cell differentiation, transport, motility, gene expression, metabolism, cell cycle effects, cell division, and pathogenesis (11). to date, a number of bacterial surface proteins have been implicated in various calcium transport mechanisms (11). in s. aureus, this includes a biofilm-associated protein (bap), a s. aureus surface protein essential for biofilm formation. studies have shown that s. aureus biofilm matrices are assembled by bap proteins (16). under controlled conditions, bap proteins are also shown to become inhibited by results and discussion to equilibrate to ambient temperature (20˚c). agar plates (100mm) were purchased “pre-poured” from teknova (hollister, ca usa). mannitol salt agar (msa) plates were used for s. aureus inoculations, luria broth (lb) agar plates were used for e. coli cultivation, and yeast mannitol (ym) agar plates were used for c. albicans. agar plates were inoculated using a spreading technique to achieve lawn growth throughout with use of sterile cotton wool swabs instead of glass spreaders. thirty millipore absorbent disks (25mm) were then sorted appropriately, three disks per eb water-type, and three disks per cb water-type (hence 30 total) with 1ml of the appropriate correlating batch. this was to ensure duplicate and triplicate measurements were taken. all appropriate general lab practices were followed to prevent cross-contamination of one batch to another during disk saturations. once disks became saturated they were placed in the center of the inoculated agar plate, one disk per plate (3 inoculated plates per batch type, 30 total). plates were then covered and placed into a norlake scientific incubator at 37˚c for 72 h. agar plates were then removed from the incubator and zone of inhibitions were measured with a standard metric ruler. measurements included the diameter of the clearings end-to-end, disregarding the disk in the center. all zone of inhibition assays were carried out in triplicate and analyzed statistically using 1-way anova (p<0.05). 44 • fine focus, vol. 2 (1) calcium treatments, thus preventing any bap-mediated biofilm formation (4) and allowing s. aureus to become susceptible to any number of exposures that can lead to cellular complications. as the concentration of calcium increases, so do the zones of inhibition by s. aureus. this result seems to be a response specific to the calcium concentrations seen on the icp-oes in the water-types, and not ph alone, as the cbs were used as ph controls. s. aureus, when exposed to the cbs, saw zero detectable inhibition, which tells us that a ph of 3.0 does not inhibit s. aureus. calcium could be playing a direct role, either through a synergistic relationship with the ph or with the other cations present (mg2+ & na+) or a combination of these effects. changes in ph during fermentation the changes in ph during fermentation for the cbs and ebs are shown in fig. 3. in the first three days of fermentation, cbs observed the greatest change in ph, 4.5 to 3.3, a 1.2 ph difference, while the ebs maintained their initial ph throughout the 8-day period, suggesting no microbial contamination occurred. the initial cbs ph drop could be relative to the high availability of glucose for yeast and bacteria, which ultimately leads to the production of acetic acid and gluconic acid, decreasing the ph. as glucose gets consumed, the availability becomes limiting, thus resulting in a slower change of ph. antimicrobial activity of the ferments the antimicrobial activity of the eb and cb treatments against bacteria and yeast strains are shown in figs. 4 and 5. results indicate that high-ion (ca2+, mg2+, na+) content water (well water) resulted in the largest zones of inhibitions at 35.7 mm for s. aureus, with a 12.3 percent difference when compared to near zero ion content water table 1: results of the icp-oes analysis on the water-types evaluated in this study (+/sd). water type well water artesian water city water type-ii water distilled water al (ppb) 4.3 ± 1.0 6.3 ± 0.8 8.8 ± .0.8 1.0 ± 1.5 6.5 ± 0.6 ba (ppb) 42.5 ± 0.8 105.0 ± 1.2 7.2 ± 0.3 0.3 ± 0.5 0.0 ± 0.5 ca (ppb) 103716.7 ± 231.4 72122.0 ± 214.3 22332.0 ± 54.4 117.9 ± 3.7 -1.7 ± 11.2 fe (ppb) 13.1 ± 0.9 1.0 ± 0.9 10.0 ± 0.9 1.6 ± 0.7 0.0 ± 1.2 k (ppb) 1201.8 ± 11.7 1219.0 ± 7.8 2561.1 ± 9.9 4.0 ± 0.6 5.0 ± 4.6 mg (ppb) 56462.0 ± 53.3 23091.0 ± 166.1 6518.4 ± 21.3 7.8 ± 0.7 3.1 ± 8.7 mn (ppb) 8.3 ± 0.2 4.8 ± 0.1 0.3 ± 0.1 5.6 ± 0.1 0.2 ± 0.2 na (ppb) 77140.0 ± 145.8 3546.2 ± 28.2 16581.3 ± 36.7 9.0 ± 71.8 -26.3 ± 9.1 p (ppb) 4.4 ± 3.8 0.2 ± 3.8 281.1 ± 5.6 2.3 ± 1.6 -0.1 ± 2.4 si (ppb) 8398.4 ± 45.7 5778.7 ± 32.4 4352.1 ± 8.6 5.0 ± 1.4 59.0 ± 4.0 sr (ppb) 88.2 ± 0.6 121.7 ± 0.8 54.9 ± 0.2 0.2 ± 0.0 0.2 ± 0.5 pathogens and antimicrobial factors • 45 (type-ii water) at 31.3 mm. e. coli maintained a constant zone of inhibition regardless of water-type or batch-type, while c. albicans showed no zone of inhibition outcomes. six different sets of the antimicrobial activity of kombucha eb treatments against s. aureus, (table 2) were used to calculate percent change, with the presumption that well water ebs = 100 percent zone of inhibition. relative to well water, type-ii water had the lowest standard deviation value of +/-2.07. ebs and cbs had zero detectable effect on c. albicans, as aggressive growth was seen throughout the plate, even on the absorbent disks. these results indicate a direct relationship may exist between kombucha prepared in high-ca2+ content water and the antibacterial activity of kombucha ferments against s. aureus. it is also suggestive of further study to determine how this relationship applies to other pathogenic strains. this behavior is indicative of a gradient trend, with an r2 value of 0.9604, which seems to correlate well with the ca2+ concentration levels identified within these water-types. the e. coli strain displayed constant zone of inhibitions regardless of batch-type, with a mean variance of 4349mm. both s. aureus and e. coli results were ph independent for ebs and cbs. we can conclude that something other than the acidic conditions must be inhibiting s. aureus. one possibility could be the varying levels of cations, especially ca2+, in the water-types. distilled water is the gold standard source of water when making kombucha, and as such, understanding the figure 2: comparison of the icp-oes major cation results relative to water-types. figure 3: after fermentation, cbs were adjusted to a ph of 3.0 for comparative purposes against ebs. analyte co nc en tr at io n (p pm ) 100 80 60 40 20 0 ca mg na si p k well artesian city type-ii distilled ph fermentation time (days) 5 4.5 4 3.5 3 2.5 0 2 4 6 8 10 eb ph cb ph 46 • fine focus, vol. 2 (1) zo ne o f in hi bi tio n (m m ) 60 50 40 30 20 10 0 20 15 10 5 0 well water artesian water city water type-ii water distilled water well water artesian water city water type-ii water distilled water table 2. comparison of six different eb treatment sets of the antimicrobial activity against s. aureus. s. aureus zone-of-inhibition %-change water type set-1 set-2 set-3 set-4 set-5 set-6 mean standard deviation standard error of mean (sem) well water 100.00 100.00 100.00 100.00 100.00 100.00 100.00 0.00 0.00 artesian water 0.00 5.70 0.00 12.50 14.60 7.70 6.75 6.13 1.02 city water 11.1 5.7 13.9 5.4 11.0 5.1 8.7 3.8 0.6 type-ii water 11.1 11.4 13.9 10.2 12.2 7.7 11.1 2.1 0.4 distilled water 13.9 20.0 19.4 5.4 2.4 2.6 10.6 8.2 1.4 figure 4. antimicrobial activity of kombucha ferments versus controls. figure 5: zone-of-inhibition results of eb and cb treatments against s. aureus, e. coli and c. albicans s. aureus cb s. aureus e. coli cb e. coli c. albicans cb c. albicans (a -c ) z on e of in hi bi tio n (m m ) (d ) z on e of in hi bi tio n (m m ) r2 = 0.9604 (a) s.aureus eb (b) e.coli eb (c) e.coli cb (d) s.aureus cb; candida eb & cb 50 48 46 44 42 40 38 36 34 32 30 28 26 24 pathogens and antimicrobial factors • 47 antimicrobial properties of kombucha in response to the addition of metal ions is still yet unknown and unidentified. since no detectable zone of inhibitions were observed in s. aureus cb treatments and in c. albicans eb and cb treatments, statistical data only represent the remaining treatment scenarios. because ca2+ concentrations were much higher in well water and artesian water relative to the other water. the antimicrobial effects of kombucha are reflected as having no inhibition for c. albicans, and a constant zone of inhibition for e. coli regardless of eb or cb treatments. s. aureus displayed the greatest potential, with zone of inhibitions decreasing in size linearly relative to the cationic concentrations measured by the icp-oes in each of the five water-types. this is indicative that a ca2+ relationship may exist due to the same linear fashion it displayed in its concentration levels decreasing in each of the water-types (well water > artesian water > distilled water). it should also be noted that mg2+ and si also exhibited linear drops in concentration levels relative to water-types, but due to being less than the ca2+ levels and not playing as much a role within cellular systems as ca2+, they were not the focus of this paper. every metal cation has its own unique system to support prokaryotic and eukaryotic cellular regulation and growth requirements. however, it is calcium that plays the universal role as a messenger, transmitting signals from the cell surface to the interior of the cell (6). calcium is also identified as regulating many types of cell processes relative to cell differentiation, transport, motility, gene expression, metabolism, cell cycle, cell division and pathogenesis (11). within that, a number of bacteria, surface proteins, many number of these factors have been identified and shown to be involved in various calcium transport mechanisms (11). on s. aureus one such component has been identified as a biofilm-associated protein (bap), a s. aureus surface protein that is essential for biofilm formation. one particular study shows that s. aureus biofilm matrices assembled by bap proteins protect the bacteria from antimicrobial treatments (15), and under controlled conditions, these bap proteins become inhibited by calcium treatments, thus preventing any bapmediated biofilm formation (4) and in return allowing s. aureus to become susceptible to various environmental stressors. as the concentration of ca2+ increases, so does the zone of inhibitions in s. aureus. this result appears to be a response specific to the calcium concentrations measured in the water-types, and not ph alone, as the cbs were used as ph controls, and as such, when s. aureus was exposed to the cbs, zero inhibition took place. this tells us that a ph of 3.0 is not enough to inhibit s. aureus. there must be a direct role that these cations are having on the kombucha during fermentation to allow inhibition, possibly calcium, either through synergistic relationships with the kombucha scoby, ph or with the other cations (mg2+, na+, si). no ca2+ control was conducted, so it is difficult to say with certainty how much of a role ca2+ is really having on s. aureus. it is suggested that if this study is replicated, that cationic controls be inserted. s. aureus is a major opportunistic human pathogen, able to cause a wide range of conclusion 48 • fine focus, vol. 2 (1) diseases in humans and in animals (6), which is why it is important to further investigate these relationships. further study is needed to determine how these cations are affecting kombucha fermentation, how it is interacting with s. aureus, and then whether or not the same results apply to other s. aureus strains, specifically methicillin resistant s. aureus (mrsa). acknowledgments funding provided by mctc science club with support of club advisor dr. bram middeldorp. we are grateful to dr. robin wright, who provided constant academic support and financial assistance, and dr. david kirkpatrick at umn for providing the candida albicans strain used in this study. references 1. ahantharik, p., laohawiwattanakul, s., premsuk, a., ratanapo, s., and talawat, s. 2006. efficacy of fermented teas in antibacterial activity. kasetsart j. nat sci.:40:925–933. 2. american society for microbiology (asm) press. mcfarland standards sop 5.14.1. http:// www.asmpress.org/asmpress/files/cclibraryfiles/ filename/000000000262/05.14.01.pdf. accessed february 2011. 3. ammar, e., bakhrouf, a., and battikh, h. 2012. antimicrobial effect of kombucha analogues. lwt food sci. technol.: 47:71-77. 4. amorena, b., arrizubieta, m.j., lasa, i., penadés, j.r., and toledo-arana, a. 2004. calcium inhibits bapdependent multicellular behavior in staphylococcus aureus. j. bacteriol. 186:7490-7498. 5. asai, t. 1968. acetic acid bacteria: classification and biochemical activities. tokyo: university of tokyo press. 6. barton, l.l. 2004. structural and functional relationships in prokaryotes. springer science: 793794. 7. bartram, j., and cotruvo, j., eds. 2009. calcium and magnesium in drinking water: public health significance. geneva, world health organization. 8. čanadanović-brunet, j.m., lončar, e.s., malbaša, r.v., and vitas, j.s. 2011. influence of starter cultures on the antioxidant activity of kombucha beverage. food chem. 127:1727-1731. 9. clarke, s.r., costabile, a., gibson, g.r., and sannasiddappa, t.h. 2011. the influence of staphylococcus aureus on gut microbial ecology in an in vitro continuous culture human colonic model system. plos one: 6:e23227. 10. crum, h., and lagory, a. 2001. the kombucha crisis: one year later. bevnet. http://www.bevnet. com/news/2011/the-kombucha-crisis-one-year-later accessed february 2011. 11. dominguez, d.c. 2004. calcium signaling in bacteria. mol. microbiol. 54:291-297. 12. greenwalt, c., ledford, r., and steinkraus, k. 1998. determination and characterization of the antimicrobial activity of the fermented tea kombucha. lwt food sci. technol. 31:291-296. 13. hotchkiss, j. h., mortlock, r. p., shapiro, k. b., and steinkraus, k. h. 1996. investigations into the antibiotic activity of tea fungus/ kombucha beverage. acta biotechnologica: 16:199-205. 14. gruender, k., leitzmann, c., fromme, s., and mayser p. 1995. the yeast spectrum of the tea fungus kombucha. mycoses 38:289-295. 15. knol, w., sreeramulu, g., and zhu, y. 2001. characterization of antimicrobial activity in kombucha fermentation. acta biotechnologica: 21:49-56. 16. valle, j., latasa, c., gil, c., toledo-arana, a., solano, c., penadés, j.r., and lasa, i. 2012. bap, a biofilm matrix protein of staphylococcus aureus prevents cellular internalization through binding to gp96 host receptor. plos pathogens 8: e1002843. pathogens and antimicrobial factors • 49 undergraduate research in the sciences as a series of transformative opportunities jill bange undergraduate student, senior majoring in microbiology and premedical preparations at ball state university copyright 2014, fine focus all rights reserved perspective“ expectations of undergraduates undergraduate students tend to find, from the moment of their arrival on campus until graduation, that they are held to a long series of ever-increasing professional expectations. some are curricular standards set by the university, while others are evolving objectives the students decide for themselves. students anticipate they should engage during lecture and laboratory courses, maintain high academic standings throughout their college career, and, potentially, pursue part-time employment. but perhaps one of the most pronounced expectations of an undergraduate is for the student to become socially and professionally involved on campus. the idea of campus “involvement” can be both vague and intimidating, especially to a new student. universities have numerous and varied organizations; these can be academic, social, faith-based, or service-oriented in nature, to name only a few. when a student is faced with many opportunities but has limited time, it can be a challenge to decide which of these commitments are worth pursuing with limited time. it is in an undergraduate student’s best interest to choose activities that complement his or her area of study while promoting personal and professional growth. however, a student may be more interested in finding activities which build lasting, meaningful relationships with peers. this fundamental choice does not have to be a mutually exclusive one. none of the elements mentioned above are missing from undergraduate research experiences, which is why commitment to extended study outside of the classroom is one of the most valuable uses for an undergraduate’s time. this is especially true of students majoring in biology. research allows students to apply broad concepts learned in the classroom to original research problems in the field or laboratory setting, all of which enhances content comprehension, professional development, and peer interaction. content comprehension and technical skill the most immediate benefit of an undergraduate research experience is the ability to translate what is learned in the laboratory to one’s understanding of scientific concepts learned in the classroom. a recent study by hunter et al. indicated a common gain for students after an undergraduate research experience was perceiving “increased relevance of coursework” (2007). in a science lecture, broad and sometimes overgeneralized ideas are taught first, and eventually the finer details are covered. research, however, begins by trying to answer a very specific question or solve a particular problem. for example, my first research experience involved determining the effects of different concentrations of carvacrol (a bactericidal extract from oil of oregano) on bacillus cereus, a toxigenic bacterium associated with foodborne illness and ocular infections. using a nematode model, caenorhabditis elegans, i was able to quantify the effects of bacillus toxins because the nematodes would ingest the bacteria and become infected. although i had no background in cell biology or genetics at that point in my college career, my research advisor was able to build from my knowledge of basic biology and teach me about the organism i was studying. often, i encountered information while working in the lab before i had taken a course which covered those ideas—part of my bacillus project involved transforming the bacterium with a specific plasmid vector that my advisor and i had designed. when i took genetics a few semesters later, i studied how bacteria are naturally competent. research for me became a balance of relating concepts from the classroom to my project, and relating 78 • fine focus, vol. 1 my research back to the classroom to realize the real-world implications of what i was learning. this learning style does not stress memorization as much as application, which is more valuable considering scientific “facts” may change with breakthroughs (aaas, 2011). translating knowledge between the lab and the classroom allowed me to appreciate the complexity and importance of what i was studying, while giving me a better, more complete understanding of some of the more challenging theories. as classroom content is applied to a realworld setting, students performing research also begin to increase their technical skill set in the lab. some of the first aspects of my research experience were becoming oriented with the lab and learning proper execution of basic bench skills, such as using aseptic technique or performing polymerase chain reaction (pcr). bench work and instrumentation revealed the reality of research: it can often be tedious. but the practical experience was worthwhile in learning what the process of designing, executing, and analyzing an experiment is like from start to finish. one of the most valuable skills learned in research is the ability to troubleshoot problems when they arise. in the early phases of my bacillus study, one nematode was to be placed in an individual well with agar on a 96 well plate. then, each individual nematode could be studied separately as the bacillus toxins began to take effect. isolating a microscopic animal, however, turned out to be extraordinarily difficult. it was hard to avoid picking up multiple nematodes at a time, so the methodology for the project had to be amended. while this may sound like it would have been a frustrating experience, it was actually exciting and eye-opening. the difference between a real undergraduate research experience and a “canned” lab experiment that a student encounters in a basic biology class is that no one knows the “right” way to execute a research project. this gives the student ownership of the entire experiment and the freedom to be creative when adjusting for problems encountered during the process, and the end results are that much more rewarding when the project is completed. the three-hour labs designed for a classroom setting may give students some practice in bench techniques, but these skills are only applied to a piece of an overall research experiment. in an immunology lab, i read through a three-part protocol that stated parts one and two had been done for the students. this is not a criticism of the immunology course; it simply illustrates that students have a limited perspective of the goals in a research experiment and the process involved to acquire the end results in a short lab period. furthermore, students may find it difficult to imagine application of methods they are using to solve real problems, even if they understand the concept being illustrated in a classroom lab. this is the advantage of undergraduate research: students are exposed to the scientific method from beginning to end, including the planning of the project and ““ research for me became a balance of relating concepts from the classroom to my project, and relating my research back to the classroom... perspective • 79 the presentation of results. after participating in undergraduate research, i no longer look at a graph and see only the data, but also have an appreciation for the months and years of work that producing only one figure required. i have a more realistic conception of what research is like, and i am more able to understand how other scientists arrived at their results and conclusions because i have a sense of what they may have done in their own processes. in fact, one study indicated students who perform research-based activities rather than lab-based activities gain confidence in interpreting data (brownell, 2012). when i have questions about a concept in microbiology, i can imagine how a scientist may have approached discovering an answer because i have been exposed to a number of techniques and instruments used in my field. research enables a student to think critically within his or her own field rather than simply accepting facts in a classroom without being able to put the “pieces” together in a broader understanding of the world. performing undergraduate research shifts a student’s outlook on aspects of his or her own specific area of study. but the research process may also give students a new appreciation for other natural sciences as well, primarily because students will discover that subsets of science are not separated by as distinct of boundaries as course curricula may indicate. while i primarily use techniques i have learned in microbiology courses in the research lab, i also find myself referring to knowledge i acquired in chemistry or physics classes to execute my project. for example, purifying the plasmids necessary for transformation of bacillus requires a number of reagents. i relied on general chemistry knowledge to make these solutions at appropriate concentrations. and while not a topic i studied directly, having knowledge of the laws that govern forces and energy because of my physics education also helps me to understand the living systems in which i was interested. physics, chemistry, and biology all build upon each other, something that is not stressed in lecture. therefore, it was difficult for me to see how necessary my understanding of all of these disciplines was until i had research experience. undergraduate research gives a student appreciation for all of the “core curricular” sciences, but for students studying microbiology, research also allows for a better understanding of the relationship between the various disciplines within biology. for example, my research in environmental microbiology involved taking measurements such as ph, dissolved oxygen, water table height, and temperature of the water in which the algae of interest was growing. these data were considered when studying nutrient effects on algae because they can influence algal metabolism, as well as the presence of other microbes, and interactions between these communities also impact algal biomass and metabolism. evaluating the influence of the environment on microorganisms helped me appreciate that the toxigenic bacterium i was studying in the food microbiology lab also changed depending on the conditions in which the cells grew, even though my work was in the laboratory and not in the field. i appreciated more the ability to carefully control variables and i became a more conscientious scientist. while working with bacillus, i learned the importance of handling samples with precise, sterile techniques, and this training prepared me to more efficiently process hundreds of water samples in the environmental microbiology lab. my involvement in two laboratory projects has exposed me to the details within a subdiscipline, but has also enabled me to think critically about the broader concepts and implications of the subjects i am studying, and the problems and diagnoses i will make in my future training and career. 80 • fine focus, vol. 1 “soft” skills and professional development developing a deeper understanding of the biological sciences through research is a critical and valuable undergraduate experience, and a student undertaking a research project might expect this to be an outcome of the process. what students may find surprising is that they also grow interpersonal skills immensely while engaging in research. communication of scientific concepts becomes more comfortable as a student has more practice both reading and writing scientific literature. utilizing primary literature—peer-reviewed publication of original scientific findings—is helpful in learning background information for a project, but it also adjusts a student to thinking and speaking in scientific terminology. as scientific studies produce information much faster than editions of textbooks can be produced, relying on scientific articles for supplemental detail of a broader classroom concept can be a critical piece of an undergraduate science education (hoskins, 2007). the first time i read a seven-page piece of primary literature about bacillus, i spent several hours deciphering the dense writing. i found later that this was a valuable investment of my time; i became more confident in speaking about my research to professors and other students because i understood the “language.” with enough practice, i could read a scientific article as fast as i could read anything else, and this gave me a sense of belonging to the scientific community. the more a student reads primary literature, the better he or she will be able to compose a poster presentation, oral presentation, or manuscript in the future, and the more insightful their questions will become. likewise, delivering an oral or poster presentation requires much practice to convey the essential information to an interdisciplinary audience. successfully transferring the salient aspects of your work to a mixed audience involves not only a thorough understanding of your project on all levels, but a realization for how to “teach” and engage your audience as well. this concept is becoming more important with each passing year as new specialty areas develop within each subdiscipline of the life sciences. without consideration of the audience at hand when rehearsing a presentation, the implications of a student’s finding may be lost on those who are not familiar with the jargon of a subspecialty. it is critical that a student presents his or her findings in a way that allows the scientific community to learn from the results and build from them in future studies. with careful preparation, especially in the background content of a presentation, a student can successfully and confidently convey findings from a study without overestimating the audience’s background, and without running overtime, two of the most common errors among students and experienced researchers alike. as a student gains more experience presenting, these presentations become less rehearsed and more of a conversation between the student and the audience. this is an exciting transformation, because students can begin to share ideas with peers about each other’s projects, and they become more interested and engaged in each other’s work as the conversation progresses. i encountered this at the 2014 indiana academy of science conference, where a professor was presenting a poster on her study of the nervous system of the same nematode model which i used for my bacillus project. as the conversation progressed, i was both learning from this professor and offering valuable information for her; it was a discussion that felt more collegial than instructional, which is atypical compared to most of my interactions with professors. communicating and sharing ideas in this way builds a sense of fellowship between students and professors, so the student starts to feel less perspective • 81 like a science major and more like a scientist through this process of contributing and collaborating. collaboration is, in fact, an important piece of the research process. even if a student is working on an individual project, he or she will often rely on peers who have more research experience for advice and wisdom. this student-centered learning, with the advising professor assuming the role of a facilitator rather than an instructor, builds students’ prowess in the lab and willingness to give input as to the direction of the research projects discussed. teamwork in the lab makes the research projects more successful, but it also allows a students to form valuable friendships with others of their own discipline. another research experience which i undertook relied heavily upon collaboration. during the summer of 2013, i studied in the bonanza creek experimental forest in fairbanks, alaska for three weeks with a professor and graduate student. we were assessing the effects of warming and nutrient addition on algal biomass and metabolism. this experiment had many components, and at times, it was difficult to keep the “big picture” in mind when i was focused on my comparatively small set of data. i was able to rely on the graduate assistant for help when i was trying to make sense of the results. she helped me have a better appreciation for the role of algae as primary producers, and i was able to keep the end goal of the experiment in mind because of her explanations. i began to see her as a mentor, but also as a friend, because we worked very closely over the course of those three weeks. but these friendships form regardless of the length or location of the project. i interact with students working in the same research labs as i on a more regular basis than many other students. not only do we collaborate on our research together, but we have many of the same classes together as well, so some of the best connections i have had with peers during my college career have resulted from research experiences. my relationships with my faculty advisors have also grown and become more valuable than i anticipated as i have become more involved with research. at the beginning, i was being told what to do and how to work at the bench. i was being taught in the traditional way i was used to in a classroom, although it was one-on-one interaction. as my skills grew and i relied on my professors less for technical instruction, i felt more confident in expressing my take on the data or my ideas for amending the methodology. my advisors respected what i had to offer; i felt trusted and accepted as a scientist, even while i was still their student. beyond that, my advisors have been incredible resources to me in realms outside of the research laboratory. they have written recommendation letters for me and edited my research presentations and posters, but they’ve also given me advice throughout my undergraduate career, which has been what i value most about out interactions. i can share experiences i’m having in class or in the process of applying for medical school, and they encourage me and give me a sense of what to expect as i move forward in my college years. having a faculty member support me as i work to accomplish my goals has increased my confidence and improved my work, and has been easily the best aspect of my undergraduate research experiences. by mentoring undergraduate students, faculty engage in service to their profession by training future scientists. of course, the student is helping further that research project, but there is a great deal of commitment to the training of that student and investment in that student’s future given by the most dedicated faculty before those results emerge. “service to the profession” has been heavily emphasized in my own 82 • fine focus, vol. 1 research training; for example, i have been especially encouraged to be on a journal editorial board someday so that i can aid my peers in science by critiquing their manuscripts. as an undergraduate, students are prepared for this service through research training. students will often read and discuss primary literature with each other or an advisor and learn how to critique an author’s work thoroughly while still communicating the errors respectfully. following an advisor’s example, more advanced students can also facilitate the training of some of the novice students in bench technique and general concept comprehension. commentary on each other’s poster presentations and talks also models a professional conference, in which a scientist would field questions from colleagues and engage in dialogue about the study. in addition to professional service, students also serve their community through volunteerism. our lab community, for example, organizes a fundraiser for next generation nepal, which is a non-profit dedicated to returning trafficked children to their families. we use the “penny war” method of collecting donations and involve the science professors and students in the process. while not directly connected to our lab work, this collaboration for a greater cause on the part of a few research students has allowed us to contribute to society in both an academic and social capacity. personal growth and identity as students begin to build relationships with peers and faculty who are also involved in research, these students are engaging in a socialization process into the scientific community. students in an undergraduate research experience are integrating the role “scientist” as part of their identity, and they are learning that a scientist is so much more than someone who executes an experiment. as i enter into my last year of undergraduate research, i find that i begin to take on the role of a peer mentor while still being guided by advisors and other students. mentorship is so closely connected to research because science involves a great deal of collaboration to be successful. the characteristics that i have appreciated in my own advisors—patience, enthusiasm, and respect—i have attempted to implement in my own attitude when working with other students. for example, when consulting a lab partner on methodology for the bacillus project, i noticed that she had difficulty recalling some of the math concepts from general chemistry. i was able to find a new way of explaining the calculations that she hadn’t heard before which made sense to her. at the same time, my lab partner organized the methodology into a list and was able to walk me through what needed to be done. she saw the bigger picture of the project and how we needed to progress through each phase, whereas i was focused on the details of a particular step. we both assumed different roles in the partnership and were able to teach each other different aspects of the same research project, which was valuable leadership practice. in the future, my career as a physician will require a great deal of patience and commitment to mentorship of medical students and resident physicians. these partnerships are most successful when the members rely on each other’s strengths, even though one is the “mentor” and one is the “mentee.” it is difficult to be engaged and “ “ my advisors respected what i had to offer; i felt trusted and accepted as a scientist, even while i was still their student. perspective • 83 invested in one’s own learning if one does not have an active hand in the learning process. as a physician, i would expect my mentees to offer input regarding the subject material and i, as a mentor, would be willing to let the students take ownership over solving the problem at hand with guidance from me. i know this method has worked for me while i have been a mentee, and i think it is important to deviate from the traditional lecture-based learning to some degree so the students feel like a valued member of the class or group. this is what research does, and i believe my peer mentorship experience from research will translate easily to the medical field. beyond mediating discussion and encouraging my peers in science, research has increased my interest in developing methods of communicating scientific findings to the general public. through my involvement as an editor for fine focus, i collaborate regularly with a marketing team, while my role is primarily for handling manuscript submissions. the interdisciplinary project has revealed to me the importance of packaging content in a way that is appealing and understandable for a target audience. this is a new concept to me; i am familiar with marketing products, but the intricacies of marketing information have become a more immediate challenge to me as someone striving to publish in the sciences. the frustration that scientists can feel when their findings are lost on an under-informed audience is expressed by volpes’ the shame of science education (1984): public understanding of science is appalling. the major contributor to society’s stunning ignorance of science has been our educational system. the inability of students to appreciate the scope, meaning, and limitations of science reflects our conventional lecture-oriented curriculum with its emphasis on passive learning. i would argue that while the public may have a limited view of some current scientific studies, scientists also have a minimal understanding of how to convey that information to a broad audience. scientists write and talk for other scientists in the system of publication that currently exists. these are valuable data and analyses, but it is not for everyone. i would argue that undergraduate participation in research begins to encourage students to think about science from other perspectives so that the student can communicate to individuals of various educational backgrounds. for example, an ecology professor of mine once played an npr interview of a paleontologist; this is a perfect example of an instance in which language had to be carefully tailored to speak to a particular audience, and this particular interviewee did so effectively. undergraduates may, in their future careers, encounter situations in which they need to convey findings to the media or other public entities. collaboration in research is a small step in developing these communication skills, because students are only working with other science majors. nonetheless, students are bound to encounter diversity even within the sciences, and this studentcentered, active learning process is excellent practice for conveying scientific content to a number of audiences. to be certain, communicating scientific concepts is both exciting and challenging for any student new to research. a student is bound to encounter road blocks throughout the research process which will require critical thinking and problem solving, especially when the original methods fail to produce acceptable results. these frustrations are combatted by the desire to satiate one’s own curiosity as to how living systems operate, which develops as one becomes more attached to the research project, and more empowered in knowing that research can allow these discoveries to be 84 • fine focus, vol. 1 had. this desire for understanding motivates a student to be flexible as he or she copes with the challenges associated with running an experiment. i felt tested when attempting to run a successful polymerase chain reaction (pcr) for my bacillus experiment. pcr, like other tools in the arsenal of a microbiologist, involves sensitive reactions and is timesensitive as well. it was important for me to be precise when working with small volumes of dna, primers, and reagents. even though i knew i had handled the samples carefully, it still took several attempts to generate copies of the plasmid i needed, and i was embarrassed i might have had poor technique. my advisor reassured me i was doing well, and that successful pcr is determined by a variety of factors, some of which may have been outside my control. with this in mind, i was able to be more patient with myself as i made more attempts at pcr, and this shift in attitude has translated over to my classroom work as well. i am less likely to get frustrated if, for example, i am trying to solve a chemistry problem that i don’t understand. instead, i look for creative approaches to the question and persist until i find an explanation for the concept that makes sense to me. this patience and flexibility is crucial to the mindset of a college student, because balancing schoolwork can be difficult. training in perseverance through the research process helps a student better face this obstacle. once i was able to solve problems on my own in the lab, i began to feel more ownership over the project which had been assigned to me. i was more comfortable working without supervision and i felt responsible for performing quality work, even though there would be no “grade” assigned to my research. this intrinsic motivation is harder to feel in a classroom setting. classroom learning is passive, and students may not know how to integrate information that seems surface-level (lopatto, 2009). the knowledge a student gains in a lecture doesn’t feel as though it “belongs” to the student because it is so readily given. but new knowledge generated in research almost has an emotional attachment associated with it, because the student knows first-hand the work required to discover this information. in this way, research is its own reward, and it fosters a desire for understanding in other realms of a student’s life. the personal satisfaction and comprehension of scientific content are only gained, however, if the student is producing original results at the end of the research process. a research project which does not add new knowledge to the scientific community does injustice to both the student and fellow scientists. a typical classroom science lab, when the results are known at the process is designed to “work”, is helpful in illustrating a concept but does little to prepare a student for the reality of research as a career, in which results are elusive and methodology often needs revision (chmielewski, 2009). furthermore, if a student is not striving to solve unanswered problems through research, the student does not have new information to publish, and the opportunity to grow scientific writing and presenting skills is lost. one way to ensure that a student is building on prior studies but is developing novel results is by reading primary literature. consulting scientific journal articles, whether for a course or for research, begins to feel more like participating in a dialogue than tedious work. i became more interested in scientific discovery as my research progressed, making me more willing to ask questions of my teachers and advisors when i was confused. throughout primary and even secondary education, there is this fear associated with “being wrong” which can prevent students from engaging in classroom conversation. this anxiety quickly becomes outweighed during undergraduate education by the perspective • 85 desire to know more as a student becomes more involved with research. this internal drive brought on by research has allowed me to overcome the fears associated with the risk of trying something unusual. adapting the attitude that a new experience will enable me, even if it may seem intimidating at first, has been a direct lesson of my undergraduate research experiences. i have learned not to feel anxious when i don’t know what to anticipate from a class or a job, because i have experience encountering “the unexpected” in the lab. for example, in the bacillus experiment, the plasmids were designed with the addition of the gfp gene, so that once the bacteria transformed the vector, fluorescence would be an indicator of expression of products on the vector. we used a flow cytometer to measure fluorescence, and we expected stressed bacillus to express a particular gene on the vector and therefore fluoresce. we also expected our control bacteria not to fluoresce because they did not have the vector with gfp. however, our control bacillus did fluoresce. the experiment was repeated, because it was assumed that we mislabeled our samples or some other aspect of the methodology went wrong. but again, the control bacteria fluoresced. making sense of the unexpected was challenging and exciting, and it was concluded that when stressed, bacillus must produce a primary metabolite that fluoresces. an experience that could have been frustrating ended up being enlightening, and it has allowed me to readily embrace new challenges. broad impacts engaging in an undergraduate research experience is a large undertaking. scientific discovery involves active learning and adapting to new findings, a process initially uncomfortable to students accustomed to lecture-style lessons and rigid syllabi. yet these challenges enable a student to grow in ways that a standard course could not allow. students learn the complexity of the scientific method, and are able to appreciate and understand published literature after going through the process themselves. students collaborate with faculty and peers to better communicate their findings and learn from the experience of others. students come to realize that they are more capable in understanding and performing science than they could have known. the contributions which undergraduate research students make to the body of scientific knowledge are rewarding and stimulate further interest and motivation in scientific work. in my own experience, research has allowed me to feel immersed in the process of doing science and has made me more invested and interested in my own education. my undergraduate career would have been incredibly different without research as a tool to enhance my core understanding of science and improve my confidence in professional settings. i highly encourage all students participate in an undergraduate research experience to realize their full potential as a scholar and scientist. ““... undergraduate participation in research begins to encourage students to think about science from other perspectives... 86 • fine focus, vol. 1 perspective • 87 1. american association for the advancement of science. vision and change in undergraduate biology education: a call to action. washington, dc: 2011. 2. brownell, s. e., kloser, m.j., fukami, t., & shavelson, r. (2012). undergraduate biology lab courses: comparing the impact of traditionally based ‘cookbook’ and authentic research-based courses on student lab experiences. j. coll. sci. teach, 41:18-27. 3. chmielewski, j. g., & stapleton, m. g. (2009). the biologists’ forum: the undergraduate research experience: it’s really not for everyone, students and faculty alike. bios, 80: 53-58. 4. hoskins, s. g., stevens, l.m., & nehm, r.h. (2007). selective use of the primary literature transforms the classroom into a virtual laboratory. genetics, 176: 1381-1389. 5. hunter, a., laursen, s. l., & seymour, e. (2007). becoming a scientist: the role of undergraduate research in students’ cognitive, personal, and professional development. science education, 91: 36-74. 6. lopatto, d., & tobias, s. (2009). science in solution: the impact of undergraduate research on student learning. tucson: research corporation for science advancement. 7. volpe, e.p. (1984) the shame of science education [abstract]. american zoologist, 24:433-441. works cited 9 objective lens john l. mckillip, ph.d managing editor, fine focus ball state university perspective“ objective lens • copyright 2018, fine focus. all rights reserved. professor of biology, 10• fine focus, vol. 4(1) this issue is likely our most substantial yet in terms of content volume and variety. this issue reflects the dedication of dozens of students, faculty, and collaborators who have been active in managing fine focus, all while developing new partnerships with universities in the us and europe over the last several months. the primary visible outcome of much of this effort is reflected in the individual perspectives at the end of this issue, written by undergraduates at the university of detroit mercy, whom we have most recently adopted as a campus ambassador. the stem outreach described in our summer 2017 issue (fine focus vol. 3(2)) is the means by which these activities have been accomplished. this semester has been an intense but rewarding experience at many levels. the mini-documentary film produced by the most recent fine focus editorial staff, captures many of these students’ personal stories, and will soon be available and linked to our website finefocus.org. we hope you find their spoken perspectives to be inspiring and memorable. last autumn, fine focus hosted our first annual executive committee meeting at the virginia b. ball center for creative inquiry (www.bsu.edu/vbc). our executive committee consists of kyla adamson, a scientist at eli lilly & co., in indianapolis, in; emma kate fittes, a journalist for the indianapolis star; saara-maria kallio, a marketing and salesforce consultant at t-systems multimedia solutions in dresden, germany; and indira komanapalli, who holds her ph.d. in molecular biology and has agreed to join our team as a fundraising specialist. during our 2 ½ day meeting, we covered a great deal of material, and identified a few key priority areas that i would like to share with you here. first, we would like to introduce prospective authors to the concept of overleaf, a new platform that will allow submitting authors to format their manuscripts during submission to fine focus exactly how they will appear if published. this system is user-friendly, and will greatly streamline copyediting and proofing of the final manuscript after acceptance. our next issue will contain additional information and instructions on how this new system will be used by authors. speaking of our web presence, we are in the midst of a complete website update to bring fresh content and a refined new look to the international microbiology research community. if this new site is not yet up by the time you read this issue, it will be within the next few weeks. with assistance from saara kallio and emma kate fittes, we are also updating our marketing plan to incorporate aspects of the new campus ambassador program (cap). the cap 11 will consist of selected universities around the world that maintain a strong emphasis on undergraduate research. we aim for this program to increase our submissions, enabling us to produce a more impactful journal as we progress. it is important to also note that while these partners will provide fine focus with submissions, they will also serve an important role as the extension of our global presence and will strengthen our domestic network. lastly, we organized a plagiarism task force over last fall and winter, and now have a standardized system in place to use for screening incoming manuscripts for plagiarism. these checks will be run as part of our standard vetting stage prior to each new submission going out for double-blind peer review. we always welcome your suggestions and feedback by email at finefocus@bsu.edu -jlm objective lens • undergraduate perspective • 135 perspective“undergraduate perspectives from the fine focus student team, fall term 2017, university of detroit mercy 136• fine focus, vol. 4(1) working with fine focus this semester has been a truly insightful and fulfilling experience. upon hearing about the opportunity to work with ball state university’s fine focus, i was immediately drawn in and excited for what was ahead. as an undergraduate student in research, i understood the importance of the work behind publishing a paper, but from this experience i learned what happens on the other side of submission, the great effort put into creating a quality research journal, and what many journals look for in research articles. the experience with the fine focus group was not only educational, but also personal. the students at detroit mercy were able to connect with those from ball state; hearing each other’s stories and learning hands-on from one’s peers was a personable learning experience for all of us. the work that fine focus is doing has bounds of benefits to students from all places. with the fine focus journal, they are recognizing hardworking undergraduates within the stem sciences and spreading this new science across the world. under the grant for the educational outreach, fine focus is teaching important aspects of research that is often gone unseen (peer-review and publication) and diversifying the publication industry by reaching out to different student groups. in a field that needs more diversity, inclusion, and input, fine focus’ mission and work is vital to the future of science. we rebuildetroit students that have collaborated with fine focus value diversity within the stem fields, and we are ecstatic to see the fire for teaching others and diversifying the field ignite with a group organization like fine focus. perspective is everything, and i can only say “thank you” to the fine focus group for teaching us about the perspective of reviewers and publishers and for valuing our perspectives. sarah buhay biology (pre-medical) “ “ fine focus is teaching important aspects of research that is often gone unseen (peer-review and publication) and diversifying the publication industry by reaching out to different student groups. undergraduate perspective • 137 ball state university has an incredible group of undergraduate students that call themselves fine focus. fine focus’s mission is to give undergraduate researchers from all over the world an opportunity to publish their hard work. that is something fantastic and every university should fragment a similar group with similar ideologies. before i attended my first fine focus meeting, i was very hesitant, especially because i was one of the few rebuildetroit students who were randomly selected to be a part of this special opportunity. i had no idea what the ball state group was about, or why they chose the university of detroit mercy (which is in a completely different state) as a place to communicate and distribute their knowledge. after my first two meetings, i fell in love with everything they were doing. we sat through various presentations and participated in many engaging activities that were meant to help us better understand the process of the editorial board. the members of the editorial board are the ones who review journals; in this case, microbiology journals. i learned a lot from fine focus. i learned that the editorial board does a double-blind peer review. a double-blind peer review is something many journals do, including fine focus, so that the identity of the authors and the reviewers are never revealed to each other. something cool i learned was that fine focus receives many submissions from authors all over the world, including third world countries. many of the authors of these countries don’t have english as their first language, so when their manuscripts go through the editorial process, rejections are often given out due to a plethora of errors. errors may include maintaining a consistent idea that is brought up in the paper, or too many grammatical errors. with all the hard work fine focus is doing, i hope that we can establish a similar group on our campus who can publish undergraduate research from the detroit area or even from all parts of the world. i appreciate fine focus for taking their time and driving all the way to detroit to educate us in the stressful work that goes into editing journals. i am so thankful for this experience. ali issa biology i’m christina jones, a rebuildetroit scholar and biology major at the university of detroit mercy. i joined this program as a community college student with little research experience and a lot of determination. since my transition to udm, i have gained extensive research experience working in a bacterial pathogenesis lab at the school of dentistry. i work christina jones biology (pre-dental) 138• fine focus, vol. 4(1) with yersinia pestis, the causative agent of plague. i have also become a published author through my work under the sea-phages program, a discovery-based undergraduate research course administered by the university of pittsburg and the howard hughes medical institute. collaborating with the ball state fine focus group has been a far more insightful and enjoyable experience than i would have imagined. i was surprised to learn about all that goes into the peer review process and feel that this knowledge will help me when i draft my own report for publication. i enjoyed having the opportunity to share my experiences with fellow undergraduate students and to give feedback on an actual article being reviewed by the fine focus team. this collaboration has also helped to reinforce my passion for research—i love being a part of the scientific community and each new experience that i have as a rebuildetroit scholar brings me that much closer to where i want to be in life. as a biology/pre-dental major and senior at udm, i am excited to continue my journey in research in the field of dentistry. i hope to pursue a dds/phd or dmd/phd degree so that i may continue publishing my work and contributing to scientific knowledge. “ participating in fine focus was a chance for me to enhance my researching skills and learn all that goes into the making of a scientific journal. from this experience, i learned the do’s and don’ts of writing a scholarly article, was able to have intellectual conversations with peers about issues that arise in article reviewing processes, and gained friendships. being around a group of individuals who also share a passion for research was a valuable and unique experience for me. one that i brag about. “ shelbi matlock engineering & science this collaboration [with fine focus] has helped to reinforce my passion for research—i love being a part of the scientific community and each new experience that i have as a rebuildetroit scholar brings me that much closer to where i want to be in life. undergraduate perspective • 139 from this, i have gained confidence in my own scholarly article writing, assurance in knowing what scholarly journals look for, and an abundance of information that i can use towards my own research and article writing experiences. another thing is the pleasure of knowing that i have created a network with young researchers like myself who i can turn to if i need advice or help. i am extremely thankful and forever grateful for the collaboration of rebuildetroit and fine focus of ball state. my experience with fine focus this last semester was one that i didn’t think i would enjoy as much as i did. i was expecting a cut and dry set of lectures about the peer review process and all the behind-the-scenes work that goes on in a publication. there was always good food to be eaten, stories to be shared, and laughs to be heard throughout the building. not only did our team of build scholars learn a lot from this experience, but over the course of several months, we became friends. the crew from muncie, indiana would always seem eager to trek out to detroit, michigan, just so they could teach us and graciously involve us in their magazine. believe me when i say, the energy and tenacity that these people have is amazing, and i’m glad we were given the opportunity to come together and put the work in with them to get the last issue out. it was also a treat feeling like a hollywood celeb, being interviewed underneath all the hot lights, with microphone wires running up and down our shirts, being able to talk about our experiences in the world of science, research, and eventually publishing and disseminating some incredible novel research. working with fine focus has not only given me an inside view of the workings of a scientific journal, but has also helped me forge friendships that span across the country, which is a true blessing. i give my best to the whole ball state crew, and hope to see everyone again soon! channing sesoko engineering & science nicotine: its stimulating and inhibitory effects on oral microorganisms aubrey e. dubois*1, zachary c. bennett*1, umara khalid2, ariba khalid1, reed a. meece1, gabrielle j. difiore3, and richard l. gregory4 1department of biology, school of science, purdue university, indianapolis, in; 2department of psychology, school of science, purdue university, indianapolis, in; 3department of kinesiology, school of physical education and tourism management, indiana university, indianapolis, in; 4department of oral biology, school of dentistry, indiana university, indianapolis, in *a.e.d. and z.c.b. are joint first authors of this work. copyright 2014, fine focus all rights reserved manuscript received 15 september, 2014; accepted 30 october, 2014 tobacco users are much more susceptible to dental caries and periodontal diseases than nontobacco users. research suggests that this increased susceptibility may be due in part to nicotine, a primary active component of tobacco. five bacterial species and one yeast species commonly found in the human oral cavity, lactobacillus casei, actinomyces viscosus, actinomyces naeslundii, rothia dentocariosa, enterococcus faecalis, and candida albicans respectively, were utilized to investigate if any correlation existed between exposure to various concentrations of nicotine ranging from 0 to 32 mg/ml and the growth of each microorganism. the minimum inhibitory concentration (mic), minimum biofilm inhibitory concentration (mbic), and planktonic growth were measured. the mic was determined to be 16 mg/ ml for all organisms except e. faecalis, which had an mic of 32 mg/ml. nicotine had a varying effect on planktonic growth across the different species. a distinct upward trend in biofilm formation was found in a. viscosus, l. casei, e. faecalis, and c. albicans through 8 mg/ml. nicotine also enhanced r. dentocariosa biofilm formation in all concentrations through 8 mg/ml but was most enhanced at 1 mg/ml. alternatively, a. naeslundii exhibited a complete downward trend through 32 mg/ml. the mbic was found to be 16 mg/ml in all organisms studied. these findings further support research suggesting that the increased susceptibility to oral health diseases experienced by tobacco users may be caused in part by an upregulation in biofilm formation of these oral pathogens. abstract corresponding author richard l. gregory rgregory@iu.edu keywords • dental caries • periodontal disease • biofilm • nicotine • tobacco • lactobacillus casei • actinomyces viscosus • actinomyces naeslundii • rothia dentocariosa • enterococcus faecalis • candida albicans 64 • fine focus, vol. 1 introduction periodontal disease is a condition that causes inflammation of the gums including diseases such as gingivitis, in which gums become swollen and bleed easily. periodontitis, another form of periodontal disease, is an advanced form of gingivitis in which the gums pull away from the teeth and form pockets where dental plaque begins to grow. this infection can lead to both severe tissue and bone damage due to the increased exposure to previously protected areas. periodontal disease is also bacterial host interactions • 65 closely linked with dental caries. the occurrence of both periodontal disease and dental caries can be associated with several factors. some of the most common risk factors include smoking, diabetes, autoimmune disorders, genetics, and obesity (14). the use of tobacco products remains the largest risk factor for periodontal disease. environmental tobacco smoke has been associated with the increased likelihood of caries formation in children (2). recent studies in this laboratory demonstrated increased biofilm formation of s. mutans with increasing nicotine concentrations (20). the average nicotine content in a single cigarette has been found in amounts up to 2 mg (12). a recent study tested over 40 smokeless tobacco products, such as snuff and chew, and it was discovered that they contained anywhere from 3.6-25.3 mg of nicotine per gram of smokeless tobacco (33). nicotine contents of cigars have been found to vary greatly, ranging from 5.9 mg per cigar to 335.2 mg per cigar (18). studies have demonstrated that when smoking cigarettes, an average of approximately 1 mg of nicotine per cigarette is absorbed by the body, however, the amount of nicotine within oral biofilm of smokers has not yet been explored (5). this study used the following opportunistic pathogens, commonly found in the human oral cavity, to investigate possible effects on growth and biofilm formation. lactobacillus casei lactobacillus casei is a gram-positive, facultative anaerobic bacterium. they are nonmotile and have a rod shape. like other strains of lactobacilli, l. casei produces a significant amount of lactic acid, allowing it to remain viable under various ph levels. poor diet, weak tooth enamel, previous plaque buildup, and acidic conditions contribute to an optimal environment for l. casei to thrive (22). l. casei alone lacks adequate strength to adhere to tooth enamel, however, when co-cultured with streptococcus mutans, l. casei gains the ability to synthesize glucans thus improving tooth enamel adhesion. because of this, l. casei along with s. mutans have been found to be the most prevalent bacteria leading to dental caries (19). actinomyces viscosus actinomyces viscosus is a facultative anaerobic, gram-positive bacterium that is often isolated from the oral cavity, especially from subgingival plaque and along the teeth and gums. a. viscosus is rod-shaped and filamentous. distinct characteristics of the filamentous shape include branching with swollen, rounded, or clavate ends (7). a. viscosus is a severely cariogenic bacterium that acts as an agent in promoting and initializing carious lesions (15). studies have shown that the bacterium has adhesins which allow it to bind to complementary receptors on a substrate. a. viscosus binds to salivary acidic proline-rich proteins (prps) adsorbed onto the apatitic surfaces of the tooth. cryptic segments exposed in adsorbed molecules are identified by the adhesins, which allow a. viscosus to efficiently attach to teeth while planktonically suspended in saliva (1). actinomyces naeslundii actinomyces naeslundii is a gram-positive bacterium characterized by its rod shape and prominent fimbriae that allow it to bind to the tooth surface. a. naeslundii is facultatively anaerobic, nonmotile, nonspore forming, and commonly grows in the oral cavity. (6) a. naeslundii is also a major component of dental plaque and it is known to cause dental caries, root canal infections, and periodontal disease (27). it is one of the specific species of the genus actinomyces capable of causing the chronic bacterial infection, actinomycosis, in humans (8). a. naeslundii plays a role in actinomycosis by initiating infections in carious teeth, as well as causing infections in places where the mucosal integrity of the tooth is compromised (32). these infections are the precursor of the actinomycosis, however, the bacterium itself exhibits very low pathogenic properties. these properties are enhanced by the presence of microorganisms such as species of prevotella, staphylococcus, and streptococcus (35). rothia dentocariosa rothia dentocariosa is a facultative anaerobic, gram-positive, pleomorphic bacterium that is commonly found in either filamentous or coccoid forms (24). it is common in the oral cavity, and has been associated with many types of bacterial infections. although r. dentocariosa was first isolated in dental caries, endocarditis is the most common infection caused by the organism. cases have also been reported associating r. dentocariosa with intrauterine fetal death (16,25,28). r. dentocariosa is commonly associated with dental caries and periodontal disease. studies have demonstrated the ability of r. dentocariosa to induce the release of tnf-a by macrophages, thereby eliciting inflammatory responses within the gingiva (36). enterococcus faecalis enterococcus faecalis is a facultative anaerobic, gram-positive cocci bacterium that is typically found in pairs or short chains. the most common locations for this microorganism in humans are the gastrointestinal tract, the vagina, and the oral cavity. e. faecalis is considered to be the most prevalent bacterium found in root canal infections (23). it is able to resist nutritional deprivation in part due to its ability to utilize a number of energy sources including malate, citrate, lactate, carbohydrates, and a number of amino acids. it is also able to withstand extremely basic environments, high salt concentrations, desiccation, and the presence of many antibiotics (17). these attributes allow e. faecalis to thrive in deep root canals. additionally, scientists recently discovered e. faecalis has the ability to suppress lymphocyte function as well as produce lytic enzymes which aid in destruction of gingival tissues (9). candida albicans candida albicans is a ubiquitous pleomorphic fungus that colonizes the gastrointestinal, epithelial, and mucosal tissues of over 70% of the human population (26). whether c. albicans takes on hyphal, pseudohyphal, or yeast form is dependent upon environmental factors such as temperature, ph, and presence of serum. c. albicans is an opportunistic pathogen that is often the cause of infection in immunocompromised patients, such as those with hiv/aids or indwelling medical devices. imbalances in normal body flora due to antibiotic and steroid use have also been shown to provoke the overgrowth of candida, causing candidiasis (21). oral candidiasis, commonly known as thrush, can exacerbate already existing oral diseases. c. albicans has also been found within dental caries and is a contributor to periodontal disease (26). many proteins within saliva have been found to promote adherence of c. albicans to gingival tissues (10). additionally, tissues already colonized by streptococcal strains greatly enhance the ability of c. albicans to colonize the oral cavity (10). 66 • fine focus, vol. 1 microbial strains and media one yeast and five bacterial strains were utilized for this investigation. all microorganisms were obtained from the american type culture collection (atcc), manassas, va. the bacterial strains used were: lactobacillus casei (atcc 393), rothia dentocariosa (atcc 17931), enterococcus faecalis (atcc 29212), actinomyces naeslundii (atcc 12104) and actinomyces viscosus (atcc 43146). the yeast strain used was candida albicans (atcc 10231). four of the bacterial strains were grown in tryptic soy broth + 1% sucrose (tsbs; difco laboratories, detroit, mi), while actinomyces naeslundii was grown in brain heart infusion broth (bhi; difco). all bacteria were incubated at 37°c in 5% co2 for 24 or 48 hours. c. albicans was grown in yeast peptone dextrose broth (ypd; difco) at 30°c in 5% co2 for 48 hours. the design of the experiment is consistent with other studies that have been both published and conducted in this laboratory (20). minimum inhibitory concentration and planktonic growth quantification nicotine dilutions were prepared using a 1 g/ml nicotine stock solution (sigma chemical co., st. louis, mo) in tsbs or bhi for the bacteria and in ypd for the yeast. an initial dilution of 32 mg/ml of nicotine was prepared and a 1:2 serial dilution series was made through 0.25 mg/ ml. additionally, a control was prepared containing only 200 μl of media with no nicotine. diluted aliquots of 190μl were transferred into a sterile 96-well microtiter plate along with 10 μl of an overnight culture of the respective organism. the bacterial microtiter plates, excluding a. naeslundii, were incubated at 37°c in 5% co2 for 24 hours and c. albicans and a. neaslundii were incubated under the same conditions for 48 hours. after incubation, the absorbance of each well in the plates was measured in a spectrophotometer (spectramax 190; molecular devices, inc., sunnyvale, ca) at an optical absorbance of 595 nm (od595) to determine the minimum inhibitory concentration (mic). (20) to quantify the planktonic growth, 120μl aliquots of planktonic culture fluid was removed from each well into corresponding wells in a fresh 96-well microtiter plate and the absorbances determined in a spectrophotometer at 595 nm. absorbance values greater than .05 units of the control would be considered a significant change. anything equal or less to .05 absorbance units would be considered insignificant. minimum biofilm inhibitory concentration ninety six-well plates of the organisms in varying concentrations of nicotine were prepared as described above. following incubation, the remaining planktonic culture fluid was disposed of and 200μl of 10% formaldehyde (fisher scientific co., pittsburg, pa) was added to each well for 30 minutes at room temperature. the plate was then washed in deionized (di) water before adding 200μl of 0.3% crystal violet (sigma) to each well. the plate was allowed to incubate for an additional 30 minutes and then washed in di water. 200μl of isopropanol (fisher) was then added to each well to allow extraction of the crystal violet from the biofilm cells. following a one-hour incubation in isopropanol at materials and methods bacterial host interactions • 67 results table 1 indicates the mic and mbic of each microorganism tested. all organisms had an mic of 16 mg/ml of nicotine except e. faecalis, which had an mic of 32 mg/ ml of nicotine. similarly, the mbic of all organisms tested was 16 mg/ml of nicotine. the total growth of the organisms in each nicotine concentration is presented in fig. 1. this data represents the total of biofilm and planktonic growth. l. casei, a. viscosus, r. dentocariosa, and c. albicans had an increase in growth through 8 mg/ml of nicotine. e. faecalis growth increased through 16 mg/ ml but growth of all other organisms was decreased at this concentration of nicotine. a. naeslundii growth decreased through 8 mg/ ml and demonstrated complete inhibition beginning at 16 mg/ml. additionally, all organisms displayed a decrease in total growth at 32 mg/ml of nicotine. fig. 2 denotes the planktonic growth of each organism in nicotine. decreased growth occurred at 32 mg/ml of nicotine in all organisms tested. additionally, l. casei, a. viscosus, and r. dentocariosa demonstrated statistically significant inhibition of planktonic growth at 16 mg/ml. an increase in growth occurred at 8 mg/ml of room temperature, the optical density of the biofilm was recorded at 490 nm. using this method, both r. dentocariosa and a. naeslundii were unable to form tight biofilms in their 96-well plates. therefore, instead of utilizing formaldehyde, crystal violet and isopropanol, the biofilm of r. dentocariosa and a. naeslundii were resuspended in 200μl of a 0.9% saline solution and the optical density was measured at 595 nm. statistical analyses all experiments were carried out in quadruplicate wells at least three times. means, standard deviations, and statistical significance were calculated using excel. student t-tests were used to determine p values, which were considered significant if p<0.05. data was transferred to sigmaplot 12.0 to graph and further analyze results. 68 • fine focus, vol. 1 organisms l. casei a. viscosus a. naeslundii r. dentocariosa e. faecalis c. albicans mic (mg/ml) 16 16 16 16 16 32 mbic (mg/ml) 16 16 16 16 16 16 minimum inhibitory concentration (mic) and minimum biofilm inhibitory concentration (mbic) of nicotine on microorganisms table 1 minimum inhibitory concentration (mic) and minimum biofilm inhibitory concentration (mbic) of nicotine on microorganisms fig. 1: total growth of nicotine-treated microorganisms in quadruplicate wells. after incubating with various concentrations of nicotine the od was measured at 595 nm. the od value is represented as (mean + sd). each experiment was repeated at least three times. asterisks indicate statistical significance (p< 0.05) compared to the 0 mg/ml nicotine control. lactobacillus casei nicotine concentration (mg/ml) o pt ic al d en si ty a t 5 95 n m 0.000 0.200 0.400 0.600 0.800 0 0.25 0.5 1 2 4 8 16 32 actinomyces viscosus nicotine concentration (mg/ml) o pt ic al d en si ty a t 5 95 n m 0.000 0.200 0.400 0.600 0.800 0 0.25 0.5 1 2 4 8 16 32 1.000 rothia dentocariosa nicotine concentration (mg/ml) o pt ic al d en si ty a t 5 95 n m 0.000 0.200 0.400 0.600 0.800 0 0.250.5 1 2 4 8 32 1.000 16 candida albicans nicotine concentration (mg/ml) o pt ic al d en si ty a t 5 95 n m 0.000 0.200 0.400 0.600 0.800 0 0.25 0.5 1 2 1.000 4 8 16 32 actinomyces naeslundii o pt ic al d en si ty a t 5 95 n m nicotine concentration (mg/ml) 0.000 0.200 0.400 0.600 0.800 0 0.25 0.5 1 2 16 32 1.000 4 8 enterococcus faecalis nicotine concentration (mg/ml) o pt ic al d en si ty a t 5 95 n m 0.000 0.200 0.400 0.600 0.700 0 0.250.5 1 2 8 32 0.500 0.300 0.100 164 a. c. e. f. d. b. bacterial host interactions • 69 fig. 2: planktonic growth of nicotine-treated microorganisms in quadruplicate wells. after incubating with various concentrations of nicotine for 48 hours, the supernatant was removed and the od of the supernatant was measured at 595 nm. the od value is represented as (mean + sd). each experiment was repeated at least three times. asterisks indicate statistical significance (p< 0.05) compared to the 0 mg/ml nicotine control. lactobacillus casei nicotine concentration (mg/ml) o pt ic al d en si ty a t 5 95 n m 0.000 0.050 0.100 0.150 0.200 0 0.25 0.5 1 2 4 8 16 32 actinomyces viscosus nicotine concentration (mg/ml) o pt ic al d en si ty a t 5 95 n m 0.000 0.100 0.150 0.200 0.250 0 0.25 0.5 1 2 4 8 16 32 0.300 0.050 actinomyces naeslundii o pt ic al d en si ty a t 5 95 n m nicotine concentration (mg/ml) 0.000 0.010 0.020 0.030 0.040 0 0.25 0.5 1 2 16 32 0.050 4 8 rothia dentocariosa nicotine concentration (mg/ml) o pt ic al d en si ty a t 5 95 n m 0.000 0.050 0.100 0.150 0.200 0 0.250.5 1 2 4 8 32 0.250 16 enterococcus faecalis nicotine concentration (mg/ml) o pt ic al d en si ty a t 5 95 n m 0.000 0.020 0.040 0.060 0.070 0 0.250.5 1 2 8 32 0.050 0.030 0.010 164 candida albicans nicotine concentration (mg/ml) o pt ic al d en si ty a t 5 95 n m 0.000 0.005 0.010 0.015 0.020 0 0.25 0.5 1 2 0.025 4 8 16 32 a. c. e. f. d. b. 70 • fine focus, vol. 1 fig. 3: biofilm formation of nicotine-treated microorganisms in quadruplicate wells. after incubating with various concentrations of nicotine for 24 or 48 hours, the biofilm was either stained with crystal violet or suspended in saline and the od was assessed at 490 nm or 595 nm, respectively. the od value is represented as (mean + sd). each experiment was repeated at least three times. asterisks indicate statistical significance (p< 0.05) compared to the 0 mg/ml nicotine control. lactobacillus casei nicotine concentration (mg/ml) o pt ic al d en si ty a t 4 90 n m 0.000 0.500 0.750 1.250 1.500 0 0.25 0.5 1 2 4 8 16 32 1.000 0.250 actinomyces viscosus nicotine concentration (mg/ml) o pt ic al d en si ty a t 4 90 n m 0.000 2.000 3.000 4.000 0 0.25 0.5 1 2 4 8 16 32 1.000 actinomyces naeslundii o pt ic al d en si ty a t 4 90 n m nicotine concentration (mg/ml) 0.000 0.200 0.400 0.600 0.800 0 0.25 0.5 1 2 16 32 1.000 4 8 rothia dentocariosa nicotine concentration (mg/ml) o pt ic al d en si ty a t 4 90 n m 0.000 0.050 0.100 0.150 0.200 0 0.250.5 1 2 4 8 32 0.250 16 enterococcus faecalis nicotine concentration (mg/ml) o pt ic al d en si ty a t 4 90 n m 0.000 0.050 0.150 0.250 0.300 0 0.250.5 1 2 8 32 0.200 0.100 164 candida albicans nicotine concentration (mg/ml) o pt ic al d en si ty a t 4 90 n m 0.000 0.200 0.400 0.600 0.800 0 0.25 0.5 1 2 1.000 4 8 16 32 a. c. e. f. d. b. bacterial host interactions • 71 table 1 indicates the mic and mbic of each microorganism tested. all organisms had an mic of 16 mg/ml of nicotine except e. faecalis, which had an mic of 32 mg/ ml of nicotine. similarly, the mbic of all organisms tested was 16 mg/ml of nicotine. the total growth of the organisms in each nicotine concentration is presented in fig. 1. this data represents the total of biofilm and planktonic growth. l. casei, a. viscosus, r. dentocariosa, and c. albicans had an increase in growth through 8 mg/ml of nicotine. e. faecalis growth increased through 16 mg/ ml but growth of all other organisms was decreased at this concentration of nicotine. a. naeslundii growth decreased through 8 mg/ ml and demonstrated complete inhibition beginning at 16 mg/ml. additionally, all organisms displayed a decrease in total growth at 32 mg/ml of nicotine. fig. 2 denotes the planktonic growth of each organism in nicotine. decreased growth occurred at 32 mg/ml of nicotine in all organisms tested. additionally, l. casei, a. viscosus, and r. dentocariosa demonstrated statistically significant inhibition of planktonic growth at 16 mg/ml. an increase in growth occurred at 8 mg/ml of nicotine for a. viscosus and r. dentocariosa. moreover, e. faecalis planktonic growth was enhanced at 8 mg/ml and 16 mg/ml of nicotine. significant inhibition was observed in 1 mg/ml of nicotine for a. viscosus and for 4, 8, and 32 mg/ml of nicotine in c. albicans. a. naeslundii along with other bacteria in this study depict a high sensitivity to nicotine. the planktonic readings varied between trials, which made interpretations of planktonic response to nicotine inconsistent at times and difficult to interpret. biofilm formation of the organisms in each nicotine concentration is indicated in fig. 3. an upward trend in biofilm growth through 8 mg/ml of nicotine was observed for l. casei, a. viscosus, e. faecalis, and c. albicans. r. dentocariosa exhibited the most growth at 1 mg/ml of nicotine but also had increased growth in 0.25, 2, and 8 mg/ml. inhibition of biofilm formation for all organisms was indicated at 16 and 32 mg/ml of nicotine. additionally, a. naeslundii demonstrated statistically significant inhibition beginning at 1 mg/ml and continuing through 32 mg/ml of nicotine. discussion significant inhibition occurred with all tested organisms in nicotine concentrations of 16 and 32 mg/ml except e. faecalis, providing a mic of 32 mg/ml for e. faecalis and a mic of 16 mg/ml for the remaining organisms. similar studies demonstrate other oral microorganisms, most notably s. mutans, also have a mic of 16 mg/ml. (7)(20) it is possible that the mic exists at nicotine concentrations between 8 and 16 mg/ml but further studies are required to determine the validity of this supposition. research by huang et al. (2014) also demonstrated stimulatory effects by nicotine on streptococcus gordonii (34). another study investigating nicotine effects on oral microorganisms like actinobacillus actinomycetemcomitans, porphymonas gingivalis, fusobacterium nucleatum and s. gordonii demonstrated no effect could be observed in concentrations less than 1 mg/ ml (13). the research conducted with these microorganisms is consistent with this study and demonstrates significant variations in activity at higher concentrations of nicotine. alternatively, pavia et al. (2000) were able 72 • fine focus, vol. 1 to demonstrate that nicotine concentrations between 100 and 250 ug/ml reduced growth of escherichia coli, listeria monocytogenes, candida albicans, klebsiella pneumoniae and cryptococcus neoformans (27). all concentrations of nicotine exerted some inhibition of planktonic growth, particularly at 16 and 32 mg/ml, when compared to the non-nicotine-treated controls for both l. casei and c. albicans. previous research suggests that planktonic bacteria are more susceptible to various chemicals and antibiotics and so it is possible that the presence of nicotine induced this effect on the microorganisms studied (4). no reports were found on planktonic yeasts relating to chemical susceptibility, but it is plausible that similar effects as those observed in planktonic bacteria might also exist in yeast. all species displayed planktonic inhibition at 32 mg/ ml. furthermore, at concentrations of 16 mg/ ml both r. dentocariosa and l. casei were significantly inhibited. c. albicans growth was also inhibited in nicotine concentrations of 4 and 8 mg/ml. l. casei, a. viscosus, r. dentocariosa, e. faecalis, and c. albicans demonstrated no statistically significant differences in non-nicotine treated planktonic organisms compared to those treated with nicotine concentrations up to 2 mg/ml. a. viscosus, r. dentocariosa and e. faecalis had planktonic growth enhancement in concentrations of 16, 8, and 8 through 16 mg/ ml, respectively. all od values measured from the planktonic growth were much lower than od values recorded from biofilm growth. visual analysis of the microtiter plates also signified greater biofilm growth when compared to planktonic growth. it is known that bacteria prefer existing in biofilms rather than as unicellular planktonic cells and it has been suggested that nearly 90% of bacteria exist as biofilms in nature. (31) the present study further supports this theory as well as extends the observation to c. albicans due to its perceived preference to grow as a biofilm rather than as a planktonic yeast phase. as fig. 3 indicates, a notable upward trend in the biofilm formation of l. casei, a. viscosus, a. naeslundii, e. faecalis and c. albicans was demonstrated through 8 mg/ml of nicotine. biofilm growth was most notably enhanced in 8 mg/ml of nicotine in the aforementioned organisms. r. dentocariosa presents a unique demonstration of enhancement in concentrations of nicotine ranging from 0.25 to 8 mg/ml but was most prominently enhanced at 1 mg/ml. in addition, studies have shown that an increase in adhesion to surfaces occurs over time when c. albican, s. mutans, and s. pneumonaiae are exposed to cigarette smoke condensate (csc). (4)(29) csc is a crude aqueous extract of tobacco and contains approximately 2.4% nicotine by weight along with many other chemical components of tobacco. it is plausible that one of the attributing factors to the increase in adhesion is due to the nicotine contained within the csc, which would thus account for the results observed in this study. additionally, antunes et al. 2012 (3) found that oxidative stress caused increased biofilm formation of p. aeruginosa. this study identified hydrogen peroxide as one causative agent of oxidative stress in cigarette smoke. other research has indicated nicotine can also act as an oxidative stressor, which might account for the increased biofilm formation seen in many of the nicotine concentrations used in the current study (11). the mbic of nicotine for all organisms studied was determined to be 16 mg/ml as indicated in fig. 3. as with the mic, it is possible the mbic also exists at some concentration between 8 and 16 mg/ml of nicotine, but further studies are needed to explore this probability. in this present study, concentrations at which enhanced growth was demonstrated correspond to average nicotine concentrations found in many of the tobacco bacterial host interactions • 73 products discussed previously. it is believed that the findings of this study support the hypothesis that the increased risk of oral health issues faced by many tobacco users is caused in part by the stimulation of oral microorganism biofilm formation by nicotine and other tobacco components. this study can be used to provide confirmation that further research on the relationship between oral microorganisms and nicotine is needed. under similar growth conditions, the organisms could be exposed to nicotine intermittently throughout a specified length of time, much like how a smoker smokes periodically throughout the day. this study emulates the nicotine exposure which occurs to the oral flora of a smoker. additionally, because microbes often exhibit different behaviors when interacting with multiple species, it would be beneficial to conduct a study which assesses the biofilm formation of multiple bacterial species in the mouth. this would more closely simulate an in vivo environment in an attempt to simulate dental caries, oral biofilm, periodontal disease and other diseases present in the mouth. furthermore, investigating the types of interactions which occur between nicotine, proline-rich proteins, and bacteria would allow for a more thorough understanding of the specific mechanisms which cause biofilm enhancement in the presence of nicotine. acknowledgements this study was financially supported by the center for research and learning, indiana university purdue university at indianapolis through a multidisciplinary undergraduate research institute (muri) grant. assistance and advice provided by mentors drs. richard gregory, fengyu song, angela bruzzaniti, and jack windsor was greatly appreciated. further thanks are also 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infection 39:177-179. 36. stuart, c.h., schwartz, s.a., beeson, t.j., & owatz, c.b. 2006. enterococcus faecalis in root canal treatment failure. j. endodontitis 32:93-98. bacterial host interactions • 75 the occurrence of myxomycetes from a lowland montane forest and agricultural plantations of negros occidental, western visayas, philippines julius raynard d. alfaro, donn lorenz ivan m. alcayde, joel b. agbulos, nikki heherson a. dagamac*, thomas edison e. dela cruz* department of biological sciences, college of science, university of santo tomas, españa, manila, philippines copyright 2014, fine focus all rights reserved manuscript received 22 september, 2014; accepted 19 december, 2014 higher floral and faunal biodiversity is expected in multi-species-covered mountainous forests than in mono-typic agricultural plantations. to verify this supposition for cryptogamic species like the plasmodial slime molds, a rapid field survey was conducted for myxomycetes and substrates in forest floor litter and agricultural plantation were collected in negros occidental, philippines. morphological characterization identified a total of 28 species belonging to the genera arcyria, ceratiomyxa, collaria, comatricha, craterium, cribraria, diderma, didymium, hemitrichia, lamproderma, physarum, stemonitis, trichia and tubifera. the myxomycete species arcyria cinerea was the only abundant species found both in the agricultural and forested areas. the majority of collected species were rarely occurring. in terms of species composition, more myxomycetes were recorded in the mountainous forest (27) compared to agricultural sites. furthermore, aerial leaf litter collected in the forests had the highest number of records for fruiting bodies but in terms of species diversity, twigs yielded higher value based on shannon index. findings in this study verify that a habitat with more heterogenous plant communities yields higher species of myxomycete assemblages. this research is the first study to report myxomycetes from negros occidental. abstractcorresponding authors *nikki heherson a. dagamac nhadagamac@gmail.com institut für botanik und landschaftökologie ernst – moritz – arndt universität greifswald, soldmannstrasse 15 d-17487 greifswald, germany *thomas edison e. dela cruz tedelacruz@mnl.ust.edu.ph department of biological sciences, college of science, and fungal biodiversity and systematics group, research center for the natural and applied sciences university of santo tomas, españa 1015 manila, philippines keywords • abundance • amoeboid eukaryotes • diversity • fruiting body • sclerotia myxomycetes, commonly known as true slime molds, are acellular, phagotrophic, eukaryotic organisms under the kingdom protista (9). these organisms have been known to exhibit both fungal and protozoan characteristics but through an amoeboid phase, they feed on other microorganisms, including bacteria and yeast (9). these microorganisms are distributed worldwide and usually occur on dead substrata such as bark, twigs, and dried leaves of plants (29). several studies regarding the taxonomy and ecology of myxomycetes have been conducted but most of this research was carried out in temperate regions, such as north america (35, 36), south america (16), and europe (8,10). numerous studies have also been completed in the tropics, such as costa rica (30), puerto rico (23) and mexico (17). despite the number of the studies that were executed and the high potential of biodiversity introduction 08 • fine focus, vol. 1 in tropical systems, little is known about them particularly in the tropical southeast asia such as the philippines. thus, myxomycete profiling in the philippines is still considered incomplete. as a result, there is limited knowledge on the ecology and taxonomy of myxomycetes found in such tropical areas where there are abundant forested areas serving as excellent habitats for myxomycetes (25). previous studies on philippines myxomycetes in the late 1970s and early 1980s encompassed the most comprehensive listing for the country (24). however, most of these publications during that time were merely extensive annotated lists. but in recent years, myxomycete diversity and ecology studies in the philippines has progressed, as several reports have accounted on myxomycete distribution and occurrences in several habitat types, e.g. in selected forest parks (7, 19), in coastal forests (12, 18), and lowland mountain forests (3,4). these papers surveyed different forest habitats in the luzon main island, which is just a small portion of the large archipelagic geography of the philippines. no previous reports had ever documented myxomycetes on the scatter islands of the visayas region, or reported myxomycete occurrence in large agricultural plantations in the country. as such, the findings from this research paper on myxomycete composition in lowland forests will serve as a baseline reference for the profiles of myxomycetes in a local scale of negros occidental, but will also contribute to understanding their distribution in the whole of the philippines. thus, the objectives of this paper are to (1) collect myxomycetes using opportunistic sampling methods in the field, and moist chamber cultures; (2) determine the collected species of myxomycetes; (3) measure the sampling strategy used in the survey; (4) assess the occurrence of each myxomycete species; (5) calculate the species diversity of the myxomycetes from the different substrates; and (6) compare the similarities of myxomycete assemblages between mountainous forests and agricultural plantations. materials and methods study sites and its collecting localities field survey and substrate collection were carried out during may 2013 in negros occidental, western visayas. the province is part of the whole negros island, the third largest island in the philippines. it is estimated that the province is approximately 375 kilometers long from north to south with basically volcanic vegetation, making its arable land ideal for cultivation of economically important crops, especially sugarcanes (http://www.negros-occ. gov.ph). based on the philippine atmospheric, geophysical and astronomical services administration – department of science and technology (pag-asa dost) climatological data, the whole area is characterized as having two distinct seasons: dry from december to may and wet from june to november. along the study area, two different habitat types, namely, a lowland forested area and an agricultural plantation were chosen. the descriptions of each habitat types and its collecting localities (fig. 1) are further described below. a. forested areas (mt. kanlaon national park, 10° 24.787n, 123° 07.982e). this area rises to a height of 2,465 m (7987 ft.) and is located in the province of negros occidental, western visayas. it is characterized by low serrated mountain ranges. the forest can be applied/environmental • 09 described as a moist tropical disturbed rainforest dominated with large dipterocarp trees and non-vascular plants. within this forest area, six sites were randomly selected along an established 1000 m accessible forest trail that serves as a transect. b. agricultural area (sugarcane plantations, saccharum officinarum). only sugarcane plantations along the road in the northwest part of the province were selected for this study. three collecting localities characterized as dry and with extensive light exposure were selected to collect substrates that were subjected for moist chamber cultures. dried substrata are ideal for the preparation of moist chambers, as these substrates retain spores better. the collecting localities are: silay city (sc1, 10°46’46.18”n 123°0’24.80”e), bacolod city (sc2, 10°42’29.30”n 122°58’56.72”e), and bago city (sc3, 10°32’37.29”n 122°51’47.51”e). field collection of myxomycete specimens fruiting bodies of myxomycetes directly observed in the field were immediately placed in clean compartmentalized plastic collecting boxes. these specimens were brought back to the laboratory and after several days of air drying, the specimens was glued in herbarium trays and placed inside matchbox-sized herbarium boxes for permanent storage. moist chamber preparation from substrate collected in the two habitat types ninety samples each of ground leaf litter (gl), aerial leaf litter (al), twigs (tw), 60 samples each of ferns (f), and 30 samples of vines (v) from mountain forests, and 90 sugarcane leaf litter (sc) from agricultural plantations were collected, accounting for a total of 450 substrates used for this study. the collected substrates were placed inside dry paper bags, labeled, and transported back to the laboratory. collection of samples was done following the methods described by stephenson (36). samples of ground floor litter were gathered at 3-5 m regular intervals. these samples consisted of mixtures of leaves. twigs < 1.0 cm in diameter in size were also collected. samples of aerial litter were collected from dead twigs or leaves still attached to branches of plants and trees. the specimens were wrapped gently in paper before being transported to the laboratory, where they were placed in small boxes for storage to prevent insects from getting into the samples. the samples were air dried for one week to prevent the growth of molds. to avoid pseudoreplication, a single moist chamber (mc) was prepared for each substrate collected. the moist chambers used consisted of disposable plastic petri dishes, 10 cm in diameter and 4 cm deep, lined with filter paper. samples were moistened with sterile distilled water. after 24 h, excess water was removed up to the point adequate enough for the chamber to be moist, and the ph of each of the substrate was checked using a ph meter (sartorius pb-11). following the incubation conditions of dagamac et al. (3), moist chambers were kept at room temperature (22-25°c) in diffuse daylight. when necessary, a small amount of water was added to each culture to maintain moist conditions. determination of myxomycete species the specimens obtained from the moist chamber cultures were identified using a dissecting microscope three times per week (two day intervals) for a period of up to three months, by comparing the color, size, and structure of the myxomycete plasmodia, 10 • fine focus, vol. 1 types of fruiting bodies (e.g. sporangium, aethalium, pseudoaethalium, and plasmodiocarp), and spores of myxomycetes in the descriptions stated in the standard monographs of myxomycetes (21, 32). web-based electronic databases, e.g. eumycetozoan project (http://slimemold.uark.edu), were also utilized for verification of some morphological features. nomenclature used for the identified myxomycetes follows the names used in nomenyx (http://nomen.eumycetozoa. com). for specimens that could not be fully identified with strong certainty due to some malformed specimens but with distinguishing character enough to separate as a species, the abbreviation “cf” was used in the taxon name. all specimens listed herein are deposited in the myxomycete herbarium of the fungal biodiversity and systematics group of the research center for the natural and applied sciences at the university of santo tomas in manila, philippines. evaluation of data to evaluate the sample effort of the myxomycete survey in this study, an individual-based rarefaction curve was established. using the rarefaction formula that computes for a number of estimators for species richness of the free downloadable program estimates (version 9, colwell 2013, 100 randomizations), species accumulation curves were initially constructed. in accordance with unterseher et al. (39), the chao2 estimator was then chosen as the best estimator to use and was calculated using the classical settings of estimates. the estimated value for the sampling effort in the study area was then determined using the formula of ndiritu et al. (22) by dividing the actual number of species recorded by the mean number of species expected as estimated by the chao 2 estimator. additionally, a hyperbolic regression in the form of coleman rarefaction curve according to the michaelis-menten formula y = ax/(b+x), where x represents the number of samples, y is the number of species recorded and the parameter a giving an estimate for the maximum number of species to be expected at this kind of substrate resulting in a very close curve shape (magurran 20) was applied to the dataset. for the assessment of species occurrence of myxomycetes, species composition was initially determined for the collection site. occurrence refers to the frequency of the presence of a particular species of myxomycetes in a positive mc. a moist chamber positive for having a fruiting body of a particular species was considered as one positive collection. a collection was then considered as a single unit. the number of collections reflects the abundance of myxomycetes in negros occidental and was expressed as relative abundance. the relative abundance for every species of myxomycetes were then calculated and reported as abundance index (ai) by stephenson et al. (1993). each species were then categorized as: (1) abundant if their relative abundance (ra) is>3% of the total collections, (2) common if ra is >1.5% but <3% of the total collections, (3) occasionally occurring if ra is >0.5% but <1.5% of the total collections, and (4) rare if the myxomycetes had an ra of < 0.5% of the total collections. to further determine the myxomycetes diversity for the different substrates, species diversity was also calculated using three different diversity indices provided in magurran (20). shannon diversity index (hs) measures species diversity with respect to both species evenness and richness. this index assumes that individuals are randomly sampled from an infinitely large community and that all species are represented in the sample (14). the gleason index (hg) measures the species diversity in relation to species richness. richness is defined as the number of different species applied/environmental • 11 found in a biota. pielou’s species evenness index (e), on the other hand, quantifies how equal communities are in a given sampling area. these indices are computed as follows: equation 1: shannon index of diversity (hs) = −_i(pi ln pi), where pi = the total number of individuals in the ith species. equation 2: gleason index (hg) = np − 1/ln ni, where np = the total number of species and ni = the total number of individuals in the ith species. equation 3: pielou’s index of species evenness (e) e = hs/hmax where hs = shannon index of diversity and hmax= the maximum value of hs. the similarities of myxomycete assemblages between the mountainous forest and agricultural plantation were also compared by sorensen’s coefficient of community index and the percentage similarity index. the equation for sorensen’s coefficient is based on the presence or absences of species. equation 4: coefficient of community (cc = 2c/(a+b)), where a = total number of species in the first habitat, b = total number of species in the second habitat, and c = no. of species common to both habitat. the value of cc ranges from 0 – 1 where 0 is if there are no species present in both habitat and 1 when all species are present in both habitat. on the other hand, the percentage similarity (ps) index considers not only the presence or absence of species but their relative abundance. the ps value was computed as follows: equation 5: ps = σ min (a, b, … x) where min = the lesser of the two percentage compositions of species a, b, c, … x in the two communities. using the combined opportunistic sampling in the field and moist chamber culture preparation, a total of 193 records of myxomycetes were noted for this survey. from these 193 records, 42 were fruiting body records in the field and 151 were recovered either as plasmodia or fruiting body records in the moist chamber cultures. in terms of the field survey, there were no field specimens that were observed in the agricultural plantations. moreover, a higher yield of myxomycetes was noted among moist chambers in forest litter than agricultural litter. only two bright-spored myxomycetes species (arcyria cinerea and tubifera ferruginosa) were recorded in the sugarcane litter. a total of 32 morphospecies were identified from the 193 myxomycete records. however, four of these species were only determined to the genus level (arcyria, comatricha, didymium, and stemonitis) because they were recovered from moist chambers wherein most of the fruiting bodies were already withered. the list of species presented hereafter has a total of 28 species belonging to 14 genera. to evaluate the sampling effort used in this study, an individual based species accumulation curve was constructed using the software estimates and showed that the mean chao 2 estimator reached a constant value of 59 (fig. 2). using the formula of ndiritu et al. (22) to calculate the exhaustiveness of the sampling effort for the whole study, our results gave us a computed sampling effort of 54.2% for the present study. assessing the occurrence of the 28 determinable myxomycete species, two species were reported to be abundant, results 12 • fine focus, vol. 1 fig. 1. study sites: mt. kanlaon national park (forested areas) and the agricultural plantation (sc) in negros occidental, western visayas, philippines, may 2013. namely arcyria cinerea and didymium nigripes. nine species were common, four were occasional and 13 were reported to be occurring as rare (table 1). comparing the composition of myxomycetes from the different substrates collected from two habitat types, 27 species were found in forested areas that were characterized to have heterogenous plant litter and only two species were accounted in the sugarcane plantations. in terms of productivity of the microhabitats tested in this study by using the moist chambers, 121 of the 450 mcs (27%) were positive for growth of myxomycetes either as plasmodia or as fruiting body. all of the moist chambers prepared had a relatively acidic mean ph condition. highest percent yield applied/environmental • 13 fig. 2. individual based species accumulation curve smoothed by cole rarefaction for the myxomycetes collection in negros occidental. a b c d e f g h i j k l fig. 3. some representative myxomycetes collected in negros occidental: (a) arcyria cinerea, (b) arcyria denudata, (c) ceratiomyxa fruticulosa, (d) collaria arcyrionema, (e) craterium leucocephalum var. cylindricum, (f) didymium squmolosum, (g) diderma effusum, (h) hemitricha calyculata, (i) physarum bogoriense, (k) stemonitis fusca, and (l) trichia decepiens. 14 • fine focus, vol. 1 was observed from the aerial litter (72%), and consequently had the most number of records of myxomycetes (table 2). the other substrates, such as the twigs and vines had the next highest number of percent yield. however, these substrates had a relatively low number of determinable records due to the fact that most of the substrates were recorded as positive culture because of the appearance of plasmodium during the incubation period. these mcs were unsuccessful in developing into fruiting bodies (table 2). lowest percentage yield (18%) was observed in sugarcane litter randomly collected in the agricultural plantations of the study area. moreover, among the six substrates collected in the two types of habitats (forest and agricultural), twigs had the highest species diversity as calculated using gleason index (hg table 1. occurrence of myxomycetes in negros occidental showing the number of records accounted from the rapid field survey and the use of moist chamber cultures applied/environmental • 15 table 2. statistics of the different substrate types used in the moist chamber =2.12) and ferns had the highest species evenness based from pielou’s evenness index (e =1.00). however, using the shannon index that considers species diversity and species evenness, twigs gave the highest value (hs = 0.75). comparing the assemblages of myxomycetes between the two habitat types, a cc value of 0.08 and ps value of 0.48 were computed in the study (table 3). these results show that species similarities between the two sites were only 8.3%, which is relatively low since the only species of myxomycetes that was present in both sites was arcyria cinerea. discussion in terms of biodiversity, the philippines is considered to be one of the most diverse countries in the asia pacific basin. however, microbial diversity assessments in the country are under-investigated. this particularly holds true among the less explored fungus-like protists like the myxomycetes where a generally tropical condition would seem to be favorable for their growth and development (15). in fact, in recent years, most investigations on myxomycetes in the philippines were concentrated only among the forest vegetation and coastal habitats of the luzon main island (3,18). thus, findings in this research paper are the first intensive diversity report for the visayas group of islands of the philippine archipelago. productivity of myxomycetes in moist chamber cultures the use of a moist chamber culture in assessing the occurrences of myxomycetes was a vital component for this study. current studies from arid environments in china (28) and submerged plant materials in the big thicket national preserve (40) employed the usage of this technique to recover species of myxomycetes not easily seen on the field. in our study, a total of 450 moist chambers were prepared wherein only 27% were positive for myxomycete growth. after almost 15 weeks of incubation, 11% showed positive results for fruiting bodies, while 16% were positive for plasmodial growth. this now shows that the majority of 16 • fine focus, vol. 1 the plasmodium specimens were not able to develop to fruiting bodies. in comparison to related local studies, a similarly low yield was observed by dagamac et al. (5), wherein 17.5% yielded plasmodia and only 5.1% yielded fruiting bodies from different bark samples collected from the luzon islands. however, kuhn et al. (13) had a percentage yield of 51%, or 214 out of 420 moist chamber cultures containing 40% positive for plasmodia and 23% positive for fruiting bodies in six highland areas in luzon. substrates collected in a protected ecopark by macabago et al. (19) had a percentage yield of 51%, or 121 out of 240 moist chambers. it seems now that most studies of myxomycetes in the philippines that used moist chambers always supported a higher level of plasmodium yield than recovering fruiting body phenologies. perhaps the fast dessication of most of the moist chambers during the incubation time can be a factor here, since a suitable moist environment is needed to allow for the plasmodium to successfully develop into fruiting bodies. it is important to note that in doing myxomycetes biodiversity and distribution studies by means of the moist chamber technique, fruiting bodies are more important as compared to plasmodium or sclerotia, since most species identification is based on the determinable morphologies of the fruiting body (11, 31). moreover, among the six substrates collected in our study, aerial leaf litter yielded the highest level of success. the highest productivity yield from aerial leaf litter was also recorded from other studies in the tropics, including rojas & stephenson (27) who reported 93% in the coco’s island, costa rica, and in a more recent comparative species listing of dela cruz et al. (6) between substrates collected in the tropics and the temperate ecoregions. this may be attributed to specimen exposure to open air where aerial litter has a higher potential in catching spores. this supposition was already demonstrated by the studies from schnittler & stephenson (30) where the authors noted that slight breeze can cause the myxomycetes spores to be dispersed more than one kilometer from the starting point. perhaps aerial litter from our study has a higher probability to trap spores dispersed by wind. species composition of myxomycetes in negros occidental in this study, 28 morphospecies of myxomycetes were collected from lowland montane forests and sugarcane plantations in the northern part of negros occidental. this number is similar in comparison to other lowland montane vegetation area studies conducted in the luzon main islands, including mt. arayat national park (3) and in mt. makulot (1), which reported 30 and 28 morphospecies of myxomycetes, respectively. albeit this number of morphospecies is not yet reflective of the overall number of myxomycetes that can be accounted in negros occidental as was suggested by the 54.2% sampling effort for this study, findings from this research paper serve as a good starting basis for future directives in understanding the distribution of myxomycetes in a local setting. to expand the sampling effort, it is recommended to increase the distance covered during intensive surveys and to add other substrates, i.e. barks of living deciduous trees, dung of herviborous animals, and inflorescences where myxomycetes can also thrive. in terms of species composition in the whole study area, arcyria cinerea was noted to be the only abundant species found in both the agricultural and forest habitats, with the other species occurring relatively rarely. stephenson (35) had the highest percentage yield of a. cinerea in moist chambers (85% in the upland temperate forest of southwestern virginia, usa). similar results in terms of occurrence were also obtained from the studies conducted by rojas et al. (26) in the northern neotropics and applied/environmental • 17 kuhn et al. (12) in anda island in pangasinan, philippines. our findings now support other previous results that also showed arcyria cinerea to be of cosmopolitan distribution worldwide, since it is widely known to be tolerant to many environments. myxomycete distribution in agricultural litter is more limited than in forest litter most of the related studies on myxomycetes in the philippines always used litter from the forest floor. to the best of our knowledge, the findings in this paper are the first report for the philippines attempting to evaluate myxomycetes in a sugarcane plantation where the decaying litter and vegetation is generally specific and to compare it to the myxomycete communities in forest litter where decaying litter is more heterogenous. in contrast to related studies in the paleotropics, our findings seems to contradict the observations of tran et al. (38), which intensively evaluated distribution of myxomycete assemblages in agricultural ground litter and the forest floor. their results showed a relatively higher productivity among agricultural litter than the forest floor litter during both the rainy and dry seasons. perhaps the smooth surface of the sugarcane leaf is not a favorable spore trap for other myxomycetes species in contrast to the pubescent surfaces of the three agricultural litters used in thailand (banana, mango and corn plantations). nonetheless, findings from our study supports the theory that diversities of plant communities and litter heterogeneity (37) in a study area influence the composition of myxomycete assemblages, as evident from a higher number of myxomycete occurrences in the forest floor litters in negros occidental. myxomycetes from negros occidental as baseline information an understanding of the distribution for myxomycetes in the philippines is still far from complete. many ecological factors and/ or unexplored landscapes in the country still need to be investigated. despite the findings presented in this study, it is still significant to note the limitations of a descriptive study like this are associated with the sampling efforts in collecting the substrata used in the study. the most noteworthy contribution of this paper relates to the fact that it increases the knowledge about the local ecology of myxomycetes in an ecoregion of the world where investigations about myxomycete diversity is still considered to be in its infancy. 18 • fine focus, vol. 1 the authors are grateful to the department of environment and natural resources (denr, pamb), negros occidental, region vi for the gratuitous permit granted for the collection and field survey in mt. kanlaon nation park. moreover, jra, dla, jba and tedc are indebted to the philippine society of microbiology (psm) for the undergraduate thesis subsidy grant used for this research study. nhad would also like to thank the deutscher akademischer auslandtausch dienst (daad) for the scholarship grant. furthermore, the authors would like to thank angelica rea – maminta and sittie aisha macabago for all the technical assistance and to the anonymous reviewers that helped in improving this manuscript. acknowledgements references 1. cheng, c.b.t., yu, k.n.t., campos, m.l., adora, j.m.v., pascua, g.c.p., pangilinan, m.v.b., buaya, a.t. & dela cruz t.e.e. 2013. occurrence and diversity of of myxomycetes (plasmodial slime molds) along the northern slope of mt. makulot, cuenca, batangas, philippines. asian j. biodivers. 4:65-83. 2. colwell, r.k. 2013. estimates: statistical estimation of species richness and shared species from samples. version 7. user’s guide and application published at http://purl.oclc.org/estimates (accessed 23.01.2014). 3. dagamac, n.h.a., stephenson, s.l., & dela cruz, t.e.e. 2014. the occurrence of litter myxomycetes at different elevations in mt. arayat, national park, pampanga, philippines. nova 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potts, and rafael torres-cruz. division of occupational health and safety, office of research services, national institutes of health, bethesda, maryland, usa copyright 2015, fine focus all rights reserved manuscript received 11 may, 2015; accepted 29 may, 2015 *corresponding author finaljournal.indd 153 9/25/15 11:38 am introduction each year thousands of student researchers pursue honors theses and practicums at university laboratories in preparation for future careers. while many will learn the nuances of performing biomedical research, a majority will likely graduate without knowing much about biosafety. as a new generation of scientists emerge from academia, it is crucial that they learn about the value of biosafety and begin to develop safe work practices to protect themselves, others, and the environment. researchers must practice laboratory techniques and safety skills with equal diligence. individual responsibility is the foundation for developing a culture of safety in biomedical research. what is biosafety? biosafety is a form of risk management. the ultimate goal of biosafety is to decrease the incidence of laboratory-associated infections (lais) by lowering the risk of accidental exposure to biological agents through a combination of risk assessment and mitigation strategies. there is never a total reduction in risk, but the probability of an undesired outcome can be reduced to almost zero with an appropriate understanding of the risks presented by the agent, the procedures involved in the research, and the facilities where the work is conducted. the practice of biosafety has progressively become a mainstream discipline and an international profession in itself. several national and international biosafety associations exist to enable biosafety professionals to share best practices, inform policy discussions, and provide guidance on standards. biosafety professionals work in occupational health and safety programs at universities, corporations, nonprofits and government agencies. they perform risk assessments and establish controls on practice and procedures to protect the worker, public, and environment. hazard analysis, applied research, engineering, program management, emergency response, microbiology, molecular biology, and even investigative work are common elements of the biosafety profession. what should be considered in a risk assessment? a risk assessment is a formal consideration of the potential hazards associated with a particular agent and its use. when conducting a biosafety risk assessment, one must be able to identify possible sources and routes of infection to the worker. once the hazards are anticipated, specific controls can be put into place to reduce the risk of lais. there are many factors that must be considered when conducting a standard risk assessment, but they can generally be categorized as either agent hazards or laboratory hazards. agent hazards are the intrinsic or modified characteristics of a biologic that may have an adverse impact on health. the type of species or strain used, virulence, transmissibility, resistance, infectious dose, tropism, and other factors are crucial elements to consider when evaluating the inherent risk of an agent to cause disease. the potential outcomes of natural mutation and genetic engineering must also be considered when assessing the hazards of an agent. a determination as to whether a biological agent is harmful depends on the host as well. what may be a harmless microorganism to a healthy individual could be a formidable pathogen leading to disease in another individual who has a weakened immune system. for example, the yeast, candida albicans, is an opportunistic pathogen commonly found in human flora. individuals who are immunocompromised, such as those with autoimmune diseases or 154 • fine focus, vol. 1 (2) finaljournal.indd 154 9/25/15 11:38 am who undergo chemotherapy treatment may develop serious disease when infected with this organism. the risk of an lai also depends on laboratory hazards. laboratory hazards are vulnerabilities in the lab environment that increase the risk of injury or illness. unsafe practices such as recapping a syringe may lead to a sharps injury. common practices like pipetting, vortexing, sonication, and other aerosol-generating procedures may create bioaerosols leading to a potential inhalation exposure. faulty equipment such as a cracked seal in a centrifuge cup have been known to release aerosols. among the most common of all laboratory hazards is the failure to wear the right attire and personal protective equipment (ppe) appropriate for the laboratory procedure to be performed. although risk assessment often involves considering many factors at the same time, its purpose is not to confound but to clarify. its careful application reveals potential hazards and vulnerabilities before research begins. preventive action can be taken to protect the worker and others. no matter how virulent or transmissible the microorganism, it cannot cause disease if proper biosafety measures are implemented to prevent exposure. how is risk reduced? once the agent and laboratory hazards are assessed, steps must be taken to reduce the likelihood of potential harm to the worker. in the biosafety field, it is standard practice to categorize research with biological hazards according to a “biosafety level”. based on the risk assessment, work with a pathogen can be assigned to biosafety levels (bsl) 1 through 4, with bsl-4 offering the highest protection to the worker and the environment. each biosafety level builds upon the safety requirements of the previous level. an appropriate combination of microbiological practices, safety equipment, personal protective equipment, and facility design according to each biosafety level reduces the risk of potential biological exposures. an undergraduate microbiology teaching laboratory may be considered a bsl-1 laboratory. bsl-1 typically involves work with agents that are not known to cause disease in healthy adults. bacillus subtilis, naegleria gruberi, escherichia coli (k12) are examples of agents that may be used in a bsl-1 laboratory. the protection afforded by bsl-2 practices and procedures should be utilized when working with moderate-risk agents that can be acquired through mucous membrane exposure, percutaneous injury or ingestion. examples of agents that may be used in a bsl-2 laboratory include staphylococcus aureus, pseudomonas aeruginosa, and the measles virus. in the case of measles, bsl-2 is appropriate because adequate vaccines are available. biosafety level 3 (bsl-3) is assigned for potentially lethal agents that are known to be transmitted via aerosols and may or may not be indigenous to the region. mycobacterium tuberculosis, highly pathogenic avian influenza a virus, and francisella tularensis are a few common examples of agents worked with in bsl-3 laboratories. when there are no treatments available for life-threatening infectious diseases caused by exotic pathogens, and there is a high individual risk to the worker, particularly through the aerosol route, these agents are handled under bsl-4 conditions. these laboratories are usually referred to as maximum-containment facilities and provide the highest levels of protection for the laboratory worker. ebola virus, marburg virus, crimean-congo hemorrhagic fever virus and nipah virus issues in biosafety • 155 finaljournal.indd 155 9/25/15 11:38 am 156 • fine focus, vol. 1 (2) are examples of pathogens that require bsl-4 containment. in addition to these standard biosafety levels, there are specific containment designations depending on the activity being performed. for example, infectious disease work involving animals are designated as animal biosafety levels 1, 2, 3 and 4 (absl). the challenges presented by the animal model are taken into consideration when performing a risk assessment. animals can be unpredictable. they can bite, scratch, kick, shed fluids, produce splashes and aerosols, and increase the risk of accidental exposure to the worker. some animals may even be infected naturally with zoonotic agents that can cause dangerous, sometimes deadly infections. research involving large or loose-housed animals is conducted in specially designed bsl-3-agriculture (bsl-3 ag) laboratories. in this scenario, the risk assessment places greater emphasis on preventing the pathogen from escaping into the environment. if there were a release of an agricultural pathogen, the economic implications could be significant. for example, an accidental release of a pathogen that infects cattle could potentially impact a $44 billion industry. a useful resource for learning more about biosafety levels is the cdc/nih publication, biosafety in microbiological and biomedical laboratories 5th edition (4). the value of biosafety careful adherence to biosafety principles protects individuals, reputations, and research. perhaps the best way to communicate the value of biosafety is through the tragic example of a laboratory-associated infection that occurred at yerkes regional primate center at emory university. on october 29, 1997, a young researcher was conducting a routine procedure that involved moving a non-human primate in caging. the primate, a rhesus macaque (macaca mulatta), was unknowingly infected with a zoologic virus known as macacine herpesvirus (formerly cercopithecine herpesvirus 1 [chv-1]). the pathogen can be present in monkey saliva, urine, fecal matter, and conjunctival fluid. although disease is usually mild in primates, it is frequently deadly in humans who are exposed to the virus. during the transfer, the researcher suffered an ocular exposure to hazardous macaque fluids. despite having flushed her eyes and later seeking medical attention, it was not enough to save the worker’s life. just 42 days later, she died due to refractory respiratory failure from this seemingly minor exposure. a subsequent cdc/osha investigation found that the primate center believed the risk of chv-1 exposure was thought to be low for the activity, that eye wash first aid was not conducted for at least 15 minutes, and that medical reporting and subsequent treatment was delayed. these factors contributed to a terrible outcome (1, 5). seemingly innocuous microorganisms can lead to serious lais as well. in 2009, an associate professor at the university of chicago became the first person to die from an accidental exposure to an attenuated yersinia pestis strain. the researcher worked with pigmentation-negative kim d27, a strain considered avirulent because of its iron-acquiring limitations. however, host factors in the researcher appear to have made the laboratory strain virulent. hemochromatosis is a hereditary medical condition in which the body absorbs too much iron and deposits excess amounts in organs. it is believed that after the researcher experienced a percutaneous or mucosal exposure in the laboratory, the kim d27 strain was able to establish an infection due to elevated iron levels in finaljournal.indd 156 9/25/15 11:38 am issues in biosafety • 157 his body. an investigation revealed that the researcher did not wear appropriate personal protective equipment and did not have current biosafety training. this case highlights the importance of consistently practicing biosafety recommendations to mitigate unknown risk factors (2). it is clear that improper adherence to biosafety practices and resulting lais could have a devastating impact on the individual. however, it is worth taking a moment to appreciate the bigger picture: the impact unsafe practices in the laboratory could have on co-workers, the general public, the environment, and the overall research enterprise. recently, the centers for disease control and prevention (cdc) has been in the news media about several incidents where biosafety protocols and procedures have been breached. most notably, the unintentional exposure of personnel to potentially viable anthrax and the cross-contamination of non-pathogenic avian influenza virus with the highly pathogenic avian influenza virus strain h5n1. in both cases, the lack of proper adherence to biosafety protocols led to potential exposures outside the laboratory. as a result, research activities of the laboratories involved in this incident were suspended. significant time and funding were spent to investigate the incidents, reinspect inventories, and retrain personnel. one could argue that the most important loss in the ensuing months has been the public trust and confidence in the scientific enterprise to conduct biomedical research safely with high-risk pathogens (3, 6). how can this trust be regained? how can future incidents be prevented? diligent adherence to biosafety practices and procedures while working in the laboratory will ensure that workers are safe, the community and environment is protected and the progress of vital scientific research is not interrupted. how can students contribute to biosafety? students can protect themselves and others by taking laboratory safety seriously. they should expect their departments to provide safe work spaces and comprehensive laboratory training. before beginning research, students should make sure their project has undergone a formal risk assessment and that they feel comfortable performing it safely. students should learn how to safely operate any equipment before using it and always follow standard operating procedures. personal protective equipment must always be worn. know what to do and whom to contact when an accident occurs, such as a spill, injury, or illness. if students have safety questions or concerns, they should make them known to their faculty or institutional safety office. student researchers may even consider contributing to applied biosafety research, for example, by studying potential routes of contamination in a laboratory using a harmless surrogate or by evaluating the effectiveness of the institutional biosafety program. summary biosafety is a multi-faceted discipline, a form of risk management designed to minimize the risk of biological exposures and prevent laboratory-associated infections. biosafety professionals protect the research community’s health and safety by assessing research and setting risk reduction strategies. for these strategies to be successful, student researchers and faculty advisors must commit to incorporating them into their daily research activities. those who understand the value of biosafety will not only protect themselves and others but will also advance research by realizing that safe science is good science. finaljournal.indd 157 9/25/15 11:38 am 158 • fine focus, vol. 1 (2) references 1. centers for disease control and prevention (1998). fatal cercopithecine herpesvirus 1 (b virus) infection following a mucocutaneous exposure and interim recommendations for worker protection. morbidity and mortality weekly report, 47(49), 1073. 2. centers for disease control and prevention (2011). fatal laboratory-acquired infection with an attenuated yersinia pestis strain--chicago, illinois, 2009. morbidity and mortality weekly report, 60(7), 201. 3. centers for disease control and prevention (2014). report on the potential exposure to anthrax. 4. chosewood, l. c., & wilson, d. e. (eds.). (2007). biosafety in microbiological and biomedical laboratories (bmbl) 5th edition. centers for disease control and prevention. 5. elizabeth r griffin research foundation (2014). retrieved from http://www.ergriffinresearch.org. 6. monaco, l.o. and holdren, j.p. (2014). enhancing biosafety and biosecurity in the united states [memorandum]. united states homeland security council. office of science and technology policy. the white house. washington, dc. finaljournal.indd 158 9/25/15 11:38 am detection of borrelia and ehrlichia in rhipicephalus sanguineus rosa vasquez-espinoza and david l. beck* department of biology, tennessee technological university, cookeville, tn, usa copyright 2015, fine focus all rights reserved manuscript received 30 april, 2015; accepted 6 july, 2015 finaljournal.indd 109 9/25/15 11:38 am rhipicephalus sanguineus, the brown dog tick, is endemic throughout the world wherever domestic dogs are present. it has been recently reported by some veterinarians in the city of laredo, texas, usa, that lyme disease, the most common tickborne disease in the northern united states, is present in local domestic dogs. fully engorged r. sanguineus ticks were collected and their dna was purified. the ticks were screened to determine the prevalence of borrelia, rickettsia and ehrlichia species. sequences related to borrelia burgdorferi in 9.8% (n=11/112), “candidatus borrelia lonestari” in 16.9% (n=19/112) and ehrlichia canis in 12.5% (n=14/112) were detected by pcr. sequencing has confirmed the presence of dna from ehrlichia canis and “candidatus b. lonestari”, corroborating that borrelia and ehrlichia are present in domestic dogs in south texas. abstractcorresponding author *david l. beck assistant professor of biology department of biology tennessee technological university box 5063 cookeville, tn 38505 phone – (931) 372-3375 fax – (931) 372-6257 email – dbeck@tntech.edu keywords • borrelia • ehrlichia • rhipicephalus sanguineus • lyme disease • stari rhipicephalus sanguineus, known as the brown dog tick, is the most widely distributed tick in the world (13). this tick is a known vector of ehrlichia canis (23), the causative agent of canine ehrlichiosis (1,31). the symptoms of chronic e. canis infection in domestic dogs may include thrombocytopenia, anemia, weight loss, bleeding, fever, inflammation of the eye, and anoxic hepatitis (35). acute ehrlichiosis in domestic dogs may result in loss of appetite, lethargy, shortness of breath, bruises, joint pain and depression (25). r. sanguineus is also thought to be a vector of rickettsia rickettsii (13,22), the cause of the rocky mountain spotted fever (14). although r. sanguineus ticks typically prefer to feed on domestic dogs, they have been reported to parasitize humans as well (20,33). the presence of a disease agent in the domestic dog population can indicate that the disease could also be present in humans (29). b. burgdorferi sensu stricto (39) has been identified as the sole etiologic agent of lyme disease in north america (24,38). lyme disease is the most common vector-borne illness in the northern united states (8) and is considered an emerging infectious disease (32). the main vectors are ixodes scapularis (17,18,37) and ixodes pacificus (34). the agent has also been detected at a lower incidence in r. sanguineus (9,21,42) and amblyomma inornatum ticks (30). however, the vector potential of these ticks has not yet been characterized. it has been previously reported that b. burgdorferi in domestic dogs may result in arthritis, similar to introduction 110 • fine focus, vol. 1 (2) finaljournal.indd 110 9/25/15 11:38 am humans suffering from lyme disease (27). symptoms in domestic dogs from the acute form of lyme disease may include fever, swelling, pain, lameness, lymphadenopathy and malaise (12). acute renal failure, myocarditis, cardiac arrhythmia, peripheral edema, neurological syndrome and arthritis have been described as clinical signs found in the chronic form of lyme disease in domestic dogs (2). a lyme disease-like illness has also been described in the southern united states since the 1980s (41). this condition is referred to as the southern tick associated rash illness (stari), and is thought to be caused by “candidatus b. lonestari” (40) after being bitten by amblyomma americanum (3). the symptoms of “candidatus b. lonestari” infection in domestic dogs have not been determined. a veterinarian in laredo, texas, usa has reported that lyme disease is present in local domestic dogs (dr. sandra leyendecker, personal communication). b. burgdorferi has been previously detected in coyotes in webb county (7), texas. however, to the extent of our knowledge it has not been detected in domestic dogs. house pets, including dogs, have an increased exposure to ticks, and can serve as sentinel organisms for some diseases that occur in humans (29). given the limited information available on vector-borne diseases in south texas counties, there is a need for tick and pathogen surveillance in the area. this surveillance can help define areas at high risk for transmission (26) of infection to mammals, including humans. identifying areas at high risk of transmission can increase awareness, potentially leading to the implementation of better diagnosis and prevention methods. in addition, as laredo is the largest land-based port of entry in the united states, there is the movement of a large number of people and animals to and from this city into the rest of united states (11). for example, truck drivers frequently travel to laredo, tx with their domestic dogs to warehouses in the city. they may have to wait a day or two before leaving to their destination. the purpose of this study was to determine the prevalence of dna from tick-borne disease agents in domestic dogs from laredo, texas, by investigating the prevalence of borrelia, ehrlichia and rickettsia species in r. sanguineus ticks. pathogens and antimicrobial factors • 111 tick collection and identification fully engorged adult r. sanguineus ticks were collected at multiple sites in laredo, texas. the ticks were collected from the walls of dog kennels, or from a co2 trap placed in the laredo animal shelter. ticks that were removed while grooming dogs were also collected from animal caregivers/ owners. the researchers had no contact with any animal in the study. the ticks were counted and individually examined under the microscope to identify them to the species level using a published key (10). dna extraction a total of 124 r. sanguineus ticks (55 males and 69 females) were used for dna extraction using the e.z.n.a. mollusk dna isolation kits (omega bio-tek, norcross, ga, usa). a previously reported protocol was followed and modified as previously described (30). briefly, the tick was homogenized in 300 µl lysis buffer. materials and methods finaljournal.indd 111 9/25/15 11:38 am 112 • fine focus, vol. 1 (2) table 1. primers and thermal cycler settings used in this study primers amplicon length pcr conditions gene name sequence (5’ → 3’) specificity denaturing annealing extension cycles 12s rrna 85f ttaagcttttcagaggaatttgctc 140 bp 95°c, 30sec 45°c, 30sec 72°c, 1min 40 225r tttwwgctgcaccttgacttaa not reported flab flals aacagctgaagagcttggaatg 353 bp 95°c, 1min 55°c, 1min 72°c, 1min 36 flars ctttgatcacttatcattctaatagc borrelia genus bl-fla522f ggtacatattcagatgcagacagaggg 660 bp 95°c, 1min 55°c, 1min 72°c, 1min 46 bl-fla1182r gcacttgatttgcttgtgcaatcatagcc “candidatus b. lonestari” bl-fla662f aactgctgaagagcttggaatgc 198 bp 95°c, 1min 55°c, 1min 72°c, 1min 36 dsb bl-fla860r agctggttgaactccttcctgttgt “candidatus b. lonestari” ehr-dsb-330f gatgatgtctgaagatatgaaacaaat 398 bp 95°c, 1min 55°c, 1min 72°c, 1min 46 16s rrna ehr-dsb-728r ctgctcgtctattttacttcttaaagt ehrlichia genus b16s-fl gactcgtcaagactgacgctaagtc 131 bp 95°c, 15sec 58°c, 30sec 72°c,30sec 40 b16s-r gcacacttaacacgttagcttcggtactaa borrelia genus bl-16s5f cagtgcgtcttaagcatgcaagtcagacgg 481 bp 95°c, 1min 60°c, 1min 72°c, 1min 36 bl-16s486r ctgctggcacgtaattagccgggg “candidatus b. lonestari” b16s-23s-igsf gtatgtttagtgaggggggtg variable 94°c, 30sec 56°c, 30sec 74°c, 1min 35 b16s-23s-igsr ggatcatagctcaggtggttag borrelia genus b16s-23s-igsfn aggggggtgaagtcgtaacaag variable 94°c, 30sec 60°c, 30sec 74°c, 1min 40 b16s-23s-igsrn gtctgataaacctgaggtcgga borrelia genus ecan-f atttatagcctctggctatagga 383 bp 94°c, 30sec 52°c, 30sec 72°c, 1min 36 e. canis he1-f caattgcttataaccttttggttataaat 383 bp 94°c, 30sec 52°c, 30sec 72°c, 1min 36 e. chaffeensis rompa he3-r tataggtaccgtcattatcttccctat ehrlichia genus 94°c, 30sec 52°c, 30sec 72°c, 1min 36 rr190 70p atggcgaatatttctccaaaa 532 bp 95°c, 1min 55°c, 1min 72°c, 1min 46 rr190 602n agtgcagcattcgctccccct rickettsia genus the tick was crushed for 5 minutes using a sterile microtube and pestle. after adding proteinase k, the samples were incubated at 55°c for 3h. the sample purification was then completed following the manufacturer’s protocol. polymerase chain reaction (pcr) the samples were screened using pcr for the tick 12s rrna gene as previously described (30). samples positive for tick rdna (n=112) were subjected to pcr for amplification of borrelia, “candidatus b. lonestari”, ehrlichia and rickettsia bacteria species (43,44). finaljournal.indd 112 9/25/15 11:38 am pathogens and antimicrobial factors • 113 table 1 (ctd.). primers and thermal cycler settings used in this study primers amplicon length pcr conditions gene name sequence (5’ → 3’) specificity denaturing annealing extension cycles 12s rrna 85f ttaagcttttcagaggaatttgctc 140 bp 95°c, 30sec 45°c, 30sec 72°c, 1min 40 225r tttwwgctgcaccttgacttaa not reported flab flals aacagctgaagagcttggaatg 353 bp 95°c, 1min 55°c, 1min 72°c, 1min 36 flars ctttgatcacttatcattctaatagc borrelia genus bl-fla522f ggtacatattcagatgcagacagaggg 660 bp 95°c, 1min 55°c, 1min 72°c, 1min 46 bl-fla1182r gcacttgatttgcttgtgcaatcatagcc “candidatus b. lonestari” bl-fla662f aactgctgaagagcttggaatgc 198 bp 95°c, 1min 55°c, 1min 72°c, 1min 36 dsb bl-fla860r agctggttgaactccttcctgttgt “candidatus b. lonestari” ehr-dsb-330f gatgatgtctgaagatatgaaacaaat 398 bp 95°c, 1min 55°c, 1min 72°c, 1min 46 16s rrna ehr-dsb-728r ctgctcgtctattttacttcttaaagt ehrlichia genus b16s-fl gactcgtcaagactgacgctaagtc 131 bp 95°c, 15sec 58°c, 30sec 72°c,30sec 40 b16s-r gcacacttaacacgttagcttcggtactaa borrelia genus bl-16s5f cagtgcgtcttaagcatgcaagtcagacgg 481 bp 95°c, 1min 60°c, 1min 72°c, 1min 36 bl-16s486r ctgctggcacgtaattagccgggg “candidatus b. lonestari” b16s-23s-igsf gtatgtttagtgaggggggtg variable 94°c, 30sec 56°c, 30sec 74°c, 1min 35 b16s-23s-igsr ggatcatagctcaggtggttag borrelia genus b16s-23s-igsfn aggggggtgaagtcgtaacaag variable 94°c, 30sec 60°c, 30sec 74°c, 1min 40 b16s-23s-igsrn gtctgataaacctgaggtcgga borrelia genus ecan-f atttatagcctctggctatagga 383 bp 94°c, 30sec 52°c, 30sec 72°c, 1min 36 e. canis he1-f caattgcttataaccttttggttataaat 383 bp 94°c, 30sec 52°c, 30sec 72°c, 1min 36 e. chaffeensis rompa he3-r tataggtaccgtcattatcttccctat ehrlichia genus 94°c, 30sec 52°c, 30sec 72°c, 1min 36 rr190 70p atggcgaatatttctccaaaa 532 bp 95°c, 1min 55°c, 1min 72°c, 1min 46 rr190 602n agtgcagcattcgctccccct rickettsia genus the pcr mixture was a 25 µl reaction volume containing 0.25 µl of gotaq polymerase (promega, madison, wi), 1x gotaq buffer, 160 ng/µl bovine serum albumin, 1.0 mm mgcl2, 200 µm of each dntp, 2 pmol primers, and 5µl of template (1 µl for nested reactions). amplifications were performed on a bio-rad mycycler thermal cycler (bio-rad, carlsbad, ca) (30). different thermal cycler settings, as indicated in table 1, were used for different primers due to different optimal annealing temperatures. an initial denaturation step (95°c) of 5 min. and a final extension step (72°c) of 5 min. were used. finaljournal.indd 113 9/25/15 11:38 am 114 • fine focus, vol. 1 (2) nested pcr procedures were performed for borrelia and “candidatus b. lonestari” using 1µl from the initial reaction as a template. amplification of target sequences was performed in a bio-rad mycycler thermal cycler (bio-rad, carlsbad, ca) with several denaturing, annealing, and extension times and temperatures (table 1). for each pcr assay, 5µl of sterile distilled water was used instead of template dna as a negative control. the positive controls, when used, were added using separate hoods and pipettors to reduce the risk of cross contamination. visualization and sequencing of pcr products five microliters of each pcr reaction was subjected to gel electrophoresis, using 2% agarose gels stained with ethidium bromide in 0.5x tbe (45 mm tris, 45 mm boric acid, 1 mm disodium ethylene diamine tetraacetic acid) with 0.00005% ethidium bromide. the gels were run at 100 v for 40min.. after electrophoresis, the gels were examined under uv light. positive samples were purified using spinprep pcr clean-up kits (novagen, la jolla ca, usa) following the manufacturer’s protocols. each purified pcr product was sent for sanger sequencing at eurofins (alabama, usa) or mclab (san francisco, ca) using the primers used for pcr. nucleotide sequence accession numbers the dna sequences were visualized using finch tv, geospiza, inc software version 1.5.0 and compared to reported sequences in the ncbi genbank using blast. the assigned genbank accession numbers are: kr183798-kr183823. phylogenetic analysis initial alignments for borrelia and ehrlichia genes were executed using the multiple sequence comparison by log-expectation (muscle) (16) as performed by the european bioinformatics institute’s table 2. detection of bacterial dna in adult r. sanguineus ticks by pcr b. burgdorferilike species “candidatus b. lonestari” e. canis rickettsia b. burgdorferilike species +“candidatus b. lonestari” e. canis + b. burgdorferilike species male 12.2% (6/49) 16.3% (8/49) 8.2% (4/49) 0% (0/49) 2.0%(1/49) 4.1% (2/49) female 7.9% (5/63) 17.5%(11/63) 15.9% (10/63) 0% (0/63) 1.6%(1/63) 0% (0/63) total 9.8% (11/112) 16.9% (19/112) 12.5% (14/112) 0% (0/112) 1.8% (2/112) 1.8% (2/112) 2005 9/85 16/85 11/85 0/85 2/85 2/85 2006 1/7 1/7 0/7 0/7 0/7 0/7 2009 1/11 2/11 1/11 0/11 0/11 0/11 2010 0/4 0/4 1/4 0/4 0/4 0/4 2011 0/5 0/5 1/5 0/5 0/5 0/5 total 11 of 112 19 of 112 14 of 112 0 of 112 2 of 112 2 of 112 finaljournal.indd 114 9/25/15 11:38 am pathogens and antimicrobial factors • 115 b. turdi d82851 b. garinii kf422823 borrelia sp. e49z02-m1 “candidatus b. lonestari” a25b-m16 “candidatus b. lonestari” ay442142 b. burgdorferi kf422803 “candidatus b. lonestari” ay166716 borrelia sp. a46bb-f1 borrelia sp. b17z10-f1 b. tanukii d82848 “candidatus b. lonestari” a25b-f5 “candidatus b. lonestari” a32b-f2 borrelia sp. a25b-m2 borrelia sp. km458251 borrelia sp. a25b-m18 b. parkeri ay604980 borrelia sp. a46bb-f3 b. miyamotoi jq926187 “candidatus b. lonestari” ay237721 “candidatus b. lonestari” a25b-m11 b. microti jf825472 “candidatus b. lonestari” a32c-f5 b. burgdorferi cp009656 b. hermsii dq855534 b. lusitaniae kf836507 b. andersonii d83764 “candidatus b. lonestari” ay237722 “candidatus b. lonestari” b17z09-f2 b. valaisiana jf732880 “candidatus b. lonestari” ay850063 borrelia genomosp. 1 kf918619 b. duttonii ab113314 “candidatus b. californiensis” kf422809 “candidatus b. lonestari” a25b-m17 b. crocidurae jx292925 borrelia sp. km458263 “candidatus b. lonestari” a35a-m3 b. americana eu081293 b. spielmanii hm802196 b. microti jf708951 b. burgdorferi eu220791 “candidatus b. californiensis” eu076499 b. turicatae ay604979 “candidatus b. lonestari” a46bb-f4 b. sinica ab022137 borrelia sp. km458264 b. burgdorferi hm345910 “candidatus b. lonestari” ay237706 b. recurrentis dq346831 b. japonica d82852 b. americana hm802231 borrelia genomosp. 2 kf422812 b. anserina dq849626 b. hispanica gu357616 borrelia sp. a25b-m1 b. afzelii kf422796 borrelia sp. a25b-m19 50 100 99 88 97 100 97 62 82 90 100 100 88 100 100 100 99 92 85 79 100 51 fig. 1. baysian inference consensus tree inferred from flab of borrelia species. node support is indicated by the posterior probabilities at the node. the name of the species is followed by the genbank accession number. a25b-f5, a25b-m1, a25b-m2, a25b-m11, a25b-m16, a25b-m17, a25b-m18, a25b-m19, a32b-f2, a32c-f5, a35a-m3, a46bb-f1, a46bb-f3, a46bb-f4, b17z09-f2, b17z10-f1 and e49z02-m1 represent amplicons from r. sanguineus and are underlined. the scale bar indicates the mean number of nucleotide substitutions per site. finaljournal.indd 115 9/25/15 11:38 am 116 • fine focus, vol. 1 (2) muscle server (http://www.ebi.ac.uk/ tools/muscle/). default settings were used, with posterior manual adjustments if needed. bayesian inference phylogenetic analyses were performed using mrbayes v.3.2.5 (36) using two runs, for 10,000,000 generations each, using eight chains and a temperature coefficient of 0.1, and trees sampled every 5,000 generations. determination of the appropriate model for each genus was completed via jmodeltest 2 (15): gtr + i + ґ for borrelia and ehrlichia. the gamma distribution included six categories for all models obtained. after analysis was completed, the first 25% of trees from each run were discarded as burnin. the consensus trees were observed in figtree v1.4.2 (http://tree.bio.ed.ac.uk/ software/figtree/). e. species ab024928 e. canis a17s-m1 e. canis a17s-f5 e. ruminantium af069758 e. canis a17s-f7 e. muris u15527 e. canis kc479022 spain e. canis a25b-f22 e. species gu075696 e. canis kp844663 mexico e. canis a17s-f3 e. canis kj659037 china e. chaffeensis u60476 e. canis a25b-f16 e. species ab196303 e. canis a17s-f1 e. species af497581 e. canis a25b-f19 “candidatus ehrlichia shimanensis”ab074459 e. canis kj995841 brazil e. species ab028319 e. species dq324547 e. ewingii m73227 e. canis u54805 south africa 72 100 100 99 93 fig. 2. baysian inference consensus tree inferred from 16s rdna of erhlichia species. node support is indicated by the posterior probabilities at the node. the name of the species is followed by the genbank accession number. a17s-m1, a17s-f1, a17s-f3, a17s-f5, a17s-f7, a25b-f16, a25b-f19 and a25b-f22 represent amplicons from r. sanguineus and are underlined. the scale bar indicates the mean number of nucleotide substitutions per site. finaljournal.indd 116 9/25/15 11:38 am pathogens and antimicrobial factors • 117 results pcr detection of borrelia and ehrlichia of the 112 positive samples for tick rdna, 44 ticks (18 males and 26 females) were positive for b. burgdorferi-like species, “candidatus b. lonestari” or ehrlichia bacteria species (table 2). “candidatus b. lonestari”, the most commonly detected tick-borne pathogen, was detected in 16.9% (n=19, 16.3% of males (8/49) and 17.5% (11/63) of females) of all ticks. b. burgdorferi-like species were detected in 9.8% (n=11, 12.2% (6/49) of males and 7.9% (5/63) of females) of all ticks. e. canis was detected in 12.5% (n= 14, 8.2% (4/49) of males and 15.9% (10/63) of females) of all samples. two male ticks (4.1%, n=2 of 49) were positive for both e. canis and b. burgdorferilike species, indicating a 1.8% co-infection rate (2 of 112) of all ticks. one male and one female tick (male: 2.0%, n=1 of 49; female: 1.6%, n=1 of 63) were positive for both “candidatus b. lonestari” and b. burgdorferilike species, indicating a 1.8% co-infection rate (2 of 112) of all ticks. no tick was positive for the spotted fever group rickettsial rompa gene. phylogenetic analysis samples with strong bands by gel electrophoresis were prepared for sequencing. sequencing confirmed the pcr results. a bayesian inference tree including these sequences and other published sequences are shown in figs. 1 and 2. phylogenetic analysis showed that “candidatus b. lonestari” amplicons were in the same clade as known sequences of “candidatus b. lonestari” and b. burgdorferilike species amplicons clustered with b. burgdorferi species complex (fig. 1). “candidatus b. lonestari” sequences were all identical except a25b-f5, which was polymorphic at one position. three of the b. burgdorferi-like species sequences (b17z10-f1, a25b-m1 and e49z02-m1) were different at one position. ehrlichia amplicons were in the same clade as known sequences of e. canis (fig. 2). discussion the ticks used for this study were fully engorged. thus, the detection of pathogen dna either represents the most recent blood meal or potentially a prior infection of the tick. we detected the presence of b. burgdorferi-like species, “candidatus b. lonestari” and e. canis dna in r. sanguineus ticks from laredo, texas. our study does not address the issue of vector competency of r. sanguineus ticks in regard to b. burgdorferilike species and “candidatus b. lonestari”. the detection of pathogens in canines can indicate a potential risk for infection of humans (28,29). we have detected b. burgdorferi-like species dna in 9.8% of ticks collected from the local animal shelter, as well as ticks submitted by pet caregivers. the main vector for b. burgdorferi in the northeastern united states is i. scapularis (17,18,37). this tick is present throughout much of texas and northern mexico. b. burgdorferi was previously detected in 45% of tested i. scapularis ticks (19). however, in webb county tx, no i. scapularis were identified in the combined collection of over 70,000 ticks (5). b. burgdorferi-like finaljournal.indd 117 9/25/15 11:38 am 118 • fine focus, vol. 1 (2) species have been previously reported in a. inornatum from webb county (30) and a. mixtum from northeastern mexico (21). we also detected “candidatus b. lonestari”, which is thought to be the cause of stari (40), in 16.9% of ticks. cohen et al. (1990) reported a 5.5% seroprevalence for borrelia in domestic dogs from texas. however, of the dogs in their study 0 of 5 dogs that came from webb county were seropositive (9). likewise, bowman et al. (2009) reported b. burgdorferi in central and northern texas, but did not have any results for south texas. this is the first report of borrelia from r. sanguineus ticks and domestic dogs in webb county, texas. r. sanguineus is the only known vector of e. canis (23), and is widely distributed throughout the united states (6). we detected e. canis in 12.5% of ticks. however, all of the ticks that were positive for e. canis were collected from the laredo animal shelter. many pet owners acquire their pet from the animal shelter. these dogs are at a high risk for acquiring e. canis at this location. however, domestic dogs in the city of laredo appear to be at a low risk for acquiring e. canis. the previously reported seroprevalence for e. canis (2.0%) was higher in texas than in much of the united states (4). we did not detect e. chaffeensis and e. ewingii in r. sanguineus, even though they are present in the area (30). we also did not detect any spotted fever group rickettsia. this would suggest that any spotted fever group rickettsia are either absent from the area or were present at a very low prevalence. further research on r. sanguineus distribution and the prevalence of b. burgdorferi-like species, “candidatus b. lonestari” and ehrlichia species in south texas is needed. further research will help elucidate if r. sanguineus is a vector of a b. burgdorferi-like species. this additional research would allow for better and more accurate diagnosis of tick-borne illnesses, ultimately leading to better treatment and health care for domestic dogs and humans. this research supports the observation of dr. sandra leyendecker and suggests that domestic dogs should be screened for lyme disease if they present with appropriate symptoms. rosa vasquez-espinoza was supported by the ureca! grants program. david l. beck was funded for this project by faculty development funds. we thank g.t. pugh and m. weems for technical assistance. acknowledgements references 1. aguiar, d. m., cavalcante, g. t., pinter, a., gennari, s. m., camargo, l. m. a., & labruna, m. b. 2007. prevalence of ehrlichia canis (rickettsiales: anaplasmataceae) in dogs and rhipicephalus sanguineus (acari: ixodidae) ticks from brazil. j. med. entomol. 44: 126-132. doi:10.1093/jmedent/41.5.126 2. appel, m. j., allan, s., jacobson, r. h., lauderdale, t. l., chang, y. f., shin, s. j., thomford, j. w., todhunter, r. j., & summers, b. a. 1993. experimental lyme disease in dogs produces arthritis and persistent infection. j. infect. dis. 167: 651-654. finaljournal.indd 118 9/25/15 11:38 am pathogens and antimicrobial factors • 119 3. bacon, r. m., gilmore, r. d., quintana, m., piesman, j., & johnson, b. j. 2003. dna evidence of borrelia lonestari in amblyomma americanum (acari: ixodidae) in southeast missouri. j. med. entomol. 40: 590-592. doi:10.1603/0022-2585-40.4.590 4. beall, m. j., alleman, a. r., breitschwerdt, e. b., cohn, l. a., couto, c. g., dryden, m. w., guptill, l.c., iazbik, c., kania, 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national academies press. isbn:0-309-10897-7 29. mead p., goel r. & kugeler k. 2011. canine serology as adjunct to human lyme disease surveillance. emerg infect dis. 17: 1710-1712. doi: http://dx.doi. org/10.3201/1709.110210 30. medlin, j.s., cohen, j.i. & beck, d.l. 2015. vector potential and population dynamics for amblyomma inornatum. ticks tick-born dis. 6:463-472. doi:10.1016/j.ttbdis.2015.03.014 31. ndip, l. m., ndip, r. n., ndive, v. e., awuh, j. a., walker, d. h., & mcbride, j. w. 2007. ehrlichia species in rhipicephalus sanguineus ticks in cameroon. vectorborne zoonotic dis. 7: 221-227. doi:10.1089/vbz.2006.0608 32. parola, p., & raoult, d. 2001. ticks and tickborne bacterial diseases in humans: an emerging infectious threat. clin. infect. dis. 32: 897-928. doi: 10.1086/319347 33. perez, m., bodor, m., zhang, c., xiong, q., & rikihisa, y. 2006. human infection with ehrlichia canis accompanied by clinical signs in venezuela. ann. ny. acad. sci. 1078: 110-117. doi/10.1196/ annals.1374.016 34. piesman, j. & sinsky, r. j. 1988. ability of ixodes scapularis, dermacentor variabilis, and amblyomma americanum (acari: ixodidae) to acquire, maintain, and transmit lyme disease spirochetes (borrelia burgdorferi). j. med. entomol. 25: 336-339. doi:10.1093/jmedent/25.5.336 35. reardon, m. j., & pierce, k. r. 1981. acute experimental canine ehrlichiosis i. sequential reaction of the hemic and lymphoreticular systems. vet. pathol. online. 18: 48-61. 36. ronquist, f. & huelsenbeck, j.p. 2003. mrbayes 3: bayesian phylogenetic inference under mixed models. bioinform. 19: 1572-1574 37. stafford, k. c., cartter, m. l., magnarelli, l. a., ertel, s. h., & mshar, p. a. 1998. temporal correlations between tick abundance and prevalence of ticks infected with borrelia burgdorferi and increasing incidence of lyme disease. j. clin. microbiol. 36: 1240-1244. 38. steere, a. c., coburn, j., & glickstein, l. 2004. the emergence of lyme disease. j. clin. inv. 113: 1093-1101. doi:10.1172/jci21681 39. steere, a. c., & sikand, v. k. 2003. the presenting manifestations of lyme disease and the outcomes of treatment. new england j. med. 348: 2472-2474. doi:10.1056/nejm200306123482423 40. stromdahl, e. y., williamson, p. c., kollars, t. m., evans, s. r., barry, r. k., vince, m. a., & dobbs, n. a. 2003. evidence of borrelia lonestari dna in amblyomma americanum (acari: ixodidae) removed from humans. j. clin. microbiol. 41: 55575562. doi: 10.1128/jcm.41.12.5557-5562.2003 41. varela, a. s., luttrell, m. p., howerth, e. w., moore, v. a., davidson, w. r., stallknecht, d. e., & little, s. e. 2004. first culture isolation of borrelia lonestari, putative agent of southern tick-associated rash illness. j. clin. microbiol. 42: 1163-1169. doi: 10.1128/ jcm.42.3.1163-1169.2004 42. whitney, m. s., schwan, t. g., sultemeier, k. b., mcdonald, p. s., & brillhart, m. n. 2007. spirochetemia caused by borrelia turicatae infection in 3 dogs in texas. vet. clin. path. 36: 212-216. doi: 10.1111/j.1939-165x.2007.tb00213.x 43. wilhelmsson, p., fryland, l., börjesson, s., nordgren, j., bergström, s., ernerudh, j., forsberg, p., & lindgren, p.e. 2010. prevalence and diversity of borrelia species in ticks that have bitten humans in sweden. j. clin. microbiol. 48: 4169-4176. doi: 10.1128/jcm.01061-10 44. williamson, p. c., billingsley, p. m., teltow, g. j., seals, j. p., turnbough, m. a., & atkinson, s. f. 2010. borrelia, ehrlichia, and rickettsia in ticks removed from persons, texas, usa. emerg. infect. dis. 16:3, 441-446. doi: 10.3201/eid1603.091333 finaljournal.indd 120 9/25/15 11:38 am sean michael scully university of akureyri, faculty of natural resource science, borgir v/v nordurslod, 600 akureyri, iceland copyright 2016, fine focus all rights reserved perspective“exploiting an interdisciplinary approach using undergraduate research introduction corresponding author sean michael scully scully@unak.is keywords • undergraduate research • transferable skills • cross-sectorial coming out of high school, i chose to pursue a career in chemistry on the basis of it being regarded as the “central science”. my logic was that if i wanted to change directions, i could easily go into geology, biology, or physics. i knew from an early age that i wanted to go into science for the purpose of doing research, so going to university to pursue a career in chemistry seemed an obvious choice. as a child, i observed that i tended to learn things best by trying to connect new knowledge to things that i already knew, and topics with a “hands-on” component were particularly appealing. what i did not count on, however, was falling deeply in love with chemistry, which was further cemented by my experiences in research, a stint in the chemical industry, and other opportunities to apply bench craft to new challenges. what came as an even larger surprise was that microbiology turned out to be an exciting outlet that would prove to be every bit as rich and interesting as chemistry and that my background in laboratory work and chemistry would serve as a catalyst to further enhance my appreciation of other disciplines. as an undergraduate, my research experiences in both fields altered my way of thinking and the way in which i approach science. i started my university career in the united states majoring in chemistry at a fairly large institution. two of my professors strongly encouraged me to seek out undergraduate research opportunities and to get involved in education rather than being a passive element in the process of higher education. heeding their advice, i sought out other opportunities to further my scientific knowledge which turned out to be one of the best things that i could have done for my scientific career. one problem in pursuing an education in the sciences is the risk of over-specialization; there is an old adage that states that all problems look the same when you are only given one way of approaching problems. fortunately, a diversity of experiences in different types of professional labs as an undergraduate has shaped my interdisciplinary approach towards tackling research problems while giving a diverse skill set with which to take on new problems. in my 8 years as an 117 • fine focus, vol. 2 (2) perspective • 118 packing your toolbox using an interdisciplinary approach undergraduate, i have done stints working in two different research groups in organic chemistry, two groups in microbiology, and a three and a half year stretch working as a lab technician in the chemical industry, as well as two years working in a clinical lab. while this may be quite extreme, i strongly believe that even a few limited interdisciplinary adventures can greatly benefit anyone that is serious about a career in science. when it comes to enriching your education, there are lots of options, most of which provide excellent opportunities to advance your knowledge, enhance your ability to communicate science, or give back to the community. it has been my experience, both personally and now as someone in charge of supervising the research work of others, that real laboratory experience puts you ahead in terms of laboratory performance (i.e. actually standing at a bench and doing science). here i will go through some of the lessons that i learned being an undergraduate involved in research and cross-sectorial activities that i feel really enhanced my abilities at the bench as i further explore the intersection of chemistry and microbiology. one of the analogies that were frequently used in my undergraduate education in the u.s. was the “toolbox”. i used every opportunity to learn as many techniques, whether “soft” skills or more specialized methods, as possible and try to apply them to new situations. this is particularly relevant when you are transferring skills from one field of research to another. over time, i found that this interdisciplinary approach was exceptionally useful and rewarding. being able to use old knowledge and techniques to acquire new knowledge and solve problems is incredibly rewarding. beyond having an interdisciplinary approach, cross-sectorial training is also a highly valuable experience. there is more than one way to do science and the ultimate focus of that science very much informs the way in which day-to-day operations are approached. experiencing the way a clinician approaches science is often very different from the way someone working in an industrial field would. the most often used piece of my kit that i picked up somewhere along the way is a little mental experiment called “the truck test”. the truck test posits a simple question: could someone pick up and carry on your work from this given moment if you got run down by a truck? the degree of success to which you can answer this question revolves around several soft skills that i have found often go unaddressed in undergraduate (and often graduate) settings. traditionalists often tote the utility of the laboratory notebook as a tool for reproducing exactly what you have done. unfortunately, your experiment is more than just what you were doing; it is highly informed by the matrix in which you were doing it. 119 • fine focus, vol. 2 (2) why do undergraduate research? during my undergraduate work in the u.s., i and many others routinely heard about why we should do undergraduate research. professors often proclaimed that having research on your resume could make or break an application to a graduate program. while this certainly caught the ears of many students thinking about highly competitive careers as a medical doctor or pharmacologists, selling these opportunities to those bound for careers in science seemed to relegate doing science for the sake of science to the back burner. the traditional rationale for doing undergraduate research often includes benefits such as the following: • resume building • networking • money • class credit • publications • letters of recommendation all of these are nice, but much of this has very little to do with standing at the bench and doing actual science (which did not deter me from getting involved at the earliest opportunity!). i went into science to do science rather than chasing a career with a large paycheck attached to it. in my mind, the real benefits of doing undergraduate research really have to do with why you are presumably studying science in the first place: to learn to do actual science while being tied to a lab bench. part of doing science is venturing into the darkness of the unknown and trying to figure out what the room looks like and how stuff works. i am a firm believer that we learn by doing; thus, the best way to learn how to become a scientist is to do science. working at the bench with a more accomplished master is a fantastic way to learn techniques, carefully plan, and execute experiments, and interpret the results to answer questions and hopefully ask new questions. some of the often overlooked reasons to pursue undergraduate opportunities include the following: • cycle of science (the “real” scientific method) • confidence • bench skills • quality control and streamlining • data and project management • innovative problem solving • communication of scientific concepts • cultivating curiosity the cycle of science as it turns out, the daily grind of science is quite different from the pre-programmed laboratory experiments that you are exposed to in your general course work or the nicely displayed algorithms of the scientific method. one of the biggest takeaways from my undergraduate research experiences is mastering the workflow of real laboratory work and that successfully applying the scientific method requires a good deal of perspective • 120 creativity. in many ways, the analogy of a craftsman and an apprentice fits quite well. in undergraduate laboratory exercises, more often than not in my experience, a lot of the thinking and preparatory work has been done for you. procedures are often step-by-step and have been fined-tuned to a degree of precision that leaves no room for actual “discovery”. in real life, the flow of a research project has a “tick-tock” type rhythm whereby the results of one round of experiments directly informs the design of the next set of experiments. furthermore, most if not all of the preparatory work has been done for you in advance: plates have been poured, reagents are ready to use, and instruments are in working order and ready to use. this creates the illusion of ease because in the real world, it is unlikely that things are to be in such a state that you can simply walk into the lab and walk out with publishable results. the amount of preparation that goes into executing actual experiments can often take longer than the experiment itself. when i started my research in iceland, no one had touched one of the hplcs in years and no one knew how to operate the new one. fortunately, the three and a half years that i spent in the chemical industry as an intern lab technician (tied to the hplc and gcs) taught me everything that i needed to know to strip down an hplc and get it running in short order. this gave me a tremendous advantage to carry on with my own research projects while supporting the on-going work of researchers in other groups. confidence the psychological benefits of being professionally active during your undergraduate training are not to be underestimated. building up your selfconfidence to tackle complex, new, and highly involved research projects is critical to your ability to successfully execute laboratory operations. furthermore, having a track record including a number of failures is also important as it demonstrates that not only is botching a procedure okay, it is often part and parcel of the day-to-day grind of doing research. even after only having worked a few months one summer in a research lab radically altered the way that i approached standard coursework. as i have transitioned more towards a teaching role, i continue to find that students that engage in research early on in their careers are often much more capable when it comes to tackling their later coursework efficiently as well as conducting actual work with a high degree of reproducibility. data and project management one of the lessons that you learn from working on a research project is how to manage the planning and execution of a project as well as keeping data organized in such a way that it is actually useable. without a lot of prior experience, it is easy to become overwhelmed with the scope and interrelatedness of a series of experiments and end up dropping the ball due to disorganization. while the use of laboratory notebooks receives a tremendous degree of credit, in the electronic age, a lot of your data is captured electronically in excel worksheets or other formats. at least in my experience, data sets in “real world” research projects tend to be quite large with measurements from multiple instruments over time needing to get kept in order. an extremely useful strategy for dealing with the inflow of data is to design an excel sheet in parallel while designing the experiment. furthermore, working with an outline of 121 • fine focus, vol. 2 (2) the report or manuscript and plugging in data and discussions as you go rather than handling data after the fact can be a very efficient way of working with a data set. communication of scientific concepts stepping out of the classroom and into the real world of research showed me that scientific writing comes in more flavors than producing reports and scientific articles. in order to be an effective scientist, writing for different audiences is a skill that must be thoroughly mastered. an often overlooked backdoor route to improving your scientific communication skills is to carefully dissect the work of others. this has the advantage of being something that you can start early in your career before you know much of anything but can still be of use even if it is catching a potentially embarrassing grammatical error. early in my career, tearing apart manuscripts prior to submission was a favorite past time among those in the research groups with which i was involved. when i transferred my focus more towards the biological, i found that my background in chemistry and having english as a native language was very helpful with improving the manuscripts of other more advanced researchers. at some point, revising the work of others lead to conversations regarding ways to improve experimental designs or upgrading methodology to be higher throughput or more robust which eventually lead to my first few co-author credits. one of the aspects of real-world science that i found most surprising was the grant writing process. i had the (mis)fortune of becoming involved in writing applications for several ideas that i had while i was still an undergrad. the amount of time that goes into constructing well-thought-out grant proposals can typically run into the hundreds of man hours depending on the size of the application and the number of people involved. thus, learning how to effectively multitask while managing the contributions of others is absolutely essential. one aspect of communicating science was how to organize your ideas into do-able units which are often referred to as “work packages” consisting of well-defined tasks and subtasks. writing grant proposals that are reviewed by experts and non-experts alike requires careful balance. my earliest experiences writing grant applications were an abysmal failure: i found it difficult to overcome the formal, highly technical writing style that was drilled into my head. after several rejections and heeding the careful comments of peer-review, i started to have more success as i moved through my undergraduate career. furthermore, writing up your scientific work to be broadly understood by the public is another highly valuable writing skill. another often under-emphasized aspect of communicating your scientific work, either verbally or in writing, to the broader public. effectively communicating the importance of advanced science to laymen with little or no scientific training presents a tremendous challenge. i have found that a good place to start is by talking to friends about your work in such a way as they do not lose interest. this can often be accomplished by talking about the broader implications and impacts of your work. understanding the impacts of your own work are also of great importance when going before an opponent critically reviewing your thesis work or when an application for funding is being scrutinized by a panel of experts. another potential target to practice your communication skills include younger school children. while this often involves a healthy degree of generalization and making analogies to things perspective • 122 with which can be related to everyday life, i have often found that if you can adequately explain a scientific concept to a 5 year old, you can probably effectively communicate it to anyone else. learning how to communicate visually with effective figures is often a highly useful skill. while data visualization is a topic all by itself, taking the time to explore a few entry level texts (refer to the further reading section) can be rewarding. for instance, showing the interrelationship between tasks with a simple flow chart can add clarity to complex experimental designs and quickly confer the just of what you are doing to a reader. bench skills standing (or sitting) at a lab bench and performing tasks is the bread and butter of working in a laboratory in any context. while the individual techniques and practices that you learn may ultimately have value, the real benefit stems from your ability to rapidly assimilate a new methodology and put it into practice. i found that having a broad base of analytical techniques to choose from really enhanced my work. as you spend more time at the bench, you will observe that laboratory life is like a small ecosystem unto itself and one of the most useful elements that i gained from being involved in professional laboratories was becoming attuned to the heart beat of the lab. in many ways, any lab is like an ecosystem with an inflow of consumables and an outflow of data and waste. furthermore, each piece of equipment has its own “vital signs” and becoming attuned to the needs of each piece of equipment is a lot like the dating process. many weekends of being alone in the lab troubleshooting instrument problems became a somewhat regular phenomenon. learning to listen to equipment’s “vital signs” is often a good place to start. being attuned to what something is “supposed to sound like” can often quickly alert you to something being wrong. developing solid troubleshooting skills can save costly visits from instrument specialists as well as greatly reduce unnecessary downtime. during slow moments, i often found it useful to flip through the “troubleshooting” and “maintenance” sections of instrument manuals. also, learning how to keep equipment in proper working order by following a regular maintenance cycle can often prevent problems, especially since many consumable parts wear out in a predicable fashion with regular usage. developing a streamlined approach and quality management during the “undergraduate era” of my career, which spanned clinical, research, and industrial labs, i noticed that turnover had a profound effect on efficiency. every time that one of the more senior members of a research group left it was like cutting off an arm. most of the time, it was just basic routine tasks that suffered most, not to mention the specialized knowledge that seemingly evaporated overnight. highly efficient enterprises (like multinational corporations) tend to treat people like replaceable cogs and have a system in place to deal with retaining knowledge. in my experience, this is something that research groups do quite poorly. larger research groups seem better able to maintain some sort of procedural continuing while the loss of a senior member in a small research group is devastating. re-inventing the wheel can be costly and laboratories performing 123 • fine focus, vol. 2 (2) the same tests as other labs need to be able to demonstrate that they get comparable and highly reproducible results. in an industrial lab, especially one that operates a quality management system (qms) such as iso 17025 or iso 9001, commonly performed procedures have a high degree of standardization; procedures are written and made centrally available. while qms topics often illicit groans from academics, i learned firsthand their efficacy which was further confirmed as i put lessons from qmss into practice in my own research. while iso is often considered to be too paper heavy for research groups that need to maintain a high degree of fluidity, some of the major concepts can and should be readily integrated into your daily work. iso principles include using a process approach, systematic approach to management, striving for continual improvement, and taking a factual approach to decision making. key concepts within these principles are particularly relevant such as maintaining traceability, good records keeping, document control, and employee training. standard operating procedures (sops) maintained by one person can easily be disseminated to new hires or visiting researchers. having a system for routine procedures saves time and often money. i have found that having bottles for individual assays or media components with labels that include dummy-proof directions are a fantastic way to go. not only does it take the guess work out of preparing reagent solutions, the directions are right in front of you. as it turns out, reproducibility problems are the achilles heel of experimental results. thus, developing good quality control practices, even in a research setting, is absolutely vital to ensure that you are producing trustworthy results. fortunately, you do not need to have completed any education to start filling the gaps without much experience in quality management. after spending a few years working in the chemical industry, i found that my awareness of these procedural gaps was heightened and all that you really need to do is recognize the need to fill a gap. as someone with experience with a particular method, you can start writing sops and specific methods yourself and make them centrally available to your group. i have observed countless times in my work in research labs so-called “procedural drift” whereby a specific procedure is passed down from senior researcher to new inductee, who will then transfer their version of a procedure to someone in the future. this can result in large deviations in practice and often result in disastrous mishaps costing valuable time and deteriorating the quality of the output result. you can similarly apply the creation of standardized documentation for standard control charts or reagent logbooks. not only will this make life easier for your supervisor, it will help those that come after you more quickly adapt to their new surroundings while making it easier to catch instrumentation problems or operator errors. an added benefit of taking the initiative writing procedures and creating standardized forms is that you can build your own personal repertoire of methodologies. over my career to date, i have written dozens upon dozens of such procedures adapted from the scientific literary, crafted de novo, or “borrowed” and adapted to my own liking. this gives you a tremendous degree of power and you can easily pass this knowledge to a colleague in need with a few mouse clicks. a common topic within many qmss is continuous improvement. as you begin to develop your own sops and systematic documents, you perspective • 124 can also improve them based upon flaws or amended ways of doing things. aside from the iso quality management systems, there are many other quality management systems that may be more relevant to the constantly evolving environment you find in research. a background in good “anything” practices (gxp) such as good manufacturing processes (gmp), good laboratory practices (glp), or good clinical practices (gcp), will serve you very well as you move through your coursework, practical training, or first stint in a research lab. despite having different names, many of the practices in gmp, glp, and gcp share many commonalities. innovative problem solving innovation is not just a business buzzword; it describes ways of thinking in terms of problem solving with limited resources. doing research with limited resources, while challenging, is rewarding. the reality of science, at least as i have experienced it thus far in my career, is that you never have “ideal” conditions to do the work that you want. you might be missing a piece of equipment or full funding for a particular project, or extra pairs of hands. stated more succinctly, you have to make do with what you have or can easily get your fingers on. fortunately, this is something that time at the bench as an undergraduate can quickly teach you how to deal with using a bit of innovation and problem solving. having to deal with equipment on a daily basis, i quickly learned that being well acquainted with instrument manuals and having a supply of texts on specific techniques to be an excellent first line of defense to dealing with problems. instrumentation, whether in a lab with a chemistry focus or a biological one, is absolutely critical to being able to consistently turn out quality results. instrument downtime is an absolute killer of efficiency. in order to avoid downtime, not only should you learn the ins and outs of your equipment, you should get into the practice of learning preventative and routine maintenance procedures. i found time and again that maintaining a pool of spare parts, whether it be replacement consumables parts of an hplc’s pumps or on occasion, asking around for spare parts and solutions is a cost-effective solution and can also serve as a catalyst for professional networking. but when all else fails, you must sometimes rely on your wits to solve problems especially when replacement parts are weeks away or there is no money in the lab’s budget for a service call. the basis of all problem solving skills is a combination of highly disciplined and systematic step-by-step investigations into a problem with a bit of innovation thrown in. when i first moved to iceland, my background was mostly in chemistry. two of the first major challenges i faced in working in a microbiology lab involved patching together a water distiller with a cracked boiling housing and resuscitating a mistreated hplc. as both pieces of equipment were absolutely vital to doing any sort of microbiology, i got to work. the cracked boiler housing was a bit of a challenge. i decided to try carefully applying electrical tape, which is reasonably heat resistant. much to my surprise, it worked and the boiling chamber actually managed to run nearly continuously until the electronics failed a number of years later. coaxing the hplc back to working order, on the other hand, required several weeks of methodical troubleshooting. the biggest tell that there was a problem in the first place was incredibly high back pressure (in excess of 125 • fine focus, vol. 2 (2) 40 mpa!) at only 10% of the recommended flow rate. unfortunately, there was no “log book” detailing the instrument history so i started by patiently unraveling the history of the instrument, who had used it, and when the problem started. i slowly and patiently worked the problem backwards by eliminating one variable at a time only to find out that a series of problems was to blame. upon the instrument’s initial installation, the wrong diameter guard column had been installed and the steel tubing had been kinked by a careless technician. to make matters worse, someone had injected unfiltered biological samples onto the column. learning how to maintain, repair, and keep equipment in working order all represent a highly valuable transferable skill. once you learn how to keep one piece of equipment in order, adding another is less of a mystery. it has been my observation that hiring managers, whether interviewing to fill a research job or a job in the private sector, tend to value these types of transferable skills over an applicant’s ability to perform specific procedures. by paying close attention to parallel disciplines, you can often transfer ways of handling science to your own work. furthermore, by diversifying your work experience by venturing into an industrial or clinical setting, you can gain valuable insights into areas such as quality management which are often neglected in research. cultivating curiosity through involvement in interdisciplinary studies beyond developing transferable competencies, the opportunity to do research at the undergraduate level also allows you the rare opportunity to explore your own interests beyond the highly pre-programmed nature of the vast majority of undergraduate work. i found that i naturally gravitated towards organic chemistry as a discipline while enjoying related aspects in the fields of biochemistry and microbiology. as it turns out, the intersection of these disciplines is incredibly fascinating and acted as a catalyst for my further learning. while i was in my initial undergraduate period, i had stumbled across a few papers by george whitesides in which he used enzymes as catalysts for organic synthesis. this planted the idea in my head and i caught on fire. as a result, i decided to dual major in biology; at that time, green chemistry was up and coming and had not reached the level of awareness that it currently enjoys. while the use of enzymes in niche applications including in food processing, textiles, and organic chemistry, to name a few. while standing at the bench in a research lab as an undergraduate will often reinforce the lessons taken from your course work, it is also a fantastic opportunity to go beyond the book and expand upon your own intellectual curiosity. appreciating nature’s organic chemists going through undergraduate coursework, i could not help but stand in awe of the great synthetic masters such as karl fisher, whitesides, and e.j. corey. their insights into the way that molecules behave are nothing short of a marvel of our species’f ability to understand. at some point, however, i came across a quotation from francois jacob that goes as follows: nature is a tinkerer, not an inventor perspective • 126 as it turns out, nature has been at the synthesis game a lot longer than we have been on the scene. biochemical reactions have been around on this planet for at least 3.5 billion years, and evolution has spent millions and millions of years finetuning numerous reactions to specific conditions ranging from highly saline and icy cold to well beyond the boiling point of water. not surprisingly, enzymes are highly efficient catalytic systems given this protracted development time. to put their efficiency in perspective, a typical catalyst for a reaction uses between 0.1 to 1 mole percent (2). enzymes on the other hand, can catalyze reactions in the range of 10-3 to 10-4 mole percentage! fortunately, we are not limited to “natural” reactions. many enzymes will accommodate other substrates, a trait commonly referred to as enzyme promiscuity. biocatalysis is a broad and highlyinterdisciplinary field (fig. 1) that looks at the use of living systems, in part or in whole, to perform reactions. thus, the whole gamut from cultures of microbes, crude extracts, to purified enzymes can be used in various applications. biocatalysts have traditionally been touted for their high catalytic efficiency, mild operating conditions, high selectivity (chemo-, stereo-, and regio-selectivity), immobilization, and biodegradability. their drawbacks, however, are likely the figure 1. biocatalysis is a multidisciplinary field calling on many other disciplines biocatalysis microbiology biochemistry bio technology green chemistry organic chemistry green chemistry principle traditional synthesis biocatalysis prevent waste x ü maximize atom economy x ü minimize toxicity x ü (preserve functionality/efficacy) x/ü ü minimize auxiliary substances x ü minimize energy input x ü renewable feedstocks x ü minimize protecting group usage x/ü ü use catalysts x/ü ü design for biodegradability x/ü ü real-time analysis ü ü minimize accidents x/ü ü table 1. green chemistry (adapted from references 1 & 2) 127 • fine focus, vol. 2 (2) reason for their relegation to specialized roles; enzymes are frequently prone to inhibition phenomena, cofactors, solvent compatibility, lack of commercial availability, and their selectivity. for these reasons, talk of the use of biocatalysts is frequently regarded with suspicion and viewed as a black box due to the complexity of working with living organisms or parts thereof. with the push for “greener” chemistry and industrial practices, however, the use of biocatalysis in many applications is beginning to gain a foothold. of the commonly cited twelve principles of green chemistry (table 1) originally put forth by the epa, biocatalysts are capable of being compatible with all twelve principles. microbes provide a vast library of enzyme chemistries just waiting to be explored. given their ubiquitous nature and adaptations to broad environmental conditions, they are a potential treasure trove. one particularly interesting aspect of enzymes is their inherent stereopreference opening the doorway for their use as chiral catalysts in synthetic applications. chirality: the handedness of life while taking organic chemistry, i became enamored with the “handedness” of life which i found to be a bit of an abstraction at first. chirality (greek for “handed-ness”) is of fundamental importance in the biological sciences and poses a number of serious problems in organic synthesis. enantiomers are stereoisomers that are mirror images of one another (as shown in fig. 2) whereas diastereomers are stereoisomers that are not mirror images of one another. most biomolecules have inherent chirality yet we take this for granted. sugars typically have multiple chiral centers. at some point during my long undergraduate tenure, i took an interest with 1,2-propanediol (1,2-pd, figure 3). this three carbon diol has a single stereocenter at c2 and thus exists as two enantiomers. while being an extremely simple molecule, separating the two enantiomers of 1,2-pd is incredibly challenging and caught my attention. exploiting the chirality of enzymes enzymes, being proteins, are composed of l-amino acids. as such, enzymes are highly specialized organic chemists that often demonstrate chiral preferences and in many cases can accept nonfigure 2 enantiomers are moleculer mirror images figure 3 1,2-propanediol and its two enantiomers. note the difference in configuration at c2 perspective • 128 figure 4. a pseudomonas cepacia lipase (pcl) catalyzed kinetic resolution of 1-bis(4methylphenyl)methyl-propan-2-ol; b – kinetic resolution of racemic 1,2-propandiol derivative using pcl monitored by high performance liquid chromatography native substrates (a feature often referred to as “catalytic promiscuity”). as i neared graduation, i decided to use the opportunity to combine my love of organic chemistry with my new found appreciation for microbiology. the work i did for my b.sc. thesis examined the use of lipases from psychrophilic or psychrotolerant microbes, such as pseudomonas cepacia (pcl), to resolve partially protected 1,2-propanediol derivatives. the approach i chose was two pronged: a regioselective protection using a variety of tin(ii) halide catalyzed etherification reactions that had shown to have a high degree of regiopreference in partially protected carbohydrate diol systems followed by an enzymatic resolution of the two enantiomers (fig. 4). given my extensive background with high performance liquid chromatography (hplc), i decided to monitor the reactions in near-real time to ensure that only one of the enantiomers was indeed reacting. while the pcl-catalyzed reaction portion worked very well, there were issues with the regioselectivity of the initial protection step necessitating an extra derivatization step. while the project as a whole was only partially successful, it gave me crucial insights into the challenges of separating biologically relevant enantiomers and gave me the impetus to pursue other lines of thought to reach my goal of separating a racemic mixture. oh oac oh + o o o starting material partially protected racemic 1,2-proapnediol mono-ether product i 2r-acetate derivative pseudomonas cepacia lipase (pcl) diisopropyl ether vinyl acetate product ii 2s-free oh derivative a 100 90 80 70 60 50 40 30 20 10 0 0 2000 4000 time (min) c on ce nt ra tio n (% ) 6000 8000 b starting material product (2-r-ac) 129 • fine focus, vol. 2 (2) figure 5. professor johann orlygsson (university of akureyri) demonstrating sampling technique in geothermally heated stream while several undergraduate researchers look on. bioprospecting for extreme enzymes and chiral products since finishing my master’s degree (albeit in a project focusing on carbohydrate chemistry), i have had the good fortune to manage a number of projects all of which have given undergraduates research opportunities at the intersection of microbiology and chemistry. the focus of this work has been on two fields: prospecting for enzymes with biotechnological potential and bacteria producing (r)or (s)-1,2-propanediol. the logic behind these two approaches is that it is easier to produce an enantiomerically pure compound such as 1,2-pd rather than trying to separate a mixture of the two enantiomers after the fact and that producing these compounds from enzymatically treated abundant biomass (such as lignocellulose) would be the most sustainable approach. the early work involved leading a few groups of willing undergraduate into the field to collect samples from iceland’s various geothermal areas (fig. 5). once in the lab, the laborious process of enriching samples and isolating pure cultures began and continues to this day (some 3 years later). one of the reasons for going after thermophiles was that it has not been widely reported that they are capable of producing 1,2-pd. screening for 1,2-propanediol producing bacteria and enzymes of biotechnological potential has since expanded to new environs fueled by the initiative of a new generation of undergraduate researchers. watching students enrich and screen isolates from environments and finding new and exciting bugs and expanding the knowledge base has been rewarding in its own right. parting words science is very much an active endeavor, and one’s ability to succeed can be very much enriched by making connections by different disciplines and moving out of your comfort zone. once piece of advice i like to give my students is to fervently seek out new opportunities in science, whether at home or abroad, and to strive to create a culture of learning. there are many ways of enriching your undergraduate experiences beyond research including doing science outreach (a great activity for sharpening those communication skills while giving back to the community) as well as teaching, whether it be acting as a teaching assistant for undergraduate course work or tutoring high school students. perspective • 130 i would like to express my gratitude to those that have supported my career including the faculty members at the university of toledo and the university of akureyri with a special thank you to professor sigthor petursson who gave me a high degree of freedom to pursue my interests. additionally, i would like to thank the staff of perstorp polyols for the opportunity to work in their quality lab. a special thanks is due to eva maria ingvadottir for editing this manuscript. knisely, k. (2013). a student handbook for writing in biology (4th ed.). sunderland: sinauer associates, inc. dean, j.r., et al. (2011). practical skills in chemistry (2nd ed.). london: pearson education. tufte, e.r. (2001). the visual display of quantitative information (2nd ed.). new york: graphics pr. 1. anastas, p. t., & warner, j. c. (1998). green chemistry: theory and practice. new york: oxford university press. 2. faber, k. (2011). biotransformations in organic chemistry: a textbook. springer. 3. liese, a., seelbach, k., & wandrey, c. (eds.). (2006). industrial biotransformations (2nd ed.). weinheim: wiley-vch. 4. wong, c.-h., & whitesides, g. m. (1994). enzymes in synthetic organic chemistry. oxford: pergamon. acknowledgments further reading references striking up the conversation: quorum sensing in fungi brooke martini, cody orr, and ginny webb* natural sciences and engineering, university of south carolina upstate, spartanburg, sc, usa brooke martini and cody orr contributed equally to this work copyright 2015, fine focus all rights reserved manuscript received 26 february, 2015; accepted 2 july, 2015 finaljournal.indd 139 9/25/15 11:38 am quorum sensing is a form of communication observed in different species of microbes. numerous studies have shown the ability of bacteria and fungi to carry out quorum sensing by releasing specific molecules to enable communication in a large population. quorum sensing has been shown to influence growth, morphology, and other factors pertaining to virulence in pathogenic microbes. in this review, we address three important fungal species and explain how each fungus has a unique and dynamic way of communicating. candida albicans is an opportunistic pathogen, or one that is part of the normal microbiota that can become pathogenic and cause several diseases. here, we address two quorum sensing molecules (qsms) identified by investigators. these chemicals are tyrosol and farnesol, which act together to control cellular growth, morphology and biofilm production. another opportunistic fungal pathogen, cryptococcus neoformans, has been shown to display quorum sensing activity by using pantothenic acid as well as a peptide called quorum sensing-like peptide 1. these molecules have both been shown to control growth rates of c. neoformans. saccharomyces cerevisiae is another dimorphic fungus that uses qsms, although it is nonpathogenic. using two aromatic alcohols, phenylethanol and tryptophol, s. cerevisiae can alter pseudohyphal growth in diploid cells as well as invasive growth in haploid cells. by understanding more about the ways these organisms communicate, we present the potential for new and better targets for the treatment of fungal infections. abstract corresponding author *ginny webb gwebb@uscupstate.edu keywords • quorum sensing • fungi • c. albicans • c. neoformans • s. cerevisiae many microbes use a form of communication known as quorum sensing, which is a function of population density. this phenomenon was first discovered in the bacteria vibrio fischeri and vibrio harveyi in the 1970s. these bacteria were shown to luminesce only when high numbers of bacteria cells were present (30, 31). in 2001, hornby et al. reported evidence of a quorum sensing mechanism in the eukaryotic yeast candida albicans (15). similar to the process used by bacteria, fungal cell density was found to play a major role in communication (18, 25). fungi, along with introduction 140 • fine focus, vol. 1 (2) finaljournal.indd 140 9/25/15 11:38 am review • 141 other organisms, secrete compounds known as quorum sensing molecules (qsms) into their environment to communicate with neighboring cells. qsms, also known as autoinducers, regulate many functions of the population as a whole. qsms must accumulate to reach high concentrations to have an effect on the organism. a large population of microbes, therefore, must be present to produce high enough levels of qsms to have an effect (3). the qsms bind receptors on the microbe’s cell and when enough receptors are bound they activate signaling pathways. the induction of these signaling pathways affects gene expression in the organism, which in turn controls various cellular processes, including cell growth, biofilm formation, motility, cell morphology and secretion of virulence factors (1, 2, 8, 14, 25, 27, 45). much work has been done to uncover the various mechanisms that regulate quorum sensing by identifying the pathways and signaling proteins involved. for example, kruppa et al. were the first to report a twocomponent signal transduction pathway in eukaryotic cells that mediates morphology, phenotype, and biofilm formation (18). other recent studies have determined ras1, cyclic amp (camp), and mitogen-activated protein kinase (mapk), among others, to be important factors for quorum sensing in both c. albicans and cryptococcus neoformans (10, 20, 22). although more is known about quorum sensing in bacteria, this method of communication in fungal microbes has been an area of great interest in recent years. quorum sensing in candida albicans is perhaps the best understood example of quorum sensing in fungi (3, 7, 10, 15, 35, 41, 45). in addition, cryptococcus neoformans and saccharomyces cerevisiae have also been identified as fungal species that utilize this cellular communication (2, 6, 21, 22). this review will discuss the quorum sensing mechanisms that c. albicans, c. neoformans, and s. cerevisiae employ in regulating the cellular population, as these are the species in which fungal quorum sensing has been most thoroughly studied. although other fungal species have been shown to have quorum sensing activities, the qsms in these other species have not yet been identified. the significance of fungal quorum sensing will also be addressed as it applies to fungal signaling, the pathogenicity of fungi, and treatment of fungal infections. candida albicans candida albicans is a dimorphic fungus that has been extensively studied due to its unique ability to cause diseases even though it is part of the normal human microbiota. this fungus causes thrush, genital candidiasis, and bloodborne invasive candidiasis (16, 17, 20). an important attribute contributing to the virulence of c. albicans is its dimorphism, or the ability to switch morphological forms between mycelium (hyphae) and budding yeast. the mycelium form of fungi embeds into tissue, while the yeast form is associated with initiating infections and dissemination (32, 38). two qsms, farnesol and tyrosol, have been determined to have an effect on this dimorphism phenomenon as well as other cellular processes (7, 10, 15, 18, 20, 33). farnesol was discovered in 2001 and was the first qsm identified in a eukaryotic cell (fig. 1) (15, 34). farnesol, an isoprenoid, was identified as a qsm in c. albicans by observing the effects that supernatants from spent media (cell-free, filtered supernatant from a previously grown culture) had on the differentiation and mycelial growth of the fungi. the results of the assays showed that the highest amount of mycelial growth occurred when no supernatant was added. finaljournal.indd 141 9/25/15 11:38 am 142 • fine focus, vol. 1 (2) table 1. summary of quorum sensing in fungi figure 1. schematic diagram illustrating fungal quorum sensing molecules and their effects. organism quorum sensing molecule effect of quorum sensing molecule references candida albicans farnesol prevent mycelial formation reduce biofilm formation increase resistance to oxidative stress induce apoptosis 15 35 10,45 41 candida albicans tyrosol increase growth rate increase mycelial formation increase biofilm formation 7 7 3 cryptococcus neoformans pantothenic acid increase growth rate increase gxm release increase melanin synthesis 2 2 2 cryptococcus neoformans quorum sensing-like peptide 1 induce cell growth increase capsule formation decrease melanin synthesis 21 22 22 saccharomyces cerevisiae phenylethanol induce hyphal growth 6 saccharomyces cerevisiae tryptophol induce hyphal growth 6 quorum sensing molecules tyrosol • increase growth rate • increase mycelial formation • increase biofilm formation tryptophol • induce hyphal growth pantothenic acid • increase growth rate • increase gxm release • increase melanin synthesis quorum sensing-like peptide 1 • induce cell growth • increase capsule formation • decreased melanin synthesis phenylethanol • induce hylphal growth c. albicans s. cerevisiae c. neoformans farnesol • inhibit mycelial formation • reduce biofilm formation • resistance to oxidative stress • induce apoptosis finaljournal.indd 142 9/25/15 11:38 am review • 143 when a high concentration of supernatant was added, the amount of mycelia was substantially decreased. this indicated qsms had a dose-dependent effect on the morphology (15). to identify the qsm in the supernatant, solvent extraction of spent media was performed and coupled with thin-layer chromatography (tlc) and gas chromatography-mass spectroscopy (gc-ms). the use of these techniques combined with the elimination of other potential qsms resulted in the identification of farnesol as the qsm responsible for the effect on mycelial growth of c. albicans. commercially prepared farnesol also prevented mycelial formation; further suggesting that farnesol was the qsm responsible for the observed effect on morphology (table 1). growth rates were also observed when farnesol was added to c. albicans cultures and it was concluded that farnesol did not have an effect on yeast budding (15). the discovery of a molecule that inhibits mycelial formation is important because it is the mycelium form of the organism that allows it to enter the bloodstream, resulting in invasive candidiasis (15). researchers have demonstrated that farnesol inhibits the mycelial phase and promotes the yeast phase through the regulation of many different signaling pathways as depicted in fig. 2. chk1p was identified as a possible two-component signal transduction protein that acts with an unidentified upstream protein to mediate farnesol sensing. authors tested a wild type strain and a chk1 mutant strain of c. albicans and found that the chk1 mutant germinated, resulting in mycelial growth, in the presence of farnesol while the others did not. this could be valuable knowledge because two-component signaling genes are not found in humans and could provide a potential therapeutic target for this fungal pathogen (18). in 2011, it was reported that farnesol suppresses hyphal formation through the inhibition of the ras1cyr1-camp signaling pathway (10, 23, 34). in fact, cells with increased camp signaling are more resistant to the morphology effects of farnesol (23). tpk1/2, czf1, and efg1 were discovered to be required for the morphology effects of farnesol. these findings are in line with the previous studies, as tpk1/2, efg1, and czf1 are downstream of the ras1-cyr1-camp pathway (20). farnesol signaling was discovered to lead to resistance to oxidative stress, also due to inhibition of the ras1-cyr1-camp pathway (10). the discovery of farnesol enabling the cell to resist oxidative stress provides an additional mechanism farnesol uses to aid in the pathogenicity of c. albicans (10, 45). evidence also suggests that the transcription regulator tup1 plays a role in the inhibition of mycelial formation in response to farnesol. although tup1 seems to play a role in the effects of farnesol signaling, the signaling pathway linking tup1 to farnesol-mediated morphology effects is still unclear (20). the involvement of tup1 in quorum sensing of c. albicans is especially interesting because tup1 is also involved in quorum sensing in c. neoformans, which will be discussed later in this review (20, 21). furthermore, nrg1 was discovered to have a crucial role in the farnesol-dependent inhibition of hyphal formation (25). nrg1 inhibits hyphal formation and therefore must be absent for hyphal initiation. the absence of nrg1 is controlled by two mechanisms: 1) the ras1-cyr1-camp pathway has a negative effect on nrg1 transcription and 2) protein degradation of nrg1. farnesol inhibits the ras1-cyr1-camp pathway, resulting in the expression of nrg1 which results in inhibition of hyphal formation. in addition, farnesol blocks nrg1 degradation by stabilizing the cup9 transcriptional repressor, which in turn represses sok1 expression. finaljournal.indd 143 9/25/15 11:38 am 144 • fine focus, vol. 1 (2) sok1 leads to the degradation of nrg1, therefore a repression of sok1 results in inhibition of nrg1 degradation thus allowing nrg1’s presence to inhibit hyphal formation. interestingly, both the ras1-camp pathway and the cup9-sok1-nrg1 pathway are required for farnesol to elicit its effects on the morphology of c. albicans (fig. 2) (25). additional effects of farnesol on c. albicans include the induction of apoptosis via a caspase-dependent pathway (table 1) (41). in addition, farnesol was shown to induce apoptosis and decrease proliferation in human oral squamous carcinoma cells (39). the ability of farnesol to affect cell death may have important implications for the development of therapies for fungal infections and for cancer (39, 41). tyrosol is another qsm produced by c. albicans that regulates growth and morphology (table 1) (7). tyrosol increases fungal growth by substantially decreasing the length of time the fungus is in lag phase. this was observed when chen et al. (6) compared the growth of dilute overnight c. albicans cultures in the presence and absence of tyrosol over an 8-hour period. they also demonstrated that tyrosol is continuously released into the medium during growth and that it promotes filamentous morphology. tyrosol was shown to promote germ tube formation, stimulating the conversion of yeast cells to hyphae (fig. 1). the effect tyrosol has on cell growth may be due to tyrosol stabilizing transcripts that encode proteins involved in dna synthesis and the cell cycle, therefore affecting growth by regulating the processes of dna synthesis and the cell cycle (7). these results suggest that tyrosol and farnesol work together to control cellular growth and morphology of c. albicans (7). another important factor contributing to the virulence of some pathogens is biofilm figure 2. a diagram depicting the pathways that have been implicated in farnesol signaling in c. albicans. farnesol hyphal initiation biofilm formation ? ? chk1p ras1 cyr1 camp tpk1/2 efg1 czf1 hyphal inhibition cup9 sok1 nrg1-tup1 caspasedependent pathway apoptosis induction sensitivity to oxidative stress finaljournal.indd 144 9/25/15 11:38 am review• 145 formation. biofilms play an important role in natural and medical environments. biofilms are organized microbial communities that adhere to surfaces, posing a serious health threat for patients with medical devices like stents and catheters (35). the mycelium form of fungi is crucial for biofilm development of c. albicans; therefore, inhibiting mycelial formation could be important for controlling fungal biofilms (35). studies done by ramage et al. and kruppa et al. demonstrated that farnesol had a direct effect on biofilm formation (table 1) (18, 35). the amount of biofilm formation was reduced as the concentration of farnesol increased. there was a 60% reduction in biofilm formation when the wild type strain of c. albicans was exposed to 25 µm and 250 µm of farnesol (18). ramage et al. hypothesized that the reduction in biofilm formation was due to the fact that farnesol decreased mycelial formation, which is crucial for biofilm development of c. albicans. they concluded that this was a mechanism the organism uses to control overgrowth and limit competition (35). microarray analysis comparing c. albicans biofilms inhibited by farnesol to natural c. albicans biofilms shows expression changes in genes controlling hyphal formation, drug resistance, cell wall maintenance and iron transport (4). interestingly, when tyrosol was added to c. albicans during different stages of biofilm formation, there was no effect observed. when farnesol and tyrosol were added together, the effect was dependent on the combination of the individual concentrations of the two qsms. tyrosol eliminated the inhibitory effect of low concentrations of farnesol. however, tyrosol was unable to overcome farnesol’s inhibition of biofilm formation when 1 mm of farnesol was used. in addition, biofilms exposed to both farnesol and tyrosol were almost entirely in the yeast form suggesting that farnesol has a dominant effect. these discoveries could lead to farnesol as a possible treatment for fungal biofilms (3). as shown in table 1, the qsms produced by c. albicans affect multiple aspects of its pathogenicity, including: morphology, biofilm formation, and resistance to oxidative stress. these quorum sensing molecules require additional study to further investigate this common opportunistic fungus. cryptococcus neoformans cryptococcus neoformans is another dimorphic fungal microbe and can cause life-threatening meningitis, particularly in aids patients (5, 19). quorum sensing is not as well understood in c. neoformans; however, it appears to play a significant role in the regulation of several cellular processes of the fungus (fig. 1). it was observed that when the gene for the transcriptional repressor tup1 is disrupted, c. neoformans colonies were less than 1% of the normal size and number compared to cells expressing tup1 (21). the authors also discovered that growth of tup1 mutants (tup1δ) was dependent on the density of cells. this cell density dependent phenotype is an indication c. neoformans participates in quorum sensing. when a low cell number of tup1 mutants were plated, no growth occurred. even though a growth or nogrowth phenotype had not previously been described as an effect of quorum sensing, the effects of tup1 on growth would seem to indicate quorum sensing is involved. the effect tup1 has on the growth of c. neoformans was shown when tup1δ strains were grown on conditioned medium (filtered supernatant from a previously grown culture) from cultured tup1δ cells. at low cell numbers, the cells placed in conditioned medium formed colonies, while cells plated at the same low cell number, finaljournal.indd 145 9/25/15 11:38 am 146 • fine focus, vol. 1 (2) but without conditioned medium failed to grow (21). the molecule in the conditioned medium responsible for this activity is quorum sensing-like peptide 1 (qsp1) (table 1). the investigators artificially synthesized qsp1, placed it in media with tup1δ strains and observed that growth patterns were just as strong as the tup1δ strains grown in conditioned medium (21). in addition to growth regulation, tup1 has been determined to play a role in the pathobiology of c. neoformans. for example, lee et al. demonstrated that tup1 regulates iron and copper utilization, which are known modulators of melanin synthesis, a pigment produced by c. neoformans that contributes to its pathogenicity (22). melanin plays a role in the protection of the yeast from oxidative stress (24). melanin production is controlled by the laccase enzyme, which requires copper ions to function. gene mutations resulting in a loss of metal ion homeostasis can therefore result in a lack of melanin production, which can be overcome by the addition of copper. lee et al. observed a reduction in melanin synthesis in the tup1δ strain that was restored after the addition of 10 µm cuso4 (22). this suggests tup1 has an effect on metal ion homeostasis and therefore melanin synthesis. additional studies also showed melanization was cell density-dependent, indicating a quorum sensing effect. melanin production began when the cell density reached 4 x 107 cfu/ml. it was observed that the rate of melanization increased as the cell density increased (11). the capsule, which plays a role in the pathogenicity of c. neoformans, was also shown to be regulated by tup1. lee et al. reported a significant increase in capsule size of the tup1δ strain compared to the wild-type strain (22). the capsule, composed primarily of glucuronoxylomannan (gxm), protects the yeast from phagocytosis and aids in dissemination (24). finally, the tup1δ strain of c. neoformans was demonstrated to be less virulent than the wild-type strain. mice were infected with both strains and observed. mice inoculated with the wildtype lived for 9 days while those inoculated with tup1δ lived for 20 days (22). thus, the effect on virulence is a result of the multiple effects tup1 has on c. neoformans (fig. 1). albuquerque et al. described quorum sensing in c. neoformans after they observed regulation of fungal activity dependent on cell density (2). they determined quorum sensing was involved in the regulation and production of several cellular components of the yeast such as the capsule and melanin. investigators took conditioned medium from c. neoformans cultures and placed it in fresh c. neoformans cultures. conditioned medium caused increased growth rates, gxm release, and melanin synthesis. pantothenic acid was identified as a key component of these observed quorum sensing effects (table 1) (2). pantothenic acid was identified by conducting mass spectrometry and nmr. when pantothenic acid was placed in cultures, the same quorum sensing effects seen with conditioned medium were observed. it was found that higher concentrations of synthetic pantothenic acid were required to reproduce the effect seen from the concentration of pantothenic acid naturally found in conditioned medium. the authors also found the effects caused by pantothenic acid alone were not to the same extent as conditioned medium, indicating that some other molecule plays a role in the quorum sensing activity. pantothenic acid was found to be an important bioactive molecule that most likely works in concert with one or more other molecules to produce the quorum sensing effects (2). at this time, there is not a definitive identification of the finaljournal.indd 146 9/25/15 11:38 am review• 147 other qsm(s) that may be utilized by c. neoformans. interestingly, the growth rate of c. neoformans was also increased, to a lesser extent, by conditioned medium produced by cryptococcus albidus, candida albicans, saccharomyces cerevisiae, sporothrix schenckii, and blastomyces dermatitidis suggesting there may be qsms produced by other fungi that have an effect on c. neoformans (2). c. neoformans produces gxm and melanin, two important cellular components of the yeast that contribute to its pathogenicity. the qsms addressed are capable of increasing gxm production as well as decreasing the time required for both gxm and melanin production. they have also been shown to control growth (fig. 1) (2, 21). a virulence factor of c. neoformans that is known to be regulated by the environment is antiphagocytic protein 1 (app1) (46). one possible future study would be to test if quorum sensing has an effect on the expression of app1 or other factors of the yeast. c. neoformans appears to have a complex quorum sensing system, but more research is needed to identify all of the strategies this yeast uses to survive inside the host. saccharomyces cerevisiae saccharomyces cerevisiae is a nonpathogenic, dimorphic fungus. its morphology has been shown to be auto-controlled by qsms. when s. cerevisiae cells were placed in conditioned medium produced from cultures in the stationary phase, the flo11 gene used for hyphal formation was upregulated fivefold (6). the two qsms discovered to be causing this effect are two aromatic alcohols, phenylethanol and tryptophol (table 1). during nitrogen starvation, phenylethanol and tryptophol induce pseudohyphal growth in diploid cells and invasive growth in haploid cells (figure 1). filamentous growth was observed even with low concentrations of each molecule; however, when the molecules were combined the effect on filamentation was even higher (6). tpk2p, a component of the pka signal transduction pathway, and flo8p, a transcription factor, are required for the upregulation of flo11. these findings indicate that s. cerevisiae uses the pka pathway to control morphology. in addition to nitrogen starvation, cell density also affects the release of qsms. high density cultures produce more phenylethanol and tryptophol per cell than do low density cultures. in addition, this work discovered a link between quorum sensing and nutrient sensing (6). although this yeast is non-pathogenic, it is important to learn more about how the organism communicates at a population level, to add to the body of knowledge of fungal quorum sensing. quorum sensing is a complex phenomenon that allows microorganisms to communicate and regulate many processes. communication is accomplished through molecules, known as quorum sensing molecules, which are released into the extracellular environment. qsms must accumulate to reach a concentration threshold in order to have an effect on the organism. these molecules bind to receptors on the organism’s cells and once enough receptors are bound they activate signaling pathways. many fungi utilize significance and conclusion finaljournal.indd 147 9/25/15 11:38 am 148 • fine focus, vol. 1 (2) quorum sensing to affect pathogenicity by controlling growth rates, morphology, gene expression, biofilm formation, and virulence factors, all at a population level. the understanding of these mechanisms is of great importance in today’s world and needs extensive attention due to the prevalence of fungal infections and the need for new and better antifungal therapies. table 1 gives a summary of the qsms discussed in this review. in c. albicans, farnesol aids in altering the morphology of the fungi by inhibiting hyphal formation, while tyrosol promotes hyphal formation and increases growth rates (7, 15, 35). c. neoformans qsms are not as well-known as those of other microbes. pantothenic acid was identified as a qsm in c. neoformans; however, it appears other molecule(s) also play a role in the quorum sensing effects of this organism. pantothenic acid was found to affect the growth rate, melanin production, and gxm release of c. neoformans (2). peptides have been known for some time to have quorum sensing properties in bacteria; the peptide qsp1 is able to have quorum sensing-like effects on the growth of the fungal species c. neoformans (21). in s. cerevisiae, the qsms that have been discovered are the aromatic alcohols phenylethanol and tryptophol. these molecules induce hyphal growth (6). quorum sensing was a significant discovery as many of the processes it affects are required for the pathogenicity of microbes. one of the most interesting hypothesized reasons bacteria use quorum sensing is to remain hidden from the host’s immune system until a large population of the pathogen has grown. this allows the bacteria to grow in number and be ready to overcome the host’s immune response before releasing virulence factors that activate the host’s immune response (12). it has even been observed that host organisms have the ability to inhibit quorum sensing in bacteria by mimicking qsms that the bacteria secrete. these host organisms include the macro alga delisea pulchra, as well as several plant species (26, 43). macroalgae and plants have been shown to inhibit the most well-known qsm, n-acyl-homoserine lactone (ahl), which is produced by many gram negative bacterial species. the inhibitor molecules were found to compete with ahl molecules for their receptor, which is the luxr receptor. by binding and blocking the receptor, the inhibitors prevent ahl-dependent signaling. this finding was further supported by findings that these inhibitor molecules also inhibit ahl-dependent processes in v. fischeri, p. aeruginosa, v. harveyi, and e. carotovora. the discovery of these natural methods of inhibiting quorum sensing led to studies aimed at developing drugs that will inhibit quorum sensing (12). there are three potential mechanisms to inhibit quorum sensing systems: a) inhibit the production of the qsms, b) degrade the qsms, and c) block the qsm receptor (36). more recent studies have resulted in the identification of compounds that inhibit quorum sensing in pathogenic bacteria (9, 37, 42, 44). the use of quorum sensing inhibitors is of great interest as potential therapeutics to bacterial infections due to the increasing rate of resistance to conventional antibiotics. in this review, we discussed the quorum sensing processes and qsms in fungi. knowledge of how fungi participate in quorum sensing has great potential for improved medical treatments and preventions. for example, farnesol produced by c. albicans shows potential as a preventative option against biofilm formation, which could be important, as biofilm formation on medical devices is an emerging problem in patient care (35). farnesol was demonstrated to have protective effects in mice with oral candidiasis finaljournal.indd 148 9/25/15 11:38 am review• 149 (13). another study showed farnesol to aid the effects of certain antifungal drugs against c. albicans and alter drug efflux (40). while these studies have shown farnesol to result in a less severe infection, other studies have shown farnesol increases virulence of c. albicans. one group discovered that farnesol given to mice with systemic candidiasis resulted in an increase in virulence (28). the effect farnesol had on the virulence of c. albicans was a result of modulation of the host’s cytokine response (29). these contrasting studies on the effect of farnesol on pathogenicity demonstrate the complexity of the effect of qsms on fungi and fungal infections. in particular, farnesol may have a different effect on the pathogenicity of localized c. albicans infections compared to systemic infections. due to the growing threat of fungal infections, new and better therapeutics are needed for these infections. the modulation of quorum sensing in fungi represents a promising potential mechanism of new antifungal therapies. this treatment approach is especially promising due to the ability to target fungal cells with the potential for little to no effect on mammalian cells. more research is needed to understand quorum sensing in fungi and uncover methods of regulating these processes. many current antifungal treatments are toxic and costly. thus, exploration into quorum sensing may be very beneficial in this area (2). quorum sensing in bacteria has become a growing area of research due to the increasing number of drug resistant bacteria. inhibiting cell-to-cell communication could be beneficial by targeting one type of bacteria in a host’s microbiota while not harming the whole population (12). this strategy could potentially be used to treat fungal infections as well, while potentially preventing the overwhelming side effects of the commonly used antifungals. many qsms overlap in different microbes. this may conceivably represent a form of competition that may be exploited to target pathogenic fungi. insights gained from studies on fungal quorum sensing provide potential for the development of new therapeutics aimed at treating and preventing fungal infections. references 1. albuquerque, p., & casadevall, a. 2012. quorum sensing in fungi--a review. med. mycol. 50(4):337-345. 2. albuquerque, p., nicola, a. m., 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engineered control of cell norphology in vivo reveals distinct roles for yeast and filamentous forms of candida albicans during infection. euk. cell 2(5):1053-1060. 39. scheper, m. a., shirtliff, m. e., meiller, t. f., peters, b. m., & jabra-rizk, m. a. 2008. farnesol, a fungal quorum-sensing molecule triggers apoptosis in human oral squamous carcinoma cells. neoplasia 10(9):954-963. 40. sharma, m., & prasad, r. 2011. the quorum-sensing molecule farnesol is a modulator of drug efflux mediated by abc multidrug transporters and synergizes with drugs in candida albicans. antimicrob. agents chemother. 55(10):4834-4843. 41. shirtliff, m. e., krom, b. p., meijering, r. a. m., peters, b. m., zhu, j., scheper, m. a., . . . jabra-rizk, m. a. 2009. farnesol-induced apoptosis in candida albicans. antimicrob. agents chemother. 53(6):2392-2401. 42. tan, s. y., chua, s. l., chen, y., rice, s. a., kjelleberg, s., nielsen, t. e., . . . givskov, m. 2013. identification of five structurally unrelated 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molecular techniques to better understand the roles of rumen microbiota in cattle feed efficiency jun hong liu and le luo guan department of agricultural, food and nutritional science, university of alberta, edmonton, ab, canada manuscript received 18 march 2016; accepted 14 august 2016 copyright 2016, fine focus all rights reserved feed efficiency, simply expressed as less feed inputs versus animal production outputs, can be measured in several ways, such as feed conversion ratio (fcr) and residual feed intake (rfi). fcr is a common measurement in beef cattle operations, and is the ratio of feed intake to live-weight gain. rfi is defined as the difference between actual and predicted feed intake after taking into account variability in maintenance and growth requirements. rumen microbiota, which inludes bacteria, archaea, protozoa, and fungi, play an essential role in the digestion of lignocellulosic plant biomass, and can provide more than 70% of the host ruminants energy requirements via the production of volatile fatty acids (vfas). methane, a potent greenhouse gas (ghg), is produced in large quantities by the rumen microbiota, and is a known contributor to the global increase in ghg emissions. studies have shown a negative relationship between methane emission and feed efficiency. therefore, there is a need to study the feed efficiency from a rumen microbiome perspective and explore the probability of improving feed efficiency and hence reduce methane production in cattle by manipulating the rumen microbiome. the development of highthroughput sequencing technologies incuding metagenomics and metatranscriptomic analyses in the past decade has led to a sharp increase in understanding the rumen microbiota and associated function. as such, this mini-review will focus on the new findings during the last decade in cattle feed efficiency and the rumen microbiome. abstract corresponding author le luo guan lguan@ualberta.ca keywords • rfi • rumen microbiota • pyrosequencing • next generation sequencing • metagenomics the term feed efficiency implies a ratio of outputs to inputs. therefore, feed efficiency of beef cattle is the relative ability of the animal to turn feed nutrients into animal products (8). feed efficiency in cattle is important since it is directly associated with economic profit and enteric greenhouse gas emission from agriculture sectors. for farm owners, 66% of costs in calf management are spent in feed, rising to 77% in yearling finishing systems (1). fox et al. (11) estimated that while a 10% improvement introduction 83 • fine focus, vol. 2 applied & environmental microbiology • 84 the cattle rumen is the largest of the four compartments of the stomach; with the other three being reticulum, omasum and abomasum. the rumen is a fermentation chamber where fibers are broken down into smaller digestible components by symbiotic microbiota (28). rumen epithelium can efficiently absorb lactic acid, electrolytes, water and volatile fatty acids (vfas). vfas produced by rumen fermentation can meet more than 70% of the ruminant’s energy requirement, and are absorbed across the ruminal epithelium for metabolism in the liver (28). rumen function in daily gain would increase profitability by 18%, and improving growth efficiency by 10% could increase profits by 43% in feedlot cattle. another study demonstrated that improvement in feed efficiency has 7-8 times greater economic impact than similar improvements in daily gain (27). other than the economic effect, improving feed efficiency can reduce ghg emission, as cattle with higher feed efficiency are reported to produce 20% to 30% less methane than inefficient ones under the same conditions (33). given its importance in production systems, measuring the feed efficiency trait in cattle is important. several different measurements of feed efficiency have been developed and used by industry, such as feed conversion ratio (fcr) and residual feed intake (rfi). fcr is a common measurement in beef cattle operations, which is the ratio of feed intake to liveweight gain (8). while fcr is useful for evaluating management, feed quality, and environment on efficiency in growing and finishing cattle, it has limited value with genetic improvement. fcr has a strong correlation with growth traits, meaning that the selection by lower fcr will increase cow mature size, rather than reduce feed inputs (8). nowadays, researchers use rfi more generally than fcr for feed efficiency measuring in selecting beef cattle. the concept of rfi is defined as the difference between actual and predicted feed intake after taking into account variability in maintenance requirement and growth; therefore, when cattle consume less feed than expected for their body size and rate of gain, they are considered to have a negative rfi, which means a higher feed efficiency status as compared to positive rfi (17). compared to fcr, selection by rfi would produce efficient offspring in all segments, because the progeny would be similar to their low rfi parents in yearling weight and average daily gain after almost two generations, but the progeny have been reported to have a lower feed intake (4). the moderate heritability of rfi indicated that selection from the low rfi herd will result in progeny that consume less feed than the high rfi herd (3). feed efficiency in cattle is influenced by multiple factors, such as variation in breeding, feed formulation and the rumen microbiota. this review will focus on the role of rumen microbiota in cattle feed efficiency, and will explore the feasibility of improving feed efficiency in beef cattle by modifying the rumen microbiota. 85 • fine focus, vol. 2 rumen microbiota the rumen microbiota, comprised of bacteria, archaea, protozoa and fungi, play an essential role in the digestion of recalcitrant lignocellulosic plant matter. consequentially, examination of this microbiota has been of interest for many years. wilson and briggs applied a counting based method in 1955 by obtaining material from diluted rumen content and found that there were about 108-1010 microorganisms per gram in rumen contents (36). wilson and briggs results were similar to a more recent study, in which the mean population densities of bacteria, archaea, protozoa, and fungi were reported as 1010-1011, 107109, 104-106, and 103-106 (cells/ml rumen conten), respectively (33). this population, however, can be affected by many other factors, such as the time of the day, host, and diet. the microbes of the rumen may be separated into three distinct populational niches: solid adhered, free in the fluid, and attached to the epithelium wall (16). bacteria are the most diverse microbes in rumen content, and are largely involved in digesting lignocellulosic feed and producing vfas for host maintenance and growth, most notably acetate, propionate and butyrate (33). fibrobacter succinogenes, ruminococcus flavefaciens, and ruminococcus albus are some of the main species involved in cellulose and hemicellulose digestion (32). recently, henderson et al. (13) studied rumen microbiota composition in ruminants from 35 countries using a deep sequencing approach, and found that the dominant microbes in rumen change with diets, host species, and geography. despite this, their results also showed the existence of a core rumen microbiota, with the 30 most abundant bacterial groups present in over 90% of the samples, regardless of the factors mentioned above. the most abundant bacterial groups in all samples included prevotella, butyrivibrio, and ruminococcus, as well as unclassified lachnospiraceae, ruminococcaceae, bacteroidales, and clostridiales. methanogenic archaea are also established members of the rumen microbial community, with the phylum euryarchaeota dominating (33). henderson et al. (13) found that rumen archaea are much less diverse than rumen bacteria because the two largest groups, methanobrevibacter gottschalkii and methanobrevibacter ruminantium clades, were found in almost all samples, accounting for 74% of all archaea. together with a methanosphaera sp. and two methanomassiliicoccaceae-affiliated groups, the five dominant methanogen groups comprised 89.2% of the archaeal communities. protozoan species of the rumen microbiota are in lower abundance than the prokaryotes, but may account for as much as 50% of the microbial biomass, due to their larger size (34). protozoa exist in close association with other microbial groups by scavenging oxygen, transferring nitrogen from bacteria to the host, and regulating microbial population through predation (33). the majority of known rumen protozoa have been assigned to one of 12 genera (13). the rumen protozoal community structure has strong host individuality (35). anaerobic fungi in the rumen are also significant players in plant fiber digestion, degrading the lignocellulosic biomass by invasive rhizoidal growth and production of polysaccharide-degrading enzymes (18). applied & environmental microbiology • 86 rumen microbiota and feed efficiency rumen microbes are different in functions, but they also interact with each other by digestion. fungi can assist bacteria and other microbes by the initial colonization of fiber. bacteria, fungi and protozoa break down the indigestible lignocellulosic and release hydrogen, which archaea can utilize. other fermentation end-products, carbon dioxide, formate and methylcontaining compounds are important substrates for methanogenesis by archaea (9). because of the importance of rumen microbiota, there are emerging studies focused on understanding its role in feed efficiency in cattle. the first attempt to link rumen microbiota and cattle efficiency was reported by guan and colleagues, who showed that bacterial profiles detected by fingerprint of l-rfi animals were grouped together, which was distinct from h-rfi animals (12). guan et al.’s study indicated that specific bacterial groups may only inhabit efficient steers and host genetics may play an important role in rumen microbial structure. hernandez et al. and zhou et al. confirmed the difference in rumen bacteria and methanogens between hand lrfi beef steers under both low and high energy diets (14,15,37,38). hernandez et al. found the abundance eubacterium spp. was significantly (p<0.05) different between rfi groups that were only on the high-energy diet and observed correlations between the abundance robinsoniella sp. and rfi (p<0.05) for h-rfi animals (15). zhou et al. found methanobrevibacter gottschalkii was linked to the low-energy diet, whereas methanobrevibacter smithii and methanobrevibacter sp. abm4 were associated with the high-energy diet (38). for rfi groups in zhou et al.’s study, methanosphaera stadtmanae was detected more frequently in l-rfi animals, and methanobrevibacter ruminantium more likely to appear in h-rfi animals with methanobrevibacter smithii was observed only for h-rfi animals (38). a later study by carberry et al. (6) showed that prevotella abundance was higher (p<0.0001) in inefficient animals and other bacterial populations had relationship with different diets. archaea in rumen are responsible for methane production by phylum euryarchaeota, which is usually the only phylum found in rumen (33). studies have showed that methanogens were greatly affected by different diets (high energy vs low energy), as well as feed efficiency (l-rfi vs h-rfi) (36). carberry et al. (7) also conducted a study that focused on the rumen methanogen microbiota of cattle divergent for phenotypic rfi across two contrasting diets (high forage vs high grain). results showed that methanobrevibacter spp. was the dominant methanogens in rumen, with methanobrevibacter smithii being the most abundant species. the abundance of methanobrevibacter smithii and methanosphaera stadtmanae were detected from the low forage diet group; but irrespective of diet, methanobrevibacter smithii was different between h-rfi and l-rfi animals, which was significantly overrepresented in h-rfi animals. 87 • fine focus, vol. 2 other than bacteria and rchaea, researchers have also tried to examine the relationship between cattle rumen feed efficiency and fungi or protozoa. in carberry et al.’s study (6), there was no evidence that the total abundance of fungi could be influenced by feed efficiency or diet, but a positive association between the abundance of fungi and ch4 emission was observed in the study. carberry et al. (7) also observed a negative relationship between protozoa and propionate concentration and positive relationships between protozoa and butyrate, isobutyrate, and acetate propionate (a:p) ratios. since decreased a:p ratio is associated with decreased methane emissions, and propionate provides most energy requirement for weight gain as a major contributor to gluconeogenesis (5), improving feed efficiency in cattle may be implemented by removing protozoa (defaunation), thereby increasing propionate concentrations and reducing a:p ratio. however, newbold et al. (26) studied the role of protozoa in rumen and the results suggested that the main drawbacks of defaunation is decreasing feed digestibility, since defaunation could limit the feed intake and feed utilization efficiency. with the understanding of relation between rumen microbiota and feed efficiency, improving feed efficiency may be implemented by regulating the rumen microbiota. since cattle with higher feed efficiencies are reported to produce 20% to 30% less ch4 (33), it was thought that reducing methanogen populations in rumen would lead to the improvement of feed efficiency. however, zhou et al. (38) found that total methanogen population did not correlate with differences in feed efficiency, diet, or metabolic measurements. li et al. (20) tried to reduce the methane production during the fermentation in an in vitro continuous culture system (rusitec) with eremophila glabra, a native australian shrub. after 33 days fermentation, the results showed that the total gas production, methane and volatile fatty acid concentrations were significantly reduced with the addition of e. glabra. the overall methane reduction was 32% and 45% with 150g/kg dm and 250g/kg dm respectively, compared to the control group. though the total bacterial numbers did not change, the total methanogen population decreased by up to 42.1% (with 400g/kg dm) when compared to the control group. this suggests that reducing methane emission by changing the fibrous substrate is feasible. however, studies to date have shown a trend of short term effectiveness of dietary intervention, and there are practical impediments to on-farm use of ingredients that may be hard to obtain in some regions. the evolution of next generation sequencing (ngs) technology over the last 10 years has led to a sharp increase in studying the gastrointestinal microbiota in production animals, without the need for time-consuming cultivation studies. the first of the “next generation” sequencing technologies to emerge was 454, commercialized by roche and based on pyrosequencing mechanism (22). pyrosequencing can detect the pyrophosphate release upon nucleotide incorporation in real time, the pyrosequencing relies on the technology used for studying rumen microbiota applied & environmental microbiology• 88 cooperation of four different enzymatic reactions (fig. 1) (2,32). pyrosequencing was initially used for single-nucleotide polymorphism (snp) based genotyping, rather than standard dna sequencing, because of the short read-lengths (31). 454 sequencing is a parallelized version of pyrosequencing, which was the first ngs available as a commercial product. such technologies have dramatically increased the throughput of microbiota studies, as hundreds of samples may be multiplexed on a single sequencing run. for example, the latest version of 454 sequencing named “gs flx titanium xl+” can provide up to 1,000 base pairs (bps) read length with 700 mbps throughput in a single 23-hour run, compared to sanger sequencing which can only provide 1000-1200 bps each time with the limitation of electrophoresis (24,29). mccann et al. (23) reviewed the recent bovine rumen metagenomic publications and found that the roche 454 flx platform is commonly used today because of the longer read lengths. however, the increased throughput and lowered cost of illumina platforms (miseq, hiseq), has led to increases in their use. illumina platforms also allow for paired end sequencing, as an alternative to the expensive long-read system employed by 454. fouts et al. (10) pointed out that next generation sequencing technologies provide promises to help us better understand how rumen microbial community structure and function affects ruminant feed efficiency, biofuel production, and environmental impact. metagenome is the dna sequence information of a community as a whole (21), which is commonly used today to study rumen microbiota. the complex nature of the rumen environment is difficult to replicate in the laboratory. in determining an accurate rumen microbiome, a whole microbial community database would be the most promising option (25), therefore, a metagenome system is essential in the study. ross et al. (30) developed a reference metagenome to compare rumen metagenomic profiles for individual cattle. when the reads from the study were aligned to a rumen metagenome figure 1. the template strand is represented in red, the annealed primer is shown in black and the dna polymerase is shown as the green oval. incorporation of the complementary base (the blue “g”) generates inorganic pyrophosphate (ppi), which is converted to atp by the sulfurylase (blue arrow). luciferase (red arrow) uses the atp to convert luciferin to oxyluciferin, producing light (32). ppi atp luciferin light oxyluciferin atp + + 89 • fine focus, vol. 2 conclusion acknowledgments references in summary, with the evolution of new molecular biology techniques, researchers can determine more about rumen microbiota composition, function, and its relationship with feed efficiency, as well as attempting to improve feed efficiency by manipulation of rumen microbiome. however, it is still hard to permanently change the rumen microbiome. while this review has focused solely on literature of the rumen microbiota and its relationship with feed efficiency, future studies should examine microbiota throughout the git, as intestinal microbiota also play important roles in feed efficiency. the interaction between the host and resident microbes also warrants further study in terms of feed efficiency. also, studies of beef cattle rumen microbiota mainly focused on feedlot cattle, the differences between feedlot cattle and free grazing cattle rumen microbiota are still unknown. 1. anderson, r. v., rasby, r. j., klopfenstein, t. j., & clark, r. t. 2005. an evaluation of production and economic efficiency of two beef systems from calving to slaughter. j. animal sci. 83:694-704. 2. ahmadian, a., gharizadeh, b., gustafsson, a. c., sterky, f., nyrén, p., uhlén, m., & lundeberg, j. 2000. single-nucleotide polymorphism analysis by pyrosequencing. analytical biochemistry 280:103-110 3. arthur, j. p., & herd, r. m. 2008. 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2014. evaluation of automated ribosomal intergenic spacer analysis for bacterial fingerprinting of rumen microbiome compared to pyrosequencing technology. pathogens 3:109-120. 17. koch, r. m., swiger, l. a., chambers, d., & gregory k. e. 1963. efficiency of feed use in beef cattle. j. animal sci. 22:486-494. 18. kumar, s., indugu, n., vecchiarelli, b., & pitta, d. w. 2015. associative patterns among anaerobic fungi, methanogenic archaea, and bacterial communities in response to changes in diet and age in the rumen of dairy cows. frontiers in microbiology 6. 19. langille, m. g., zaneveld, j., caporaso, j. g., mcdonald, d., knights, d., reyes, j. a., clemente, j.c., burkepile, d.e., thurber, r.l.v., knight, r., & beiko, r. g. 2013. predictive functional profiling of microbial communities using 16s rrna marker gene sequences. nature biotechnology 31:814-821. 20. li, x., xixi, l., zoey, d., shimin, l., mcsweeney, c. s., & vercoe, p. e. 2014. eremophila glabra reduces methane production and 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m., hernandez-sanabria, e., & guan, l. l. 2009. assessment of the microbial ecology of ruminal methanogens in cattle with different feed efficiencies. appl. environ. microbiol. 75:6524-6533. 38. zhou, m., hernandez-sanabria, e., & guan, l. l. 2010. characterization of variation in rumen methanogenic communities under different dietary and host feed efficiency conditions, as determined by pcr-denaturing gradient gel electrophoresis analysis. appl. environ. microbiol. 76:3776-3786. 19applied & environmental microbiology • characterization of a 1,2-propanediol producing escherichia strain isolated from a geothermally heated intertidal pool in northern iceland birta líf fjölnisdóttir1*, pálína haraldsdóttir1, eva maría ingvadóttir1, sean m. scully1,2, and jóhann örlygsson1 1university of akureyri, faculty of natural resource sciences, borgir, nordurslod 2, 600 akureyri, iceland 2university of iceland, sæmundargötu 2, 101 reykjavík, iceland manuscript received 11 august 2016; accepted 29 july 2017 copyright 2018, fine focus. all rights reserved. 20 • fine focus, vol. 4(1) the ability of a mesophilic isolate, strain cc5c, isolated from a temperate geothermally-heated intertidal pool in northern iceland is described herein. strain cc5c belongs to the family enterobacteriaceae with greater than 99.1% similarity to escherichia marmotae and shigella dysenteriae based upon 16s rrna gene analysis. strain cc5c is a facultative anaerobe exhibiting growth between 5 and 50°c with an optimal growth at 40°c, initial ph values between 4.0 and 9.0 with an optimum at ph 7.5. strain cc5c, unlike its nearest phylogenetic neighbors, degrades starch, dulcitol, and sucrose as well as potentially cellobiose which are uncommon features among these genera. under aerobic conditions the catabolism of l-rhamnose and l-fucose revealed that the dominant product was 1,2-propanediol while under anaerobic conditions it was a mixture of acetate and 1,2-propanediol. the effect of increased initial substrate concentration was investigated for glucose, l-fucose, and l-rhamnose with inhibition apparent at concentrations above 20 mm under both anaerobic and aerobic conditions. although, differences in end product formation were observed between aerobic and anaerobic conditions for l-rhamnose. strain cc5c rapidly catabolizes rhamnose and produces 1,2-pd at a rate of 3.41 mmol/h on l-rhamnose and 2.37 mmol/h on a mixture of glucose and rhamnose with up to 96% of the theoretical yield on l-rhamnose. abstract birta líf fjölnisdóttir birtaliff@gmail.com pálína haraldsdóttir haraldsdottir@hotmail.com correspondence should be addressed to birta líf fjölnisdóttir and pálína haraldsdóttir keywords • deoxy sugars • culture conditions • propylene glycol • enterobacteriaceae knowledge regarding the production of 1,2-propanediol (1,2-pd) by bacteria from methylpentoses has been widely limited to clinical isolates and soil bacteria with work on organisms from extreme environments as well as the marine ecosystems being lacking. geothermally-heated intertidal pools represent unique environments that are an interface between the marine and freshwater environments. the tides bring a regular influx of nutrients, with some serving as carbon sources for multiple microbe species. organic material deposited in intertidal pools can include terrestrial and marine materials, introduction 21applied & environmental microbiology • which provide a source of protein, lipids, and carbohydrates to microbes existing within the pool. macro algae often contain methylpentoses not commonly found in terrestrial biomass; the methylpentoses l-fucose and l-rhamnose composition of a given macro algae species is highly dependent upon the type of macro algae. for example, green algae within the genus of ulva contain ulvan, which is composed of l-rhamnose, while brown algae species, including laminaria, fucus, and ascophyllum species, contain fucoidan, which is comprised on l-fucose. given the influx of macro algae into geothermally-heated intertidal niches, this is a logical environmental niche in which to look for organisms degrading macro algae components. 1,2-pd (α-propylene glycol) is a chiral threecarbon diol with two enantiomers, (r)-1,2pd and (s)-1,2-pd, although it is commonly sold commercially as a racemic mixture. in its pure form, 1,2-pd is clear, colorless, and highly viscous which is also odorand tasteless. 1,2-pd is generally regarded as benign and has been accepted as safe status (gras). 1,2-pd has many applications and is used in antifreeze products, disinfectants, lubricants, artificial smoke, as a solvent, or as a moisturizer in cosmetics, plastics, paint, pharmaceuticals, and food products (10, 24). commercially available rac-1,2-pd is produced by the hydrolysis of propylene oxide which is a product of petroleum reformation, while enantiomerically pure (s)-1,2-pd can be produced microbially from l-fucose and l-rhamnose and enantiomerically pure (r)-1,2-pd can be produced from glucose via the methylglyoxal bypass (24). during methylpentose metabolism, l-rhamnose and l-fucose are converted to their corresponding phosphate intermediates, fuculose-1-phosphate and rhamnulose-1phosphate, by inducible parallel pathways each including a permease, isomerase, kinase and aldolase, which are then converted to dihydroxyacetone (dhap) and l-lactaldehyde. under anaerobic conditions, l-lactaldehyde is reduced to (s)-1,2-pd via the action of propanediol oxidoreductase. under aerobic conditions, l-lactaldehyde is oxidized to l-lactate via lactaldehyde dehydrogenase and then further to pyruvate. the methylglyoxal pathway is an offshoot branch of glycolysis where dhap is converted to methylglyoxal and then d-lactaldehyde. the end-products, depending on oxygen conditions, are either (r)-1,2-pd or pyruvate (2, 3). the catabolism of sugars leading to 1,2-pd production has been examined in several organisms of which the methylpentose metabolism of escherichia coli strain k-12 was extensively studied (4, 5). under aerobic conditions, active lactaldehyde dehydrogenase is induced in both fucoseand rhamnosegrown cells and a basal level expression of inactive propanediol oxidoreductase is observed in fucose-grown cells. this allows for much quicker adaptation of fucosegrown cells to anaerobiosis, although final enzymatic activities are the same. the propanediol oxidoreductases produced by l-rhamnose-grown cells and fucose-grown cells are immunologically identical, which strongly suggests that they are products of a single gene located in the fucose locus (fuco) and when disrupted, l-rhamnose (as well as l-fucose) fails to induce propanediol oxidoreductase (11). 22 • fine focus, vol. 4(1) materials and methods the role of phosphate limitation in the regulation of the methylglyoxal pathway and subsequent production of (r)-1,2-pd has as well been detailed for clostridium sphenoides as described by (31). conversion of sugars, including glucose and xylose to 1,2-pd has also been reported by salmonella typhimurium under aerobic conditions (5) and by the strictly anaerobic thermophilic mutant of thermoanerobacterium thermosaccharolyticum strain hg-8 (2, 8). more recently, mention of the ability of extremely thermophilic caldicellulosiruptor species to catabolize l-rhamnose to 1,2-pd has also been described (6) and found to be a common feature in the genus (14). the aim of this study was to examine escherichia strain cc5c isolated from geothermally-heated intertidal pools north of húsavík, north central iceland, which preliminary screening identified as a 1,2-pd materials all reagents were acquired from sigmaaldrich unless stated otherwise. brilliant green agar (bga), eosin-methylene blue agar (emb), macconkey agar (mac), mannitol salt agar (msa), yeast extract (ye), reasoner’s 2a agar (r2a), and plate count agar (pca) were obtained from difco. l-fucose and fucoidan were obtained from dextra (reading, uk). azure-linked β-glucan and xylan were obtained from megazyme. nitrogen used for headspace gas contained less than 5 ppm of oxygen. e. coli (dsm 30083) was obtained from deutsche producing organism from methylpentoses. since the ecosystem of the these intertidal pools consists largely of methylpentoserich macro algae, serving as a renewable carbon source, it was hypothesized that the microbes within that ecosystem would be able to ferment common sugars to 1,2-pd while demonstrating a broader tolerance to environmental stresses due to the highly variable geophysical conditions associated with both geothermally heated fresh water and the tidal influx of saline water. sammlung von mikroorganismen und zellkulturen (germany). cultivation medium anaerobic cultivations were performed in basal mineral medium (bm) as previously described by (21). the culture medium consisted of (per liter): nah2po4 2.34 g, na2hpo4 3.33 g, nh4cl 2.2 g, nacl 3.0 g, cacl2 8.8 g, mgcl2 x 6h2o 0.8 g, yeast extract 2.0 g, resazurin 1 mg, trace element solution 1 ml, vitamin solution 1 ml and nahco3 0.8 g. carbon and energy sources were 20 mm or in the case of polymeric 23applied & environmental microbiology • substrates, 2 g l-1. the trace element solution consisted of (per liter): fecl2 x 4h2o 2.0 g, edta 0.5 g, cucl2 0.03 g, h3bo3, zncl2, mncl2 x 4h2o, (nh4)6mo7o24, alcl3, cocl2 x 6h2o, nicl2, all 0.05 g, and 1 ml of concentrated hcl. the medium was reduced with a solution of na2s x 9h2o, 0.3 g. the vitamin solution was prepared according to dsm141. media was autoclaved for 60 minutes after which vitamins, trace elements, and carbon sources were added through a syringe filter (whatman, pes 0.22 µm) from sterile stock bottles. all experiments were performed at 35°c at ph 7.0 with a liquidgas phase ratio of 1:1 without agitation unless otherwise noted. aerobic cultivations were performed using bm medium with the omission of sodium sulphide and cysteine solution. the inoculum volume was 2% (v/v); anaerobic cultures were cultivated without shaking while those grown aerobically were shaken at 125 rpm unless otherwise noted. all experiments were performed in triplicate. 2.2 isolation of strain cc5c a geothermal fluid sample from a geothermally heated intertidal pool (66° 03.451 n, 17° 21.466 w, 40.3°c, 5.7 ms) was collected with a sampling pool in a sterile 50 ml polypropylene sample tube in an area north of husavik (north central), iceland in june, 2015. the sample was enriched using a 2% (v/v) inoculation volume in basal mineral medium (bm) that contained 0.2% (v/v) rapeseed oil and incubated at 40°c under aerobic conditions without shaking. the culture was subcultured three times prior to streaking on pca. a single isolated colony was selected and repurified two additional times by streaking on pca. the culture was maintained on pca and cryopreserved at -20°c in 30% (v/v) glycerol. 2.3 strain characterization and substrate spectra strain cc5c was evaluated using api 20e, api 20ne, api 50ch, and api zym test strips (biomerieux, france) according to manufacturer’s directions using e. coli dsm 30083 as a control. voges-proskauer, 3% koh string test, and peroxidase test were performed using standard methods (30). oxidase activity was evaluated using test discs (sigma-aldrich) according to manufacturer’s directions. gram staining was performed according to (30). oxidative fermentation (of) tests were conducted according to (30). characteristics on selective and differential agars (30) were performed in accordance with the manufacturer’s directions. the utilization of starch, casein, and cellulose was evaluated on pca plates containing 2% (w/v) of the target substrate and visualized according to (30). alginate utilization was evaluated on r2a plates prepared with the inclusion of 2% (w/v) sodium alginate with the exclusion of starch and glucose; visualization was performed using gram’s iodine solution. plate-based inorganic phosphate solubilization was evaluated using nbrip medium according to the method of nautiyal (20). chitinase degradation was evaluated using the method of (13) except with the omission of agar for the detection of 24 • fine focus, vol. 4(1) chitin degradation in liquid medium. azurelinked β-glucan and xylan (0.2% w/v) was used to evaluate the degradation of β-glucan and xylan by supplementing reasoner’s 2a agar. the temperature range was determined qualitatively by incubating streaked pca plates at temperatures ranging from 5 to 60°c in 5°c increments. growth curves were generated by performing kinetic experiments in triplicate using tryptic soy broth (difco, tsb), in microtiter plate from a stock culture in the exponential growth phase; a 2% (v/v) inoculation volume was used in all cases. the increase in od was measured using a bioscreen c (growthcurves ltd, finland) as independent triplicates at each temperature from 20 to 65°c in 5°c increments. maximum specific growth rate (µmax) and generation time (g) were calculated according to (1). ph and sodium chloride growth range and optima were examined in tsb containing 50 mm phosphate buffer (from ph 4 to 9 in 0.5 unit increments) and sodium chloride (from 0 to 9% w/v) at the topt of the strain in the manner described above. 2.2 fermentation of single substrates hexoses, pentoses, methylpentoses, sugar alcohols, and polyalcohols were used at a concentration of 20 mm while disaccharides were used at a concentration of 10 mm. all polymeric substrates were used at a concentration of 0.2% (w/v). experiments were performed anaerobically in serum bottles at ph 7 with liquid-gas phase ratio of 1:1 for all experiments. 2.3 effect of initial substrate concentration the effect of initial substrate concentration was evaluated for three substrates (glucose, l-rhamnose, and l-fucose) in batch cultures from 10 to 60 mm. the cultures were incubated at 35°c for five days under both aerobic and anaerobic conditions. after incubation the end products were analyzed. 2.4 kinetic experiments kinetic experiments were performed both aerobically and anaerobically in 125 ml erlenmeyer flasks or serum bottles without agitation. substrates were used at a concentration of 20 mm glucose, l-rhamnose, l-fucose, rac-1,2-pd, and control. fermentation broths were sampled (1 ml) and analysed for optical density over a period of 46 hours; withdrawn samples were stored at -20°c for further analysis. 2.5 analytical methods optical density was determined using a shimadzu uv-1800 uv-visible spectrophotometer at a wavelength of 600 nm and a pathlength of 1 cm. cell-free solutions were prepared by centrifugation at 13000 g for 5 min. volatile end products, such as ethanol and volatile fatty acids were measured using a clarus 580 (perkin elmer) gas chromatograph equipped with a fid as described by orlygsson & baldursson (18); samples were prepared by mixing 100 µl 25applied & environmental microbiology • formic acid (25% w/v), 100 µl crotonic acid (0.1% w/v), and 600 µl dh2o with 200 µl of cell-free sample. hydrogen was analysed using a autosystem xl (perkin elmer) gas chromatograph equipped with a tcd using the method of (21). glucose concentration was determined colorimetrically using the anthrone method described (18) with minor modifications; 50 µl cell-free sample was mixed with 250 µl of 0.1% (w/v) anthrone in concentrated sulphuric acid and incubated for 20 minutes at 100°c, cooled to room temperature and then read on a microplate reader (bioscreen c, growth curves ltd, finland) at 600 nm against a water blank. methylpentoses were analysed colorimetrically according to the method of (12). 1,2-propanediol was analysed using the colorimetric method of jones (15) modified for use in microplates; 250 µl of concentrated sulphuric acid was added with 50 µl of cell-free sample in a microplate which was then incubated at 70°c for 20 min; after cooling to room temperature, 10 µl of a 3 % w/v ninhydrin solution in 5% w/v sodium bisulfite was added and the plate was mixed (200 rpm, 60 sec) and allowed to develop for 1 hr in the absence of light and then read on a microplate reader (bioscreen c) at a wavelength of 600 nm. lactate was quantified colorimetrically according to the method of taylor (28) with the following modifications: 300 µl of concentrated sulphuric acid was added with 50 µl of sample in an eppendorf tube. the sample was incubated in a boiling water bath (~100°c) for 10 minutes and then cooled to room temperature, after cooling 5 µl of 4% (w/v) copper(ii) sulphate and 10 µl of 1.5% (w/v) p-phenyl phenol (in 95% v/v ethanol) was added. the sample was vortexed and incubated at room temperature for 30 minutes. samples were read at 580 nm against a water blank. 2.6 molecular methods the 16s rrna gene from escherichia strain cc5c was sequenced (1507 nt). the sequence was originally compared to sequences in the ncbi (national center for biotechnology information) database using the nucleotide blast. the top sequences with the highest scores were then selected for the calculation of pairwise sequence similarity using a global alignment algorithm which was implemented at the eztaxon server (23). the most similar sequences obtained were aligned with sequencing results in mega6 (27) and the maximum likelihood method based on the tamura-nei model was used to generate a phylogenetic tree (26). genomic dna extraction was carried out, by using ultraclean™ microbial dna isolation kit from mobio laboratories, according to the manufacturer’s protocol. pcr mediated amplification of the 16s rdna and purification of the pcr product was carried out with 519f (cagcagccgcggtaatac) and 926r (ccgtcaattcctttgagttt) universal primers as previously described (21). the amplified pcr products were purified with exo-sap reaction; substrate preparation was prepared by mixing 0,025 µl of exonuclease i (neb, england), 0,05 µl of antarctic shrimp phosphatase (neb, england) and 9,925 µl of mq water for each sample. 10 µl of the reagent substrate was 26 • fine focus, vol. 4(1) results then mixed with 20 µl of amplified pcr products. the reaction was carried out with ptc-200 (mj-research) for 3 min at 95°c for the initial denaturation, 30 sec at 95°c for denaturation, 30 sec at 50°c for annealing, 90 sec at 68°c for extension, and a final extension time of 7 min at 68°c followed by strain isolation and 16s sequencing strain cc5c was isolated from a geothermally-heated intertidal pool north of husavik in north central iceland. the strain produced creamy circular colonies that are off-white in color when grown on pca and r2a. the strain was more than 99% similar to escherichia coli based upon analysis of partial 16s rdna (1507 nt) using nucleotide blast. further analysis of the sequence with ez taxon indicates that cc5c is highly similar to escherichia marmotae (99.13%), shigella dysenteriae (99.13%), escherichia fergusonii (99.06%), escherichia coli (98.99%), shigella flexneri (98.99%), and shigella sonnei (98.85%), respectively. preliminary screening of the strain revealed that the isolate cc5c was capable of producing 1,2-pd from l-rhamnose and was chosen for further investigation. storage at 4°c. purified pcr products were then transferred into 96-well microamp plate, with either 519f or 926r primer, since the products were sequenced from each end, and sent to macrogen (netherlands) for sequencing. train characterization and substrate spectra strain cc5c is gram-negative, string test positive, oxidase, and catalase positive. strain cc5c is both oxidative and fermentative based upon positive oxidative fermentation tests. colonies grown on pca had an offwhite circular morphology. colonies of cc5c produced positive reaction on brilliant green agar, growth on macconkey’s with colonies presenting as pink, and produced purple colonies with a metallic green sheen on emb agar plates characteristic of e. coli. no growth was observed on mannitol salt agar. growth on starch impregnated plates followed by staining with gram´s iodine solution indicated starch being degraded. cc5c has a growth range from 5 to 45°c with a temperature optimum of 40°c (figure 1). growth was observed between ph 4.0 and 9.0 with an optima of ph 7.5, and between sodium chloride concentrations between 0 and 7% w/v with optima of 0.5% (figure 2). maximum specific growth was 1.10 h-1 with a generation time of 37.8 min. 27applied & environmental microbiology • figure 1 temperature optima of strain cc5c (black line) and e. coli (red line, dsm 30083) grown in tsb. values represent average of triplicates with the standard deviation presented as error bars. figure 2 optimization of ph and nacl for maximum specific growth rate at topt (40°c); values represent the average of triplicates (standard deviation not shown). 28 • fine focus, vol. 4(1) table 1 – comparison of biochemical characteristics between strain cc5c and closely related species. strains: 1, strain cc5c (this study); 2, escherichia marmotae ht073016t; 3, escherichia coli; 3a, escherichia coli (dsm 30083, this study), 4, escherichia albertii lmg 20976t; 5, escherichia hermannii cip 103176t; 6, escherichia vulneris atcc 33821t; 7, shigella dysenteriae; 8, shigella sonnei. data for escherichia spp. from (19) and (17) and references therein, data for shigella spp. is from (25). a onpg, o-nitrophenyl-β-dgalactopyranoside; b data from (29); +, more than 90% of strains are positive; 10% or less of strains are positive; nd, not determined; nr, not reported; d, 11-89% of strains are positive. *weak growth observed at 7% w/v nacl. strain cc5c degrades a variety of hexoses, pentoses, l-methylpentoses, and sugar alcohols as summarized in (supplementary tables 1-3). strain cc5c also has various enzyme activities including alkaline and acid phosphatase activity as well as c4 and c8 esterase activity the substrate spectra of cc5c was evaluated using api test panels in parallel with the type strain of e. coli (dsm 30083); results for (supplementary table 4). additionally, strain cc5c degrades starch, casein, and β-glucan, a weak positive reaction on chitin, but does not degrade cellulose or xylan. strain cc5c solubilizes phosphate on plates containing inorganic phosphate. commonly used taxonomic features of closely related escherichia and shigella type strains are presented in table 1. 29applied & environmental microbiology • 30 • fine focus, vol. 4(1) figure 3 end products of selected substrates after fermentation by strain cc5c grown under anaerobic conditions; initial concentrations were 20 mm for monosaccharides and organic acids and polyalcohols, 10 mm for dissacharides, and 0.2% w/v for polymers. values represent the average of triplicates; standard deviation is not shown. 3.3 fermentation of single substrates single substrates and polymers present in various terrestrial and marine biomass were examined in anaerobic batch culture. hexoses, pentoses, disaccharides except cellobiose, and starch gave a mixture of end products consisting of ethanol, acetate, lactate, and hydrogen. the sugar alcohols (mannitol and xylitol) yielded ethanol as a major end product with traces of acetate, lactate, and hydrogen. strain cc5c fermented methylpentoses (l-fucose and l-rhamnose) and yielded 1,2-propanediol as the dominant end product with acetate and lactate present in lower concentrations. laminarin, a β-1,3 glycan, yielded acetate and lactate above controls while other polymers, the end product formation patterns, from various substrates, were evaluated, and is presented for each individual substrate in figure 3. 1,2-propanediol was only observed as a fermentation product when l-fucose and l-rhamnose were used as substrates. lactate, and rac-1,2-pd were not degraded. 3.4 effect of initial substrate concentration and oxygen on 1,2-propanediol yields to investigate the production of 1,2-propanediol by strain cc5c, the strain was evaluated at different initial substrate concentrations up to 60 mm on d-glucose, l-fucose, and l-rhamnose under both aerobic and anaerobic conditions. 31applied & environmental microbiology • figure 4 end product formation of strain cc5c on glucose, l-rhamnose, and l-fucose under aerobic (a) and anaerobic (b) conditions. values represent the average of triplicates with standard deviation shown as error bars. end products were evaluated after 5 days of incubation as shown in figure 4 for both aerobic (4a) and anaerobic conditions (4b). substrate inhibition was observed on all substrates at concentrations greater than 20 mm as evidenced by the decreased substrate utilization and levelling off of end product formation. 32 • fine focus, vol. 4(1) a nm ) figure 5 – utilization of selected substrates over time by strain cc5c. values represent the average of triplicates with standard deviation presented as error bars. a: 20 mm glucose, b: 20 mm l-rhamnose, c: 20 mm glucose and 20 mm l-rhamnose, d: 20 mm rac-1,2-propanediol; ■ – 1,2-propanediol, ♦ optical density (600 nm). 3.5 kinetic experiments kinetic experiments were performed aerobically on 20 mm of d-glucose, l-rhamnose, l-fucose, a mixture of d-glucose and l-rhamnose (20 mm each), and 20 mm of rac-1,2-propanediol as shown in figure 5a-d, as well as a control containing only yeast extract. the fermentation characteristics for the production of 1,2-pd are summarized in table 2. table 2 – fermentation characteristics of 1,2 propanediol production on selected substrates substrate (20mm) maximum 1,2-pd yield maximum 1,2-pd production rate (% of theoretical) (mmol/hr) d-glucose 9.93 0.99 l-rhamnose 96.89 3.41 glucose + rhamnose 75.84 2.37 rac-1,2-pd 69.90 -1.86 table 2 – fermentation characteristics of 1,2 propanediol production on selected substrates substrate (20mm) maximum 1,2-pd yield maximum 1,2-pd production rate (% of theoretical) (mmol/hr) d-glucose 9.93 0.99 l-rhamnose 96.89 3.41 glucose + rhamnose 75.84 2.37 rac-1,2-pd 69.90 -1.86 table 2 – fermentation characteristics of 1,2 propanediol production on selected substrates substrate (20mm) maximum 1,2-pd yield maximum 1,2-pd production rate (% of theoretical) (mmol/hr) d-glucose 9.93 0.99 l-rhamnose 96.89 3.41 glucose + rhamnose 75.84 2.37 rac-1,2-pd 69.90 -1.86 33applied & environmental microbiology • strain cc5c is very closely related to a number of organisms within the genera of escherichia and shigella, the latter of which are widely regarded as likely being representatives of e. coli clones (25). other techniques, such as dna-dna hybridization are required to determine conclusively if strain cc5c represent a novel species given the high degree of similarity among species within these genera. strain cc5c’s topt matches the temperature of the geothermal pool (40°c) from which it was isolated from. similarly, e. marmatoae can grow at 25–37°c, and with 0–6 % nacl with optimum growth conditions are 37°c and 1 % nacl. interestingly, enteric bacteria are not typically associated with a highlysaline marine environment or geothermal feature as escherichia species are typically associated with the digestive tract of mammals and, less commonly, other animals. however, there is at least one example of an organism within family enterobacteriaceae being found in intertidal hot springs, that being the moderately thermophilic halophile alterococcus agarolyticus which was isolated in taiwan (32). their secondary presence in other environments is often the result of fecal contamination (32), thus the presence of this organism could be the result of nesting birds near the sampling location observed at the time that the sample was taken. the high conductivity of the sample taken from the pool, which is equivalent to strain cc5c rapidly catabolizes glucose and rhamnose and produces 1,2-pd at a rate of 3.41 mmol/h on l-rhamnose and 2.37 mmol/h on a mixture of glucose and rhamnose with up to 96% on l-rhamnose. approximately 3.1% sodium chloride is less saline than seawater. as members of the family enterobacteriaceae are typically not halophilic (brenner, 2005), it is somewhat surprising that strain cc5c persists in this environment. strain cc5c may be thriving in this environment due to the influx of fresh geothermal water into the intertidal pool from the geothermal system and due to the abundance of marine biomass in the pool such as protein and carbohydrates from macro algae. metagenomic studies of these intertidal pools over the course of multiple seasons are needed to resolve whether strains such as cc5c are stable members of the geothermal intertidal community or not. strain cc5c is phenotypically similar to e. coli and other closely related species with several notable differences in substrate utilization and growth conditions as compared to related species; the majority of the biochemical tests reveal traits similar to other enteric bacteria that are facultative anaerobes within the family of enterobacteriaceae. from a purely taxonomic standpoint, a lack of lysine decarboxylation is a defining feature of the genus shigella, and therefore it is suggested that cc5c is a member of the genus escherichia. strain cc5c has a broader temperature range than both e. marmatoa (reported as only growing between 25 and 37°c) and e. coli while demonstrating higher specific growth rates at 40 and 45°c as compared to e. coli (dsm given a solution of rac-1,2-pd, strain cc5c degrades approximately half of the initial 1,2-pd concentration at a rate of 1.86 mmol/h. discussion 34 • fine focus, vol. 4(1) 30083) under equivalent conditions. while strain cc5c is not thermophilic, it does seem better adapted to growth at elevated temperatures than the reference strain used (e. coli dsm 30083). additionally, cc5c´s growth rates at elevated salt concentrations are higher than the type strain of e. coli. this may suggest that cc5c has adapted to its environmental niche (40°c or more with regular saline intrusion). cc5c has a broader substrate spectra than closely related species as it degrades hexoses, pentoses, methylpentoses, sugar alcohols, and disaccharides as well as some polysaccharides. four substrates stand out at differentiating strain cc5c from its nearest phylogenetic neighbours, namely the utilization of starch, sucrose, dulcitol, and possibly cellobiose. strain cc5c degrades starch as evidenced by positive tests on both the api 50ch test strips, starch plate assay, as well as the appearance of organic acids under fermentative conditions suggesting the strain has the required amylase activity and potentially an import mechanism for maltose, which is supported by growth on maltose, or higher order oligosaccharides. this stands in contrast to other organisms within the genus of escherichia, which cannot degrade starch but can utilize maltose. remarkably, strain cc5c utilizes cellobiose and sucrose, which is uncharacteristic for e. coli and other closely related enteroacteriaceae. a follow-up experiment examining the fermentation of cellobiose found that no end products were detected suggesting that the result of the api 50ch test strip was a false positive or utilization of cellobiose is limited to aerobic conditions. the utilization of dulcitol, the sugar alcohol formed from the reduction of galactose, is a somewhat unusual feature. as can be seen in table 1, cc5c´s substrate spectra is more similar to e. coli than e. marmatoa, its nearest phylogenetic neighbour based on analysis of the 16s gene but also possesses features somewhat similar to e. coli with some of the aforementioned differentiating characteristics. the number of divergent features may suggest that cc5c is a novel species within the genus. as the environment from which cc5c was isolated is rich with macro algae species with high protein fractions as well as polysaccharides containing methylpentoses and other sugars not common within the terrestrial biosphere, the strain will likely thrive in this substrate-rich niche. the strain also has acid and alkaline phosphatase activities suggesting that cc5c might serve a role mobilizing phosphate in the environment. cc5c also degrades casein and gelatin but lacks trypsin and chymotrypsin activities suggesting other protease chemistries are present. the strain showed esterase activity but not lipase activity, which is surprising since this bacterium was enriched on rapeseed oil; this might suggest that cc5c was utilizing the yeast extract present in the medium. it is worth noting that cc5c possessed at c8 esterase activity, unlike e. coli (dsm 30083). cc5c also showed phosphatase activity which may help the strain in scavenging phosphate from inorganic or polyphosphates in the environment to support growth. to further examine the end products produced by cc5c, the fermentation of selected substrates was examined. under anaerobic conditions, the dominant end products are a mixture of ethanol, acetate, and hydrogen on most substrates examined, with the exception of l-fucose and l-rhamnose, which yielded 1,2pd. the observed substrate spectrum and end product formation patterns observed for strain cc5c is quite similar to other members of the family of 35applied & environmental microbiology • enterobacteriaceae, with members such as e. coli being well-established mixed acid producers as well as producers of 1,2-pd in the presence of methylpentoses. to further investigate the production of 1,2-pd from cc5c, the strain was cultivated in the presence of d-glucose as well as the methylpentoses l-fucose and l-rhamnose at initial concentrations ranging from 10 mm to 60 mm. strain cc5c was clearly inhibited by high initial substrate concentrations of all three monosaccharides examined under aerobic and strictly anaerobic conditions. 1,2-pd was the primary end product when cc5c was grown on l-rhamnose and l-fucose under both aerobic and anaerobic conditions. interestingly, very little 1,2-pd was produced from l-rhamnose under anaerobic conditions at all concentrations. under aerobic conditions, 1,2-pd yields were much higher although inhibition phenomena were apparent at higher substrate concentrations. this stark shift in end product formation could implicate the importance of oxygen in the ability of the strain to produce 1,2-pd. it is known that during the production of 2,3-butanediol by organisms such as paenibacillus polymyxa, that the oxygen concentration is of critical importance (22). furthermore, studies in the 1980s examined changes in the regulation, expression and activity of propanediol oxidoreductase by e. coli under specific oxygen conditions (5, 7). it is possible that reducing potential from rhamnose metabolism is being shifted to other end products or that highly reduced conditions interfere with enzymes necessary for rhamnose catabolism that are specific to rhamnose regulation but do not affect the enzymes involved in fucose catabolism. the presence and organization of the rhamnose and fucose operons, if present, should be investigated via whole genome sequencing or specific assays to confirm the presence and functionality of specific gene products. analysis of cc5c’s transcriptome, as well as the activities of specific enzymes, such as propanediol oxidoreductase as a function of oxygen concentration, should also be considered in future work. at high glucose concentrations, the amount of 1,2-pd increased to 7.9 mm at an initial glucose concentration of 60 mm as compared to only 0.3 mm of 1,2-pd at an initial glucose concentration of 10 mm. this may be due to the rapid metabolism of glucose to glycolysis intermediates such as dihydroxyacetone phosphate (dhap), while further metabolism to other end products was inhibited. to proceed through glycolysis, dhap must be converted to glyceraldehyde3-phosphate (g3p), which serves as a metabolic bottleneck resulting in carbon flowing into the methyglyoxal bypass. this pathway is well known to produce (r)1,2-propanediol in e. coli and yeasts and could be active in thermoanaerobacterium saccharolyticum hg-8 (3). phosphate limitation could also be a factor at higher initial substrate concentrations as phosphate limiting conditions is known to influence the flow of carbon into the methylglyoxal pathway as has been demonstrated with clostridium sphenoides (31). there have been many recent efforts to engineer (r)-1,2-pd producing pathways into e. coli to improve yields although efforts to improve (s)-1,2-pd production have been largely ignored. a kinetic study of 1,2-pd production by strain cc5c reveals that the methylpentoses are rapidly consumed while 1,2-pd production lags. this suggests that the metabolism of intermediates involved in methylpentose catabolism is slower than that of the glycolytic intermediates of cells grown on glucose. 36 • fine focus, vol. 4(1) conclusions cc5c partially degraded rac-1,2-pd to roughly 50% of its initial concentration. this suggests that the strain may only degrade one enantiomer of 1,2-pd. as the 1,2-pd concentrations were stable in the experiments using l-fucose and l-rhamnose as a carbon source cc5c likely produces the s enantiomer. it is likely that cc5c can only degrade the r enantiomer or degrades one enantiomer at a faster rate. this may have potential biotechnological applications in the production of enantiomerically pure 1,2-pd from inexpensive commercially available racemic 1,2-pd as has been done with strains of baker’s yeast (16). further work is needed to confirm the enantiomeric selectivity of 1,2-pd degradation by cc5c. the high yields of 1,2-pd observed on l-fucose are promising although l-fucose is an expensive substrate. thus, inexpensive sources of l-fucose should be examined, potentially including hydrolysates of components found in brown macro algae. further work to evaluate the ubiquity of methylpentose metabolism resulting in 1,2pd production by members of the genus requires further examination. additionally, bioprospecting efforts to find 1,2-pd strain cc5c is a gram-negative, facultative anaerobe isolated from a geothermally-heated intertidal pool in husavik, iceland, and is closely related to escherichia marmotae (99.13%) and shigella dysenteriae(99.13%). the strain had optimum growth conditions of 40°c, ph 7.5 and 0.5% nacl with a maximum specific growth rate of 1.10 h-1. the strain has a broad substrate spectrum and grew on a number of substrates (dulcitol, cellobiose, starch, producing strains adapted in situ to more extreme environments as well as possessing expanded substrate spectra would be of potential interest to the industry. further work is needed to investigate the influence of culture parameters, such as the partial pressure of oxygen and hydrogen, effect of ph and temperature, as well as the effect of phosphate limitation on the production of 1,2-pd on specific substrates. the molecular biology involved in the changes in 1,2pd production from l-rhamnose under aerobic versus strictly anaerobic conditions warrants further study. the response of cc5c to inhibitory compounds such as 5-hydroxylmethylfurfuraldhyde and 2-furfuraldehyde, and production of 1,2-pd from biomass, containing methylpentoses should be investigated. due to the observed substrate inhibition, other fermentations modes such as fed batch or continuous culture should be investigated. also, the inhibitory effects of end products, such as acetate, lactate, and 1,2-pd, should be investigated to better understand whether the observed inhibition is solely to the initial concentration of the substrate or another type of inhibition. and sucrose) unlike a number of closely related species potentially suggesting that cc5c is a novel isolate. strain cc5c is also a potent 1,2-propaendiol producing organisms fermenting methylpentoses to 1,2-pd in good yield (>95% of theoretical) although some 1,2-pd was observed on glucose under high initial substrate concentrations (aerobic versus anaerobic). strain cc5c had a maximum production rate of 3.41 mmol/l/h from l-rhamnose under aerobic conditions. 37applied & environmental microbiology • references 1. almarsdottir, a. r., tarazewicz, a., gunnarsson, i., & orlygsson, j. 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(2013). mega6: molecular evolutionary genetics analysis version 6.0. molecular biology and evolution, 30(12), 2725–2729. 28. taylor, k. a. c. c. (1996). a simple colorimetric assay for muramic acid and lactic acid. applied biochemistry and biotechnology, 56, 49–58. 29. tilly, b., gonthier, a., & gue, v. (2001). optimal growth temperature of o157 and non-o157 escherichia coli strains, 352–356. 30. tindall, b. j., sikorski, j., smibert, r. a., & krieg, n. r. (2007). phenotypic characterization and the principles of comparative systematics. in c. a. reddy (ed.), methods for general and molecular microbiology (3rd ed., pp. 330–393). washington, d.c.: asm press. 31. tran-din, k., & gottschalk, g. (1985). formation of d(-)-1,2-propanediol and d(-)-lactate from glucose by clostridium sphenoides under phosphate limitation*. archives of microbiology, 142, 87–92. 32. welch, r. a. (2006). the genus escherichia. in m. dworkin (ed.), the prokaryotes (3rd ed., pp. 60–71). new york: springer. characterization of a mucoid-like pseudomonas aeruginosa biofilm brandon m. bauer, lewis rogers, monique macias, gabriella iacovetti, alexander m. woodrow, melissa j. labonte-wilson and kathleen g. tallman* department of biology and chemistry, azusa pacific university 901 e. alosta ave., azusa, ca, usa copyright 2015, fine focus all rights reserved manuscript received 2 april, 2015; accepted 29 june, 2015 finaljournal.indd 121 9/25/15 11:38 am pseudomonas aeruginosa biofilms are implicated in chronic infections. a key element of p. aeruginosa pathogenicity is the formation of a biofilm, a community of bacteria encased in an exopolymeric substance (eps) that shields the bacteria from the host immune response and antibiotic treatment. a crucial step in biofilm production is a switch in motility from freely swimming, planktonic bacteria to twitching movement and then to attached and sedentary bacteria that develop into a mature pillar-shaped biofilm. a mucoid biofilm produces an excess of alginate and is clinically the most pathogenic and the most resistant to antibiotics. biofilms from patients exhibit a wide variety of structure, motility, and levels of attachment. in vitro biofilms do not exhibit such a wide variety of structure and physiology. the difference between in vivo and in vitro biofilms has made the translation of in vitro studies into in vivo treatments difficult. under different growth conditions in our lab, the p. aeruginosa strain pao1 demonstrates two phenotypes: a non-mucoid and a mucoid-like phenotype. confocal laser scanning microscopy (clsm) indicates the mucoid-like phenotype is intermediate in height to the non-mucoid phenotype and biofilms formed in a once-flowthrough chamber. both mucoid-like and nonmucoid phenotypes exhibit a significant increase in twitching between 24 and 72 hours of development. the mucoid-like phenotype had greater attachment at 72 hours compared to non-mucoid phenotype. therefore, the two phenotypes observed in our lab may represent the effect of environment to stimulate development of two types of biofilms by pao1. abstract corresponding author *kathleen g. tallman ktallman@apu.edu keywords • psl • pel • alginate • type iv pili • flagella pseudomonas aeruginosa (pa) is an opportunistic pathogen implicated in chronic infections in cystic fibrosis (cf) (15, 17, 33) and chronic wounds (4, 8, 17). its virulence is due, in part, to formation of biofilms which confer resistance to both the innate immune system and antibiotics. biofilms recovered from the sputum of cf patients introduction 122 • fine focus, vol. 1 (2) finaljournal.indd 122 9/25/15 11:38 am can exhibit a widespread variability in biofilm formation both in structure and virulence (1, 10, 27, 33). biofilm formation biofilms are understood to have several steps in their formation (8, 33). there is an initial attachment to a surface followed by microcolony formation. the biofilm may then mature to a pillar-shaped structure which can release planktonic (freely moving) bacteria to colonize new areas. inside biofilms, cells in different regions exhibit differences in gene expression as well as functional heterogeneity, indicating a complex community of cells within biofilms (8). the time span for the formation of a pillar-shaped biofilm depends on many factors, but has been estimated to take 5-7 days at minimum (33). the triggers for biofilm formation are complex, but one trigger is the formation of a quorum sensing (qs) signaling cascade. one qs molecule, n-(3-oxodo-decanoyl)-l-homoserine lactone (3oc12-hsl) is constitutively released from p. aeruginosa. when p. aeruginosa has multiplied and reached a large enough population (i.e. a “quorum”), 3oc12-hsl levels reach a threshold and signal extensive changes in gene expression through the lasr-lasi system (8, 9, 28, 33). one of these changes includes the formation of a second qs molecule, n-butyryl-l-homoserine lactone (c4-hsl). c4-hsl acts through the rhlr-rhli system independently and in a coordinated fashion with 3oc12-hsl further influences gene expression. in fact, blocking qs molecule action has been one target of therapeutic interventions, but with limited success (2). exopolymeric substance (eps) biofilm maturation requires the formation of an exopolymeric substance (eps) which confers antibiotic resistance and protection from host immunity by the biofilm (4, 8, 33). the eps in a p. aeruginosa biofilm is composed of three components: alginate, psl, and pel, although other polymers such as certain proteins, lipids, and extracellular dna also contribute to biofilm structure (12, 24, 25, 36). psl, composed of rhamnose, mannose, and glucose, is required for maintenance of any biofilm structure, and for biofilm construction (31, 36). overproduction of psl promotes mushroom-shaped biofilms (36). pel, a glucose-rich component of the eps, is required for initiation of any biofilm, as well as for the growth of air-liquid interface biofilms (31, 36). the final eps component, alginate, is a complex polysaccharide composed of two different sugars, ß-d-mannuronate and l-glucuronate, and is one of the most studied and well understood portions of the matrix. over-expression of alginate causes what has been termed the mucoid biofilm phenotype for its mucus-like appearance (31). mucoid biofilms have been shown to be more virulent and are often found in chronic infections (11, 23). mutant strains of p. aeruginosa which produce mucoid biofilms can be studied in vitro; however, the transformation of wild-type p. aeruginosa into a mucoid biofilm is not well understood (7). because of the homogeneity of in vitro biofilms grown in the laboratory setting and the heterogeneity of in vivo biofilms as recovered from cf patients, it has been difficult to study these pathogenic mucoid biofilms within the lab. this heterogeneity of biofilms recovered from cf patients is well documented. they differ structurally as demonstrated pathogens and antimicrobial factors • 123 finaljournal.indd 123 9/25/15 11:38 am 124 • fine focus, vol. 1 (2) in scanning electron microscopy as pillar-shaped, knobby, or flat (10). the pao1 within biofilms are also functionally distinct as seen in the qs molecules released, their virulence, the types of motility they exhibit, their ability to attach to a surface, and their response to treatment (1, 27, 33). in recent years several strategies have been employed to characterize p. aeruginosa isolates from patients in order to identify the most effective treatment. virulent strains of p. aeruginosa form biofilms, but also have decreased motility and decreased production of virulence factors such as pyocyanin (1, 27). it has been hypothesized that a lack of production of flagella or type iv pili might help p. aeruginosa evade the host immune system since both innate and adaptive immune systems respond to epitopes on flagella and type iv pili (1, 27). a lack of motility may also increase the ability of p. aeruginosa to attach to a surface and produce a biofilm. these studies also demonstrated a decrease in production of virulence factors such as pyocyanin (1, 27). virulence factors are common in acute infection and may spur the immune system response. therefore, eradicating or decreasing the production of virulence factors may decrease stimulation of the host immune system by p. aeruginosa, increasing the ability of p. aeruginosa to evade the host immune response. finally, the production of biofilms increases the ability of p. aeruginosa to evade the host immune system by creating the exopolysaccharide (eps) matrix that limits the ability of the host immune system to reach p. aeruginosa. clinical isolates of p. aeruginosa biofilms can be classified by their motility patterns and biofilm structure (1, 10, 27). mulet et al. (2013) demonstrated that high risk clones of p. aeruginosa from cf sputum contained greater drug resistance as well as increased biofilm production, decreased twitching, and decreased production of pyoverdine and pyocyanin, two virulence factors. therefore, changes in motility from swimming to swarming to twitching are a standard part of the formation of biofilms. these changes allow p. aeruginosa to attach to a surface, form a microcolony, and finally, form a biofilm. however, the emerging clinical picture indicates that motility patterns are being used to classify and characterize clinical isolates based on their virulence and ability to develop chronic infections (10, 27). in the clinical setting then, motility may not only serve a role in biofilm formation, but may also serve as an additional marker that may lead to identification of the most effective methods for treatment of chronic p. aeruginosa infections. motility p. aeruginosa exhibits swimming, swarming, and twitching types of motility. twitching is mediated by type iv pili which act as retractable arms that can pull or “slingshot” cells across a surface (5, 19). twitching is usually seen in more viscous types of media or environments. swimming through a liquid medium is performed by the use of flagella (18, 36). swarming movements use both type iv pili and flagella resulting in a movement in between swimming and twitching (14, 36). to test motility, assays are used measuring distance displaced by p. aeruginosa on plates of media with different fluidity (5, 10). as previously stated, the motility of p. aeruginosa within a biofilm can be used to identify virulent strains that are more likely to cause chronic infection. however, the correlation between motility and its role in biofilm formation is not entirely clear. swimming motility may not be required finaljournal.indd 124 9/25/15 11:38 am pathogens and antimicrobial factors • 125 for biofilm formation, but may be important in allowing p. aeruginosa to reach an environment with greater nutrients or to colonize a new area (36). therefore, although it may not be directly involved in the stage of biofilm formation, swimming motility may play a role in determining sites where p. aeruginosa can travel to form a biofilm. perhaps the most critical type of motility for p. aeruginosa during biofilm formation is the ability to twitch. this is how bacteria move on a solid surface, perhaps as they are attaching to a surface to form a microcolony (10, 36, 37). psl may be secreted to coat a solid surface as bacteria twitch in a spider web fashion. other bacteria then attach to this surface to form a microcolony, which is one of the first steps in biofilm formation (36, 37). there is evidence that bacteria favor the use of flagella to swim or type iv pili to twitch; once there is a switch to a different type of movement, it may be difficult for the bacteria to revert to the previous type of motility (35). swarming, which uses both flagella and type iv pili, may represent p. aeruginosa in transition from swimming to attachment in which twitching will then predominate. originally, research indicated that swarming is only mediated by flagella (10); however, more recent data indicate the use of both flagella and type iv pili are required (5, 36). therefore, swarming may be an intermediate form of motility for p. aeruginosa between swimming which requires only flagella and twitching which requires only type iv pili. in fact, data indicate that bacteria can twitch or swim, but rarely do both well (35), indicating that modes of motility vary according to stimuli. two phenotypes of pao1 develop under different environmental conditions pao1 is a strain of p. aeruginosa commonly used in laboratory experiments. we have been able to grow pao1 in our lab with two distinct phenotypes, which have named non-mucoid and “mucoid-like” phenotypes. we wanted to investigate the differences in structure, motility, and surface attachment between these two phenotypes. it is our hypothesis that pao1 in the mucoid-like phenotype produce a biofilm closer to a “mucoid” biofilm with greater twitching motility, greater attachment to a surface, and greater height and pillar-shaped structure, when compared to the non-mucoid phenotype of pao1. to investigate these two phenotypes we used confocal scanning laser microscopy, motility assays, and a surface attachment assay. if the mucoid-like phenotype exhibits greater twitching behavior, surface attachment, and more pillar-shaped morphology than the non-mucoid biofilm, these environmental conditions may be replicated to study different phenotypes of pao1 biofilms. finaljournal.indd 125 9/25/15 11:38 am 126 • fine focus, vol. 1 (2) materials and methods growth of mucoidlike and non-mucoid cultures: pao1 (attc baa-47) cultures were grown overnight in 10 ml tryptic soy broth (tsb) (difco laboratories, sparks, md) in standard 16 ml glass test tubes with a plastic cap (vwr, 89000-482) before being streaked for isolation on tryptic soy agar (tsa) plates (difco laboratories, sparks, md). single colonies were used to inoculate tsb under two environmental conditions to produce biofilms with either mucoid-like or non-mucoid phenotypes. mucoid-like phenotype pao1 biofilms were grown in 10 ml tsb in standard 16 ml glass test tubes with a plastic cap (vwr, 89000482) on a shaker incubator at 37°c and 225 rpm. non-mucoid phenotype pao1 biofilms were grown in a 250 ml erlenmeyer flask (vwr, 29136-060) with 100 ml tsb and a foil cap at the same conditions. the shaking was set so that the biofilms did not grow on the surface of the culture or on a solid surface, but throughout the media. the two phenotypes were easily distinguished. the mucoid-like phenotype developed strings of a mucous-like substance that spun down toward the bottom of the test tube. the shaking was necessary to keep the mucoid-like strings suspended in the tsb and maintain oxygenation of the tsb. without shaking, the mucoid-like strings of biofilm sank to the bottom of the test tube and died (data not shown). the non-mucoid biofilms developed a thick, consistent composition throughout the tsb. previous studies have implicated nutrient or oxygen deprivation in initiating biofilm formation (13, 16, 32, 34). samples were grown for 24, 48, or 72-hour time periods. growth of biofilms using once flow through chambers: mature biofilms are most fully developed in vitro in a flow cell apparatus with a constant flow of media over the biofilm. as a positive control for biofilm development, a three-chambered flow cell apparatus (ibi scientific, peosta, ia) was set up as previously described (6, 24). tsb at room temperature was pumped through the individual chambers at a rate of 2.5 ml/minute. prior to inoculation of the apparatus chambers, a single colony of pao1 was inoculated into 10ml tsb overnight at 37°c. the 10 ml sample was diluted until an absorbance of 0.5 at od600 was reached using a protocol outlined in deligianni et al. (10). each chamber was inoculated with 0.5 ml of the diluted sample. during the inoculation period, chambers were inverted and flow was stopped for one hour while bacterial cells were given time to attach (6). each of the three chambers was assigned to a 24, 48, or 72 hour sample. one chamber was inoculated each day for three days. all three chambers were stopped on the same day, 24 hours after the inoculation of the final chamber. biofilms were first fixed with 100 ml of 4% paraformaldehyde (electron microscopy sciences, hatfield, pa) for 30 minutes. flow chambers were then rinsed with 50 ml pbs for approximately 30 minutes before embedding. the embedding gel was a 20% 200:1 acrylamide:bis-acrylamide polyacrylamide gel that was prepared with 20 µl of ap s (10 mg/ml), 8 µl temed, and 1 ml of 20% polyacrylamide 200:1 acrylamide:bis solution. the gel was injected into each flow chamber using a 1ml tuberculin syringe. (biorad, hercules, ca) (6, 26). flow direction was marked before the glass which covered each chamber was carefully removed and the fixed biofilms embedded in the polyacrylaminde gel were kept in pbs until stained for microscopy. finaljournal.indd 126 9/25/15 11:38 am pathogens and antimicrobial factors• 127 staining samples for examination by confocal light scanning microscopy: cultures of mucoid-like or non-mucoid bacterial cells were allowed one hour to attach to the surface of poly-l-lysine (sigma-aldrich, st. louis, mo) coated cover slips before being fixed with 4% paraformaldehyde. biofilms grown in the flow cell apparatus were stained through the polyacrylamide gel. all biofilms were stained with a 1:300 dilution of dapi (invitrogen, grand island, ny), which was used as a counterstain to label nucleic acids. a fitc conjugated antibody to lectin from hippeastrum amaryllis (hha) (ey laboratories, san mateo, ca, f-8008-1), which binds to 1,3or 1,6mannosyl units in polysaccharides of the psl component of the biofilms (25), was then applied at 0.2 mg/ml. slides were rinsed, and mounted using an anti-fade reagent (prolong, life technologies, grand island, ny). flow cell samples were too thick to add a coverslip and were maintained under wet conditions for microscopy. imaging of biofilms using confocal laser scanning microscopy a zeiss lsm 700 confocal laser scanning microscope was used to image the previously stained bacterial samples for dapi label of nucleic acids or fitc-hha label of the psl component of pao1 biofilms. single images of the biofilms were taken for dapi or fitc and image j was used to generate composite images (30). z-stacks were also taken (data not shown) and used to give an estimate of biofilm height (20). as a negative control coverslips with mucoid-like pao1 were examined without any stain or fitc-hha label. these were viewed through the dapi filter or the fitc filter to check for autofluorescence of p. aeruginosa. motility assay motility assays were conducted as previously described (5, 10). briefly, swim medium contained 0.982% tryptone, 0.295% agarose, and 0.491% sodium chloride. this is the most fluid medium that allows the easiest movement. swarm medium contained 0.786% nutrient broth, 0.491% agar, and 0.491% glucose. this is a thicker medium that restricts movement. twitch plates contained 0.976% tryptone, 0.9763% agarose, and 0.976% sodium chloride. these plates have the highest concentration of agarose and the most restricted movement. non-mucoid and mucoid-like samples for the motility assay were prepared as described above and given 72 hours to develop. at 72 hours, an inoculating needle was sterilized in alcohol and flame, inserted into the non-mucoid or mucoid-like sample just beneath the surface of the biofilm, and each motility plate was stab inoculated to the bottom of the plate, according to deligianni et al. (10). the point of inoculation was marked on the bottom of the plate and the plates were incubated at 37°c. growth of colonies was measured 24, 48, or 72 hours after inoculation; images were taken after 48 hours. the sample size was three to six plates per time point and plate type. data were analyzed using two comparisons. the purpose of the first analysis is to evaluate changes in motility over the 72 hour period of the motility assay. to do this, values for each type of motility (swimming, swarming, or twitching), were statistically compared to the 24 hour mucoid-like phenotype with an unpaired t-test using graphpad. the second analysis determines if there is a difference in motility between the two phenotypes at each time point. in this analysis, an unpaired t-test compared mucoid-like to non-mucoid samples at each time point. finaljournal.indd 127 9/25/15 11:38 am 128 • fine focus, vol. 1 (2) surface attachment assay surface attachment assays were conducted as previously described (21). briefly, bacterial cultures were grown under either mucoid-like or non-mucoid conditions for 24, 48, or 72 hours and then diluted down to an absorbance of od600 of 0.5. the cultures were then diluted 1:2 in tsb and 0.5 ml of each diluted culture was placed in an individual well of a poly-l-lysine coated 24 well plate for 1.5 hours. culture fluid was removed before the cultures were fixed with 70% methanol for 30 minutes and stained with 0.5% crystal violet for 30 minutes. excess crystal violet was then washed from the wells using pbs. digital images of the plates were obtained. non-mucoid and mucoid-like phenotypes an example of non-mucoid and mucoid-like biofilms developed in our lab is shown in fig. 1. as can be seen in fig. 1a, the non-mucoid phenotype grown for 72 hours is concentrated with a consistent composition. the mucoid-like biofilm seen in fig. 1b has thick, mucous-like strings that emanate from the surface and spread interior into the tsb. if reresults figure 1: non-mucoid and mucoid-like pseudomonas aeruginosa biofilm. the non-mucoid biofilm (a) was grown in 100 ml tsb in a 250 ml erlenmeyer flask and has a consistent composition. the mucoid-like biofilm (b) was grown in 10 ml tsb in a 16 ml test tube and demonstrates strings of mucoid-like biofilm. all samples were incubated at 37°c on a rotary shaker at 225 rpm for 72 hours. the specific differences in environment conditions leading to the formation of two phenotypes was not investigated. however, studies have shown that oxygen deprivation can induce biofilm formation; thus, it is possible the low surface-tovolume ratio in the test tube may lead to favorable conditions for biofilm development. b finaljournal.indd 128 9/25/15 11:38 am pathogens and antimicrobial factors• 129 moved from the test tubes, these mucous-like strings are easily disrupted and disseminated. however, in the test tube, they are discrete from the surrounding media. the observation of these two phenotypes is the basis of our examination. when cultured for longer than 72 hours, pao1 in the mucoid-like phenotype died, perhaps from a lack of nutrients. confocal laser scanning microscopy (clsm) fig. 2 a-d demonstrates a composite image of a 72 hour mucoid-like biofilm labeled with fitc-hha (a), dapi (b), and the composite image (c). in confocal images of pillar-shaped biofilms, it is common to view the eps matrix figure 2. a-d is an example of a 72 hour mucoid-like biofilm stained with dapi and hhafitc shown with the fitc filter (a), the dapi filter (b) and the composite image (c). d is the corresponding negative control without dapi or hha-fitc antibody. pockets, indicated by dark “holes” within the image, appear to be formed throughout the eps matrix of the biofilm. at these pockets a higher concentration of nucleic acids are present as seen by the dapi fluorescence. these “pockets” filled with pa may be the beginning of pillar-shaped biofilms. scale bars are 25µm. a c d b finaljournal.indd 129 9/25/15 11:38 am 130 • fine focus, vol. 1 (2) surrounding a “hollow-like” center that may contain bacteria (8). our biofilms contain similar structures. fig. 2a demonstrates “holes”, black spaces in the biofilm surrounded by fitc-labeled matrix. these “holes” are, however, “filled in” with dapi label in fig. 2b, indicating that bacteria are within this structure. the composite image, fig. 2c demonstrates the separation of the matrix from the bacteria with the pattern of blue dapi stain surrounded by the fitc-hha label for the matrix. fig. 2d is a negative control of mucoid-like pao1 biofilm indicating some background label; however, it is much less than the positive label seen in fig. 2a-c. figure 3. confocal microscopy of pa biofilms. pa samples were prepared as described in the methods. at 24 hours, all samples demonstrated biofilm growth (a-c). the flow cell samples (c) are slanted in the direction of media flow through the chamber and difficult to obtain focused images. at 48 hours, the mucoid-like and flow cell samples demonstrate greater growth (d, f) compared to the non-mucoid sample (e). the spherical structures in d could be the beginning of pillar-shaped biofilms. in the 72 hour samples (g-i), the mucoidlike sample (g) shows string-like biofilms structures typical of the mucoid-type phenotype compared to the punctate labeling seen in the non-mucoid sample (h). the flow cell sample (i) has circular structures indicative of pillar-shaped biofilms. calibration bars are 25µm. 24 hour mucoid non mucoid flow cell 48 hour 72 hour a d g b e h c f i finaljournal.indd 130 9/25/15 11:38 am pathogens and antimicrobial factors• 131 in order to compare biofilm development between the non-mucoid and mucoid-like phenotypes generated in this lab, biofilms were also grown in once-through flow cell chambers. these chambers have been shown in the past to provide optimal conditions for biofilm development (3, 29). fig. 3 compares non-mucoid, mucoid-like, and flow cell biofilms 24, 48, and 72 hours after inoculation of a chamber. biofilm development was greatest in the mucoid-like and flow cell samples at 72 hours (fig. 3g and i). there was some labeling in the 72 hour non-mucoid sample (fig. 3h), but it is much more punctate and does not show the elaborate psl network seen in the mucoid-like or flow-cell apparatus biofilms. in fig. 3i, the 72 hour flow-cell biofilm sample demonstrates circular structures similar to the pillar shaped biofilms shown in fig. 2c. the mucoid-like biofilms (fig. 3d, 3g) demonstrate biofilm structure that has the psl matrix as observed by the fitc-hha label, but is not always a discrete or circular structure (fig. 3g). however, there is much greater development of the psl matrix than in the non-mucoid biofilm which is a more consistent, punctate fitc-hha label indicating a much less complex and fully formed biofilm matrix in the non-mucoid sample. to measure biofilm height, z-stacks of 72 hour samples were used since biofilms at this point are the most developed (data not shown). non-mucoid phenotype biofilms were smaller and flatter with an average height about 6 µm. mucoid-like phenotype biofilms were taller with an average height of 6-12 µm. flow figure 4. motility assay. shown are images of swim (a-b), swarm (c-d), and twitch plates (e-f) for non-mucoid and mucoid phenotypes. all plates were inoculated with 72 hour samples and allowed to develop for 48 hours. swarming showed the greatest motility of the three types of movement as indicated by the increased diameter. all three types of movement demonstrated similar diameters, and therefore similar amounts of movement, between the non-mucoid and mucoid-like phenotypes. swim non mucoid mucoid-like swarm twitch a c e b d f finaljournal.indd 131 9/25/15 11:38 am 132 • fine focus, vol. 1 (2) cell biofilms were the largest biofilms with average heights between 30-40 µm. biofilms grown in a flow cell chamber were slanted in the direction of the flow of media, making it difficult to focus the image. motility assay fig. 4 a-f demonstrates images of the motility assays. as can be seen swarming motility demonstrated the greatest diameters (fig. 4 c, d). visual inspection alone indicates that motility is very similar between pao1 in each phenotype. the average movements of swarm motilities at 72 hours as shown on the column graphs (fig. 5b) were 18.1 mm (non-mucoid) and 21.8 mm (mucoid-like) as compared to the swim plate values (fig. 5a) of 16.3 mm (non-mucoid) 14.6 mm (mucoid-like). swarming is the only motility in which the 72 hour mucoid-like biofilm exhibited greater motility as compared to the non-mucoid sample (fig. 5b) although there was no statistical difference. it was hypothesized that twitching movement would be greater in the mucoid-like sample (fig. 5c); however, it was slightly greater in the 72 hour non-mucoid sample (12.2 mm vs. 9.5 mm). twitching had the only statistically significant data with both the 48 hour non-mucoid and 72 hour non-mucoid being significantly higher in twitching (p = 0.0019 for 48 hour; p = 0.0031 for 72 hour) than the 24 hour mucoid-like phenotype. the 72 hour mucoid-like phenotype also demonstrated significantly more twitching than the 24 hour mucoid-like sample (p = 0.0001). changes in motility are a part of biofilm formation as p. aeruginosa moves from swimming to swarming to twitching, eventually attaching to a surface and forming a microcolony and then a biofilm. clinical data from cf patient sputum indicate that clinical isolates containing p. aeruginosa exhibit widely varying patterns of motility (10, 27). these differences in motility may indicate differences in virulence and ability to develop chronic figure 5. graphs of motility data. a-c are graphs of the average diameter for each assay. data were compared to the 24 hour mucoid sample for each motility using an unpaired t-test (graphpad). twitch motility demonstrated a significant increase from the 24 hour sample for the 72 hour mucoid, 48 hour non-mucoid, and 72 hour non-mucoid. this indicates that over time, both mucoid and nonmucoid phenotypes exhibited a significant increase in twitching. this indicates that both phenotypes are exhibiting characteristics of biofilm formation; however, the biofilm structure between the two phenotypes remains different as observed by a flatter biofilm structure for non-mucoid biofilms using microscopy. non-mucoid p values: p = 0.0019, 48 hours; p = 0.0031, 72 hours. mucoidlike phenotype p value at 72 hours, p = 0.0001). a. swim b. swarm c. twitch 10 8 4 10 0 0 0 20 12 20 30 16 30 24 24 24 48 48 48 72 72 72 time (hours) non-mucoid non-mucoid non-mucoid mucoid-like mucoid-like mucoid-like time (hours) time (hours) * * * * d ia m et er (m m ) d ia m et er (m m ) d ia m et er (m m ) finaljournal.indd 132 9/25/15 11:38 am pathogens and antimicrobial factors• 133 figure 6. surface attachment assay. all samples were given 1.5 hours to attach to the well before being fixed and stained with crystal violet. as can be seen, attachment decreased for both mucoid-like and non-mucoid samples between 24 and 72 hours. biofilm formation is usually associated with an increase in attachment. while the trend in attachment was not as expected, it can be noted that the 72 hour mucoid-like sample exhibited greater punctate staining; these punctate areas of staining could be biofilms that attached. there are clearly qualitative differences in the development of the mucoid-like phenotype as compared to the non-mucoid phenotype. 24 hour mucoid-like non-mucoid 48 hour 72 hour infections. motility is now being used as a method to further identify and characterize p. aeruginosa in sputum from cf patients (10, 27). these motility patterns may help identify the most effective treatments (27). surface attachment attachment decreased as one moved from 24 hour to 72 hour samples (fig. 6). there was a qualitative difference in that the mucoid-like samples attached with more punctate staining indicating clusters or colonies of pao1 whereas the non-mucoid phenotype exhibited a more diffuse, even attachment. each sample was given 1.5 hours to attach so the punctate staining represents clusters of bacteria that existed at the time of sample attachment not colonies formed through cell division after attachment. the clusters may be similar to the circular structures observed in fig. 2a-c in which a cluster of pao1 is surrounded by psl matrix. absorbance data from the surface attachment assays did not indicate any significant difference in surface attachment when compared to the 24 hour mucoid-like sample or when comparing mucoid-like and non-mucoid samples at each time point. finaljournal.indd 133 9/25/15 11:38 am 134 • fine focus, vol. 1 (2) discussion biofilm formation is a critical component of bacterial infections in a clinical setting; for example, in chronic infections in cf that increase mortality and morbidity (17). biofilms are also implicated in chronic sinusitis and chronic wounds as well as other infections (4). it is well documented that mucoid biofilms are more pathogenic (11, 23). alginate, one of the prime components of a mucoid biofilm elicits different responses from airway epithelium that attenuates the host response (7). it is also well documented that biofilms isolated from clinical patients exhibit widely different morphology and physiology with some forming pillar-shaped biofilms, some forming knobby shaped biofilms, and some with flat biofilms (10, 27). motility assays are one way in which clinical strains are analyzed to determine trends in pathogenicity. multiple studies using motility assays have found that pathogenic strains have decreased motility (1, 27). the classic biofilm structure is that of a pillar-shaped structure containing bacteria and surrounded by an exopolymeric substance (eps) composed of psl, pel, and alginate. it is hypothesized that biofilm formation may be driven by environmental conditions such as nutrient depletion, although the environmental triggers driving biofilm formation are not completely understood (1, 4, 7). a common way in which biofilms are grown within the lab is with rotation in a shaker, similar to the non-mucoid phenotype in this study using an erlenmeyer flask. this biofilm produces different ratios of quorum-sensing molecules than biofilms formed within patient sputum (33). the environmental conditions that trigger biofilm transformation from a non-mucoid to a mucoid phenotype remain unknown (7). our lab was able to grow two distinct phenotypes of pao1 biofilms under two environmental conditions that may include oxygen or nutrient deprivation. it was our hypothesis that pao1 in the mucoid-like phenotype would produce a biofilm closer to a clinical “mucoid” biofilm with greater twitching motility, greater attachment to a surface, and greater height and pillar-shaped structure when compared to the non-mucoid phenotype of pao1. based on our data, both phenotypes exhibit some markers of biofilm development such as increased twitching behavior by pao1 as well as a clearly defined matrix, indicated by fitc-hha labeling. however, the mucoid-like phenotype exhibits characteristics closer to the more fully formed, mature pillar-shaped biofilms described in literature (8, 10). the mucoid-like biofilm developing in our lab had evidence of small pillar-shaped structures that were beginning to form (fig. 2 a-c), but did not reach the height or complexity of biofilms formed in a flow-through chamber (fig. 3g and 3i). biofilms are associated with a decrease in motility, both swimming and swarming (10, 27). swimming utilizes flagella to propel bacteria to a new location, possibly to obtain a better food source. twitching utilizes type iv pili to slowly move across a surface and is associated with surface attachment and the beginning stages of biofilm formation. swarming is one of several intermediate forms of motility that utilizes both flagella and type iv pili as well as a bacterial-secreted rhamnolipid that acts as a surfactant to create a more fluid surface for bacterial movement (5, 22, 35). pao1 in our mucoid-like phenotype exhibited swimming and swarming at 24, 48, and 72 hour time points (fig. 4f); however, the only significant differences finaljournal.indd 134 9/25/15 11:38 am pathogens and antimicrobial factors• 135 in motility when compared to the 24 hour mucoid-like phenotype were in twitching. at both 48 hours and 72 hours the non-mucoid phenotype had significantly greater twitching than the 24 hour mucoid-like phenotype (fig. 5c, p = 0.0019, 48 hours; p = 0.0031, 72 hours). at 72 hours, the mucoid-like phenotype had significantly greater twitching than the 24 hour mucoid-like (fig. 5c, p = 0.0001). these data signal an increase in twitching across a 72 hour period and support that both of these phenotypes may be progressing through the early stages of biofilm development. surface attachment is also an established and necessary component for biofilm formation (8, 17). this may be facilitated by a switch from swimming motility to swarming and then to twitching as the bacteria become sedentary. in our phenotypes, attachment consistently decreased from 24 to 72 hours in both the mucoid-like and non-mucoid phenotypes. this is counter to the generally accepted view that surface attachment of bacteria increases as biofilms form. however, there were also qualitative differences, especially in the mucoid-like phenotype which exhibited more punctate staining. these punctate spots could be small biofilms (fig. 6) which would be consistent with biofilm formation. in conclusion, our data indicate both phenotypes are in the process of biofilm formation due to fitc-hha label and increased twitching. however, there are also differences between the phentoypes as shown in clsm images in which the mucoid-like biofilm has more pillar-shaped biofilms and in the increased level of punctate staining in the surface attachment assay. not much is known about the environmental factors that drive the formation of the wide variety of biofilms observed in clinical isolates. nutrient and oxygen deprivation have been implicated in the formation of mucoid-like biofilms (16, 32, 37). the role of specific environmental triggers was not investigated in this study; however, knowing that environments as simple as using a test tube or an erlenmeyer flask can produce two different phenotypes of biofilms might lead to further identification of the triggers that lead to the development of pathogenic biofilms in patients. the authors would like to gratefully acknowledge funding for this project from the following sources: azusa pacific university faculty research council, beta beta beta national honor society research foundation, and the department of biology and chemistry at azusa pacific university. we would also like to thank megan prosser and skyla herod for their valuable feedback in preparing this manuscript. acknowledgements references 1. bianconi, i., milani, a., cigana, c., paroni, m., levesque, r. c., bertoni, g., & bragonzi, a. 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(2013). psl trails guide exploration and microcolony formation in pseudomonas aeruginosa biofilms. nature, 497(7449), 388-391. finaljournal.indd 137 9/25/15 11:38 am rhizofiltration of lead contaminated soil by helianthus annuus amended with bacillus megaterium and edta kaitlin m. pearce, alexandra kurtz, and rebekah j. ward* georgia gwinnett college 1000 university center lane lawrenceville, ga, usa copyright 2015, fine focus all rights reserved manuscript received 24 april, 2015; accepted 2 july, 2015 finaljournal.indd 95 9/25/15 11:38 am heavy metal contamination causes numerous adverse effects to public health and the environment. sources of heavy metal contamination are widespread, especially in urban environments. certain plants such as sunflower (helianthus annuus) have been shown to sequester heavy metals in their root systems, thus filtering contaminants such as lead (pb) from soil, a process termed rhizofiltration. in the present study, bacillus megaterium was applied to the root system of sunflowers growing in pb-contaminated soil and the efficiency of rhizofiltration was examined. lead levels in the rhizosphere of the bacillus megaterium and edta amended plants were almost 100 mg/kg soil higher than those without treatment, suggesting the amendment may have been effective in augmenting lead sequestration. in order to further elucidate these leadsequestering communities, preliminary phylogenetic assays were conducted on the soil with and without the presence of the plant. although complete coverage of the community phylogeny was not possible, there was evidence indicating that the rhizosphere may have induced changes in the composition of the bacterial community. these studies offer simple methods for enhancing bioremediation in agriculture. abstract heavy metals are defined as metals that have a specific density > 5g/cm3 (17). elevated concentrations of heavy metals in soil can have a devastating effect on human health and the environment. this is especially true for gardens located in urban areas. human health is most adversely affected by certain heavy metals, namely cadmium, mercury, arsenic, and lead (17). excess pb is especially toxic to humans because of the effects it has on kidneys and the nervous system, which can cause headaches, weakness, cramps, anemia, and may lead to mental health disorders (1). according to the u.s. environmental protection agency (epa), lead can be found in the air, soil, water, and in homes because of its presence in gasoline, industrial facilities, paint, ceramics, pipes, batteries, cosmetics, ammunition, and even food (30).these make lead a priority heavy metal for study in urban environments. the problem of heavy metal soil contamination can be addressed using a technique known as bioremediation. bioremediation is the process of introducing organisms, such as bacteria, into a contaminated environment in order to introduction corresponding author *rebekah j. ward georgia gwinnett college 1000 university center lane lawrenceville, ga 30043 email: rward1@ggc.edu keywords • bioremediation • sunflowers • rhizofiltration • lead • phylogenetics 96 • fine focus, vol. 1 (2) finaljournal.indd 96 9/25/15 11:38 am applied and environmental microbiology • 97 remove the pollutants. phytoremediation is a subset of bioremediation that exploits plants in order to clean up contamination. certain plants termed hyperaccumulators can store the heavy metals in tissues or the rhizosphere (root system and surrounding soil), therefore taking it out of biological circulation. this process is termed rhizofiltration. this study was conducted to quicken the remediation process and increase its efficiency without hampering cost effectiveness. previous studies (13, 15, 18, 20) have indicated that adding chemicals or biological elements to the soil may help plants sequester metals in the tissues. this, in turn, reverses some of the effects of the metals by lowering the concentration in the surrounding area, allowing more plants that are less metal-tolerant to grow. after a rhizosphere absorbs as much metal as it can, the plant can be uprooted, allowing for removal of contaminants from the soil. this can alleviate some of the harmful impacts of metals on the environment. however, one of the problems with phytoremediation is that it is a slow process that produces a low yield (10). one plant that has been shown to sequester heavy metals in the rhizosphere is the sunflower (helianthus annuus) (18). sunflowers are considered hyperaccumulators and have been used for various environmental cleanup projects, (7). sunflowers also are more tolerant to ph variation than many other common plants, capable of growing in soil ranging from ph 5.7 to over 8.0, while optimal soil ph for other plants is 6.4 (24). sunflowers also produce more roots, shoots, and total biomass than many other common plants. this means that they can potentially hold larger amounts of pollutants and fewer plants are required to recover the same amount of pollutants, thereby becoming economically sustainable. bioavailability of phosphate also has an influence on the effectiveness of phytoremediation. addition of ethylenediaminetetraacetic acid (edta) has shown an increased pb uptake by almost 20% in previous studies, and has also been shown to facilitate phytoremediation in plants (13, 20). edta is a common and powerful chelating agent that has been added recently into heavy metal treatment systems and works especially well with pb and copper (4). this demonstrates that certain chemical additives can help sunflowers absorb higher levels of pb, therefore ultimately reducing the cost, amount of land used, and amount of plants needed to grow in a particular plot of land when used for bioremediation. bacteria that reside in rhizospheres of plants can play a role in reducing the toxic effects of heavy metals on the plants (15). these microorganisms can protect the plant from damage and, in return, benefit from living in the rhizosphere systems, therefore creating a mutualistic relationship with the plant. in this study, bacillus megaterium was used because this bacterium has been shown to absorb and store pb intracellularly, therefore making it resistant to elevated levels of pb (25). this species is also a common soil bacteria that is considered part of plant growth promoting rhizobacteria (pgpr), which helps improve growth by releasing a key auxin (indole-3acetic acid) to encourage cell proliferation (3). bacterial cultures were added to germinating seedlings to help improve the health of the plants for rhizofiltration and increase the concentration of pb in the rhizosphere through the intracellular sequestration used by b. megaterium. the purpose of this experiment was to analyze the impact of the addition of bioavailable phosphate and heavy-metal tolerant bacteria on the pb concentrations in highly contaminated soil. finaljournal.indd 97 9/25/15 11:38 am 98 • fine focus, vol. 1 (2) materials and methods collection, setup, and maintenance for this experiment, three five-gallon plastic containers of soil were collected from two sites in an urban garden in atlanta, georgia, usa. these samples were then transported to the laboratory at georgia gwinnett college. the soil was characterized as being a crumbly, fine soil. the soil (2.5 grams, manually homogenized) was put into 500 ml conical falcon tubes with 7.5 ml distilled h2o and stored at -80˚c for dna extraction. a sample was taken from the middle of each container prior to planting seedlings, homogenized, and sent to the university of georgia soil and water analysis lab for determination of pb concentration. a teddy bear sunflower (helianthus annuus) seed was planted one inch deep in nine 10” x 12” containers with each weighing approximately 0.95 kg per container. the samples were watered with 40 ml of tap water on alternate days for three months. after the seeds sprouted, a 40 watt growlux, wide-spectrum grow light (grower’s supply, dexter, mi) was placed on a timer for eight hours daily. unplanted soil was maintained as the control experiment. sterile luria broth (lb, 100 ml) was inoculated with bacillus megaterium atcc14581 and placed in a shaker for 24 h at 37°c. the nine containers of soil were then separated into three different categories. three control containers were watered with 40 ml tap water on alternate days. three edta containers were watered with 80µl edta once then 40 ml tap water on alternate days. three edta plus b. megaterium containers were watered with 80 µl 0.1 m edta once, 1 ml of freshly made bacterial culture once, and then watered with 40 ml tap water on alternate days. the bacterial culture was then serially diluted onto lb agar plates to determine the original cell count. sunflower height was then measured seven times for a total of three months and recorded. the measurements were taken from the base of the stem to the tip of the tallest leaf. soil filtration tubes with the most contaminated soil, according to the analysis results, were thawed, and 1 ml 0.1 m edta was added, along with 200 µl tris buffer and distilled water to balance the tubes. they were centrifuged at 1000 rcf for 1 min. at 25˚c, then the supernatant was removed and 10 ml of tris buffer was added. after thorough mixing with a vortex mixer, the tubes were weighed and water was added to balance them within 0.1 g. this was repeated three times. after the fourth centrifugation, the liquid was poured through muslin to filter out particles and 1 ml 1x tris borate edta (tbe) was added to the soil, weighed out in tubes, and centrifuged one more time at the same settings. the supernatant was transferred to two microcentrifuge tubes for storage at -80˚c for five days. dna extraction and cloning the method of tsai and olson (29) was used for dna extraction in the present study with the following modifications: tris edta was used in place of tris hcl, the tubes were stored in a -80˚c freezer instead of in dry ice, and the tubes were centrifuged at 6000 rcf for 10 min. before starting the protocol. ten new pcr finaljournal.indd 98 9/25/15 11:38 am applied and environmental microbiology • 99 samples were made with cetyltrimethyl ammonium bromide (ctab) and 5 g of soil was mixed with 3 ml distilled water and 5 mg/ml lysozyme. the mixture was placed in a shaker for 2 h. four samples were used to proceed to the transformation and cloning steps. ammonium acetate (5 mg/ml) was used instead of magnesium acetate. the phenol chloroform extraction was a 1:1 ratio of the working solution to phenol: chloroform: isoamyl alcohol (sigma, st. louis, mo). the mixture was centrifuged at maximum speed (13,000 rcf) for 10 min. the top layer was then removed using a pipette and 100 µl of ammonium acetate was subsequently added. of this volume, 350 µl were transferred into four test tubes. an ethanol precipitation was carried out, after which a tris-edta (te) suspension and dna samples were stored at -20°c. for the polymerase chain reaction (pcr) amplification, the primers used were 8f (5’-agagtttgatcctggctcag-3’) and 1492r (5’-ggttaccttgttacgactt-3’) (integrated dna technologies, coralville, ia). pcr samples were prepared as follows: 1 µl of each primer (integrated dna technologies, coralville, ia), 2 µl of dna, 46 µl distilled water (48 µl for the controls), and 50 µl of master mix (new england biosystems, ipswich, ma). twenty pcr cycles were used as follows: 94˚c for 30 seconds as a hot start and denaturation, 53˚ c for 30 seconds for annealing, 68˚c for 1 minute for elongation, and 68˚c for 5 minutes for final extension. the samples were analyzed using nanodrop 2000 by thermo scientific and polymerase chain reaction was again conducted as the above paragraph described with the following modifications: 50˚c for 30 seconds for annealing, 72˚c for 45 seconds for elongation, and the positive control used bacillus subtilis dna (48 µl). upon maturation of the remaining plants, each was removed at the base of the stem and 700 g of soil was collected from the middle of the container and manually homogenized. one gram was taken from the homogenized soil and put into a test tube with 5 ml distilled water. they were each mixed and stored at -80˚c for 30 min. lysozyme (75 mg) was then added to each tube after being thawed in a bead bath at 65˚c for 3 min., after which the tubes were stored at 37˚c for 24 h. gene cloning of pcr products was done through a tri-n-octylphosphine oxide (topo) cloning reaction with standard procedures. a series of minipreps were done from a qiaprep spin miniprep kit (from qiagen, germantown, md) using manufacturer’s instructions. pcr and agarose gel electrophoresis were completed (29). a nanodrop analysis was then conducted for all 95 control samples. after testing, the data was analyzed using finchtv, seaview, blast, clustalw, and geneious and the samples were transferred to the university of georgia genomics center to be sequenced. sequencing and phylogenetics sequences were manually analyzed for quality using finchtv. following blast searches, the most similar sequences were selected and aligned via seaview. clustalw was used to make a phylogenetic tree including all of the samples. geneious was used to make an outgroup phylogenetic tree using methanococcus voltae as the outgroup. two known organisms (16s genes of bacillus subtilis and escherichia coli) were also added to the tree. finaljournal.indd 99 9/25/15 11:38 am 100 • fine focus, vol. 1 (2) results some of the original plants did not survive, leaving only control samples and amended samples. the original cell count was determined to be 2.21 x 107 cfu/ml. the initial pb concentration was 542 mg/kg (table 1). the final concentrations of pb for the “control” plants was 531.2 ppm, while the final concentrations for the “non-vegetated” and “b. megaterium plus edta” samples were 567.0 mg/kg and 613.7 mg/kg, respectively. the phylogenetic tree data for all samples from the non-vegetated and control treatment indicate that the samples were dominated by a gram-negative rhizosphere-associated phylotype (fig. 1). figs. 2 and 3 show the phylogenetic trees for the control or non-vegetated samples, analyzed separately. the two known bacterial operational taxonomic units (otus) were included in each tree for comparison purposes. table 2 includes five different classes of bacteria in the four clades in the total phylogenetic tree. fig. 4 is a representation of the percent breakdown of each class that was discovered in the non-vegetated and control samples. these data reiterate the difference in proteobacteria between the control and non-vegetated samples. discussion the control plant did not appear to effectively sequester pb compared to the non-vegetated soil (table 1). explanations for this could include that earlier data for rhizofiltration used a different subspecies, or natural variability that could diminish with a larger sample size. however, the amended rhizosphere shows sequestration outside the range of this hypothetical variation. the final concentrations of the control and non-vegetated samples are within a 30 mg/ kg range, so that variation is unlikely to be a determinant of pb sequestration, seen in the b. megaterium plus edta samples (table 1). however, the bacillus megaterium plus edta sample had sequestered almost 75 mg/kg more than the initial concentration, or more than a 13% increase in mg/kg, as compared to the control (-1.9%) and non-vegetated (4.6%) samples. this suggests that bacillus megaterium and edta may have helped the sunflower sequester more pb as compared to the other samples. the edta-alone samples were not viable, likely due to too high of a concentration of edta, and therefore pb sequestration cannot be evaluated. for future experiments, the concentration of edta may be varied and optimized for the best results. the non-vegetated and control sequences were used because studies have shown that non-amended sunflowers can carry out rhizofiltration and by using these sequences, the variety in the bacterial community could be analyzed (1, 2, 18). the phylogenetic tree (fig. 1) is split into four clades with five distinct classes of bacteria. the first contains alphaproteobacteria, which were only found in the control samples and had four different otus (fig. 2). the alphaproteobacteria also were the main bacterial class found in the control soil (table 2). this could be because alphaproteobacteria are the common inhabitants of the rhizosphere and therefore absent in soil without plants. finaljournal.indd 100 9/25/15 11:38 am applied and environmental microbiology • 101 phylogentic tree for all samples plasmidc34 0.0048 hyphomicrobium.facile 0.00474 clostridium.aminovalericum 0.03498 bacillus.subtilis 0.00445 bacillus.thuringiensis 0.00069 bacillus.toyonensis 0.00061 pseudomonas.panacis 0.00157 pseudomonas.fluorescens 0.00342 bauldia.litoralis 0.03946 plasmidc67 0.16065 sphingomonas.jaspsi 0.00976 plasmidc63 0.01801 pandoraea.sputorum 0.03728 plasmidc51 0.0272 achromobacter.insuavis 0.00195 acinetobacter.calcoaceticus 0.00178 plasmidc59 0.00407 plasmidn38 0.01286 escherichia.coli 0.07451 plasmidc28 0.01132 granulicella.aggregans 0.07567 plasmidc20 0.06357 plasmidc68 0.01394 plasmidn15 0.01509 pseudomonas.putida 0.00341 plasmidn36 0.01056 bacillus.atrophaeus 0.00048 plasmidn40 0.01058 plasmidc64 0.00614 plasmidn8 0.00664 aciditerrimonas.ferrireducens 0.04288 plasmidc40 0.03624 telmatocola.sphagniphila 0.07971 plasmidn33 0.04637 crocinitomix.catalasitica 0.06677 plasmidn35 0.08944 plasmidc44 0.00137 rhizobium.tropici 0.00052 figure 1. phylogenetic tree for all non-vegetated (n) and control (c) samples. finaljournal.indd 101 9/25/15 11:38 am 102 • fine focus, vol. 1 (2) this class of bacteria is important because they are a large and diverse group often symbiotically associated with plants. there are also species that can fix nitrogen and can be found in legumes and other plants. legumes are hypogenous and these bacteria have adapted to create various classes of relationships with the root system, which supports the argument that they are an expected component of the rhizosphere. one otu was found in the control samples that may correspond to bacteria such as rhizobium tropici, which has been shown to fix nitrogen and be associated with legumes (22). this suggests that the rhizosphere may have recruited nitrogen-fixing bacteria to soil without the help of surrounding plants. in the second clade, two classes are found: betaproteobacteria and gammaproteobacteria. two otus which affiliate with betaproteobacteria are only found in the control samples (fig. 2). betaproteobacteria share the same relative community composition in control soils with both firmicutes and acidobacteria (18%, fig. 4). this class of bacteria consists of aerobic or facultative bacteria; they can be found in waste water and other environments and some can fix nitrogen like alphaproteobacteria. one otu found control phylogenetic tree methanococcus_voltae clostridium.aminovalericum aciditerrimonas.ferrireducens granulicella.aggregans hyphomicrobium.facile sphingomonas.jaspsi rhizobium.tropici bauldia.litoralis achromobacter.insuavis pseudomonas.panacis pandoraea.sputorum escherichia.coli bacillus.subtillis bacillus.toyonensis plasmidc64 plasmidc28 plasmidc68 plasmidc34 plasmidc44 plasmidc67 plasmidc63 plasmidc20 plasmidc40 plasmidc59 plasmidc51 0.05figure 2. outgroup phylogenetic tree for control samples. finaljournal.indd 102 9/25/15 11:38 am applied and environmental microbiology • 103 in these samples was related to pandoraea sputorum. the genus pandoraea is important to environmental health because of its known use of bio-catalytic activities, such as biodegradation (14). it is also closely related to species belonging to the ralstonia lineage, which encompasses many heavy metal-resistant betaproteobacteria (12). the otus found in these samples correlate with other samples that are found in highly polluted environments, which suggest an environmental selection for heavy metal resistance (28). gammaproteobacteria are a class of bacteria that contain environmentally important species, some of which are human pathogens. gammaproteobacteria were found in both the control and non-vegetated samples, with a more plentiful amount found in the soil without plants (table 2). for the control soil, this class is the least abundant (fig. 4). however, in the non-vegetated soil, this class was the most abundant of all the other classes. this, along with previously stated data, suggests differences between the composition between the non-vegetated and control soil communities. it also suggests that the addition of the rhizosphere enriched certain types of bacteria suggesting that certain species may be integral to the observed augmented pb sequestration (18, 26). a total of three different otus of pseudomonas were detected in the samples and pseudomonas panacis was detected in both the control and non-vegetated soil. p. panacis is root-associated and has been identified in root lesions of various plants (23). pseudomonas spp. are known for aerobic growth and association with plants. pseudomonas putida is an environmentally important species because it has been shown effective in improving the chemical and physical properties of polluted soil, bioremediating substances such as crude oil and naphthalene (11,21). pseudomonas fluorescens is also an important species that inhabits the rhizosphere and is effective in bioremediation (31). this is a highly metal-resistant species that can tolerate millimolar concentrations of selected metals (5). the possibility of an enrichment of organisms with known association to polluted environments suggests that they are adapted to it and can help plants tolerate such environments, thereby supporting the previous studies of the possible use of initial and final lead concentrations for samples soil type initial pb concentration final pb concentration total percent change c nv 542.0 531.2 -1.992% 542.0 567.0 4.613% 542.0 613.7 13.229%be table 1. initial and final lead concentrations of soil for controls (c), non-vegetated (nv), and bacillus megaterium plus edta (be) samples. finaljournal.indd 103 9/25/15 11:38 am 104 • fine focus, vol. 1 (2) bacteria in bioremediation. the third clade from fig. 1 consists of a class of bacteria known as firmicutes. many firmicutes produce endospores, have a low g+c content, and are known for carbohydrate degradation. if stress is sensed in soil, firmicutes produce endospores, which can help them survive and resist desiccation. they also are abundant in root exudates with sugars and organic acids, specifically the genera bacillus and clostridium (27). bacillus and clostridium were both detected in this experiment in both types of soil at 50% (table 2). clostridium, particularly clostridium acetobutylicum (a close relative to the obtained otu related to clostridium aminovalericum), is environmentally important, as previous studies have indicated its usefulness for degradation of large biological molecules, toxic organic molecules, and metals (8). this species has also been used in the bioremediation of soil and toxic sludge by chemically reducing and solubilizing the amount of radionuclides and toxic heavy metals (such as uranium (u), iron (fe), magnesium (mg), and (pb)) (6). this species may help in remediation of soils, particularly those in urban locations, because of its potential use around industrial sites, buildings, and waste disposal sites. three different otus of bacillus were also discovered in the samples. bacillus may be a normal part of these ecosystems, suggesting that amendment with b. megaterium would not cause serious disruption of the bacterial community. this effect may be due to the ability of bacillus spp. to survive and flourish in more hostile environments. one otu that was found, associated with bacillus atrophaeus, is a non-pathogenic, aerobic spore-forming non-vegetated phylogenetic tree methanococcus_voltae bacillus.atrophaeus bacillus.thuringiensis pseudomonas.panacis plasmidn15 crocinitomix.catalasitica pseudomonas.putida plasmidn38 acinetobacter.calcoaceticus plasmidn33 bacillus.subtilis telmatocola.sphagniphila plasmidn40 plasmidn36 pseudomonas.fluorescens plasmidn12 plasmidn8 plasmidn35 escherichia.coli 0.04figure 3. outgroup phylogenetic tree for non-vegetated samples. finaljournal.indd 104 9/25/15 11:38 am applied and environmental microbiology • 105 table 2. tables comparing percentage and species of bacteria found in each class according to the phylogenetic tree. comparison of classes found in control and non-vegetated samples control soil percent of bacteria in class 100% 100% 25% 50% 50% number of different bacterial species in class 4 2 1 2 2 class alphaproteobacteria betaproteobacteria gammaproteobacteria firmicutes acidobacteria non-vegetated soil percent of bacteria in class 0% 0% 75% 50% 50% number of different bacterial species in class 0 0 3 2 2 class alphaproteobacteria betaproteobacteria gammaproteobacteria firmicutes acidobacteria type of bacteria related to b. subtilis (9) (fig. 3). bacillus thuringiensis, known as the source for the entomopathogenic crystalline endotoxin for insect control, was found in the non-vegetative treatments. the last clade consists of acidobacteria, a newer phylum comprising a diverse range of bacteria, especially soil-inhabiting species. acidobacteria include species known to be pb-tolerant and also sometimes acidophilic. these, like the firmicutes, were found in equal proportions in the control and nonvegetated samples (table 2). one otu found, aciditerrimonas ferrireducens, has been reported to be iron-reducing, which could have impacts on other heavy metals, such as pb (16). phytoremediation, including rhizofiltration, is a vital area of inquiry because it is more cost effective and has fewer negative impacts on public health and the environment (19). these preliminary data suggest that there is a biologically helpful as well as economically viable method for increasing finaljournal.indd 105 9/25/15 11:38 am 106 • fine focus, vol. 1 (2) the efficiency of phytoextraction of pb from soil. for example, the flower could be cut and sold to sustain the removal of the pbcontaminated rhizosphere. it also could be used to address the problems of so-called ‘food deserts’ by eventually detoxifying soil in order to grow fresh produce in urban gardens. this investigation into the rhizosphere community without amendment suggests that acidobacteria and firmicutes may be a common component of pbcontaminated soils but that the presence of the rhizosphere may have shifted the relative abundance of the proteobacteria away from gammaproteobacteria and towards alphaproteobacteria and betaproteobacteria. this may help elucidate the mechanism through which rhizofiltration occurs. these data aim to contribute to the ongoing process of understanding and improving on methods for removing hazardous pollutants from the environment. for future studies, a larger sample size could be used, as well as a lesser concentration of edta in order to see if the sunflowers remain alive for a longer period of time. percentage breakdown of classes in both soils control soil non-vegetated soil 37% 18% 42% 29% 29% 9% 18% 18% alphaproteobacteria betaproteobacteria gammaproteobacteria firmicutes acidobacteria gammaproteobacteria firmicutes acidobacteria figure 4. pie charts representing the class diversity in the control soil and non-vegetated soil. finaljournal.indd 106 9/25/15 11:38 am applied and environmental microbiology • 107 we would like to thank monica ponce for providing us with the soil that was used in this experiment. references acknowledgements 1. akhtar, m. s., chali, b., & azam, t. 2013. bioremediation of arsenic and lead by plants and microbes from 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bacterial communities. fems microbiol. ecol. 77:600-610. 28. silver, s., & phung, l. t. 1996. bacterial heavy metal resistance: new surprises. ann. rev. microbiol. 50:753-789. 29. tsai, y. l. & olson, b. h. 1991. rapid method for direct extraction of dna from soil and sediment. appl. environ. microbiol. 57:1070–1074. 30. united states environmental protection agency. 2014. learn about lead. retrieved from the environmental protection agency website: http:// www2.epa.gov/lead/learn-about-lead 31. varivarn, k., champa, l. a., silby, m. w., & robleto, e. a. 2013. colonization strategies of pseudomonas fluorescens pf0-1: activation of soil-specific genes important for diverse and specific environments. bmc microbiology 13:92. finaljournal.indd 108 9/25/15 11:38 am • fine focus, vol. 3 (2) the colonization and establishment of the neonatal mammalian microbiome victoria a. kouritzin & leluo guan * department of agricultural, food and nutritional science, university of alberta, edmonton, alberta, canada manuscript received 13 april 2017; accepted 06 july 2017 neonatal mammalian microbiome • 89 copyright 2017, fine focus. all rights reserved. 88 • fine focus, vol. 3 (2) neonatal mammalian microbiome • 91 in current agriculture practices, such as the dairy industry, the use of antibiotics is being discouraged due to the occurrence of antibiotic resistant bacteria. however, antibiotics are used commonly to treat calf diarrhea, which is a serious issue that negatively influences calf health, growth, and development. recent research highlights the gut microbiota as a potential source to improve the gut health of a calf, which could minimize the antibiotic use. however, limited knowledge is available for the early life gut microbiota and its relationship with calf’s performance. it is known that the microbiota has an influence on immune system development, as well as behavioral development, and metabolic development. further, an atypical microbial population, or a microbial shift, has been linked to autoimmune, anxiety and metabolic disorders. the process of microbial and host interactions starts at birth, suggesting that mammals are initially colonized by microbes immediately following and during birth. differing modes of delivery, caesarian or vaginal delivery, and possibly the length of time of the birthing process, may determine initial colonization of the infant. further, the establishment of the microbiota can be influenced by host genetics, diet, and maternal environment. therefore, this review aims to summarize the current understanding of the neonatal mammalian microbiota obtained from human and mice studies, and to outline future research directions on microbial colonization and possible manipulation strategies that can be used to manipulate the gut microbiota in dairy calves. by understanding the process of how mammals and microbes interact it is possible to better target future research in order to solve the problem of calf diarrhea. abstract corresponding author leluo guan 410 agriculture/forestry centre, university of alberta, edmonton, ab, canada t6g2p5; tel, 780-492-2480 fax, 780-492-4265 lguan@ualberta.ca keywords • early-life microbiome • calf • colonization • establishment calf diarrhea can cause more of a financial loss than any other calf ailment (15). diarrhea in the young animal prevents absorption of fluids and nutrients, inhibiting growth and health (1). further, without immediate intervention, calf diarrhea can be transmitted between calves and can result in high mortality. currently, the common practice to prevent calf diarrhea is the use of antibiotics. yet, industry is encouraged to reduce the use of preventative antibiotics due to the increasing prevalence of antibiotic resistance. therefore, an alternative to antibiotic prevention for calf diarrhea is needed. recent research highlights the gut microbiota as an important factor in immune function development and maintaining neonate gut health, making the gut microbiota a potential source to improve the gut health of calves to reduce the prevalence of diarrhea. however, limited knowledge on the calf gut microbiota is available. additionally, the mechanisms of how the microbiota influences calf gut development and health are still largely unknown and undefined. therefore, this review aims to summarize the current understanding of the neonatal mammalian microbiota based on the findings from human and mice studies from three aspects including initial colonization of the neonatal mammals, the impact of the microbiota on gut immune function of the developing neonate mammal, and factors affecting microbial establishment during early life. further, this review aims to identify the knowledge gap of the calf microbiota in order to direct future research towards identifying potential ways to reduce the incidence of, and possibly prevent, calf diarrhea through the manipulation of gut microbiota. mammalian development affected by microbiota it was hypothesized by louis pasteur that the microbiota has an important and necessary influence on mammalian life (21). mammals have a rich and diverse microbial population that interacts with, and influences the development of biological processes in mammals like immune function and metabolic systems (21). additionally, the microbiota causes long-term impacts on emotional systems (4, 23). the following section of this review will outline specific changes in microbiota and how they can affect the immune system development, anxiety and depression, and digestive and metabolic functions. impact of the microbiota on immune system development the interaction between host and microbes is essential for proper immune function development. young animals enter a critical period soon after birth when exposure to antigens is imperative for immune development. if the exposure is delayed, immune development can be impacted. this can be explained by an experiment using germ free mice (21). firstly, it is important to note that the authors demonstrated that germ free mice have an atypical cytokine response to orally treated lipopolysaccharide (lps) when compared to conventional mice. it is important to note that lps is a large molecule found on the outer membrane of gram-negative bacteria, and the previously introduction 90 • fine focus, vol. 3 (2) neonatal mammalian microbiome • 93 figure 1. the relationship between the colonization and establishment of the gut microbiota on the early-life calf’s immune system, mood and metabolism, shown in blue, and the impact of the calf’s early diet, genetics and internal environment on the gut microbiota, shown in purple. also shown is the maternal impact on the calf by genetics and maternal environment, and the maternal impacts on the microbial colonization of the calf, shown in red. described atypical response differs from a normal response in that the atypical response is delayed and exaggerated. further, these authors noted that the juvenile mice can resume a normal cytokine response, and behave normally, normal being immediate and moderate, when treated with probiotic bifidobacterium infantis. however, when adult germ free mice were treated with the same probiotics they were unable to be converted back to the normal response. additionally, these effects on the immune system can have long-term consequences, which are still being investigated (10, 19). it has been found that antibiotic use in early life influenced fecal bacterial composition and can be linked with development of intestinal diseases later in life (17). however, there is convincing research that interruptions in the establishment of the microbiome during early life can result in allergies, asthma and other autoimmune diseases in adults (10). additionally, it was found that the development of eczema can be minimized if infants were treated with probiotics (10). after birth, the gut microbiota may contribute to mammalian immune system development through interactions between hosts. the interaction between microbe and host immune development is important, which has been demonstrated by germ free animals as described above. germ free animals do have immunities; however, they cannot cope with pathogens (22). this reveals that without the proper microbial exposure in early life, the immune system will not properly develop and can increase risk of disease in later life. further, a more diverse intestinal microbiota during the first week of life is associated with a reduced risk of subsequent eczema in infants (10). additionally, interventions that enhance microbial diversity in early life may provide an effective means for the prevention of eczema in high risk infants (10). impact of microbiota on anxiety and depression initially, the evidence of microbial impact on host behavior was gained from comparisons of germ free and bacterial colonized mice. in order to test the idea that postnatal microbial colonization may affect the development of brain plasticity, researchers compared the hypothalamic-pituitary response to differing levels of stress restraint using genetically identical germ free and specific pathogen free mice (23). in this study, the individual mice were placed into a 50 ml conical tube for 1 hour, or into a glass container lined with ether soaked absorbent paper for 2.5 minutes. the authors found exaggerated stress response in germ free mice when compared to pathogen free mice (23). further, the same authors observed a reduction in stress response in germ free mice after administration of probiotic bifidobacterium infantis. these results have demonstrated the differences in brain function between germ free and colonized mice, suggesting the potential relationship between gut microbiota and animal behavior. however, other researchers found that administration of antimicrobials to colonized mice reduced the microbial population, and increased exploratory behavior in mice (4). they also found that the same antimicrobials given to germ free mice had no effect on their behavior. furthermore, they reported that administering the microbial population from colonized mice to germ free mice reduced exploratory behavior in the germ free mice. such change of exploratory behavior suggests a higher activity of mice when treated with antimicrobials. though increased exploratory behavior proposes a contrast to a greater fear response, they may actually be complementary depending on the motivation of the exploration (23). however, it is clear that more research needs to be done to determine the impact of postnatal microbial colonization on the development of neural systems that induce stress in animals. impact of the gut microbiota on digestion and metabolic functions recent studies have found that a shift in microbiota during early life can cause obesity, as well as other metabolic diseases like diabetes in later life (19). the study on fecal microbiome of 298 stool samples from 22 children with type 1 diabetes, and 22 normal children of the same age found a significant difference in hostmicrobial interactions between two groups (6). there was a higher interaction between host and enterococcus, sarcina, prevotella, and corynebacterium in the children with type 1 diabetes (7). relatedly, the authors also found less interaction between enterococcus, sarcina, prevotella, and corynebacterium and host in children without type 1 diabetes (7). this correlation of host and bacterial interaction, may suggest that metabolism could be affected by the gut microbiota. to further identify the causal effect of microbiota on host metabolic dysfunction, genetically deficient toll-like receptor 5 (tlr5) mice were used to induce obesity, and then the obese tlr5 mice microbiota was transplanted to wild type germ free mice (24). found in the gut mucosa, toll-like receptor 5 is a class of receptor involved in innate immune responses, which plays a role in satiety. without tlr5, the mice exhibited significant hyperphagia, and developed metabolic disease (24). next, the germ free wild type mice treated with the gut microbiota derived from the tlr5 deficient mice, immediately showing distinct microbial changes, and later becoming obese (24). the change in body composition of germ free mice 92 • fine focus, vol. 3 (2) from normal to obese after colonized with a specific microbial population, suggests that the gut microbiota may impact body composition through affecting the host metabolic functions. initial colonization mammals are currently believed to be sterile when in utero and microbial colonization can be observed minutes after birth (18). initial colonization can be defined as the period during, and immediately following birth when microbes first interact with and colonize the sterile infant. because microbial colonization of the mammalian neonate happens so quickly it is assumed that initial colonization happens from the birth canal or from the first exposure to the environment in neonates delivered by caesarean section (18). mode of delivery: vaginal or caesarean section since the mode of initial colonization is thought to happen during the birthing process, it can be assumed that the two different modes of birth, caesarean section or vaginal birth, would result in different infant microbial populations. indeed, mode of delivery impacts microbial population initially, but the differences minimize with aging (18). specifically, caesarean section was associated with both lower abundance and diversity of the phyla actinobacteria and bacteroidetes, and higher abundance and diversity of firmicutes from birth to three months of life. it was also found that bifidobacterium and bacteroides genera seems to be more frequent in vaginally delivered infants compared with caesarean section delivered ones (18). however, after six months of age, the microbial differences minimized. furthermore, it was found that delivery mode was not significantly associated with childhood development of obesity through a longitudinal study from infancy to 7 years of age (2). though they noted initial differences in microbial population with the different mode of delivery, the differences did not make a lasting impact and by 7 years of age the mode of delivery no longer had an impact on microbial population. length of delivery currently, there is no research on the impact of length of delivery on microbial population. the length of delivery can be hypothesized to impact initial microbial colonization because a longer birth would mean the infant spends longer in the birth canal, meaning more exposure to birth canal microbiota. this could be the case, especially in the birthing process of calves. for example, heifers generally have a longer delivery than cows because it is their first time through the birthing process. it is possible that having calves born from heifer or cow may impact the exposure to birth canal microbiota thus impact initial microbial colonization. however, it can be assumed that length of delivery would be similar to type of delivery in that though it may be initially significant, the differences in microbial population may not last into adulthood. however, if there is a difference in length of delivery, it is important to determine if those differences last into adulthood. factors affecting microbial establishment the establishment of the microbiota is a dynamic process following initial colonization, which depends on factors such as host genetics, diet, maternal environment, early antibiotic exposure, and highly sterile environments (9, 12, 13). host genetics the genetics of the host determine the microenvironment of the gut thus affecting the suitability of the internal environment for microbial colonization. an early study in mice found host genetics had a 12% impact on microbial variation in the gut (25). additionally, different researchers found a significant link between a mutation in human genes and a corresponding shift in gut microbiota characterized by a depletion of total numbers of bacteria, loss of diversity, and major shifts in bacterial populations within the bacteroidetes, firmicutes and proteobacteria phyla (11). moreover, another different research group was able to identify a core measurable microbiota of 64 conserved taxonomic groups using quantitative pyrosequencing of the microbiota and individual host genotype had a measurable contribution to microbial variation which can be explained by litter and cohort effects (3). through further statistical analysis they found suggestive genome-wide linkage with relative abundances of specific microbial taxa, providing clear evidence for the importance of host genetic control in shaping individual microbiome diversity in mammals (3). although there have been recent linkages between host genetics and microbial population, and microbial population and body composition (2, 3, 17), there has not yet been a genetic connection made between gut microbiota and long-term growth performance such as weight gain. diet during early life differences in microbial population have been found with different diets during early life (9, 12). breast-fed infants have a gut microbiota dominated by bifidobacteria, whereas formula fed infants have a more heterogeneous composition and less bifidobacteria (9). furthermore, formula with the more closely resembles maternal milk results in a microbial population that is dominated by bifidobacteria, like breast-fed babies (9, 12, 14, 20). these results suggest that the diet of infants can change the gut microbiota population through the establishment process. maternal environment like diet, prenatal maternal stress can influence the composition of the infant’s gut microbiota. maternal stress is hypothesized to change the maternal physiological environment, including increased heart rate and introduction of stress hormones (2). additionally, stress can also change the maternal environment behaviorally by increasing or decreasing the mother’s appetite, activity level, and potentially changing the mother’s diet. such change in maternal environment can affect the mother’s microbial population, and can potentially influence initial colonization of offspring at birth (8, 26). maternal stress can cause increase in cytokine concentration and inflammation, possibly influencing the developing fetus. this maternal immune response creates a different environment for the developing fetus potentially influencing, or changing their immune function development. in addition to maternal stress, other maternal states, like maternal fitness, can impact the infant’s microbial population. additionally, researchers have found a high body mass index (bmi) of the pre-pregnancy mother had a significant impact on the development of obesity in the offspring (2). further, the same study reported that if those children of a high bmi mother were treated with antibiotics early in life, it reduced obesity prevalence in these children (2). contrastingly, children born to normal neonatal mammalian microbiome • 9594 • fine focus, vol. 3 (2) bmi mothers and treated to early antibiotics were seen to have an increased risk of becoming obese later in life (2). such influence of the maternal body composition on the fetal microbiome can potentially be due to a change in the maternal microbial population influencing initial colonization of the fetus. further, because when children born from a mother with a high bmi had a decreased chance of developing obesity when they were treated with antibiotics early in life, suggests a potential cause for microbial influence (2). understanding, and acknowledging the impact of the maternal microbiota on the offspring’s microbiota is important when it comes to management and treatment because it is clear that maternal environment has an effect on the infant’s microbiota. knowledge gaps in studying early life: microbiota in calves the microbial colonization in cattle, especially in neonates, has profound impacts on nutrition, health, animal physiology and productivity (6). recent research outlining the importance of the microbiome in maintaining neonate health makes the microbiota a potential source of treatment for calf diarrhea (1). nevertheless, information on the calf microbiota is still limited and more research is necessary before the microbiota can be used to limit instances, and possibly even prevent, calf diarrhea. additionally, food safety (e. coli o157 colonizes the young claves) may be another area of possible research into the importance of the microbiome of beef cattle (6). by understanding the process of how neonatal mammals and microbes interact, it is possible to better target future research in order to solve the problem of calf diarrhea, or other related concerns. two interesting effects of the microbiota on dairy calves worth noting are the effects of antibiotic treatment on calf behaviour, and the maternal control on the calf`s microbiota. also, another important area of investigation is the long term effects of microbial on weight gain and body composition of cattle. the effect of the microbiota on behaviour is important to highlight in calves because when calves are treated with antibiotics for calf diarrhea, the antibiotics may reduce their microbial population, which may lead to exaggerated stress response. therefore, it is important to investigate if these results can be replicated in calves, and potentially investigate a probiotic for calves to reduce their stress response. especially a link between stress and immune efficiency has also been noted (5). the implications of antimicrobials and probiotics on calf behavior may be important for calf management. based on these findings, producers may have the options to give calves the probiotics after antibiotic treatment of calf diarrhea. another important aspect is the effect of the mother on the calf microbiota colonization, including maternal genetics and maternal environment. the relationship of host genetics to microbial population is important for calves because it suggests that potentially genetic selection, together with a probiotic, could be the solution to treat calf diarrhea. also, maternal environment is important to note when investigating potential treatments to calf diarrhea because potentially a change in management of pregnant animals may reduce instances of calf diarrhea. however, more research needs to be done to see the influence of the mother’s environment, and body composition, on the microbial population of the calf. in addition, it is a common practice to remove dairy calves from their mothers at a very young age, often increasing their risk of microbial infection because of this high stress period. there are many gaps in the knowledge of the calf microbiota that need to be addressed through research before microbial manipulation can be a potential treatment of calf diarrhea. to date, the understanding of the early life colonization in dairy claves is very limited and it is therefore important to research the role of calf gut microbiome in immune system development. since beef cattle are raised in less intensive production systems than dairy, there have been different areas of focus on each of them (15). while microbial investigation of dairy cattle has focused mostly on calf diarrhea, and mastitis, or bacterial colonization of the utter, the microbial focus of beef cattle has been on weight gain and feed efficiency. though dairy and beef cattle are different in production systems, and physiology, each bovine sector may benefit from collaboration. therefore, it is important to outline some recent advances in the beef industry. firstly, rumen variation between beef cattle has been noted between animals housed in the same environment and fed the same diet (6). thus, the genetic component, or possibly another undiscovered factor, influence the establishment of the gut microbiota. however, more research is needed to explain the observed individualized microbial variation. further, a significant relationship between weight gain and rumen microbiome was discovered when comparing the most and least efficient animals (17). regardless, more research needs to be done to investigate long term impacts of microbial manipulation on gut health to prevent calf diarrhea, and growth for better weight gain. conclusion due to the financial impacts of calf diarrhea and the increased occurrence of antibiotic resistance, it is important to find way to treat calf diarrhea that does not include the use of preventative antimicrobials (14). it is common for calves in the dairy industry to be given preventative antimicrobial during early life. recent research has identified the gut microbiota as a potential tool in reducing the incidence of calf diarrhea. however, research on the calf microbiome is limited. consequently, this review summarized the current understanding of neonatal mice and human microbiota to outline the importance of the microbiota, as well as microbial colonization and establishment. in addition, the knowledge gaps in calves have been identified. however, it was determined that future research is needed to investigate the relationship between early life microbiota and calf immune system, behavior, and metabolism (figure 1). additionally, further investigation is needed to identify the response of the calf microbiome to a change in host genetics, birth length, diet, and maternal environment. though the understanding of mammalian microbiota has had many recent advances, specific information on the calf microbiome is needed before microbial manipulation can be a potential treatment to calf diarrhea. more research is needed in order to understand how the calf gut microbiota may respond to manipulation. neonatal mammalian microbiome • 9796 • fine focus, vol. 3 (2) references 1. abe, f. 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(2010). interactions between gut microbiota, host genetics and diet relevant to development of metabolic syndromes in mice. the isme journal 4: 232-241. 26. zhou, d., zhang, h., bai, z., zhang, a., bai, f., luo, x., hou, y., ding, x., sun, b., sun, x., ma, n., wang, c., dai, x., & sugong, l. (2015). exposure to soil, house dust and decaying plants increases gut microbial diversity and decreases serum immunoglobulin e levels in balb/c mice. environmental microbiology 18: 1326-1337. neonatal mammalian microbiome • 9998 letter to the editor elizabeth a. b. emmert chair, asm task committee on laboratory biosafety professor of biological sciences, salisbury university, salisbury, md usa letter“ i commend fine focus for its article highlighting the importance of biosafety for microbiology undergraduate researchers (1). biosafety in the microbiology laboratory should be of utmost concern to all involved in undergraduate microbiology education including student researchers, students enrolled in microbiology lab courses, faculty research mentors and teaching faculty. the article by schwartz et al. included much valuable biosafety material, but i believe the article omitted two significant pieces of information. one was the laboratoryassociated infections most relevant to undergraduate microbiology students and the other was the american society for microbiology (asm) biosafety guidelines specifically developed for the undergraduate microbiology laboratory (2). the article by schwartz et al. emphasizes the importance of laboratory biosafety through its description of two laboratory accidents with unfortunate and deadly outcomes for the researchers involved who did not practice all aspects of proper biosafety (1). although these cases are tragic, neither involved an undergraduate student in the laboratory. unfortunately, infections and hospitalizations linked to microbiology teaching laboratories are all too common. the centers for disease control and prevention (cdc) has documented numerous cases of undergraduate students and their close contacts developing infections from exposures to organisms in the teaching laboratory. between november 2013 and may 2014, a total of 41 cases of salmonella typhimurium infection with strains known to be used in laboratory settings were identified (http://www.cdc.gov/salmonella/ typhimurium-labs-06-14/index.html). these cases came from thirteen different states and 86% of the affected individuals interviewed were enrolled in a biology or microbiology course. thirty-six percent of affected individuals were hospitalized. in 2011 another multi-state outbreak of salmonella typhimurium linked to clinical or teaching microbiology laboratories involved 109 cases and one death (http://www.cdc.gov/ salmonella/2011/lab-exposure-1-17-2012. html). in response to the 2011 salmonella outbreak, asm developed a set of biosafety laboratory guidelines that address the unique aspects of biosafety in the undergraduate microbiology laboratory (2). unlike the universal and extensive bmbl guidelines (3), the asm biosafety guidelines are valuable to students and faculty because they are explicit to the undergraduate microbiology laboratory experience. the guidelines address personal protection requirements, laboratory physical space requirements, stock culture requirements, standard laboratory practices, training practices, and document practices. biosafety guidelines were developed for work at both biosafety level one (bsl-1) and biosafety level two (bsl-2). a risk assessment including the microbes to be used and the laboratory procedures to be performed should determine the appropriate biosafety level. although schwartz et al. state, “an undergraduate microbiology teaching laboratory may be considered a bsl-1 laboratory,” many undergraduate microbiology labs should be operating at bsl-2. if organisms such as staphylococcus aureus, proteus vulgaris, or salmonella enterica are used or if students are subculturing unknown, environmental microbes, then the lab should be operating under bsl-2 conditions. an extensive appendix accompanies the asm biosafety guidelines to clarify and expand on the guidelines. (http://www.asm.org/index.php/ microbelibrary/laboratory-safety-guidelines). although these guidelines were published in 2013, it is likely that many faculty are 62 • fine focus, vol. 2 (1) still unaware of them. the fact that an nih researcher in the biorisk management program failed to mention these guidelines in an article about biosafety for microbiology undergraduates demonstrates the unfortunate obscurity of the guidelines. while faculty want to keep their students safe in the lab, they may be ignorant of the best biosafety practices. the reasons why faculty are uninformed about the asm biosafety guidelines are numerous. at many colleges and universities, faculty without expertise in microbiology are called upon to teach microbiology labs. faculty who are not microbiologists occasionally do laboratory activities in their courses (introductory biology, cell biology, etc) that use microorganisms. older faculty trained in an era with a cavalier attitude toward biosafety may not view biosafety as a significant concern. even conscientious microbiology faculty whose students follow commonly accepted biosafety practices (washing hands, disinfecting benches, no food or drink, etc.) may not realize that students should only use institution-provided pens or pencils that are kept in the lab. when students use their own pens in the lab, they can become contaminated, allowing students to unwittingly take microbes out of the lab that could then infect them or others. the article by schwartz et al. focuses on undergraduate microbiology researchers (1). although the asm biosafety guidelines were written for the microbiology teaching laboratory, the information is also applicable to the undergraduate research laboratory. certainly undergraduates performing microbiology research should be just as concerned with biosafety as undergraduates enrolled in microbiology lab courses. ideally such students have already been instructed in the best biosafety practices while enrolled in a microbiology lab course. if a student researcher is working with microbes and has not taken a microbiology course, the student should be sufficiently trained in biosafety before beginning the research project. faculty advisors should be aware of the biosafety training their student researchers have received – especially if a student took a microbiology course at a different institution – and tailor the biosafety training for each student accordingly. as faculty we set the example for our students when we stress the importance and required diligence of consistently following best biosafety practices. i wholeheartedly agree that good science is safe science! students and faculty should work together to make sure the best biosafety practices are being followed at all times in all undergraduate laboratories. as readers interested in undergraduate microbiology education, i urge you to do your part to spread the word about the asm biosafety guidelines and the importance of following the best biosafety practices. references 1. schwartz, a., clarkson, a., baumann, r.g., gentilli, s.m., potts, j., and torres-cruz, r. 2015. safe science is good science. fine focus 1:153-158. 2. emmert, e.a.b. and the asm task committee on laboratory biosafety. 2013. biosafety guidelines for handling microorganisms in the teaching laboratory: development and rationale. j. microbiol. biol. education 14:78-83. 3. chosewood, l.c. and wilson, d.e. (ed). 2007. biosafety in microbiological and biomedical laboratories (bmbl) 5th edition. centers for disease control and prevention. letter • 63 identification of diatoms in a healthy pennsylvania stream compared to three downstream sites impacted by abandoned mine drainage christopher m. arena, john l. galebach, thomas m. mandichak, j. michael engle, merrilee g. anderson* mount aloysius college, cresson, pa copyright 2014, fine focus all rights reserved manuscript received 29 april, 2014; accepted 30 may, 2014 life in a healthy stream can be severely impacted by changes in ph and other water quality parameters. this study reveals differences in diatom diversity and water quality characteristics in a central pennsylvania stream. one healthy site was compared to three nearby sites affected by abandoned mine drainage during a july sampling in 2013. permanent slides were made and microscopically assessed for diatom identification. the healthy stream contained eleven diatom genera while the site most impacted by mine drainage showed only one diatom, eunotia exigua. data were analyzed for shannon diversity index and species richness. water samples showed differences in ph, aluminum, sulfate, and iron. this work demonstrates the use of diatoms as bioindicators of stream health. abstract corresponding author * merrilee g. anderson manderson@mtaloy.edu keywords • diatom • abandoned mine • drainage • borehole • eunotia exigua abandoned mine drainage (amd) is a prominent source of pollution in currently and previously mined areas throughout the united states. amd impacted water is saturated with metals such as iron and may be very low in ph making it an inhospitable environment for the majority of aquatic life. hughes borehole is a source of amd pollution that flows into the little conemaugh river near portage, pennsylvania. the borehole was drilled in the 1920s to release water from miles of flooded underground coal mines in the area. the borehole was capped in 1950s only to blow out due to underground pressure some twenty years later. since then, water with a ph as low as 3.08 has been bubbling out of the borehole at a rate of 800-3500 gallons per minute and blanketing the surrounding six acres with a reddish brown iron precipitate. (2) mine drainage occurs in areas where water comes in contact with exposed rocks that have a high concentration of sulfide minerals. pyrite, also known as fool’s gold, is a common mineral found with coal in the eastern united states. the oxidation of pyrite and other sulfide-rich minerals causes the release of sulfuric acid and metal ions. if a stream has a limited buffer capacity, the ph will continue to decrease, thus increasing the oxidation reactions and the precipitation of metals. when the temperature of the water increases in the summer months, gases such as oxygen become less soluble and salts become more soluble. (1) diatoms are unicellular, photosynthetic algae which can survive in a wide variety of aquatic environments. each diatom species has a specifically shaped silica cell wall, called a frustule, which is used for microscopic identification. diatom species are found in two different microenvironments, they are either suspended in water (planktonic) or growing on a substrate (benthic). environmental factors such as ph, light availability, and temperature may cause teratology in frustule morphology, introduction 30 • fine focus, vol. 1 sampling was conducted in july 2013. diatoms were collected by harvesting biofilms from benthonic sediments by scraping a three centimeter square area into a sterile 15ml polypropylene disposable centrifuge tube (fisherbrand). two samples per site were gathered and processed in a ventilation hood by placing 2530 ml of the sample into a 150ml beaker on a hotplate, then adding 10-15ml nitric acid (flinn scientific inc.). samples were then boiled to remove organic matter, leaving behind only diatom frustules per sgro and johansen (7). centrifugation, decanting of liquid waste, and suspension in 10-15ml of distilled water was performed six times. permanent slides were created by suspending diatom frustules in 70% denatured ethyl alcohol (fisher chemical) until a cloudy suspension was achieved. samples were diluted to approximately 400 frustules per field of view on low magnification (100x total magnification) to allow clear observation on permanent slides. approximately 1ml of solution was placed on a glass coverslip and the alcohol was allowed to evaporate overnight, leaving behind only diatom frustules fixed to the coverslip. coverslips were permanently mounted on slides using naphrax mounting medium (brunel). slide sets of 24 slides were created for use in laboratories such as general microbiology and water ecology. diatoms were identified to the genus level using an online database, diatoms of the united states, (8) and a diatom identification text (6). from each site, 400 total diatoms were identified under oil immersion (1000x total magnification). diatom images were captured with a zeiss axiostar plus light microscope and spot imaging system with an in-sight camera and edited with spot version 5.0 software (spot imaging solutions). water analysis was sent to g and c coal analysis lab (summerville, pa) for testing of the suspended solids, dissolved metals and ph. these data were used in conjunction with the shannon diversity statistical analysis data. relationships between water quality and diatom diversity were examined. materials and methods and particularly in harsh environments can have an effect on overall growth characteristics of the diatom. these factors, most importantly temperature, can have an effect on solubility of salts and gases found within waters especially those impacted with amd, thus leading to large overall changes in water chemistry. fluctuations in water chemistry throughout the year due to temperature change can have an effect on diatom species present as well as seasonal variation in diatom populations. each diatom species has specific growth parameters and morphology giving us the ability to identify them by their frustule, making them good bioindicators of water quality. (6) this study was undertaken to assess diatom diversity in a healthy stream and three sites downstream from the amd outflow. the first site is the healthy stream, 40 m upstream of the amd discharge with a ph of 7.12. the second site is at hughes borehole, 5 m below the source of amd discharge due to safety fencing, with a ph of 3.36. the borehole sits uphill and is completely devoid of vegetation. the third site is a naturally formed settling pond, 50 m below the discharge, where the flow of polluted water slows and has a ph of 3.24. the fourth site is at a bridge 600 m below the amd discharge; roughly 30 m from where the healthy and low ph waters mix, with a ph of 6.68. fig. 1 illustrates the four sampling sites. applied/environmental • 31 diatom samples from the healthy stream showed the most genera present, eleven in total. the healthy stream site contained low species diversity due to one genus (navicula) dominating the population (table 1). shannon diversity index data for this site showed a fairly low diversity rating of 0.855 and a low richness (evenness) rating of 0.356. diatoms of the genus navicula made up 78% of the population in this location followed by surirella at 12% and eunotia at 3.5%. one other notable genus found in the sample, staurosirella, was present in trace amounts but only found at this particular site (fig. 2). the hughes borehole sampling site contained only one genus, eunotia sp. within in this genus however, we noted morphological size differentials. we observed either short and fat or long and narrow frustule morphology (fig. 3). at all other sampling sites eunotia was observed as being uniform in length and width. because only one species was present, the shannon diversity index was 0 and evenness could not be calculated. the settling pond site exhibited the poorest water quality from which we sampled but showed eight genera of diatoms in total. the population here was dominated by eunotia sp. which accounted for 79.9% of the population. pinnularia sp and navicula sp. made up 15.4% and 2.45% of the population respectively. the remaining species of diatoms were present in trace amounts (fig. 4). shannon diversity index data from this site were similar to that of the healthy stream with a lower diversity score of 0.670 and a similar evenness score of 0.344. the bridge sampling site 600m downstream showed the most diverse and evenly distributed population of all four sites. the shannon diversity score here was 1.01 and this was the most evenly distributed population with a richness score of 0.439. navicula sp. made up 73.2% of the sample but three other genera made up more than 5% of the population, surirella (9.97 %), eunotia (6.81 %), and planothidium (6.08%). (fig. 5) water quality data, comparing ph, suspended solids, total iron, sulfate, and total aluminum concentration in mg/l (table 2) were taken at each site. the healthy stream (ph 7.12), had the lowest measured concentrations of suspended solids, total iron, sulfate and total aluminum at 8 mg/l, 0.16 mg/l, .68 mg/l and 40.9 mg/l respectively. at the bridge site (ph 6.68), water quality data was very similar to that of the healthy site; total suspended solids were 9 mg/l, total iron was 1.58 mg/l, aluminum was 70.9 mg/l and sulfate was 1.16 mg/l. at hughes borehole and settling pond sites extreme amounts of metals were detected. at hughes borehole (ph 3.36), iron was found at a concentration of 82.45 mg/l and aluminum is was found at 571 mg/l as well as total suspended solids with a concentration of 10 mg/l and sulfate of 10.25 mg/l. at the settling pond (ph 3.24), sulfate concentrations are 9.64 mg/l much higher than both the healthy stream and bridge sites. iron was 76.67 mg/l and aluminum at 9.64 mg/l slightly lower than the borehole site whereas the settling pond showed a higher concentration of total suspended solids at 14 mg/l. results 32 • fine focus, vol. 1 fig. 1. this map shows the healthy stream and three downstream sites impacted by abandoned mine drainage. applied/environmental • 33 table 1. summary of diatom and diversity findings at a healthy stream compared to three sites impacted by abandoned mine drainage. site healthy hughes borehole (5 m downstream) settling pond (50 m downstream) bridge (600 m downstream) 0.855 0 shanon diversity index 0.670 1.01 evenness 0.356 0 0.344 0.439 number of diatom genera identified 11 1 8 11 fig. 2. on the right, the total number of diatoms counted in our healthy stream sample. on the left, diatoms from the genera surirella (top left), navicula (top right), staurosirella (bottom left) and eunotia (bottom right). 34 • fine focus, vol. 1 table 2. water quality characteristics from the four sampling sites. site healthy hughes borehole (5 m downstream) settling pond (50 m downstream) bridge (600 m downstream) 8 10 suspended solids (mg/l) 14 9 total iron (mg/l) 0.16 82.45 76.67 1.58 ph 7.12 3.36 3.24 6.68 sulfate (mg/l) 0.68 10.25 9.64 1.16 total aluminum (mg/l) 40.9 571 564.6 70.9 fig. 4. on the right, the total number of diatoms counted in the settling pond sample. on the left, diatoms from the genera encyonema (top left), pinnularia (top right), pseudostaurosira (bottom left), cocconeis (bottom right) discussion we hypothesized an association between diatom distribution and water quality data, predicting that amd impacted sites would exhibit reduced diatom diversity compared to the healthy stream. the healthy stream site essentially served as a positive control site for comparisons and showed eleven genera of diatoms present. this was to be expected considering it had a neutral ph of 7.12 and was low in dissolved metals, with aluminum measured at 0.68 mg/l and iron at 0.16 mg/l, both within normal ranges for stream health applied/environmental • 35 fig. 3. eunotia sp. found at hughes borehole. fig. 5. on the right, the total number of diatoms counted at the bridge site. on the left, diatoms from the genera diatoma (top left and bottom right), navicula (top right), and cocconeis (bottom left). (4). the dominant diatom at this site was navicula sp. making up 78% of the diatom population. navicula was also found at the other three sampling locations; this not surprising considering navicula can survive in a wide range of environments but will tend to thrive where water quality is the highest, making it valuable for assessing water quality and health of aquatic environments (3). the genus staurosirella was also observed at the healthy site, but was not found at any other sites, leading us to hypothesize that it could be used as a bioindicator of good water quality. staurosirella has been shown to thrive in moderate light and nutrient situations similar to the healthy stream we sampled from (5). the two heavily amd-impacted sites, hughes borehole and the settling pond, are fairly similar in water quality. both have an extremely acidic ph; the settling pond being slightly more acidic with a ph of 3.24 compared to a ph of 3.36 at the borehole. the two locations were also very high in concentrations of total dissolved solids and sulfates both of which are characteristic of amd impacted waters that come in contact with organic sulfur compounds such as pyrite (fes2). as the amd water emerges at the borehole, the slight increase in temperature changes the solubility of oxygen, removing it from solution and producing the iron precipitate found at the borehole and outlying areas. 36 • fine focus, vol. 1 there is a large “terrace” of precipitate as the water moves about 50m to the settling pond. bottom dwelling organisms such as diatoms are particularly sensitive to amd precipitates so we were surprised to find diatoms growing at both locations. the only genus found at hughes borehole was eunotia, most likely eunotia exigua. however, there were variations in morphology between eunotia frustules found at the borehole and those found at our other three sites. eunotia frustules at the other three sites all appear to be consistent in length, but what we saw at the borehole was individual diatoms within the population varying greatly in length but still showing all other morphological characteristics of the genus. we believe this morphological variability is due to lack of nutrients present within the borehole environment specifically the availability of silica, a known limiting factor of diatom growth. it is otherwise possible that we have two separate species of eunotia here. further research into this phenomenon is ongoing using dna sequencing and other molecular techniques. at the settling pond site we observed eight different species, which was unexpected considering the similar water chemistry to the borehole. eunotia sp. dominated this population making up 79% of the total diatoms found in our sample. eunotia at the settling pond did not show variable morphology like we observed at the borehole. possible explanations for this increased diatom diversity include changes in nutrient availability, inflow of diatoms from surrounding unimpacted environments, or creation of microenvironments from increased organic matter. another factor possibly contributing to the increased diatom growth at the settling pond could be due to a slight decrease of dissolved metals in solution. the precipitate found near the borehole appears to be coarse and quickly accumulates as the waters hit the surface. as this water makes its way 50m downstream to the settling pond the precipitate appears more as a mud or fine silt. this in conjunction with the micronutrients being added to this sampling site could be a possible explanation for the increased diversity of diatoms observed. the final site, the bridge, showed the most evenly distributed population of diatoms. the water at this site was still moderately impacted by amd; white aluminum and orange iron precipitate was clearly be observed. of the eleven species present, there were very few genera that differed from the settling pond site. the precipitate observed at the bridge, like the settling pond, was very fine in texture. the population was mainly navicula sp. (73.2 %), but three other genera made up over 5% of the population. this possibly shows that the genera present, specifically surirella, can survive in harsh environments at the borehole and settling pond sites but don’t thrive in that environment like at the bridge site where water quality was more suitable for life. the shannon diversity index measures overall species diversity, as well as richness or the evenness of the population. a site with a low richness score is often dominated by one or two main genera with the others only being present in trace amounts. shannon diversity data in the healthy stream and settling pond sites were very similar as both populations are dominated by navicula and eunotia. each site contained different diatom genera, but the ecosystem structure appears to be similar based on shannon diversity calculations at each site. this suggests that the particular diatom genera present may be a better indicator of water quality than the diversity and richness of the aquatic life present. the borehole, not surprisingly, had a diversity and richness applied/environmental • 37 1. banfield, j. f., bond, p., and s. smriga. 2000. phylogeny of microorganisms populating a thick, subaerial, predominantly lithotrophic biofilm at an extreme acid mine drainage site. appl. environ. microbiol. 66: 3842-3849. 2. burgos, w. d., and t. desa. 2010. laboratory and field-scale evaluation of low-ph fe(ii) oxidation at hughes borehole, portage, pennsylvania. mine water and the environ. 29, 239-247. 3. denicola, d.m., stapleton, m.g. 2002. impact of acid mine drainage on benthic communities in streams: the realative roles of substratum vs. aqueous effects. environ. poll. 119, 303-315. 4. hounslow, a.w. 1995. water quality data: analysis and interpretation. (united states): lewis publishers. 5. michael, t.j., saros, j.e., interlandi, s.j. and a.p. wolfe. 2006. resource requirements of four freshwater diatom taxa determined by in situ growth bioassays using natural populations from alpine lakes. hydrobiologia. 568: 235-243. 6. round, f. e., and r.m. crawford. 1990. the diatoms: biology & morphology of the genera. cambridge england: cambridge university press. 7. sgro, g.v. and j.r. johansen. 1995. rapid bioassessment of algal periphyton in freshwater streams. in: butterworth, f.m.; corkum, l.d., and guzman-rincon, j. eds. biomarkers as indicators of environmental change: a handbook. plenum press, n.y. pp. 291-311. 8. spaulding, s. a. 2010. home : diatoms of the united states. home : diatoms of the united states. retrieved march, 2014, from http://westerndiatoms.colorado. edu/ funding: nsf grant 1226175acknowledgements references score of zero due to only one genus being present. the bridge site, however, showed the most evenly distributed and diverse population of diatoms according to the shannon diversity data. the population at the bridge site was still dominated by one genus, navicula, but a few other diatoms emerged in numbers significant enough to balance the population. although the bridge site was impacted by the amd discharge, a healthy aquatic ecosystem was achieved according to shannon diversity index and higher number of genera observed. this suggests that even a slight change in water chemistry and substrate can have a huge effect on aquatic life, meaning only a small amount of remediation is needed at amd impacted sites to make a huge difference in overall stream health and ecology. samples for this study were collected in mid-july when water temperatures are warmer than other times of the year, particularly at the settling pond. we know from sampling in the winter months that some seasonal variation in diatom diversity does occur. changes in temperature can impact water quality factors such as the solubility of salts. with warmer temperatures, more nutrients are found in solution which can be filtered and used by diatoms. changes in temperature can also impact the solubility of gases such as oxygen. in the summer months oxygen can be removed from the solution more quickly due to decreased solubility. this leads to the reaction and precipitate formation occurring closer to the borehole and thus better living conditions in the settling pond conducive to increased diatom diversity. during the winter, this reaction may occur more slowly and have an effect on downstream diatom growth. future research will focus on the amount of seasonal variation within our sites as well as the overall effects of precipitate formation on diatom growth. 38 • fine focus, vol. 1 objective lens hannah dewitt thesis advisor: john l. mckillip ball state university, muncie, indiana, usa objective lens • 5 copyright 2019, fine focus all rights reserved introduction benefits of undergraduate research participation “this issue i have selected to use a metaanalysis by hanna g. dewitt (b.s., biology, ball state university, 2019) to provide a bit of a status report on our journal’s impact and logistics to interested readers.” -jlm the council for undergraduate research (cur) has been supporting undergraduate research initiatives since 1978 and supports over 900 colleges and universities in their endeavors and found that the benefits of undergraduate research are numerous (1). cur published a book in 2009 entitled “broadening participation in undergraduate research: fostering excellence and enhancing the impact” in which they list twenty seven benefits in four categories: gains in knowledge and skills, academic achievement and educational attainment, fostering professional growth and achievement, and promoting personal growth (4). these benefits included a range of areas such as the increase in critical thinking and problem solving skills, enhanced oral and written communication skills, improved ability to apply knowledge from the classroom to practice, enhanced professional credentials, elevated confidence, higher graduation rates, and possibly one of the most important, higher rates of acceptance into and enrollment in graduate and professional schools. the survey of undergraduate research experiences (sure) collected data on undergraduate researches and their progress over a 9-month period. they found that of the 1135 students that they surveyed (with a 75% response rate), over 87% responded that they had plans or had already begun further education in the sciences. an overwhelming response from students showed that their undergraduate research experiences were a positive influence in this decision with 29% stating that their decision to pursue a phd was directly connected to the experiences of their research project (5). as students become more confident in their field of study they gain a better understanding of the potential work that might interest them in the future, leading them to continue their education to pursue a passion they might not have experienced without an undergraduate research opportunity. having the opportunity to explore research in a safe, structured environment allows for exploration and personal discovery. only 4.2% of students responded that their plans to pursue a postgraduate education had changed after their undergraduate research experience but even that information is valuable. this demonstrates that research experiences are key for students to be able to explore their field of study outside of the standard classroom structure to discover if research is the path they want to take. learning that a passion for research exists is equally as important for a student as learning that research might not be the career path they want to take. publishing as an undergraduate can have lasting effects on future endeavors. publishing research findings is a crucial part of expanding our understanding of the world, especially for those in the stem (science, math, engineering, and technology) field. if a student does have the opportunity to publish work they have been conducting they gain a permanent example of their commitment to their research field. publishing early can show graduate school and employers that a student has dedicated their spare time and effort into advancing their understanding of the scientific world to benefit the collective base of knowledge. this collaboration and show of commitment can enhance their professional credentials and establish a network of scientists that they can rely on for mentorship and further education (4). students also become familiar with the process of scientific peer review and publication early. this can improve their understanding of what is needed to construct a solid, publishable article before it is a requirement of their education or career field. sri international, a nonprofit scientific research institute, conducted a nationwide study of stem undergraduate research opportunities with over 15,000 respondents. their results showed that “(88%) of the respondents to the nsf follow up survey reported that their understanding of how to conduct a research project increased a fair amount or a great deal, 83% said their confidence in their research skills increased, and 73% said their awareness of what graduate school is like increased” as well as 29% reporting that they 6 • fine focus, vol 5 2019 course-based undergraduate research experiences what is fine focus? had new expectations for pursuing a phd (9). this increased confidence and awareness of scientific process elevates their potential success in future careers. what is cure? cure is defined as coursebased undergraduate research experiences. they are designed to incorporate an entire classroom of students on a research topic within the course-work itself, allowing an increasing amount of students to participate in gaining experience working on research related to their field of study (2). course-based opportunities allow institutions to provide research experience to underrepresented minorities, first generation, and at-risk students which has been found to improve retention rates in these groups significantly (7). according to the course-based undergraduate research experience network (curenet), an organization founded in 2012 to better understand the benefits and potential roadblocks to cures, there are five elements that must be present to enhance student learning: the use of scientific practices, discovery of knowledge previously unknown to the student and often the faculty member, work that was relevant or important to the field of study, collaboration amongst students and faculty, and iteration of the research conducted (3). cure has been implemented across the country with positive results. the genomics education partnership (gep) has created a structure of nearly 100 universities in the united states to provide students a unique way to explore the field of genetic research. students worked on sequencing the genome of the drosophila, commonly known as the fruit fly. as a result of their work, more than 100 students and faculty have become coauthors on a paper that compares the f element of four species of fruit fly (8). cures can also come in the form of field research, such as is the case in a program started by the university of minnesota which sought to provide students interested in ecology, animal behavior, and aquatic biology with an opportunity for hands-on work within a course-based curriculum (9). the fiveweek summer course resulted in positive feedback from the students and high-quality written reports that they will be able to use for future research and publication. scope: fine focus is a digital and print journal dedicated to showcasing the research of undergraduate students, internationally, in all fields of microbiology. fine focus is managed entirely by undergraduate students from production to print and coordinates double-blind peer reviews by our editorial board of experts from all subdisciplines of microbiology. mission: fine focus publishes original research by undergraduate students in microbiology. this includes works in all microbiological specialties and microbiology education. research in other biology disciplines will not be accepted unless the main emphasis of the work centers on microorganism(s). at a glance: fine focus is comprised of 10-20 undergraduate ball state university students under the direction of the faculty advisor, dr. john mckillip. each semester, the students serve as managing editors for the manuscripts that are submitted via the open journal system (ojs). the journal is managed entirely by undergraduate students from production to print, but utilizes an external editorial board of experts for double-blind peer review of manuscripts. submission of manuscripts to fine focus is free and acceptance into the journal carries no page charges, indicating that it qualifies as an open access journal. those who wish to read the journal or select manuscripts are able to receive free paper copies in the mail or access the material for free online at finefocus.org. manuscripts that are published in fine focus are indexed and searchable through cardinal scholar, ball state’s online institutional repository for scholarly works contributed by students and faculty. objective lens • 7 meta-analysis for this project, we focused on the data that has been collected on each manuscript submitted to fine focus, which included the author’s names, what institution they were submitting from, the time between decisions, and a list of decisions that had been made for each manuscript. we also took time to look at the composition of each journal issue. this allowed us to dissect the information into quantifiable points that showed the progress of the journal. these points included: acceptance vs. rejection rate, international submission numbers, location of international submissions, and the composition of each journal (page numbers and manuscript type). the graphs below show the results of this analysis: the acceptance rate was found to be 52%. there were no concerns with the current percentage. this number will be used to track future progress of the journal and can be used to market to potential authors who wish to publish. the number of pages per issue have varied significantly from issue to issue. the average number of pages was roughly 94, with the lowest number of pages being issue 2.2 with 62 pages and the highest being issue 4.1 with 143 pages. based on these numbers, we can suggest that if the journal were to move from publishing twice a year to just once a year, the number of pages would need to stay relatively high to ensure the quality and number of manuscripts is not sacrificed. the graph above shows the composition of each journal based on the type of manuscript that was accepted and published in that issue. manuscripts for fine focus tend to fall into two categories: applied/environmental and work conducted on pathogens. the journal also accepts reviews on topics related to microbiology as well as manuscripts on microbiology education. in the past, the journal has only accepted one review that 8 • fine focus, vol 5 2019 matched the standards of the journal. the recent issue showed a decrease in manuscripts published in the applied/environmental category. in the future, the journal hopes to increase this number by focusing efforts to encourage students and institutions that are working on research in this category to publish their work in the journal. fine focus is an international journal that not only has external reviewers from nine countries but features manuscripts from all over the world. recently, we have seen a sharp decrease in international submissions with the reason being unknown. the class of students that manage the journal have begun a program called cap, the campus ambassador program, that is seeking to establish satellite groups of students at other universities that would encourage their fellow students and faculty to submit their research to fine focus. the map above shows the distribution of international submissions that have been received by the journal from 2014-2018. the hope is to further the reach of the journal and encourage more countries to submit their research. to reach seven countries in four years is still significant and shows that the journal is a viable choice when undergraduates from across the world want an established journal to showcase their work. objective lens • 9 10 • fine focus, vol 5 2019 objective lens • 11 the chart above shows information on other undergraduate research journals in the stem fields. all the criteria is based on elements that fine focus is able to offer to its authors and readers. checkmarks represent characteristics which the journal shares with fine focus while empty spaces represent unshared characteristics. a dash (-) represents information that was unable to be obtained from the websites of each of the journals. most information was collected directly from the respective websites that each journal maintained as emails requesting clarifications were mostly left without answer. the chart demonstrates how fine focus fits within the established realm of undergraduate research journals and can help potential authors understand what the journal has to offer. the future the future of fine focus is vibrant and powerful. fine focus was the first international journal specifically for undergraduates that wish to publish work related to microbiology. according to the bureau of labor statistics, demand for microbiologists will increase by 8% between now and 2026 (11). with the growing demand for hardworking and dedicated researchers, publishing early and often has never been more important. as the journal continues to grow and develop, we hope to see an increase in submission overall as well as those from diverse countries. the class structure of the journal allows for new students to experience the publishing process, gaining a perspective on what happens to a manuscript that has been submitted for review. not only are students able to work together to achieve a common goal of managing the journal from start to finish, they are able to gain a better understanding of what it will take to have their own research published in the future. the hope for this research is to inform the students that manage the journal semester to semester, as well as the executive committee, on how the journal should proceed in the future. 12 • fine focus, vol 5 2019 1. petrella, j. k., & jung, a. p. (2008). undergraduate research: importance, benefits, and challenges. international journal of exercise science, 1(3), 91–95. 2. mccartney, m. (2018). a cure for undergraduate research. science, 360(6396), 14171418. 3. auchincloss et al. (2014). assessment of course-based undergraduate research experiences: a meeting report. 4. in boyd, m. k., in wesemann, j. l., frederick, k. a., & council on undergraduate research (u.s.),. (2009). broadening participation in undergraduate research: fostering excellence and enhancing the impact. 5. lopatto d. undergraduate research experiences support science career decisions and active learning. cbe life sci educ. 2007;6(4):297–306. 6. t. orr, k. watarai, y. watanabe, y. wakisaka, s. yamamoto and r. komiya, (2010). effective strategies for publishing as an undergraduate in science, engineering, and other fields. ieee international professional comunication conference, enschede, 289-296. 7. shaffer cd, alvarez cj, bednarski ae, et. al. (2014). a course-based research experience: how benefits change with increased investment in instructional time. cbe life sci educ. 13, 111–130. 8. lopatto d., hauser c., jones c. j., paetkau d., chandrasekaran v., et al. , (2014). a central support system can facilitate implementation and sustainability of a classroom-based undergraduate research experience (cure) in genomics. cbe life sci. educ. 13: 711–723. 9. thompson s, neill c, wiederhoeft e, cotner s. (2016). a model for a course-based undergraduate research experience (cure) in a field setting†. j. microbiol. biol. educ. 17(3):469471 10. russell, s., hancock, m., & mccullough, j. (2007). benefits of undergraduate research experiences. science, 316(5824), 548-549. 11. bureau of labor statistics (2016). occupational outlook handbook. references objective lens • 13 copyright 2017, fine focus. all rights reserved. ronda l. hamm intellectual property portfolio development patent liaison biological solutions dow agrosciences llc, indianapolis, in perspective“become a spokesperson for science 68 • fine focus, vol. 3 (1) the importance of outreach: why do it? corresponding author ronda l. hamm rlhamm@dow.com keywords • outreach • scientific literacy • stem the national science education standards defines scientific literacy to include a “greater knowledge and understanding of science subject matter” and “the role of science in society and personal life (1).” it is important for all members of society to understand the importance of science in our lives as they will be voting on issues, making purchases, and making health choices that involve scientific understanding. often these decisions directly impact the various industries and organizations we serve as scientists. there is a public perception that science is difficult and is typically done by albert einstein stereotypes in a laboratory with chemicals and strange looking glassware. this has been proven many times in classrooms where students have been asked to draw a scientist, and although there is some variation around the world, they typically depict a male with glasses or goggles and erratic hair (2). it is our responsibility as scientists to help change that perception and broaden the view of what scientists are. we also need to be prepared and able to respond when we see or hear stories that contain scientific inaccuracies being shared between friends, posted to social media or covered by various mainstream media outlets so that we can better stop the spread of misinformation. let’s explore a couple of examples. biotechnology, also known as genetically modified organisms (gmos), has been a target for both media outlets and marketing campaigns using scare tactics to try to influence consumers to purchase organic or non-gmo products. these campaigns and stories do not focus on the science, but rather imply that organic food products are healthier and non-gmo products are safer. despite the agreement of many esteemed scientists regarding the benefits of gm crops, and 121 nobel laureates who signed a letter calling upon governments around the world to not only approve but accelerate the access of farmers to the improved seed technology (3), some consumers at the grocery store continue to be perspective • 69 unwilling to trust the scientific experts when selecting food purchases. we see the same mistrust of scientists regarding the use of vaccines. the number of parents immunizing their children has decreased to about 60 percent in the u.s. (4). one of the contributing factors to this decline is recent negative media coverage, perpetuating the idea that the vaccines may cause autism or other side-effects. this leaves vulnerable members of the population at higher risk for diseases like measles that were once nearly eradicated. these kinds of stories make headlines but they are harmful to science and contribute to a decline in the general impression and trust the public has regarding science and scientists. as scientists, we can change the impact of the public’s overall impression of science and we can serve as a knowledgeable and reliable resource to answer questions. trust is an important part of this scenario. as scientists, we know the scientific process continues to ask questions on topics and the continuous testing of theories or hypotheses prove or disprove our current understanding, making the science selfcorrecting in this respect. we might not have all the answers but given the body of evidence provided we draw logical conclusions about our field of study. but how do we help the public understand this and trust this process? you can start building trust and breaking down certain stereotypes about scientists by becoming involved in community outreach. if scientists from various backgrounds serve as mentors, we can illustrate the diversity of those working in the sciences and the variety of topics we study. we do not all fit a single mold of a middle-aged male in a lab that is still too commonly thought of today (2). our research may take us anywhere—from the far reaches of the world to our very own backyard. showing the public that some scientists study geology in antarctica, others might collect house fly populations from dairy farms in australia, while some may take water and soil samples from our local rivers and pastures. illustrating the range of jobs in the scientific community and sharing stories about how this work leads to technology that can improve people’s day-today life will help begin to broaden the public’s overall understanding of the positive impact the scientific community makes to the world. it’s important to be willing to share our stories. students are one of the most important groups for scientists to focus on. in my opinion, one of the most rewarding experiences of doing outreach is when a student comes up to you and says you changed the course of their studies or influenced their career choice. as role models for future generations of scientists it’s important to note the need for growing the number of students pursuing science degrees. the u.s. bureau of labor statistics projects that there will be 9 million more stem jobs available between 2012 and 2022 (5). the president’s council of advisors on science and technology (pcast) states that 1 million more stem college graduates will be needed over the next decade (6). in addition, pcast estimates that “fewer than 40% of students who enter college intending to major in a stem field complete a stem degree” (6). by being role models for science you can contribute to improving the retention rate of students majoring in stem degrees. giving back to the community helps me see how one person can have such an impact on another. bringing scientific enrichment opportunities to those who may have none is something that has been a passion of mine. for the last several years, i have worked with the indianapolis school on wheels, an organization that provides tutoring and educational enrichment to students experiencing homelessness. at the school on wheels locations, colleagues and i present a variety of hands-on science demonstrations for this group of students. these hands-on activities were created by dow agrosciences’ volunteers—known as the science ambassadors. 70 • fine focus, vol. 3 (1) another personal experience was when i was a graduate student and worked with local educators to provide interactive hands-on activities presented by undergraduate mentors for their classes. students and classroom supervisors were motivated to learn new subject material and new ways to present or experience science in their classroom. we can all give back to our communities and serve as scientific mentors at any age or level of education. impact on developing your skills as a scientist: what do you gain? one of the best ways to illustrate your competency in a subject is to be able to effectively communicate it to others. learning to tailor messages to your audience is key. explaining what you do to a group of third graders is different than explaining your job to a group of college students—despite the general ideas of the subject matter being discussed is the same. regardless of age, if the person you are trying to reach is outside of your specific scientific field of study, scientific jargon can easily make them feel like an outsider in the discussion. one way to keep people involved is to consider everyone a learner; try to understand what information will be new and how best to present that, explaining critical vocabulary words. creativity in delivering your message can also be a way to help engage the audience during your outreach talks and activities, but it is often one of the hardest. i personally find this to be one of the most challenging aspects of science outreach. how can i create an experience for someone during my outreach interactions so they will connect with the subject? i do not merely want them to see the subject demonstrated; i want them to interact with it so that they will care about the subject longer than the one-time presentation. my undergraduate courses in education and communication have helped me in this regard. communication can come in a variety of forms: public presentations, written papers, social media, etc. my personal preference is to have face to face interactions, because it is easier to see how my message is coming across and know if i need to further explain topics based on the instant feedback of my audience’s body language and engagement—or lack thereof— in conversation. for example, i sometimes use skype to connect with students located in different parts of the country. snapchat, facebook, twitter, blogs, and other social media channels all have uses in communicating science to the public because this is where the public is easily accessible. the mode of communication you should choose comes down to what you are most comfortable with and good at. some people are better performing live demonstrations, while others have made a name for themselves doing scientific outreach through blogs and youtube videos. the main objective is to communicate your science in a relatable way, this helps support all sciences because it helps the public build trust and puts science and scientists in a positive light. outreach activities also require you to demonstrate many of the soft skills that perspective • 71 employers desire in job candidates. there is the leadership you show by getting involved and planning an outreach event or activity. building your organization, creativity, and ability to communicate your science in an understandable way are skills that are important to career success in the scientific world. scientists volunteering to do outreach activities also develop time management skills as almost all outreach is performed beyond the traditional course curriculum or work hours. outreach also forces you to think about your own research differently, from another person’s perspective. the confidence you have in yourself and your research hits a high when you see someone else understand your research through outreach. i had a student at a science day look at my vinegaroon, (a whip scorpion that i use for entomology discussions), from afar. it was obvious that she was clearly nervous but also curious. i started asking her questions about why she was nervous and assured her that he didn’t bite, pinch, or sting and that he had some interesting characteristics. we continued to talk about the fact that the only way he defended himself was to spray acetic acid. i followed that up by telling her that acetic acid is vinegar which she’s probably had on her salad. by the end of a few minutes of conversation she had approached the table and was reaching out to pet the creature that she initially feared. she even said he was kind of cute. this small breakthrough for this one person i believe will have an impact the next time she sees a “scary” looking arthropod, which in turn will impact how she sees the study of these creatures. building your network you’ve probably already heard how important networking is in the job search and in professional development. outreach not only allows you to practice talking to people and explaining your work, but it automatically expands your network. depending on the type of activities you get involved in you will be working with members of your department, across departments within your university, and your field of study across universities. the professors you work with on outreach events can be great resources for future letters of recommendation for graduate school or job applications. or, if your outreach program takes you across many different universities, you might find a faculty member with which you would like to pursue graduate studies. if the event is sponsored by an external organization, it is likely you will meet others doing public outreach for other institutions or companies. these contacts can provide mentorship and possibly introduce you to job opportunities. my job at dow agrosciences came about this way. i was involved in outreach in graduate school and attended the entomological society of america meeting. there was a symposium on outreach that i attended and several the speakers were industry representatives. after the talks, i asked the organizer if she could explain how she as an industry professional was involved in outreach, and why. she explained that it was important for industry scientists to be talking to the community about the work we do in agricultural sciences. during that same conversation, she asked me when i was graduating and i pulled out my cv—noting that i still needed to finish school that spring. the next thing i knew i received a phone call asking me to fly to their global headquarters for an interview, and now i have been an employee at dow agrosciences for 8 years. 72 • fine focus, vol. 3 (1) if you choose to participate in science outreach programs, you will also be interacting and networking with the community participating in your event. this could include students, teachers, parents, the public, media, and scientists in other fields. community events give you a significant platform to reach a large audience, but it is good to prepare before the event so you can clearly communicate your message and explain why you are there and what you are demonstrating. you want to sound confident and at ease with your subject, not nervous and unsure. the best way to overcome that is to be prepared and practice frequently. having your answer ready if you get asked will help you sound like the expert that you are. science outreach: i’m interested but how do i do it? the easiest way to get involved is with an event that has already started. for example earth day would be a great time to do a special activity in the community. if you’re a chemist, the american chemical society sponsors national chemistry week each year. in the indianapolis area, there is a large celebrate science indiana event held each october. investigate what the various clubs and organizations on campus are doing. many universities offer summer camps for children where you could volunteer. if you’re still having trouble finding an event, talk to faculty or others doing science outreach and see what suggestions they have to offer. i’m happy to help you get something started in your local area if you need help. if you want to take it a step further, demonstrate your leadership and passion for a particular area and start your own event. there’s nothing like the feeling of starting an event that becomes so popular that it remains long after you have graduated and moved on. as a graduate student, i had the opportunity to work with faculty at cornell university to develop the first ever insectapalooza, a one day open house event in the entomology department (7). this event is now in its thirteenth year and continues to grow! insectapalooza highlights the diversity of insects and entomology research. activities have included everything from insects in the movies to roach races to citizen science projects to insect physiology. the entire department is engaged, which allows undergraduates, graduate students, and faculty to work together to develop the materials and activities for the event. the day of the event everyone interacts with the public. undergraduates from other disciplines also participate. we’ve partnered with the art department to showcase insects and have communications students doing interviews for stories in the university newspaper. another option is to be a guest speaker for local schools. as an undergraduate i would often go back and talk to students at my former elementary school and high school. contact someone at your elementary, middle, or high school and see if you can come speak to a class or organize a larger assembly and bring some of your college colleagues to each do activities. if you have moved away from your hometown for college, get to know some of the teachers near your university. many teachers are eager to have students come in to talk about science. you provide a role model for students to aspire to and help the teacher with knowledge they might not have. perspective • 73 conclusions scientific outreach is an important endeavor that is often overlooked in the day to day aspect of coursework and research. work in this area impacts our community’s opinions and understanding of science. personal impacts include a sense of pride in the work you do, giving back to the community, building upon skills, and making connections with others. there’s freedom and creativity in expressing your chosen scientific field and connecting that with others. overall it’s rewarding and fulfilling to set aside a day or even an hour to talk about something you have invested your time and talents into studying. if you are looking for ways to get involved in scientific outreach talk to others with a similar interest and start or contribute to an event in your area. as nike so famously put it— “just do it!” thank you to all the professors that supported and helped develop my passion for outreach. thank you to dr. john mckillip for inviting me to contribute this publication and to dr. ryan jeske and jenna marston for assistance in reviewing this publication. thank you to the dow agrosciences corporate management team for supporting the science ambassadors program and to all the employees that participate in outreach activities. 1. (1996) national science education standards chapter 2: principles and definitions. p.21. https://www.nap.edu/ read/4962/chapter/4 2. hamm, r. l. and l.s. rayor. (2007). insectapalooza: practical suggestions for pulling off a large entomology outreach event. american entomologist 53(1): 12-14. 3. largent, m. a. (2012). vaccine: the debate in modern america (johns hopkins univ press, baltimore, md) 4. “laureates letter supporting precision agriculture (gmos).” letter to the leaders of greenpeace, the united nations and governments around the world. 29 june 2016. web. http://supportprecisionagriculture.org/nobel-laureate-gmo-letter_rjr.html. 5. mccann, f. f. and marek, e. a. (2016). achieving diversity in stem: the role of drawing-based instruments. creative education 7: 2293-2304. 6. pcast stem undergraduate working group (2012) engage to excel: producing one million additional college graduates with degrees in stem, eds gates, sj, jr, handlesmand j, lepage gp, mirkin c (office of the president, washington, d.c.) 7. vilorio, d. (2014). stem 101: intro to tomorrow’s jobs. occupational outlook quarterly. http://www.bls.gov/ careeroutlook/2014/spring/art01.pdf. acknowledgements references • fine focus, vol. 3 (2) imatinib mesylate as an effective anti-viral treatment for alphavirus infections jessica l. costlow, erika s. krow, & j. jordan steel* biology department, colorado state university-pueblo, pueblo co manuscript received 29 march 2017; accepted 14 may 2017 copyright 2017, fine focus. all rights reserved. imatinib mesylate • 141140 • fine focus, vol. 3 (2) alphaviruses are plus-strand rna viruses that are transmitted by mosquitoes. there are very limited vaccines and treatment options available to those infected with alphaviruses, resulting in significant human and animal morbidity and mortality each year. viruses are parasites of host cell metabolism and alphaviruses have been shown to increase glycolytic flux during infection to aid viral replication. imatinib mesylate is an fda-approved tyrosine kinase inhibitor that is used to treat several types of cancers. a hallmark of tumorous cells is an elevated metabolic rate and imatinib successfully slows metabolism by inhibiting tyrosine kinases that are required to activate metabolic enzymes, such as hexokinase in the glycolytic pathway. it was hypothesized that imatinib could be used to slow metabolism in virally-infected cells and reduce viral replication. alphavirus-infected cells were treated with various concentrations of imatinib and at a concentration of 6 μm, viral replication was reduced by more than 40% while cell viability was still at 100%. the efficacy of imatinib treatment at inhibiting alphavirus replication was confirmed at different times post infection (6, 12, 18, and 24 hours post infection), different levels of infection (multiplicities of infection= 0.1, 1, and 10), and within different cell lines (bhk, huh7 and hek). further analysis in mouse or other animal models is needed to confirm the utility of imatinib as a therapeutic option for treating alphavirus infection, but the data are promising and shows a significant reduction in viral replication and may represent a novel treatment option for alphavirus infections. abstract corresponding author j. jordan steel* jordan.steel@csupueblo.edu keywords • alphavirus • anti-virus • imatinib alphaviruses are enveloped, plus-strand rna viruses, that are transmitted via mosquito vectors. alphaviruses, including eastern, western, and venezuelan equine encephalitis virus, chikungunya virus, sindbis virus, and semliki forest virus, are responsible for millions of infections each year (15, 22). chikungunya virus recently spread to north, south, and centralamerica and resulted in more than 2.9 million confirmed and suspected cases, with nearly 300 deaths as of summer 2016 (11, 26). alphavirus infection results in a wide range of symptoms including rash, malaise, chronic arthralgia, and can even lead to fatal encephalitis (2, 23, 30). there are few effective treatment options available for those infected with alphaviruses and a novel method to treat and inhibit alphavirus infections is imperative to public health. traditionally, antiviral therapies have been designed and engineered to target viral proteins, instead of host cell proteins in order to prevent cytotoxicity to the host cells (17). however, due to the rapid rate of mutation and diversity within viral species, viral specific drug treatments are limited in efficacy and burdened with the rapid development of drug resistance. new strategies involve altering or limiting cellular pathways that the virus relies on and thereby reducing the viral replication capacity of the host cell (4). all viruses are parasites of the host cell metabolic pathways and depend on the host cell for all biomolecule and energy synthesis requirements. many viruses, including alphaviruses, have been shown to manipulate cellular metabolic pathways to enhance the cellular environment and make it optimal for viral replication (8, 9, 19, 29). sindbis virus (sinv) is the prototype alphavirus and is commonly used as the model virus for other alphaviruses. for sindbis virus, an elevated glycolytic rate appears to benefit the virus replication(6). it was hypothesized that drugs that inhibit or reduce metabolic rates may effectively be used to slow and inhibit viral replication. metabolic inhibitors have been studied extensively in the cancer research fields. cancer is essentially a metabolic/cell division disorder where cells grow uncontrollably with an elevated metabolic rate. many cancer drugs target this elevated metabolism and reduce or slow down metabolism to normal levels, thereby inhibiting cancer growth and metastasis (27). due to the widespread burden of cancer, there has been much research in metabolic inhibitor drugs and there are several options that are fda-approved and are used clinically to treat cancers (5, 10, 16). viral infections induce a similar elevated metabolic phenotype in host cells and some of these metabolic inhibitor cancer drugs have been repurposed as novel treatment options for viral infections. imatinib mesylate, or gleevec®, is a tyrosine kinase inhibitor that prevents the phosphorylation and activation of key enzymes in metabolic pathways (1). specifically, imatinib has been shown to reduce the activity of hexokinase, which is a key enzyme in the glycolysis pathway (3, 18). sindbis virus is dependent on glycolysis and it was predicted that inhibiting the glycolytic pathway would result in less viral replication. we hypothesized that imatinib mesylate, which is already used clinically, may be repurposed and effectively used to treat alphavirus infection. here we present our findings that imatinib mesylate successfully reduces sindbis virus replication in cell culture at low enough concentrations that do not affect host cell viability. investigating different imatinib concentrations, viral titers, infection times, treatment addition times, and various cell lines have all shown imatinib to be efficacious at inhibiting sindbis virus infection. methods cells and cell culture baby hamster kidney (bhk) cells were grown in dulbecco’s modified eagle medium (dmem) media supplemented with streptomycin/penicillin and 10% fbs. cells were incubated in t75 flasks or 24-well culture plates and kept in an incubator at 37ºc and 5% co2. cells were passaged every 3 days once 85% confluent using pbs and 0.25% trypsin. cells were counted and seeded into well plates (24-well) 24 hours before an experiment so introduction imatinib mesylate • 143142 • fine focus, vol. 3 (2) imatinib mesylate • 145 that the cells would be about 70% confluent at the time of infection/treatment (75,000 bhk cells/ml= ~37,500 bhk cells/well). human hepatoma (huh7) cells and human embryonic kidney (hek) cells were maintained in similar conditions but were passaged every 4 days. virus and viral infection the 100 +/10 mg of fresh rind and curd sasindbis virus, which belongs to togaviridae and is an old world alphavirus, was used as the model alphavirus for all experiments. a green fluorescent protein (gfp) reporter was previously inserted into the viral genome via a duplicated subgenomic promoter at the 3’ end of the viral genome. this gfp is concurrently expressed with the viral proteins and the relative gfp fluorescence serves as a direct indicator for viral replication in infected cells. these double subgenomic alphavirus reporters are well established and used widely in the alphavirology field(21, 23, 28). prior to infection, a sample well was used to count cells. cells were trypsinized, resuspended, and counted using trypan blue staining and a hemocytometer. once an accurate count of cells/well was available, the amount of virus could be calculated and added to the specified wells. cells were infected with a multiplicity of infection (moi) of 1, unless otherwise noted. moi is the ratio of infectious virus particles (plaque forming units (pfu)) to number of cells and an moi=1 will infect a majority of cells in the well. the corresponding amount of virus was added to the total volume needed and then was added to each well being infected. the plates were incubated for 1 hour at 37ºc to allow the virus to infect and enter the host cells. following 1 hour of infection, the media containing the virus was removed, the cells were washed with pbs, and fresh media (with or without the treatment) was added to the cells. the infected cells were put back in the incubator and allowed to infect for the specified time. imatinib treatment imatinib mesylate was purchased (sigma) and resuspended in h2o to make a 100mm stock. this 100mm stock was then serially diluted with two-fold dilutions to acquire stock concentrations at 50mm, 25mm, 12.5mm, 6.25mm, 3.125mm, and 1.56mm. to treat the cells, the mm stocks were diluted 1:1000 in cell culture media to provide media with 100μm, 50μm, 25μm, 12.5μm, 6.25μm, 3.125μm, and 1.56μm concentrations of imatinib in cell culture media that could be added directly to infected cells. a control treatment was prepared by diluting h2o solvent 1:1000 in media following the protocol as the drug dilutions. the dilutions of imatinib in cell culture media were always prepared fresh for experiments. for the experiments with different times of imatinib addition, the media was removed at the specified time and replaced with fresh dmem containing the corresponding dilution of imatinib or the solvent control. cytotoxicity of imatinib and plate reader cell viability was assessed using the alamarblue cell viability assay® from thermofisher according to manufacturer’s protocols. briefly, imatinib treated cells were stained with a concentration of 100μm resazurin/alamarblue and allowed to incubate for 1 hour at 37ºc. following the one hour incubation, the plates were analyzed on an enspire multimode plate reader ® from perkinelmer for fluorescence at an excitation of 540-570nm and an emission of 580-610nm to measure the viable cells. the mock/control treated cells were set to 100% and then the viability readings from the treated wells were calculated as a percentage of the control. all experiments were performed and analyzed multiple times in triplicate. quantifying virus replication and flow cytometry virus replication was quantified based on the gfp reporter inserted into the viral genome. gfp was measured in infected/treated cells using the plate reader (see above) and an excitation of 488nm and an emission filter of 509nm. additionally, cells were trypsinized, resuspended, and run over a guava easycyte® flow cytometer from millipore to assess the percent of cells infected (expressing gfp) and the relative levels of gfp fluorescence per cell. 5,000 cells were analyzed for each sample and each condition was run in triplicate. the same parameters, regions, thresholds, and gating was used for all analysis to successfully measure gfp positive cells. statistical analysis all data graphs were generated with microsoft excel. data and statistical analysis was performed using r software. student’s t-tests were performed and a p-value <0.05 indicated statistically significant differences. error bars indicate the standard error of the mean unless noted otherwise. quantifying virus replication and flow cytometry baby hamster kidney (bhk) cells were infected with a double subgenomic sindbis virus (sinv) containing a fluorescent reporter (gfp) protein inserted in the viral genome(21). virus was added to the cells to achieve a multiplicity of infection (moi) of one and after one hour of infection, imatinib was added to specified wells at various low concentrations of imatinib (0-100μm). after 24 hours of infection, the cells were analyzed for gfp fluorescence as an indicator for sinv replication either with a plate reader or with the flow cytometer. data from the flow cytometer accurately matched the data from the plate reader, but the flow cytometer allowed more specifics to be analyzed (fluorescence per cell and percent infected). treatment of sinv-infected bhk cells with imatinib showed a dose-dependent decrease in viral replication (figure 1). cytotoxicity of the imatinib concentrations was also measured on the bhk cells using alamarblue cell viability assay. imatinib started reducing cell viability at concentrations above 12μm, with a dosedependent curve of cytotoxicity at higher concentrations of imatinib. treatment with concentrations less than 12μm resulted in greater than 80% cell viability. however, at 6μm imatinib treatment, viral replication was significantly reduced by more than 50%. the ic50 and cc50 were calculated to be 4.8μm and 28.1μm, respectively, giving a therapeutic index of 5.85 for using imatinib as an antiviral for alphaviruses. ribavirin, which is a wellestablished antiviral therapy that is used in severe viral infections, has a therapeutic index of around 5-8 for sindbis virus, indicating that imatinib treatment is equally effective at treating alphavirus infection(13, 20, 24). imatinib’s effect over time and different levels of sinv infection imatinib’s effects on sinv virus replication over time was investigated next. cells were infected with dssinv-gfp at an moi=1 and then half of the cells were left untreated and half of the cells were treated with 10μm imatinib. cells were analyzed every 6 hours for a 24 hour period to detect gfp fluorescence results 144 • fine focus, vol. 3 (2) discussion imatinib mesylate • 147 figure 1: imatinib inhibition of sindbis virus and cell viability flow cytometry analysis of bhk cells infected with dssinv-gfp at an moi=1 and treated with dilutions of imatinib sinv replication (gray bars) and cell viability (black line) are shown with increasing concentrations of imatinib. both the mock/untreated samples were set to 100% and the different treatment conditions were calculated as a percentage of the mock. sinv was significantly inhibited at 6 μm of imatinib compared to mock treated (p<0.05). error bars represent standard deviation n=3. and calculate the percentage of cells infected using flow cytometry. significant inhibition of virus replication (student’s t-test p<0.05) was seen in all time points past 12 hours that were treated with imatinib (figure 2a). additionally, bhk cells were infected with an increasing amount of virus and then treated with mock solvent or 10um imatinib and allowed to infect for 24 hours. cells were analyzed on flow cytometry to determine the percentage of cells infected (figure 2b). increasing the level of infection (multiplicity of infection= moi) by up to 10 fold still showed a reduction in virus infection in cells treated with 10μm imatinib (figure 2b). time of treatment affects imatinib inhibition of sinv we sought to validate imatinib as a treatment option by investigating different times of treatment and at a higher infection level (moi=10). bhk cells were treated with 10μm imatinib at 6 or 3 hours pre infection, at the same time as infection (0hrs), or 3, 6, or 12 hours post infection. the virus was added at time 0 at an moi=10 and then the cells were allowed to infect for 24 hours before being analyzed on flow cytometry. each time point was analyzed and the treatment was set as a percentage of the untreated control (figure 3). imatinib inhibition of sinv works in multiple cell types to confirm the anti-viral effect of imatinib on infected cells, the study was expanded to include human cultured cells. human hepatoma cells (huh7) and human embryonic kidney cells (hek) were utilized by being plated and infected with dssinv-gfp at an moi=10 similar to the bhk cells. 10μm of imatinib treatment successfully reduced sinv replication in both the huh7 and the hek cells at 24 hours post infection with an moi=1(figure 4). imatinib mesylate, or gleevec®, is already fda-approved and is used regularly in cancer treatments. clinically, imatinib is administered orally at low dosages (400mg p.o. b.i.d=.800mg/ day)(5). we report that at similarly low concentrations (10 μm), imatinib reduced sinv replication in cultured cells. this novel finding suggests that metabolic inhibitors used in cancer chemotherapy may be repurposed and used for treating viral infections. imatinib mesylate works by inhibiting tyrosine kinases within the host cell(5, 18). many metabolic enzymes require phosphorylation from tyrosine kinases in order to be activated. the general inhibition of tyrosine kinases reduces the ability of the cell to have elevated metabolic rates. this limitation on cellular pathways prevents the virus from manipulating host cell metabolism and hijacking certain pathways for optimal viral replication. during imatinib treatment, the cell is maintained at lower metabolic rates and therefore limits the ability of the virus to replicate. this hypothesis has been validated and it has been shown that sindbis virus is significantly inhibited in cells treated with 10μm imatinib. this concentration of imatinib was not toxic to the cells and presents a therapeutic window where imatinib may be used effectively to limit virus replication, while maintaining host cell viability (figure 1). imatinib’s therapeutic index was similar to ribavirin, which is a clinical antiviral used to treat severe cases of respiratory syncytia virus (rsv)(12). although it would be more advantageous if imatinib had a higher therapeutic index, the proof-of-principle is still validated that anti-cancer and antimetabolism compounds may be repurposed and used for antivirals. imatinib is effective against sindbis virus and investigation of other anti-metabolism compounds may lead to the discovery of an even more effective drug that can be used to treat viral infections. this imatinib anti-viral effect was confirmed at different times during the virus replication cycle and indicates a general overall reduction in virus infection as compared to a step/cycle specific inhibition effect. over the course of 24 hours, untreated cells showed a gradual increase in percent of sinvinfected cells eventually reaching about 25% infected, which is statistically significant when compared to the imatinib treated cells that only reached about 5% of cells infected at 24 hours. significant reduction in virus replication was seen in a wide variety of infection levels, as indicated by different infection moi’s (figure 2). interestingly, the plates that were examined at different time points showed higher infection levels (up to 25%) compared to the moi plates (only up to about 17%). this may be due to the fact that the time course plates were physically moved every 6 hours, which would result in a disruption of the media and may have redistributed the virus supernatant throughout the culture. these results imply that imatinib may be successfully used to reduce viral loads at different times and levels of infection. to further assess the efficacy of imatinib to lower viral replication, the impact of treatment timing on viral infection was investigated. understanding the opportune timing for treatment is a critical component for antivirals to discover if the compound is more effective if given prophylactically prior to exposure or if post-infection treatment is more effective. the greatest inhibition of virus replication was seen in cells that received the imatinib treatment 3 or 6 hours prior to infection. adding the imatinib at the same time as infection or up to 3 hours post infection still achieved about a 20% reduction in virus replication, but adding 146 • fine focus, vol. 3 (2) imatinib mesylate • 149 figure 2: imatinib treatment at different times post-infection and sinv concentrations a) sinv-infected bhk cells (moi=1) were treated with 10μm imatinib (dotted line) or left untreated (black line) and analyzed every 6 hours for the percentage of cells infected using flow cytometry. b) bhk cells infected with increasing amounts of virus and treated with 10 μm imatinib (gray) or left untreated (black). the percentage of cells infected was determined with flow cytometry at 24 hours. significant reductions (students t-test p<0.05) in dssinv-gfp replication are indicated with an asterisk (*). figure 3: treatment with imatinib at different treatment times bhk cells were treated with 10 μm imatinib or solvent control at the times indicated and dssinv-gfp (moi=10) was added at time 0. at 24 hours post infection, the cells were analyzed for dssinv-gfp via flow cytometry. the untreated sample for each condition was set to 100%. the greatest difference was seen with a single treatment. figure 4: a) human hepatoma (huh7) and b) human embryonic kidney (hek) cells were infected with dssinv-gfp at an moi=10 and then treated with 10 μm imatinib or solvent control (untreated). at 24 hours post infection, the cells were analyzed for dssinv-gfp via flow cytometry. all data is displayed as a percentage of the untreated samples. (n=6). the imatinib 6 hours or later after infection did not result in a significant inhibition of virus infection (figure 3). the 20% reduction is not as significant as what was seen in previous experiments, but that is likely due to the higher moi and also greater movement of the plate during the infection incubation. pre-infection treatment with imatinib reduced the virus replication more effectively than treatments that were added concurrently or post infection. this indicates that imatinib may be most effective as a prophylactic drug, given prior to high risk exposure situations. however, due to the inherent side effects of anti-cancer and antimetabolic drugs, prophylactic treatment might not be feasible in a clinical setting, but postinfection treatment may be effective. although not as significant as a pre-infection treatment, even treating with imatinib at 3 hours postinfection successfully reduced virus infection and confirms the potential utility of using imatinib as a post-exposure/infection treatment option (figure 3). the imatinib inhibition of sinv infection was further confirmed in human cultured cells, both liver (huh7) and kidney cells (hek), which both showed a similar reduction in virus replication as was observed in the bhk cells (figure 4). huh7 and hek cells are common cell lines used in virology to assess virus replication in human tissue and it is promising that both cell lines show an effective reduction in sinv replication when treated with 10μm imatinib. variation in viral reduction is likely due to specific differences in the cell type, but the overall trend of viral inhibition with imatinib treatment is maintained in the different human cell lines. this validates the potential efficacy of using imatinib as a therapeutic anti-viral treatment option in human alphavirus infections. in this study, a single metabolic inhibiting cancer drug known as imatinib mesylate was investigated. imatinib has shown very promising inhibition of 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transplantation. blood 99: 17. oliveira afc da s, teixeira rr, oliveira as de, souza apm de, silva ml da, paula so de. 2017. potential antivirals: natural products targeting replication enzymes of dengue and chikungunya viruses. molecules 22:505. this work was partially funded by a seed grant from colorado state university-pueblo awarded to j. jordan steel and csu-pueblo’s c-base (communities to build active stem engagement) grant from the us department of education (pr/award # p031c160025). additional financial support was received from the american society for microbiology undergraduate research fellowship awarded to jessica l. costlow. we would like to thank dr. jeff smith for providing the hek cells, dr. rushika perera for providing the huh7 and bhk cells and dr. brian geiss for providing the virus stocks used in this study. acknowledgements references to validate and confirm these findings with imatinib in model organisms and in primary cells. additionally, there are many other 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sparks-thissen* department of biology, university of southern indiana, evansville, in usa manuscript received 1 august 2017; accepted 3 october 2017 copyright 2018, fine focus. all rights reserved. 104• fine focus, vol. 4(1) bacteriophages are obligate intracellular parasites, but many of the cellular proteins involved in replication have not been identified. we have tested the role of the inner membrane protein yhim in bacteriophage replication. yhim is a conserved (21) membrane protein in escherichia coli (e. coli) thought to be localized to the cytoplasmic membrane that is necessary for cell survival under conditions of cell stress, including acid shock, low osmolarity and high temperature. we show here that yhim is necessary for replication of the bacteriophage t4. it also plays a modest role in the replication of t1, t3, and t5 but it does not play a role in the replication of φx174. our data indicated that no replication of t4 occurs in cells missing yhim. this block in infection is due to a block in attachment of the virus to the cell surface. abstract corresponding author *r.l. sparks-thissen rlsparksth@usi.edu keywords • bacteriophage • e. coli • inner membrane protein • t4 bacteriophages are viruses that infect bacteria. they have numerous strategies to infect their host cell and require many host proteins for their replication. all viruses require a receptor on the cell surface to identify a susceptible cell and begin infection (3). the bacteriophage then depends on numerous host proteins to reproduce. for example, the bacteriophage t4 requires the e. coli genes tabb, groe and mop for proper assembly of the virus (6, 9, 28). t4 replication is also dependent on rpos (4), a sigma factor involved in regulating responses to cell stress and changes in the growth of e. coli (14). finally, growth of the bacteriophage introduction 105pathogens and antimicrobial factors • t4 is dependent on environmental conditions, including temperature (12, 27). given that the environment affects bacteriophage replication and that pathways involved in response to environmental stresses can affect viral replication, we were interested in determining whether other genes involved in responses to environmental stresses might also be involved in t4 replication. yhim is a conserved inner membrane protein in e. coli. it has 10 predicted transmembrane domains and homology to the duf 2776 family of proteins, an uncharacterized family of proteins (24). yhim expression is upregulated under conditions of cell stress, such as acid stress, high temperature and changes in osmolarity (22, 3032). yhim expression is regulated by the sigma factor rpos (1, 31). yhim is also necessary for survival during acid shock and conditions of high temperature and low osmolarity (2, 21). finally, yhim has also been shown to play a role in gaba transport (29). because yhim seems to play a role in protecting the cell during conditions of cell stress (2, 21) and bacteriophage replication can be affected by rpos, we wanted to know if yhim might also play a role in infection by bacteriophages that infect e. coli. there are several wellcharacterized bacteriophages that infect e. coli, including t1, t3, t4, t5 and φx174. these viruses belong to different virus families, have different morphologies and require different proteins from the cell for infection. t1 belongs to the siphoviridae family, has a ~49kb dna genome and uses the fhua protein for attachment (10). t3 has a short tail, a dna genome of ~40kb, and uses lipopolysaccharides (lps) for attachment to the cell surface (32). t4 is a member of the “t-even” phage group, has a large dna genome (~170kb) and uses ompc and lps for attachment to cells (20). t5 has a long non-contractile tail, a dna genome of ~121 kb, and uses the fhua receptor for attachments (15) (19). φx174 is a member of the microviridae family, does not contain a tail, has a genome of ~5 kb and uses lps for attachment. t3 and t4 use the same glucose residue for binding, whereas φx174 uses a different site on lps for attachment (23). we used two e. coli strains, the wildtype ucb strain and a previously published strain with a tn10 insertion into the yhim gene (21) to test whether bacteriophage replication is dependent on yhim. we tested a wide range of bacteriophages including t1, t2, t3, t4, t5, and φx174. these viruses were chosen because they are all well characterized and belong to different families of viruses. our data indicated that replication of the bacteriophage t4 is dependent on yhim. attachment to the cell surface is blocked by a lack of yhim, but this effect can be partially rescued by using a high virus number per cell or by prolonged incubation with the virus. this suggests that yhim mediates efficient t4 attachment at the cell surface. 106• fine focus, vol. 4(1) materials and methods strains used the e. coli strains ucb and nvu31 have been previously described (21). ucb is the parental strain of nvu31 and nvu31 has a tn10 insertion at position 167 of the yhim gene (21). strain b was obtained from bruce voyles, grinnell college, grinnell, ia. t1, t3, t4 and φx174 were obtained from presque isle cultures, erie, pa. t4 was a gift from peter gauss, western state college, gunnison, co. efficiency of plating these experiments were performed as previously described (18). briefly, ucb or nvu31 was mixed with top agar (per liter: 10g tryptone, 1g yeast extract, 5g nacl, 0.2g glucose, 7g agar) and then layered on top of bottom agar (per liter: 10g tryptone, 1g yeast extract, 5g nacl, 0.2g glucose, 10g agar). all media components were obtained from fisher scientific. ten µl of various dilutions of virus in t broth (per liter: 10g tryptone, 5 g nacl) were spotted on top of the top agar overlay. efficiencies are described relative to titer on ucb. attachment assays mid-log cultures of ucb or nvu31 were incubated at an moi=0.01, 0.1, 1 or 10 and lysed with chloroform prior to titer using strain b. for experiments performed at 4oc, cells were incubated on ice with shaking during attachment. cells were then shifted to 37oc prior to single step growth curve analysis (17). complementation assays yhim was amplified from genomic dna using the primers rst201 (5’-gtgaacatatatatcgggtgg-3’) and rst202 (5’ttattttttagcaggaaccgcttc-3) and was cloned into ptrc-histopo (invitrogen). ptrc-histopo-yhim or ptrc-histopo-lacz (invitrogen) was transformed into either ucb or nvu31. protein expression was verified by sds page. cells were induced for 2 hours with 1mm iptg prior to attachment assays. 107pathogens and antimicrobial factors • results to determine whether yhim plays a role in viral infections, we infected wildtype (ucb) and δyhim (nvu31) with various viruses to see if a lack of yhim affected virus growth (table 1). a lack of yhim had no effect on infection with t5. in contrast, a lack of yhim had a modest effect on infection of t1, t3 and φx174, with a we were interested in determining the stage of the viral lifecycle that was blocked in nvu31 cells. one possible explanation for the lack of plaques on nvu31 is that t4 can enter and produce virus particles but cannot lyse nvu31. we tested this possibility by doing a single step growth curve on both nvu31 and ucb. 10-fold reduction in efficiency of plating (eop). t4 was the most dependent on yhim, with a >106fold decrease in efficiency of plating (table 1 and fig. 1a). no plaques were seen after infection of nvu31 cells with t4 (fig. 1a). samples were lysed in chloroform before assaying the amount of virus to release any intracellular virus. our data indicated that no virus was produced in nvu31 cells (fig. 1b), suggesting that the virus may not be able to enter and/or replicate in nvu31. table 1: efficiency of plating in cells missing yhim. efficiency of plating is relative to number of plaques during infection of ucb. experiment was repeated at least 3 times. figure 1: a) efficiency of plating of t4 on nvu 31. b) single step growth curve of t4 infection of both wildtype (ucb) and δyhim (nvu 31). all samples were lysed in chloroform prior to plaque assay analysis. each experiment was conducted at least 3 times. error bars represent the standard error of the mean (sem). 108• fine focus, vol. 4(1) we next wanted to determine if the lack of replication was due to a lack of attachment. we performed attachment assays using both ucb and nvu31 cells. we found that after 10 minutes of incubation at 37oc, 50% of t4 attached to ucb while <5% of t4 attached to nvu31 cells (p=0.0013) (fig. 2a). these data suggested that the replication defect in yhim deficient cells may be due to an inhibited attachment. to show that the block in attachment was due to yhim, we constructed a plasmid that expresses yhim (ptrc-histoponot attach to cells deficient in yhim. we next determined whether this was a complete block in attachment or whether t4 attached inefficiently to the cell surface. we took two approaches to this question. first, we added increasing amounts of virus per cell to see if the addition of extra virus would complement the phenotype. addition of additional virus resulted in an average percentage attachment of 2% at 0.01 and 0.1 pfu/cell (fig. 3a and b) and increased to 7.5% at 1 pfu/cell (fig. 3c) and 9% at 10 pfu/cell (fig. 3d). our data indicated that yhim) and transformed both ucb and nvu31 cells. we also transformed both cell lines with ptrc-histopo-lacz as a control for protein overexpression. we then tested attachment on both cells (fig. 2b). consistent with previous data, the virus attached to ucb expressing lacz but did not attach to nvu31 expressing lacz (p=0.042). in contrast, overexpression of yhim restored the ability of t4 to attach to nvu31 (p=0.4119), suggesting that the lack of attachment to nvu31 is due to the lack of yhim (fig. 2b). the previous data suggested that t4 could while adding additional virus slightly increases attachment, t4 does not attach to mutant cells to the same degree as it attaches to wildtype cells even when we add 10 pfu/cell (p=0.0026) (fig, 3a-d). another possibility to explain the lack of attachment is that the virus attaches slowly to the surface of cells. because replication occurs when cells are incubated at 37oc, we decided to let the virus incubate at 4oc for 30 minutes and then shift the cells to 37oc for the rest of infection to occur. this allowed the virus to attach without replication and lysis of the host cell. we found figure 2: a) attachment of t4 to ucb and nvu 31 cells after 10 minutes. b) attachment of t4 to ucb and nvu31 cells expressing either lacz or yhim after 10 minutes. each experiment was conducted at least3 times. error bars represent sem. 109pathogens and antimicrobial factors • that while some of the virus attached to nvu31 after 30 minutes, it did not attach at the same level as ucb (p=0.0630) (fig. 4a). we next tested whether the virus that had attached to the surface of nvu31 could enter and replicate within these cells. we incubated ucb and nvu31 cells with t4 for 30 minutes at 40c to let the virus attach and then shifted the cells to 370c to allow dna entry and replication to occur. after attachment to ucb at 40c, t4 could enter and produce progeny when shifted to 370c (fig 4b). in contrast, no progeny was produced after attachment to nvu31 cells. this suggests that while the virus can attach inefficiently to δyhim cells, yhim is also necessary for a post attachment step in viral replication. figure 3: attachment of t4 to ucb and nvu 31 cells using a) 0.01 pfu/cell, b) 0.1 pfu/cell, c) 1 pfu/cell or d) 10 pfu/cell. attachment was measured at 10 minutes post addition of virus. each experiment was conducted at least 3 times. error bars represent sem. 110• fine focus, vol. 4(1) discussion these experiments describe the identification of the inner membrane protein yhim as a cellular component necessary for t4 but not t1, t2, t3, t5 or φx174 replication. t4 resistant e. coli were previously described (6,9,13,16,20,25,26,28). some of these mutations result in a block in head assembly (6, 9, 28). these mutations map to the tabb, groe and mop genes. mutations have also been characterized that result in the delay of the onset of viral dna replication and failure to produce late gene products (20, 25, 26). these mutations map to rna polymerase. some of the mutations have been linked to mutations in the lps (13). other mutations result in a block in t4 replication at increased temperature (16). bacteriophage adsorption involves three steps: initial contact, reversible binding and irreversible binding (7). if any of these three steps fail to occur, attachment will not occur and the virus will fail to replicate. our data indicated that yhim plays a role in one of these three steps. given that multiple viruses infect our yhim mutant (t1, t3, t5, φx174), it seems unlikely that major increases in capsule production are responsible for our lack of attachment. it is also possible that our mutants express abnormal levels of ompc or lps, the two receptors involved in t4 attachment on the cell surface. however, ompc is not required for t4 attachment, so it is unlikely that changes in ompc expression would lead to a decrease in t4 binding (11). t3, t4 and φx174 all use lps as a binding site for attachment but only t4 is unable to bind to cells missing yhim (8, 15). furthermore, t3 and t4 bind to the same glucose residue on lps (23), suggesting that the lps synthetic pathway is not figure 4: attachment of t4 at 4oc. a) % attachment after 30 minutes of attachment. b) t4 replication at 37oc after 30-minute incubation at 4oc. each experiment was conducted at least 3 times. error bars represent sem. 111pathogens and antimicrobial factors • significantly affected by a lack of yhim. since yhim belongs to a group of proteins regulated by rpos, we suggest that a lack of yhim may affect other pathways regulated by rpos and that these pathways are necessary for normal attachment and replication of t4. yhim is involved in regulating survival in many growth conditions, including response to increased temperature (2). t4 growth is also affected by changes in e. coli physiology and temperature (12). t4 replication is not regulated properly in the absence of rpos (4). since yhim is one of many rpos regulated proteins, one possibility is that the inhibition of replication in rpos deficient cells is dependent on a lack of yhim. this could be because the virus interacts directly with yhim to promote attachment, which seems unlikely since yhim is not on the outer membrane where attachment occurs. alternatively, yhim could be involved in other cell processes that are also necessary for t4 attachment. for instance, t4 replication is dependent on temperature (27), and previous t4 resistant mutants fail to replicate t4 at high temperatures (16). yhim’s role in mediating cell survival at high temperature (2) may indicate that t4 attachment depends on functional yhim to maintain cell viability at high temperatures, therefore allowing t4 to successfully attach and replicate within e. coli. we have shown that the inner membrane protein yhim is important for efficient replication of t4 but not t1, t2, t3, t5 or φx174 bacteriophages. this block in replication is due to a defect in t4 attachment. further studies will determine the mechanism by which yhim mediates this function. acknowledgements this work was supported by science, engineering, and education research grant award (seerga), junior faculty summer research fellowship, and endeavor awards from the university of southern indiana. we thank peter gauss for the t4 stock. 112• fine focus, vol. 4(1) references 1. altuvia s, weinstein-fischer d, zhang a, postow l, storz g (1997) a small, stable rna induced by oxidative stress: role as a pleiotropic regulator and antimutator. cell 90:43-53. 2. anderson ma, mann md, evans ma, sparks-thissen rl (2017) the inner membrane protein yhim is necessary for 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(ed), the bacteriophages, 2nd edition ed. oxford university press, new york, ny. 11. goldberg el, grinius l, lettelier l. (1994.) recognition, attachment and injection, p 347-356. in karam jd, drake jw, kreuzer kn, mosig g, hall dh, eiserling lw, black lw, spicer ek, kutter e, carlson k, miller es (ed), molecular biology of bacteriophage t4 american society for molecular biology, washington, d.c. 113pathogens and antimicrobial factors • 12. hadas h, einav m, fishov i, zaritsky a (1997) bacteriophage t4 development depends on the physiology of its host escherichia coli. microbiology 143 ( pt 1):179-185. 13. hancock re, reeves p (1976) lipopolysaccharide-deficient, bacteriophage-resistant mutants of escherichia coli k-12. j bacteriol 127:98-108. 14. hengge r (2008) the two-component network and the general stress sigma factor rpos (sigma s) in escherichia coli. adv. exp. med. biol. 631:40-53. 15. henning u, hashemol-hosseni s. (1994.) receptor recognition by t-even coliphages. in karam j, drake j, kreuzer k, mosig gh, dh, carlson k, miller es (ed), molecular biology bacteriophage t4. asm press, washington, d.c. 16. jensen jl, susman m (1980) a mutant of e. coli that restricts growth of bacteriophage t4 at elevated temperatures. genetics 94:301-325. 17. kroppinski a. (2009.) measurement of the rate of attachment of bacteriophage to cells, p 151-160. in clokie mj, kropinski a (ed), bacteriophages: methods and protocols, volume 1: isolation, characterization and interactions vol 501. humana press, new york, ny. 18. kutter e. (2009.) phage host range and efficiency of plating. in clokie mj, kropinski a (ed), bacteriophages: methods and protocols, volume 1: isolation, characterization, and interactions, vol 501. humana press, new york, ny. 19. mondigler m, holz r, heller k (1995) overproduced and purified receptor binding pb5 of bacteriophage t5 binds to the t5 receptor protein fhua. fems microbiol lett 130:293-300. 20. montgomery dl, snyder lr (1973) a negative effect of -glucosylation on t4 growth in certain rna polymerase mutants of escherichia coli: genetic evidence implicating pyrimidine-rich sequences of dna in transcription. virology 53:349-358. 21. nguyen tm, sparks-thissen rl (2012) the inner membrane protein, yhim, is necessary for escherichia coli (e. coli) survival in acidic conditions. arch microbiol 194:637-641. 22. peter bj, arsuaga j, breier am, khodursky ab, brown po, cozzarelli nr (2004) genomic transcriptional response to loss of chromosomal supercoiling in escherichia coli. genome biol 5:r87. 23. prehm p, jann b, jann k, schmidt g, stirm s (1976) on a bacteriophage t3 and t4 receptor region within the cell wall lipopolysaccharide of escherichia coli b. j mol biol 101:277-281. 24. rudd ke (2000) ecogene: a genome sequence database for escherichia coli 114• fine focus, vol. 4(1) k-12. nucleic acids res 28:60-64. 25. snyder l (1973) change in rna polymerase associated with the shutoff of host transcription by t4. nat new biol 243:131-134. 26. snyder lr (1972) an rna polymerase mutant of escherichia coli defective in the t4 viral transcription program. virology 50:396-403. 27. taj mk, ling jx, bing ll, qi z, taj i, hassani tm, samreen z, yunlin w (2014) effect of dilution, temperature and ph on the lysis activity of the t4 phage against e. coli b21. japs: journal of animal & plant sciences 24:1252-1255. 28. takano t, kakefuda t (1972) involvement of a bacterial factor in morphogenesis of bacteriophage capsid. nat new biol 239:34-37. 29. tramonti a, santis fd, pennacchietti e, biase dd (2017) the yhim gene codes for an inner membrane protein involved in gaba export in escherichia coli. aims microbiology doi:http://dx.doi. org/10.3934/microbiol.2017.1.71. 30. tucker dl, tucker n, conway t (2002) gene expression profiling of the ph response in escherichia coli. j bacteriol 184:6551-6558. 31. white-ziegler ca, um s, pérez nm, berns al, malhowski aj, young s (2008) low temperature (23 degrees c) increases expression of biofilm-, cold-shockand rpos-dependent genes in escherichia coli k-12. microbiology 154:148-166. 32. white-ziegler ca, davis tr (2009) genome-wide identification of h-ns-controlled, temperature-regulated genes in escherichia coli k-12. j bacteriol 191:11061110. isolation and characterization of halotolerant 2,4dichlorophenoxyacetic acid degrading bacteria from sulfidic, low salinity salt springs michael g. willis, david s. treves* department of biology, indiana university southeast, new albany, in copyright 2014, fine focus all rights reserved manuscript received 30 april, 2014; accepted 30 may, 2014 abstract introduction corresponding author * david s. treves department of biology, indiana university southeast 4201 grant line road, new albany, in, 47150 dtreves@ius.edu phone: 812-941-2129. keywords • 2,4-d • tfda • tfdaa • salt springs • halotolerant bacteria 40 • fine focus, vol. 1 bacteria have tremendous potential to degrade organic compounds and study of the metabolic pathways involved is a key component to more efficient environmental remediation. one well-studied organic acid degradation pathway is that of 2,4– dichlorophenoxyacetic acid (2,4-d) (4, 12, 13, 15, 19, 23). the broadleaf herbicide 2,4d was developed during world war ii by british scientists to help boost production of grass crops like corn, wheat, and rice (23). since the late 1940’s it has been one of the most popular herbicides used by industrial farms and homeowners for the control of broadleaf weeds (30). the 2,4-d degradation pathway of cupriavidus necator jmp134 (b-proteobacteria; formerly ralstonia eutropha) has received considerable attention and serves as a model system for microbial degradation (2, 3, 26). the first gene in the catabolic pathway, tfda, codes for an a – ketoglutarate dioxygenase (6, 7). the remainder of the pathway is comprised of at least five genes, e.g. tfdbcdef, that continue to break down the molecule for use in central metabolism (29). members of the a-, band y-proteobacteria are known to degrade 2,4-d and several unique degradation pathways have been identified. in the band yproteobacteria three classes of tfda genes are found while the a-proteobacteria carry tfdaa that has ~60% identity to the tfda gene of jmp134 (9, 30). a novel cadabc-based degradation the bacterial communities at two sulfidic, low salinity springs with no history of herbicide contamination were screened for their ability to grow on 2,4-dichlorophenoxyacetic acid (2,4-d). nineteen isolates, closely matching the genera bacillus, halobacillus, halomonas, georgenia and kocuria, showed diverse growth strategies on naclsupplemented and nacl-free 2,4-d medium. the majority of isolates were halotolerant, growing best on nutrient rich broth with 0% or 5% nacl; none of the isolates thrived in medium with 20% nacl. the tfda gene, which codes for an a – ketoglutarate dioxygenase and catalyzes the first step in 2,4d degradation, was detected in nine of the salt spring isolates. the tfdaa gene, which shows ~60% identity to tfda, was present in all nineteen isolates. many of the bacteria described here were not previously associated with 2,4-d degradation suggesting these salt springs may contain microbial communities of interest for bioremediation. materials and methods pathway which codes for 2,4-d oxygenase subunits (15) has also been found in a-proteobacteria (10). although there has been extensive research conducted on 2,4-d degraders in the soil (12 ,13 ,15, 23), there is less work on the removal of 2,4-d in aquatic environments of varying salinity. maltseva et al. (18) isolated three 2,4-d degrading bacteria from the family halomonadaceae in the soil of a high salinity, alkaline lake that was heavily polluted with 2,4-d and other organic acids. although these isolates came from an extreme environment, and were moderately halophilic and alkaliphilic, they still utilized the well-known tfda-based degradation pathway of jmp134 (18). the objective of this study was to isolate and characterize 2,4-d degrading bacteria from salt springs at big bone lick state park in boone county, ky. the park and surrounding area contain numerous natural springs but this area does not have a history of 2,4-d contamination. the isolates obtained, including some genera not previously associated with 2,4-d degradation, were examined for their ability to grow on 2,4d in the presence and absence of nacl and screened for commonly occurring 2,4-d degradation genes. isolation and characterization of 2,4-d degrading bacteria from novel environments, such as the salt springs in big bone lick state park, not only expands information on the diversity of herbicide degrading bacteria but also provides useful information for bioremediation of contaminated saline environments. site description and sample collection salt spring samples were collected from big bone lick state park located in boone county, ky. the area surrounding the park is a rural semi-agricultural region; there are several salt-springs inside the park’s boundary and many more springs throughout the surrounding area. two springs were the focus of this study because they are easily accessed from the park visitor center. spring samples representing both spring water and water mixed with spring sediment were collected from the small spring (n 38° 53’ 4.582”, w 84° 45’ 10.648”) and large spring (n 38° 53” 5.899”, w 48° 45” 9.928”) in sterile polypropylene containers, transported to iu southeast in a cooler (~1.5 h) and used immediately for plating experiments. plating and isolate purification a dilution series of the spring samples was done using sterile phosphate buffered saline with dilutions spread onto artificial sea water nutrient broth (asw) (17) agar plates with 5, 10 or 20% nacl to assess levels of halotolerant heterotrophs and mmo minimal medium plates (13) supplemented with 125 ppm 2,4-d / 5% nacl to assess levels of 2,4-d degraders. plates were incubated for two weeks at 28°c. single colonies were transferred to mmo 125 ppm 2,4-d / 5% nacl for purification and after several plate transfers they were grown in difco marine broth (beckton dickinson & co., sparks, md) and stored at -80°c in 15% glycerol. applied/environmental • 41 results salt spring characteristics this study focused on two salt springs located within the borders of big bone lick state park in boone county, ky. during six visits to the springs between 2006-2010 spring characteristics and water chemistry such as ph, temperature and salinity were measured (table 1). the springs are shallow (~13 cm at the deepest point) and located no more than 100 meters from one another. in general, chemistry between the two springs was similar which, along with their proximity, suggests a common groundwater source. growth assessment on 2,4-d purified isolates were streaked to mmo 2,4-d plates with and without 5% nacl and incubated at 28°c for 14 days. at regular intervals growth of the isolates was assessed visually by two independent observers and growth was scored as none (-), poor (+), moderate (++) or good (+++). dna extraction, pcr and 16s rrna gene characterization genomic dna from individual isolates was extracted using the mobio microbial dna extraction kit (mobio.com). the 16s rrna gene was amplified using 27f and 1492r primers (25), and isolates were screened for tfda using the tvu and tvl primers of hogan et al. (8). screening for tfdaa was done using alpha1 (5’-acs gag ttc ksc gac atg cg-3’) and alpha2 (5’-gcg gtt gtc cca cat cac-3’) primers using the pcr conditions described by itoh et al. (9). promega gotaq® green master mix was used for pcr with reaction mixtures prepared according to manufacturer’s protocols and thermal cycling was performed using a techne flexigene thermal cycler. the 16s rrna gene products were purified using the mobio ultraclean™ pcr cleanup kit and dna sequencing using primers 27f, 1492r and 519f (25) was performed at the iupui core sequencing facility using applied biosystems 3100 genetic analyzers and big dye terminator chemistry v3.1 (perkinelmer.com). sequences were edited using chromas (http://technelysium.com. au) and compiled in bioedit (http://www. mbio.ncsu.edu/bioedit/bioedit.html). full or partial sequences were used to query the genbank database using the basic local alignment search tool (blast) (1). salt tolerance each isolate was transferred to 2ml of asw nutrient broth (17) containing nacl ranging from 0, 5, 10, 15 or 20% and incubated at 25°c at 200 rpm for 48 hours. longer incubations of 72 and 96 hours did not yield increases in optical density. optical density was measured using a biotek elx 800 plate reader at 630nm. salt spring chemistry spring water temperature, ph, salinity, total dissolved solids and conductivity were measured using a hach sension 156 multi-parameter meter on 9 sept 2006, 8 mar 2007, 30 sept 2009, 16 oct 2011, 5 feb 2010 and 10 oct 2010. no ph data was collected from the springs during the 5 feb 2010 sampling trip as the ph probe malfunctioned on this date. 42 • fine focus, vol. 1 assessment of culturable bacterial communities of the springs spring samples representing water only and water mixed with sediment were plated onto artificial seawater medium (asw) supplemented with 5, 10 or 20% nacl to assess heterotrophic halotolerant bacterial populations (table 2). cfus were observed at all three salt concentrations although there was generally a reduction in cfu/ml as the salt concentration increased. sediment samples from both springs contained larger bacterial populations at each salt concentration compared to water-only spring samples. samples from both springs yielded cfus on mmo 2,4-d medium supplemented with 5% nacl (table 2). the sediment containing small spring sample had the most 2,4-d degrading bacteria with counts ranging from 1-2 orders of magnitude more than the other three spring samples. isolation and identification of putative 2,4-d degrading isolates nineteen isolates transferred from the original 2,4-d plates and which produced small transparent colonies on media with 2,4-d as the sole carbon source at 28°c were chosen for further characterization. using 16s rrna gene sequence data the closest match to each isolate was determined from genbank (table 3). eleven of 19 isolates closely matched the genus bacillus with halobacillus as the next most common genus (4 isolates) followed by halomonas (2 isolates). eleven of the sequences were nearly full length (1389 nts or greater) and all identities to database sequences were 99% with the exception of isolate 19 (halomonas ventosae) which showed 98% identity. growth assessment on 2,4-d to assess growth on 2,4-d as a sole carbon applied/environmental • 43 table 1. water characteristics of two big bone lick state park salt springs. 44 • fine focus, vol. 1 o pt ic al d en si ty (6 30 n m ) % nacl fig. 1. salt tolerance estimates of isolates from big bone lick state park. each bar shows the optical density at 630nm across five salt concentrations in asw broth. table 2. cfu/ml estimates of halotolerant bacteria in two big bone lick state park salt springs. table 3. identity, tfda gene presence and growth characteristics of 2,4-d degrading halotolerant isolates. applied/environmental • 45 1amplified with primers from hogan et al. (8).2 amplified with primers from itoh et al. (9). source each of the nineteen isolates was streaked onto mmo medium containing 125 ppm 2,4-d with or without 5% nacl. over the course of 14 days each isolate was visually examined independently by two investigators and the level of growth was scored as none (-), poor (+), moderate (++) or good (+++). five isolates (represented by the genera bacillus, georgenia, halobacillus) showed good growth (+++) on mmo 2,4-d plates with or without nacl and four isolates showed moderate growth (++) on both medium types (table 3). interestingly, isolates 18 & 19 (represented by kocuria and halomonas, respectively) showed no growth (-) on mmo 2,4-d medium but good growth (+++) on mmo 2,4-d medium supplemented with nacl. numerous attempts to grow the nineteen isolates in mmo 2,4-d broth failed to yield observable growth. additional attempts to encourage growth with casamino acids and to design liquid medium optimized to spring conditions also failed. presence of tfda and tfda-like genes the nineteen spring isolates were screened by pcr for two types of genes commonly found in 2,4-d degrading bacteria. dna extracts from nine of the isolates were positive for the presence of tfda and all nineteen isolates were positive for amplification of tfdaa (table 3). salt tolerance of the isolates salt tolerance of each isolate was determined by inoculating asw medium supplemented with 0, 5, 10, 15 or 20% nacl. fig. 1 shows the optical density at 630nm for each isolate across the five salt concentrations. eighteen of the nineteen isolates showed a preference for media with 0% or 5% nacl. asw without added nacl supported growth of all the isolates above 0.1 od units with the exception of isolates 9 and 19 (both halomonas). the two halomonas isolates showed a similar growth pattern with good growth at 5, 10 and 15% nacl. although isolate 18 (kocuria sp.) grew poorly on mmo 2,4-d medium without salt (table 3), it grew the best of the nineteen isolates in 0% asw and also had the highest combined optical density across the salt concentrations of any isolate (fig. 1). 46 • fine focus, vol. 1 discussion the objective of this study was to undertake a culture-based approach to examine 2,4-d degrading bacteria in salt springs at big bone lick state park. the springs in the park offer a unique environment for microbial growth. low salinity, sulfur-enriched groundwater feeds the springs throughout the year and mineral deposits regularly appear in the spring’s water and outer edges as white precipitate. this work represents the first attempt to examine 2,4-d degrading microbial communities in these springs. although the springs sampled are of low salinity previous culture-based studies have identified large populations of moderate halophilic and halotolerant bacteria in spring water and sediment samples. with this in mind, the current study was designed to isolate 2,4-d degrading bacteria that could tolerate at least 5% nacl. although the salt springs sampled in this applied/environmental • 47 study have no known history of 2,4-d contamination, samples from two salt springs at big bone lick state park showed an abundance of colonies on agar-based plates with 2,4-d as the sole carbon source. ka et al. (12) demonstrated that successive 2,4-d applications in agricultural soil can greatly enrich the soil microbial community for 2,4-d degrading bacteria. although the big bone lick springs were not obviously treated or heavily contaminated with 2,4-d, low level 2,4-d groundwater contamination from agricultural runoff cannot be ruled out as an enrichment source. varied growth levels on 2,4-d medium with and without salt were observed in the nineteen spring isolates chosen for further characterization. while some isolates grew equally well on 2,4-d medium with or without nacl, others showed a clear preference for salt-free or salt-containing medium. these diverse growth strategies make sense given that the isolates described here were enriched on 2,4-d agar-based medium rather than 2,4-d supplemented broth which may select only the fastest growing isolates. allowing the initial 2,4-d containing plates to incubate for 14 days may have also provided slow growing isolates time to become established. novel slow growing 2,4-d degrading bacteria have been found in pristine environments using an enrichment strategy involving organic supplements and low initial levels of 2,4-d (14). two of the nineteen isolates described here were identified as closely matching the genus halomonas. maltseva et al. (18) previously isolated three moderately halophilic, alkaliphilic bacteria from alkali lakes heavily contaminated with 2,4-d and numerous other aromatic compounds. these isolates were members of the halomonadaceae family and contained 2,4d degradation pathways that matched the tfda pathway from cupriavidus necator jmp134. as tfda was found in only one of the two halomonas isolates described here, more work is clearly needed to determine the nature of degradation in the non-tfda containing halomonas isolate. halomonas is a metabolically diverse genus well known for its halotolerance and biodegradative potential. for example, halomonas organivorans, isolated from the soil of isla cristina, spain grows on phenol and a wide-range of other organic acids (5). interestingly, seventeen of the nineteen isolates described here were gram positive bacteria. bacillus was the most common genus found (eleven isolates) and several of these contained the tfda gene. bacillus sp. have been previously reported to carry tfda (8) but these isolates did not degrade 2,4-d. the bacillus isolates in this study closely match recently described halotolerant species such as such as bacillus aquamaris, bacillus hwajinpoensis and bacillus baekryungensis (27, 28) and thus these isolates should be screened more fully for their degradative potential. isolates closely matching halobacillus were also identified as 2,4-d degraders in this study and three out of the four isolates contained the tfda gene. the genus halobacillus was first proposed in 1996 (24) and now contains nearly 20 species with interesting properties such as production of protective carotenoids and novel salt-tolerant enzymes of interest to biotechnology (11). this is the first report identifying 2,4-d degradation genes in this genus. two additional gram positive isolates closely matched kocuria sp. and georgenia muralis. kocuria are commonly found on the skin of humans and some species have recently emerged as pathogens in immunocompromised patients (22). georgenia muralis was first isolated from 48 • fine focus, vol. 1 m.g.w. was supported by an indiana university southeast undergraduate research fellowship for part of this work. thanks to todd young, naturalist at big bone lick state park for his help with access to the salt springs, sampling and information about the park’s history. acknowledgements a medieval wall painting in the church st. georgen in austria in 2002 (16). a novel finding of this study is that georgenia muralis and kocuria sp. have not previously been shown to degrade 2,4-d. the georgenia muralis isolate described here not only grew well on 2,4-d medium with or without added nacl but also contained the tfda gene, which has not previously been reported. all nineteen of the isolates from big bone lick state park were positive for tfdaa. tfdaa is found in proteobacteria such as bradyrhizobium and sphingomonas sp. (9, 10) and thus it is interesting that the diverse collection of isolates described here, none of which are a-proteobacteria, carry this gene. additional primer sets designed to detect novel tfda-like genes were recently described (30) and it would be informative to screen the isolates described here with these primers to further examine tfda gene diversity. nine of the nineteen isolates in this study contained both tfda and tfdaa raising the possibility that multiple degradation pathways exist in these isolates. it has been suggested that the tfda gene products serve other cellular roles besides the degradation of herbicides (8) and it would be worthwhile to screen the big bone lick salt spring isolates for other degradation abilities. one area to enhance this study lies in designing minimal broth that will support growth of the salt spring isolates. numerous attempts to grow the isolates in minimal liquid medium with 2,4-d as the sole carbon source failed to stimulate growth. additionally, liquid medium designed to replicate spring water chemistry also yielded poor growth. development of minimal broth supporting growth of the isolates described here will allow confirmation of 2,4-d degradation and provide a means for quantitative determination of 2,4-d disappearance (14). in summary, this study has identified the salt springs at big bone lick state park as a potentially novel source of 2,4-d degrading bacteria. since degradation pathways in the salt spring isolates appear to match those associated with tfda and tfdaa, it would be informative to sequence these genes to determine their relationship to other known 2,4-d degradation pathways. horizontal gene transfer of 2,4-d degradation pathways has been proposed based on incongruent phylogenies of 16s rrna genes compared to tfda (20). more recently, a transposon containing both cadab and tfd associated genes, but not tfda or tfdaa was found in a 2-methyl-4-chlorophenoxyacetic acid (mcpa) degrading 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liu, s., drake, h. l., & horn, m. a. 2010. abundance of novel and diverse tfdalike genes, encoding putative phenoxyalkonic acid herbicide-degrading dioxygenases, in soil. appl. environ. microbiol. 76:119-128. 50 • fine focus, vol. 1 41applied & environmental microbiology • bioinformatics analysis of microbial abundance and diversity in acid mine drainage from the solomon mine near creede, colorado manuscript received 6 august 2017; accepted 1 december 2017 ryan k. miller1, robert m. kirkham2, and adam j. kleinschmit1* 1department of biology and earth sciences, adams state university, alamosa, colorado 2geological solutions, alamosa, colorado copyright 2018, fine focus. all rights reserved. 42• fine focus, vol. 4(1) this study focused on characterizing the phylotypic composition of acid mine drainage (amd) communities associated with the solomon mine near creede, colorado, and its relative diversity compared to microbial communities found in the east willow creek (ewc) watershed. amd from the solomon mine adit flows into an existing passive bioremediation wetland system located next to the solomon mine adit that currently is ineffective and is under consideration for renovation. we are interested in gaining an understanding of the baseline microbial communities present in amd/ewc and to monitor them during future wetland renovation. prokaryotic community profiling was approached using ssu 16s rrna marker gene amplification coupled with next generation sequencing. bioinformatics analysis included raw read preprocessing, data visualization, and statistical testing using a combination of usearch and qiimebased scripts. a ph and conductivity gradient were observed for water moving through the currently inefficient wetland system at the solomon mine. the ewc microbiomes had statistically significant higher alpha diversity compared to the amd microbiomes. beta diversity analysis parsed the sample locations into statistically significant groups including core amd microbiomes, the wetland cell 3 microbiome, and ewc microbiomes using multidimensional scaling. taxa driving beta diversity included the phylum proteobacteria for the core amd microbiomes, the phyla firmicutes and chloroflexi for the constructed wetland cell 3, and the phyla bacteroidetes and verrucomicrobia for ewc. our data suggests that the microbial community in constructed wetland cell 3 is likely where limited sulfate reduction activity is operating at low capacity, which will be further investigated via shotgun metagenomic analysis. abstract keywords • acid mine drainage • microbial ecology • 16s rrna marker gene survey • high throughput sequencing • environmental microbiota corresponding author *adam j. kleinschmit , adams state university, alamosa, co usa akleinschmit@adams.edu 43applied & environmental microbiology • introduction microbes populate every known niche on earth including physiologically challenging environments for biological organisms, such as deep-sea hydrothermal vents and subglacial antarctic lakes, where extremophiles thrive (38, 41). environmentally, microbes are essential in the ecological biogeochemical cycling of elements (e.g., carbon, nitrogen, sulfur, phosphorus) to allow other biological organisms access to tangible forms of these elements, thus making life sustainable (18). the shear abundance of microbes makes them a major force that keeps elements circulating between biotic and abiotic entities in an ecosystem. for example, chemolithotrophs can “eat rocks” to obtain energy and electrons, while utilizing carbon dioxide as a carbon source. thus, chemolithotrophs do not need light or organic matter to proliferate and can live in extreme habitats such as deep-sea hydrothermal vents and underground caves (33). oxidation of inorganic matter by chemolithotrophs associated with volcanic caves or even exposed rock that is highly mineralized with metal sulfides can be the limiting factor for the generation of acid rock drainage (ard) (39). ard is a result of an abiotic reaction typically involving iron pyrite that can be accelerated by microbes and produces sulfuric acid as byproduct, thus dramatically lowering the ph within a watershed downstream of the ard source. under these low ph conditions, other heavy metals are easily solubilized from metallic minerals and can lead to environmental degradation within the watershed (13). ard generation can be expedited and/ or be directly generated as a result of anthropogenic activity such as civil engineering projects and mining. when ard is associated with mining activity it is more commonly referred to as acid mine drainage (amd). abandoned mine adits and associated tailing piles can bring sulfide-rich rock into contact with abiotic factors (e.g., oxygen and moisture) along with biotic factors (e.g., chemolithotrophs), which can in turn produce amd for thousands of years. this undesired impact from mining can lead to both economic and conservationbased environmental issues for biological organisms living downstream of an acidified heavy-metal leaching water source. studying microbial ecology associated with amd sources has historically been fruitful in gaining insight into microbial interactions, as the harsh conditions yield an ecosystem with low complexity (3, 22). knowledge gained from understanding microbial interactions in amd has the potential to be applied to promising fields associated with the mining industry, including biomining and bioremediation of amd (6). biomining has the potential to extract trace metals via bioleaching in an efficient and environmentally conscious way and perhaps even remine minerals that historically were not at a sufficient concentration to warrant previous efforts. bioremediation, on the other hand, can be harnessed as a desired passive approach to 44• fine focus, vol. 4(1) naturally address amd through microbial reduction of metal oxides, thus increasing ph and precipitating heavy metals. this passive approach is generally more economically sustainable compared to intensive active approaches (e.g., lime treatment, filtering) (24). for this project, we are specifically interested in characterizing the microbial ecology of microbes associated with amd produced from the abandoned solomon mine along east willow creek (ewc) near the town of creede, colorado. the solomon mine was established in 1890 along ewc in the south san juan mountains (figure 1). the mine has been worked sporadically with the primary minerals mined at the site including galena (lead sulfide), sphalerite (zinc sulfide), pyrite (iron sulfide), and minor chalcopyrite (copper and iron sulfide) up until its formal decommission in 1971 (15, 23). the mine also produced minor gold and even less silver. in 1991, a passive mine drainage treatment system was constructed by the colorado mined land reclamation division (now called colorado division of reclamation, mining, and safety) to reduce the load of heavy metals moving into ewc. the system consisted of three constructed wetland cells. amd from the adit flowed by gravity through the cells. as originally constructed, cell 1 was filled with alfalfa hay, cell 2 contained wood chips, and a mixture of spent mushroom compost and wood chips were placed in cell 3. the system worked well for a few years, but a lack of funds for upkeep and maintenance dramatically reduced the efficiency of the constructed wetland system and only very minor levels of dissolved metals currently are being removed from amd that passes through the system (47). willow creek reclamation committee (wcrc) is contemplating the renovation or rebuilding of the constructed wetland system. local geochemistry greatly influences amd microbial ecosystems; thus, it is important to characterize this site to understand the dynamics within the solomon mine amd microbiome for future intervention efforts (5, 27). we undertook an initial survey of the pre-intervention microbial phylotype composition and diversity, with the aim of identifying focused investigatory questions and monitoring future microbial phylotype evolution longitudinally after the installation of a functioning passive bioremediation pond system. experimentally, we approached the characterization of the amd phylotype composition and diversity using a cultureindependent method to gain an inclusive profile of the present microbial communities (16). preliminary community profiling was performed by targeting the ssu 16s rrna marker gene with next generation sequencing of the resulting amplicons (14) coupled with bioinformatic analysis using usearch (12) and quantitative insights into microbial ecology (qiime)-based scripts on the resulting sequence reads (7, 32). here we describe the baseline microbial community of solomon mine amd and ewc drainage by using the ssu 16s rrna marker gene to taxonomically profile associated sediment samples. 45applied & environmental microbiology • figure 1 hillshade map depicting the east willow creek watershed in southwest colorado and the solomon mine project area, which includes both the solomon mine adit and associated constructed wetlands along east willow creek. 46• fine focus, vol. 4(1) materials and methods sample collection water and sediment samples were collected on july 9, 2015 in the east willow creek drainage basin. the following sample locations mirrored locations used by willow creek reclamation committee for waterquality monitoring (marked and labeled by wood stakes) along east willow creek at site a (ew-a), site g (ew-g), from amd at the solomon mine adit portal just inside and adjacent to the bulkhead (sma), and the solomon mine wetland discharge (swd). additional samples were obtained within the solomon mine adit ~20 meters from the bulkhead (smt) and from a central position within each of the currently defunct constructed wetland system cells (cell 1, cell 2, cell 3), and in east willow creek immediately upstream of the constructed wetlands (ew-usm). the samples associated with the solomon mine were obtained with permission from willow creek reclamation committee. for the collection of water samples, sterile nalgene polypropylene bottles were rinsed three times with sample water prior to sample collection in undisturbed water. sediment samples were collected aseptically from the upper oxic zone at three points at 1 m distances triangulated and pooled as a single sample in sterile polypropylene conical tubes. water and sediment samples were immediately placed on ice in a cooler for transport to the laboratory. sediment sample aliquots were stored at -80°c in the laboratory until processed for dna extraction. sample metadata collection air and water temperature, ph, and electrical conductivity were measured in the field using a handheld combination multimeter. a handheld gps unit determined sample locations in nad83. laboratory ph and oxidation/reduction potential were measured ~3 hours after sample collection using a vwr symphony multimeter device. historical east willow creek water-quality monitoring data was obtained from willow creek reclamation committee (47). 16s rrna gene amplicon sequencing total genomic dna was extracted from 0.25 g of sediment per sample using a mobio powersoil kit following the manufacturer’s instructions (qiagen, carlsbad, ca, usa). illumina 515f and 806r itag primers were designed based on caporaso et al. (8) and used to amplify the v4 region of the 16s rrna gene. pcr reactions were composed of a final concentration of 5-10 ng of template dna, 0.8 mm dntp’s, 0.2 µm forward and reverse primers, 1x pcr buffer, and 1x promega gotaq with use of a bio-rad mycycler. duplicate pcr reactions were pooled to address pcr drift (42), were gel-purified using a qiagen kit (qiagen, frederick, md, 47applied & environmental microbiology • usa), and quantified using a high sensitivity dsdna kit with a qubit 3.0 fluorometer (life technologies, carlsbad, ca, usa). processed sediment samples were sent overnight on dry ice for sequencing preparation and service by wright laboratories (affiliated with juniata college, huntingdon, pa). wright laboratories quality checked the amplicon libraries using a 2100 bioanalyzer dna 1000 chip and performed 150 bp paired-end sequencing reads using the illumina miseq platform. bioinformatics analysis and data visualization the sequencing service carried out by wright laboratories provided the authors with the raw sequence reads via a set of demultiplexed fastq files for the forward and reverse reads. the authors remotely utilized juniata college’s hhmi computing cluster server to run a custom unixbased usearch/quantitative insights into microbial ecology (qiime) pipeline to process the raw reads. briefly, paired end reads were merged using a minimal overlap of 46 bp and a minimum quality score of 36. quality filtering of the paired reads was performed by trimming reads to 252 bp and a maximum average expected error rate of 1% using usearch v7 (12). usearch v7 was also used for sequence dereplication, de novo otu picking at 97% identity, and removal of chimeras. a combination of qiime 1.9.0 and usearch v7 scripts were used to generate an otu table, assign taxonomy using the greengenes reference database (13_8 release) clustered at 97% identity (11), and to generate a phylogenetic tree (7). the pipeline output files were further analyzed using qiime 1.9.0 via oracle vm virtualbox software on a pc for downstream otu table filtering, diversity analysis, and multivariate statistical tests. circos plots were generated using a css normalized filtered otu table with relative abundance values transformed into integers by multiplying values by 10,000 and rounding up to the nearest whole value. relative abundance values for sample location groups were averaged and formatted for circos online (26). alpha diversity box plots were generated using boxplotr, a webbased tool running r packages on a shiny server (40). statistical tests all statistical tests were performed in qiime. significance of alpha diversity between amd and ewc samples was obtained via a non-parametric t-test using monte carlo permutation to calculate the p-value. a beta diversity non-parametric multivariate statistical test was calculated with adonis (modified permanova). beta diversity distance plot p-values were generated using a paired bonferroni-corrected t-test. resulting p-values were adjusted via false discovery rate (fdr) or bonferroni method for multiple comparisons as indicated in the results. differential abundance analysis was performed using the metagenomeseq and deseq2 package via qiime (34). 48• fine focus, vol. 4(1) environmental parameters of solomon mine amd and east willow creek water-quality measurements and sediment samples were taken from the solomon mine site and neighboring ewc at selected wcrc water-quality sampling sites, and from the mine adit and constructed wetland cells (figure 2). amd water and sediment samples collected from the mine adit, wetland cell 1, and wetland cell 2 were stained with iron oxides with no obvious biological organisms present, while wetland cell 3 exhibited notable algal and aquatic plant growth (figure 3). generally, an increasing ph gradient was detected from the amd source point, through the constructed wetland pond cells, and into ewc (table 1). additionally, an increasing gradient of conductivity and oxidation/ reduction potential correlated with the ph gradient as amd moved via gravity flow through the constructed wetlands and into ewc (table 1). ewc samples were within about a half ph unit of 7 and were 2-3 orders of magnitude higher than the amd measurements. additionally, conductivity and oxidation/reduction potential measurements in the ewc samples were markedly lower than the amd samples (table 1) in the ewc samples were markedly lower than the amd samples (table 1). results 49applied & environmental microbiology • * sample id acronyms key: east willow creek site a (ew-a) or site g (ew-g) = wcrc sample site a, g, or immediately upstream of solomon mine (usm); swd = solomon mine constructed wetland discharge; sma = solomon mine adit just inside the bulkhead; smt = inside solomon mine adit, ~20 meters from bulkhead. figure 2. east willow creek and solomon mine acid mine drainage (amd) sample locations (red circles). (a) east willow creek sample locations immediately upstream (ew-usm) and downstream (ew-g) of the solomon mine and immediately upstream of the confluence of east and west willow creek (ew-a). (b) solomon mine and constructed wetlands (outlined region in panel “a”). smt (solomon mine adit, ~20 meters from the bulkhead); sma (solomon mine adit, just inside and adjacent to the bulkhead); smd (solomon mine wetland discharge). 50• fine focus, vol. 4(1) figure 3. acid mine drainage (amd) sample location images. (a) overview photograph of the constructed wetland system looking north. portal of the solomon adit is in the hillside immediately beyond the lower left side of the photograph. water draining from the solomon adit flows into the cells via a buried pipe that extends from the portal to the shutoff valve located below ground in the vertical culvert with metal lid (seen in the photograph above the right end of the pile of lumber and timbers). the buried pipe then runs from the shutoff valve into the bottom of cell 1, where the mine drainage enters the wetland system. (b) inside the solomon mine adit, ~20 meters from the bulkhead. (c-e) images of the constructed wetland cell 1 (c), cell 2 (d), and cell 3 (e) prior to sampling. note the iron-oxide “yellow boy” in cell 1 and -2 (c and d respectively), while cell 3 (e) exhibited noticeable aquatic plant and algal growth. 51applied & environmental microbiology • taxonomic composition raw illumina miseq reads were preprocessed to merge paired-end reads, quality filter to trim or remove low quality reads, add qiime sample identifiers, dereplicate, cluster into operational taxonomic units (otus), remove chimeras and a demultiplexed sequences are split across nine sample locations b rarefaction for the chao1 alpha diversity metric utilized a otu table without singletons removed to obtain a visual representation of the relative abundance differences between the nine samples for the 3,832 otus, a logtransformed heatmap was generated with the otus sorted by phylogeny along the y-axis (figure 4). the visual otu relative abundance for the amd sample subgroup exhibited a relative low abundance of otus indicated by color intensity across the set of 3,832 otus compared to the ewc sample subset. this result is indicative of low amd alpha diversity and more generally of a relatively different phylotype composition between the two environments. at the phylum level, amd and ewc subgroups exhibited differential patterns of relative abundance of major phyla (phyla with >2% relative abundance after css normalization) (figure 5). amd samples singletons, and assign otu taxonomy (table 2). across the nine samples, 3,832 otus were observed in the final otu table. depending on the specific downstream analysis, the table was either rarefied or underwent css normalization to account for the varying read count depth across samples, which ranged from 21,994-37,949 reads/ sample location. 52• fine focus, vol. 4(1) figure 4. relative abundance heat map of the 3,832 usearch clustered taxa. a css normalized relative abundance otu table was used to plot log-transformed relative abundance intensity for each of the 3,832 clustered otus for each sample location. note the relative abundance of otus in the ewc sample location microbiomes (ew-a, ewusm, & ew-g) compared to the amd sample location microbiomes (cells 1 to 3, smt, swd, & sma). generally exhibited a higher relative abundance of proteobacteria, actinobacteria, chloroflexi, and firmicutes phyla. the ewc subgroup generally exhibited a higher relative abundance of bacteroidetes and verrucomicrobia phyla. these differences in the relative abundance of phyla were also observed to drive differences in beta diversity between amd and ewc sample location groups when visualized via 3-d pcoa biplots (data not shown). differential abundance analysis was performed on the amd and ewc subgroups to identify over/under-represented taxa between the sample groups. a single statistically significant otu (bonferroni corrected; p=0.034), acidobacteria-6 iii1-15, which was identified down to the order level, was absent in amd samples, but present at low levels in the ewc samples. although not significant, the bdellovibrio and gallionella genera were notably over-represented in amd samples with gallionella more abundant in the solomon mine adit sample locations than within the other amd sampling locations. 53applied & environmental microbiology • 0% 10% 20% 30% 40% 50% 60% 70% 80% 90% 100% smt sma cell 1 cell 2 cell 3 swd ew-a ew-g ew-usm cs s n or m al ize d re la tiv e ab un de nc e sample location k__bacteria;p__proteobacteria k__bacteria;p__bacteroidetes k__bacteria;p__planctomycetes k__bacteria;p__actinobacteria k__bacteria;p__chloroflexi k__bacteria;p__verrucomicrobia k__bacteria;p__acidobacteria unassigned k__bacteria;p__firmicutes k__bacteria;p__cyanobacteria k__bacteria;p__gemmatimonadetes k__bacteria;p__armatimonadetes k__bacteria;p__wps-2 k__bacteria;p__chlorobi k__bacteria;p__spirochaetes rare taxa (<2%) figure 5. relative abundance of bacterial phyla by sample location. a css normalized and singleton filtered otu table was utilized to show phyla that make up >2% relative abundance in at least one of the sample locations. amd sample group: smt, sma, cell 1, cell 2, cell 3, swd. ewc sample group: ew-a, ew-g, ew-usm. diversity analysis the amount of diversity within each sample site was determined using a collection of alpha diversity metrics and indices. multiple rarefactions were performed to normalize the data set for alpha diversity analysis and to validate that sequencing was done at an adequate depth for fair representation of the microbial community. the raw read depth ranged from 21,994-37,949 reads/sample, thus the otu table was rarefied without replacement to a depth of 21,600. the resulting rarefactions were collated and a rarefaction plot was generated for the sample locations. the resulting rarefaction curves were indicative of quality representative samples as the curves were close to reaching the asymptote (figure 6). observed species (p=0.013), phylogenetic diversity index (p=0.021), chao1 richness estimator (p=0.006), and the shannon index (p=0.039) exhibited 54• fine focus, vol. 4(1) figure 7. alpha diversity metric box plots. error bars represent +/1.58*iqr/ sqrt(n). an unfiltered otu table was subsampled without replacement for 20 iterations using 500 sequence steps with a maximum of 21,600 sequences. (a) chao1 estimator using an otu table with the presence of singletons. (b) phylogenetic diversity, (c) shannon index, (d) heips evenness index. statistically significant differences in alpha diversity between amd and ewc sample location microbiomes (figure 7). heips evenness index (p=0.123) did not indicate a statistically significant difference between amd and ewc suggesting that although there is a marked difference in species richness between amd and ewc sample locations, that a portion of those additional taxa in ewc may not be as relatively abundant. the input otu table used for chao1 alpha diversity analysis was not filtered for singletons due to the chao1 metric requirement of needing to take rare taxa into figure 6. chao1 rarefaction curve for amd and ewc samples. solomon mine acid mine drainage (amd -red) and east willow creek (ewc blue). an unfiltered otu table was subsampled without replacement for 20 iterations using 500 sequence steps with a maximum of 21,600 sequences. chao1 alpha diversity was calculated on the directory of rarified tables, and the multiple rarefactions were collated to produce the plot. amd sample group: smt, sma, cell 1, cell 2, cell 3, swd. ewc sample group: ew-a, ew-g, ew-usm. 55applied & environmental microbiology • account for modeling. the amount of diversity between sample sites was determined through beta diversity analysis. the original otu table was filtered to remove singletons and rare taxa (<0.005% relative abundance) that might inflate the data set (4). to account for sequencing depth, the filtered otu table was normalized via cumulative sum scaling (34) and used as an input for the calculation of a weighted unifrac distance metric (29). this data reduction step was then visualized via a principle coordinate analysis (pcoa) plot along with the principle coordinates summarized via a scree plot. the scree plot indicated that the samples generated 100% of the variance within 7 dimensions, with >86% of the variance observed in the first 3 dimensions (supplemental figure s1). the amd and the ewc sample microbiomes clustered tightly in the first two dimensions of the pcoa plot, with the exception being the constructed wetland cell 3 which diverged from the other amd samples along the second principal coordinate (figure 8). beta diversity between sample location microbiomes exhibited statistical significance via the adonis permutational multivariate analysis of variance statistical test using the three clustered sample group microbiomes as an input (p<0.001). furthermore, distance comparison boxplots representing all the dimensions of variance from the distance matrix indicated a statistically significant difference when comparing diversity within and between the three categories (p<0.001) (data not shown). supplemental figure s1. scree plot of principal coordinate analysis. 100% of the variance can be explained within seven coordinates. 56• fine focus, vol. 4(1) analysis of the major taxa and their relative abundance driving the beta diversity clustering of the samples was performed via 3-d pcoa biplots. this data supports the general observed differences in relative abundance at the phylum level. further exploration of pcoa biplots down to the family level, reveals major taxa in terms of abundance that drive differences in microbial community composition between amd, ewc, and constructed wetland cell 3. amd sample locations were associated with the relative abundance of the family-level taxa acetobacteraceae and rhodospirillaceae, which are both hierarchically placed within the proteobacteria phylum. constructed wetland cell 3 beta diversity was driven by the phylum chloroflexi and the family isophaeraceae and a group of unassigned taxa. lastly, the planctomycete phylum family member germataceae and chitinophagaceae family member of the bacteroidetes phylum are particularly notable in driving beta diversity in ewc samples (figure 9). figure 8. first two principal coordinates of a principle coordinate analysis (pcoa) plot. solomon mine acid mine drainage (amd red circles), constructed wetland cell 3 acid mine drainage (cell 3 blue box), east willow creek (ewc orange triangles). 57applied & environmental microbiology • figure 9. circos plot of family-level taxa driving beta diversity between amd, cell 3, and ewc sampling locations. each family-level taxon indicated on the left side of the plot is represented with a colored ribbon. the width of each thread of the split ribbon directly correlates with the number of normalized average otu reads associated with the respective sample location that the ribbon bridges to on the right side of the figure. the outer ring segments are color coded along a percentage scale of total reads for the family member that associate with each sample location. css normalized otu table was positive filtered for the respective family-level taxa. read counts were averaged for each grouped microbiome. 58• fine focus, vol. 4(1) interestingly, a group of unassigned otus was prominent across all the sample locations after taxonomy was assigned using the greengenes reference database. in the 3-d beta diversity biplots, the unassigned taxonomy group appears to be driving beta diversity in the amd samples along principal coordinate 1 as well as forcing the wetland cell 3 sample location to diverge along principal coordinate 2. to further investigate this group, we tabulated the total number of normalized reads across samples and sorted the unassigned otus by number of reads, followed by sorting the top unassigned otus with maximum relative distribution across all samples. next, to gain insight into the unassigned identities, alpha diversity analysis indicates that the diversity within samples is relatively higher for ewc compared to amd sample locations, as is expected considering the more challenging physiological conditions associated with low ph and higher soluble concentrations of heavy metals. the lower alpha diversity in amd samples is significantly supported beyond the number of otus by species estimators and indices that took species richness, evenness, and/ or evolutionary distance into account. for example, the chao1 species richness estimator attempts to predict species saturation at the asymptote of the species accumulation curve by considering the number of rare taxa (9). this estimator is thought to provide a more realistic picture than observed species alone. the shannon index takes into account both we used the reference sequence for each respective unassigned otu as an input for the ribosome database project (rdp) classifier algorithm (44). the majority of the top greengenes unclassified otus had a higher relative abundance within the amd sample location subgroup. furthermore, the predominant phylum predicted by rdb classifier for the majority of these samples was chloroflexi, which already was shown to be driving variation along the second principal coordinate. thus, in conclusion, the unassigned taxa group is composed to a large degree with chloroflexi taxa, which dominate amd sample locations, especially wetland cell 3. taxa richness and evenness (differential abundance), as a community may have a large number of taxa but be dominated by a few select taxa (37). the heips evenness alpha diversity index does not yield statistical significance, suggesting that a proportion of additional taxa in ewc may be rare taxa. recently there has been renewed interest in rare taxa with the evidence suggesting that rare taxa can dictate and influence the dynamics of microbial communities (2). thus, evenness may be a good descriptive index but not a cut and dry indicator of alpha diversity significance. beta diversity analysis indicates that the diversity between sample locations was substantially different between ewc and amd subgroups along the first principal discussion 59applied & environmental microbiology • coordinate (54.64% of explained variance). the second principal coordinate (20.91% of explained variance) further separated the solomon mine constructed wetland cell 3 from the other sample locations, suggesting a large change in community composition compared to the core amd (smt, sma, cell 1, cell 2, and smd) and ewc groups. pcoa biplots indicate that firmicutes play a role in driving this positioning of the constructed wetland cell 3 community within the second principal coordinate. cell 3 also physically differed from the other constructed wetland cells in that it had noticeable algal and aquatic plant growth. constructed wetland cells 1 and 2 were heavily stained with iron oxides and lacked noticeable biological activity to the naked eye (figure 3). the distance matrix comparison, which took all seven collective principal coordinates into account, exhibited a statistically significant difference between amd and ewc sample location groups, suggesting that the variance between samples were statistically closer to each other than between sample locations. historically, the constructed wetland system has had only a minor effect on reducing metal loads and increasing ph after the first three years of service (47). although our budget did not allow for heavy metal analysis of the water samples, our waterquality data collected during field work (ph and conductivity), along with recent water-quality data by the wcrc suggest that heavy metals continue to leach from minerals exposed in the mine workings and enter ewc (table 3). this study’s budgetary restrictions and experimental design resulted in low statistical power, but interesting changes in relative abundance data of microbes suggests future investigation is warranted. for example, the parasitic bacterium bdellovibrio was observed to be higher in abundance in amd samples compared to ewc (fdr adjusted p=0.685). this bacterium is interesting from the standpoint that it has the potential to be used as a means for biological control of amd production by reducing populations of gram-negative chemolithotrophs through predation (20). gallionella is another interesting genus associated with amd as gallionella species are iron-oxidizing chemolithotrophs (fdr adjusted, p=0.615) (45). this microbe was observed at higher relative abundance levels within the mine adit, where chemolithotrophs can thrive (fdr adjusted, p=0.311). more generally, the phylum proteobacteria was enriched within the amd sample locations, which makes sense in that they are commonly found in acidic ecosystems (31). the phylum proteobacteria includes the acetobacteraceae and rhodospirillaceae family members (21). these alphaproteobacteria and betaproteobacteria family taxa, respectively, drove beta diversity separation between amd and ewc sample locations. acetobacteraceae are acid-tolerant aerobes that typically ferment organic material on the surface of water and are likely to thrive in amd outside of the mine adit, while rhodospirillaceae family members are purple non-sulfur bacteria that can utilize anoxygenic photosynthesis 60• fine focus, vol. 4(1) using hydrogen or low concentrations of sulfide while living in anaerobic aquatic environments, such as the amd sediment. constructed wetland cell 3 contained an abundance of bacteria within the phylum chloroflexi, which are generally composed of purple and green bacteria that may use sulfide as an electron donor for anoxygenic photosynthesis and have previously been associated with amd (48). additionally, the phylum firmicutes strongly drives the microbial community in cell 3 along the second principal coordinate axis. there are many sulfate-reducing bacteria that belong to the firmicutes, such as the alicyclobacillaceae family found in cell 3, which likely are reducing sulfate and thus reducing ph and helping precipitate heavy metals. the aquatic bacterial family isosphaeraceae, which includes a number of characterized moderately acidophilic genera, was also associated with wetland cell 3 (25, 28). acidophilic isophaeraceae members are chemohetrorophs that rely on carbohydrates yielded from plant and microbial sources, thus they may be important in the degradation of the abundant organic material observed within cell 3 (10). limitations of this preliminary analysis include low statistical power, which increases the risk of type ii error, and thus our ability to identify major taxa that are over/ under-represented in the sample groups and are likely ecosystem biomarkers. due to budgetary limitations, biological replicates from three triangulated sediment samples for each location were pooled prior to pcr. this technique has been demonstrated to often miss locally rare prokaryotes, but yielded spatially ubiquitous phylotypes at higher taxonomic classifications (30), which was our aim. future examination of the microbial populations at this project site should address this issue through use of true biological and technical replicates. a series of replicates combined with deeper sequencing will shed light on the role of rare microbial community members on dynamic interactions as well as provide us with a better idea of spatial niches (2, 35). additionally, we are interested in how seasonal changes may affect the taxonomic profile of the sampling sites. we hypothesize that microbial community composition will be correlated with geochemical variables including ph and metal concentrations as flow rates drop during the late summer/early fall as has been observed in other amd studies (1). likewise, temperature fluctuation and changes in dissolved oxygen levels during the winter in this high-elevation, alpine environment will likely drive changes in the taxonomic profile due to reduced photosynthetic activity and diffusion from the atmosphere resulting from ice and snow that cover the constructed wetland cells. taxonomic resolution is also a limiting factor in our study. we used the v4 hypervariability region of the ssu 16s rrna gene (8) with our miseq run with paired-end (pe) 150 bp reads that provided 48 bp overlap. although sufficient for pairing reads to spanning the 252 bp v4 hypervariability region, we would expect to increase taxonomic resolution by sequencing with longer pe reads (250+ bp), which would retain a greater percentage of reads by boosting quality through full overlap of 61applied & environmental microbiology • the v4 hypervariability region. alternatively, longer reads could be used with a new primer set constructed to span both the v3 and v4 hypervariability regions for longer pe reads to increase the data set size and resolution (17). either approach (increased quality or amount of data), obtained with miseq pe 250+ bp reads would likely provide a desired data set through more accurate clustering, assignment of taxonomy, and generation of a more accurate phylogenetic tree (46). biases that should be considered when interpreting our data include limitations of using a single marker gene for inferring phylogeny, in addition to biases introduced during pcr. the ssu 16s rrna gene is a well-documented marker gene and has extensive database records to associate with the data, but is likely not always the best measure of phylogeny between all organisms. pcr primer binding fidelity and the stochastic processes that occur early during amplification can introduce biases in the amplicons produced to be sequenced (36). pcr enzyme choice also has been demonstrated to effect accuracy of microbial community profiling (19). future studies could address these limitations by taking a true shotgun metagenomic approach to community dna analysis. not only would this approach provide taxonomic information through the combination of multiple marker genes, but it would also provide us with information on the functional potential of the microbes within the ecosystem (14). for future study, we are interested in investigating the taxonomic composition of biofilms found within the solomon mine adit in addition to looking at differences between amd microbial communities within the adit (no light or source of significant organic matter) to those directly towards the end of the first constructed wetland cell (exposed to light with organic matter). we hypothesize that phototrophs may play a larger role in generating organic matter, while chemolithotrophs are likely to have a reduced role in the bioremediation cells considering the more abundant environmental oxygen and nitrogen sources from organic debris present in those cells. to gain insight into the ecological role of these microbes, we plan to correlate microbe presence with dissolved oxygen levels and sources of carbon and nitrogen. further shotgun metagenomic analysis could also reveal the relative abundance of pathways for carbon, nitrogen fixation, and energy generation expected for this extreme environment. association of these geochemical parameters with differentially abundant microbes will allow us to understand symbiotic relationships between taxa in such an ecosystem (43). secondly, we are interested in determining the metagenome or metatranscriptome of the microbes found within constructed wetland cell 3 to better understand the metabolic potential or functionality of the microbial community. visual inspection alone differentiates the cell from the other amd sampling location sites considering the extensive algal growth (figure 3) and the water-quality measures of an increase in ph and reduction in conductivity suggesting sulfate-reducing bacteria may be at work. the beta diversity analysis also indicates 62• fine focus, vol. 4(1) that microbial community composition is significantly different from that of the other amd sample locations and in ewc. in conclusion, further investigation is necessary to determine if current microbial we thank the adams state university porter scholars in science & mathematics program for the support of ryan miller. gwen nelson, watershed coordinator with willow creek reclamation committee, was instrumental for access to the solomon mine adit and providing historical 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the world but particularly in southeast asia and northern australia. b. pseudomallei is inherently antibiotic resistant and therefore new therapies are needed to combat this pathogen. previous studies with the related organism burkholderia thailandensis have shown that the antibiotic ceftazidime does not eliminate all bacteria in an in vitro macrophage model, and the remaining bacteria could still pose a health threat to a potential host. due to their survival in the presence of antibiotics, we hypothesized that the remaining bacteria were one of two types of antibiotic tolerant cells: classically antibiotic resistant cells or persister cells. to test our hypothesis we isolated the bacteria that had survived ceftazidime treatment in the macrophage infection model and performed additional in vitro experiments to show that the surviving bacteria are neither antibiotic resistant nor persister cells. instead, they are still susceptible to high doses (200 µg/ml) of the antibiotic over a period of 48 hours (p<0.001). we believe the bacteria survive exposure to the antibiotic during the macrophage infection because of their ability to move between intracellular and extracellular compartments, thus avoiding the antibiotic and its deadly effects. our results provide evidence to suggest that intracellular pathogens, through movement between intracellular and extracellular compartments, may be protected from the effects of antibiotics in similar macrophage infection models. abstract keywords • persister cells • antibiotic resistance • burkholderia • macrophages • intracellular bacteria introduction burkholderia pseudomallei is a facultative intracellular pathogen and the etiological agent of melioidosis (1, 13, 14, 19). this disease is endemic in regions such as southeast asia and northern australia and causes potentially fatal infections that can present as pneumonia or septicemia (16). b. pseudomallei is inherently highly antibiotic resistant and due to the swift progression and deadly nature of the disease it causes, is considered to be a bioterrorism threat (17). there is no current vaccine to prevent this corresponding author kara mosovsky mosovskyk@moravian.edu 119pathogens & antimicrobial factors • deadly disease, and most of the treatments for melioidosis involve several weeks of intravenous antibiotics, often with the antibiotic ceftazidime (13, 19). however, even with antibiotic treatment, mortality rates still range from 20-50%, and as many as 10% of individuals experience recurrent infections, usually due to relapse of the original infection (17). the high rate of relapse infections would suggest that complete elimination of the pathogen is important for a patient’s full recovery. considering the challenges of treating melioidosis, it is essential to develop new therapies, which could potentially decrease the burden of disease. a series of previous studies have investigated one alternative treatment option in a macrophage infection model using both the virulent b. pseudomallei and also the closely related and avirulent organism burkholderia thailandensis. these studies show the ability of the antibiotic ceftazidime to interact with the macrophage activator, interferon-gamma (ifn-γ), to synergistically reduce the bacterial burden both inside the infected macrophages (intracellular compartment) and outside of the infected macrophages (extracellular compartment) (10, 12). what was surprising from this study was that the infected macrophages treated with antibiotic alone still harbored a significant amount of extracellular bacteria, even when the antibiotic concentration was approximately five times the minimum inhibitory concentration (mic) (10). since current melioidosis treatment relies almost exclusively on antibiotics alone, this result was disconcerting and warranted further investigation. such remaining bacteria may still pose a significant threat in a host and could explain why patients with melioidosis often develop recurrent infections due to relapse of the original infection. from these previous studies we developed a new research question regarding these surviving extracellular bacteria: are the remaining extracellular bacteria still sensitive to the antibiotic or have they become antibiotic tolerant? this question was the focal point of our study. there are two main types of antibiotic tolerant bacteria. classical antibiotic resistance is a permanent and heritable tolerance to antibiotics, while persistence is a temporary and non-heritable tolerance (8). persister cells are thought to be present as a small subpopulation in any large bacterial population and are characterized as slow or non-growing, dormant, bacterial cells that are temporarily tolerant to antibiotics due to their lack of replication (3-5, 8, 9). additionally, when the antibiotic is removed and persister cells resume replication, most of the progeny of persister cells are susceptible to the antibiotic, a trait that helps distinguish them from classically resistant bacteria (7, 8). while the rate of persister cell formation is typically less than 1% for many pathogens, studies on burkholderia persisters have shown that cultures of b. pseudomallei and b. thailandensis can harbor around ten times higher frequencies of persister cells compared to other bacteria such as escherichia coli, staphylococcus aureus, and pseudomonas aeruginosa (2, 6, 11, 18). we therefore had reason to hypothesize that persister cells may compose some or all of the remaining population of bacteria that had survived antibiotic treatment 120• fine focus, vol. 4(1) during the macrophage infection. the aim of the current study was to determine if the remaining extracellular bacteria were sensitive to antibiotics, antibiotic resistant, or persister cells. by further characterizing the surviving extracellular bacteria, we would have the opportunity to gain insights about the treatment of macrophages in our in vitro model and help inform future treatment options against melioidosis. materials and methods biochemicals ceftazidime hydrate was purchased from sigma-aldrich (st. louis, mo). recombinant murine ifn-γ was purchased from peprotech (rocky hill, nj). kanamycin monosulfate was purchased from mp biomedicals, llc (santa ana, ca), and luria bertani (lb) broth was purchased from thermofisher scientific (waltham, ma). bacteria burkholderia thailandensis e264 was purchased from american type culture collection (manassas, va) and used for all experiments. a single isolated colony was inoculated into sterile lb broth and incubated on an ambient air, shaking incubator at 200 rpm for 16 hours at 37°c. overnight cultures were aliquoted and stored at -80°c in 0.5 ml volumes as a 15% glycerol stock solution. a new vial of b. thailandensis was thawed and resuspended immediately prior to use in the macrophage infection assay. cell lines the murine macrophage cell line, raw 264.7, was purchased from american type culture collection (manassas, va) and used for all macrophage infections. macrophages were maintained in complete medium consisting of 1x minimum essential medium (mem; thermofisher scientific) supplemented to a final concentration of 10% equafetal bovine serum (atlas biologicals, fort collins, co), 1x mem non-essential amino acids, 0.5x mem essential amino acids, 0.075% sodium bicarbonate (sigma-aldrich), and 2 mm l-glutamine solution. in order to maintain sterility of the cells, passages of the macrophages were maintained in complete mem supplemented to a final dilution of 100 µg/ml of penicillin-streptomycin solution. all macrophage infections were performed in antibiotic-free complete mem (cmem). macrophages were maintained in a 37°c incubator supplemented with 5% co2 and 80% relative humidity. macrophage infection assay the macrophage infection was conducted as previously described (10, 12). briefly, a hemocytometer was used to appropriately count and dilute the raw cells to a density of 200,000 cells per well in 0.5 ml of cmem. after the cells were added to a 24-well plate, they were incubated at 37°c with 5% co2 and 80% humidity overnight 121pathogens & antimicrobial factors • for adherence. after this incubation period, the cells were observed under an inverted microscope to determine the general health of the cells. the cells in each well were washed one time, slowly and carefully, with 1 ml of phosphate buffered saline (pbs). a bacteria dilution of b. thailandensis in cmem was then applied to the cells at a multiplicity of infection of 5, and incubated for 1 hour. after the first hour of incubation, the cells were again slowly washed one time with 2 ml of pbs and then 1 ml of a 350 µg/ml kanamycin solution was added into each of the wells and incubated for 1 hour to remove extracellular bacteria. after this final incubation period, the cells were washed two times with 2 ml of pbs to remove the killed extracellular bacteria. then 0.5 ml of a 10 µg/ml solution of ceftazidime, was added into each of the wells and incubated for 18-24 hours. testing for antibiotic resistance after 24 hours of infection and treatments, the remaining extracellular bacteria were carefully resuspended in their wells and 100µl was directly plated onto agar with varying concentrations of ceftazidime. the agar plates contained either no antibiotic, 10 µg/ml of ceftazidime, or 200 µg/ml of ceftazidime (which equates to ~100x minimal inhibitory concentration (mic) of 1.75 µg/ml) (20). plates were incubated in an ambient air incubator for 24-36 hours at 37°c and then inspected for growth. testing for persister cell formation after the 18-hour treatment period of the macrophage infection assay, macrophages were inspected for apparent signs of infection and consistency between wells. remaining extracellular bacteria were resuspended slowly and carefully, and transferred to 2 ml tubes, combining two identical wells into 1 tube due to low numbers of remaining bacteria. tubes of bacteria were centrifuged at 12000 xg for five minutes, the supernatant was carefully removed, and a 1 ml wash with complete cmem was added to each tube. after a second spin, the pellets were reconstituted in 0.5 ml cmem and the entire tube contents were plated into separate wells of a new 24-well plate. ceftazidime was readded to a final concentration of 200 µg/ ml and returned to the incubator at 37°c with 5% co2 and 80% humidity. subjecting bacteria to very high doses of antibiotics is one way to identify persister cells (7). serial dilutions of the surviving bacteria were plated at various time points postantibiotic treatment, and bacterial density was calculated as colony forming units per milliliter (cfu/ml) after incubation of the agar plates. statistical analyses prism software version 7.01 (graphpad, la jolla, ca) was used to plot the mean and standard error of the mean (sem) for different treatment groups. one-way analysis of variance (anova) and tukey’s post test was used to compare differences between three or more treatment groups. 122• fine focus, vol. 4(1) results testing for classical resistance previously, we found that treatment of b. thailandensis-infected macrophages failed to kill all extracellular bacteria even after 18-24 hours of exposure to ceftazidime above the mic. to determine if these remaining bacteria had developed classical resistance to ceftazidime during the treatment, we first recapitulated the standard in vitro infection with appropriate treatments. we then harvested the remaining survivors from the treatment wells and plated them on antibiotic-infused agar. after incubation, bacteria were only detected on the antibiotic-free control plates, with the greatest density of bacteria coming from the untreated control wells of the macrophage infection (table 1). ceftazidime treatment of infected macrophages yielded many surviving bacteria on the antibiotic-free control plates, and the combination treatment of ceftazidime and ifn-γ yielded no detectable surviving bacteria. no extracellular bacteria grew on ceftazidimeinfused agar, regardless of the concentration and regardless of the treatment the infected macrophages had received. with these results in mind, we next asked whether or not the bacteria that were surviving 18-24 hrs of ceftazidime treatment during the macrophage infection were persister cells. 123pathogens & antimicrobial factors • table 1: remaining bacteria fail to grow on antibiotic-infused agar. raw macrophages were infected with b. thailandensis, and treated for 18 hours with ceftazidime alone (10 µg/ml) or the combination of ceftazidime (10 µg/ml) and ifn-γ (10 ng/ml). surviving extracellular bacteria were then plated onto agar plates without antibiotic, or with 10 µg/ ml or 200 µg/ml ceftazidime. this table represents similar data from two independent experiments. testing for persister cells to determine if the remaining extracellular b. thailandensis were persister cells, we again harvested the bacteria from the end of the macrophage infection to determine their sensitivity. after 18 hours of ceftazidime treatment (10 µg/ml) of the infected macrophages, the remaining extracellular bacteria were separated from the macrophages, processed, and re-subjected to a higher concentration of ceftazidime to identify persister cells. at different time points well contents were serially diluted and plated onto lb agar. as shown in (figure 1), the bacteria that had previously survived antibiotic treatment in the presence of the macrophages were now killed by high doses of ceftazidime (200 µg/ml) over the 48 hour time frame, with most of the killing occurring by 24 hours. from time point 0 to 3 hours, there was no statistical difference in how many bacteria were killed by the antibiotic. there was however, statistically significant killing at the time point for 24 hours (p<0.01). by 48 hours, we were unable to detect any viable b. thailandensis cells (p<0.001), indicating that all bacteria had been killed. 124• fine focus, vol. 4(1) figure 1: after 18 hour macrophage infection, remaining extracellular bacteria are still sensitive to high doses of antibiotic. raw cells were infected with b. thailandensis. after 18 hours of treatment with 10 µg/ml ceftazidime, the surviving extracellular bacteria were removed, washed, and re-subjected to ceftazidime at 100x the mic (200 µg/ml) in a macrophage-free system. bacterial density was determined at time points 0, 1, 2, 3, 24, and 48 hours by plating dilutions of the well contents. data is representative of three similar and independent experiments that showed similar trends in killing. means and sem were plotted for each time point. one-way anova was used to determine statistically significant differences from time point 0, with ** indicating p < 0.01, and *** indicating p < 0.001. discussion the purpose of these experiments was to determine if the extracellular bacteria that were surviving 18 hours of treatment with ceftazidime in the macrophage infection model were classically resistant or if they were considered to be persister cells. similar to titball et. al. we plated the bacteria onto antibiotic-infused media to test for antibiotic resistance due to spontaneous mutations (2). in table 1, we showed a lack of growth on the antibiotic-infused plates, which serves as evidence that the extracellular bacteria were not classically resistant. in classical resistance, the 125pathogens & antimicrobial factors • progeny of the resistant cells would also be resistant, therefore we would expect to see whole colonies of antibiotic-resistant bacteria on the plates infused with antibiotic concentrations above the mic. instead, we were only able to see growth on the antibiotic-free control plates. since we did not plate all of the volume from each of the treated wells of the macrophage infection, there is also a limit of detection to our assay, and we cannot say with certainty that the well contents did not contain any antibiotic resistant bacteria. regardless, we were able to determine that the majority of the extracellular bacteria are not resistant. previous studies found low bacterial burden in the extracellular compartment following macrophage treatment with ceftazidime and ifn-γ (10). here we were unable to detect any extracellular bacteria from the wells of infected macrophages that had received the combination treatment group, which indicates that this population of bacteria is very small, and provides further evidence that the combination therapy may provide significantly more protection than the antibiotic alone. while table 1 shows evidence that the remaining bacteria are not classically resistant, from these results alone, we cannot rule out the possibility that some, or many, of the treatment-surviving bacteria are persisters. if persisters existed following treatment in the macrphage infection model, then they would tolerate high doses of antibiotics on the agar plates, but the great majority of their progeny would be susceptible, and therefore, we wouldn’t be able to visualize any colonies on the agar plate. since this theory mirrored what we observed, we hypothesized that some remaining bacteria may be persisters. we decided to identify potential persisters as done previously by treating the bacteria with a high dose of antibiotic and looking for a plateau in the killing over time (4, 9). in such cases, the bulk of the population of bacteria is killed, while the antibiotic tolerant persisters survive. persisters are identified as the subpopulation of surviving bacteria after the bulk of the population has been eliminated. graphically, the sensitive majority of the population dies quickly over time, but then the killing curve plateaus because the remaining persisters are tolerant to the antibiotic (4, 9). previous experiments with b. pseudomallei 82 showed that bacteria exposed to cefotaxime at 100x the mic, without shaking, were killed modestly for 24 hours, after which there was no additional killing throughout the 33 hour experiment (11). in these experiments, there was less than a 10 fold overall decrease in bacteria from time 0 through the 33 hour antibiotic treatment period, showing tolerance of the bacteria to the antibiotic. we expected persister cells in our assay to show a similar trend in tolerance when exposed to 100x the mic of antibiotic over a period of time, even though we used b. thailandensis instead of b. pseudomallei and a different cephalosporin antibiotic. if most of the remaining bacteria were indeed persisters, we would have expected to see little killing or at least a plateau in the killing over time. in figure 1 we show that the remaining extracellular bacteria are still sensitive to high doses of ceftazidime over a 24 hour period, with no detectable bacteria by 48 hours. while we certainly saw significant killing over time, we cannot say definitively that the antibiotic completely sterilized the wells, due to our limit of detection. based on standardized definitions of the limit of detection (15) we estimate that the number of remaining 126• fine focus, vol. 4(1) bacteria at 48 hours post-treatment is <1 cfu/ml. overall, our evidence has suggested that the extracellular bacteria that survive the 18-24 hour treatments during the macrophage infection remain sensitive to both low (10 µg/ml) and high doses (200 µg/ml) of the antibiotic ceftazidime. previous studies have suggested that the transitioning of extracellular b. thailandensis to the intracellular space, upon uptake, or transitioning of intracellular bacteria to the extracellular space, upon macrophage lysis, may influence in vitro macrophage infection models (10). we now expand upon that hypothesis, suggesting that the dynamic movement of extracellular bacteria to the intracellular compartment may protect them from the antibiotic. this protected movement between compartments may explain why bacteria are still present in low numbers in the extracellular niche of the macrophage infection model after 18 hours of treatment, and yet are still sensitive to the antibiotic. perhaps it is the rapid movement between extracellular and intracellular compartments that accounts for their protection from the antibiotic. future studies will attempt to tease out these nuances of the macrophage infection model through visualization of real-time infections. the interactions between bacteria in the intracellular and extracellular compartments could also be studied through repeated saline washes of the extracellular compartment throughout the 18 hour infection. if there is a dynamic movement of bacteria between compartments, and if the bacteria are not able to re-infect the macrophages once released to the extracellular compartment, we would expect the intracellular numbers to fall more quickly over time upon treatment with the synergistic combination of ifn-γ and ceftazidime. even if the bacteria are neither resistant nor persister cells, there might be some difference in susceptibility of the extracellular bacteria that remain at the end of the macrophage infection compared to bacteria from untreated wells of the macrophage infection. future experiments to compare the rate of killing of the extracellular bacteria that came from antibiotic-treated macrophages to those that came from untreated macrophages, and even to bacteria never exposed to macrophages. these experiments could determine if interactions with the macrophages affords the bacteria any amount of tolerance to the antibiotic. lower doses of antibiotic could also be used against these groups to compare killing curves. in conclusion, we have shown evidence that the b. thailandensis cells that survive ceftazidime treatment for 18-24 hours in the macrophage infection model are still sensitive to high and low concentrations of ceftazidime, and they are not classically resistant nor are they persister cells. we hypothesize that the ability of the bacteria to move between intracellular and extracellular compartments during the macrophage infection likely accounts for their ability to survive long periods of time in the presence of bactericidal concentrations of ceftazidime. future studies will continue to investigate various host-pathogen interactions of this macrophage infection model. 127pathogens & antimicrobial factors • acknowledgements all research was conducted at moravian college in biosafety level-2 facilities in compliance with federal and institutional regulations for handling infectious agents. funding was provided by the beta beta beta research scholarship foundation fund as well as the department of biological sciences at moravian college. 128• fine focus, vol. 4(1) references 1. allwood, e. m., devenish, r. j., prescott, m., adler, b., & boyce, j. d. (2011). strategies for intracellular survival of burkholderia pseudomallei. front microbiol, 2, 170 2. butt, a., higman, v. a., williams, c., crump, m. p., hemsley, c. m., harmer, n., & titball, r. w. (2014). the hica toxin from burkholderia pseudomallei has a role in persister cell formation. biochem j, 459(2), 333-344. 3. dawson, c. c., intapa, c., & jabrarizk, m. a. (2011). “persisters”: survival at the cellular level. plos pathog, 7(7), e1002121. 4. fauvart, m., de groote, v. n., & michiels, j. (2011). role of persister cells in chronic infections: clinical relevance and perspectives on anti-persister therapies. j med microbiol, 60(pt 6), 699-709. 5. jayaraman, r. (2008). bacterial persistence: some new insights into an old phenomenon. j biosci, 33(5), 795-805. 6. keren, i., shah, d., spoering, a., kaldalu, n., & lewis, k. (2004). specialized persister cells and the mechanism of multidrug tolerance in escherichia coli. j bacteriol, 186(24), 81728180. 7. lewis, k. (2005). persister cells and the riddle of biofilm survival. biochemistry (mosc), 70(2), 267-274. 8. lewis, k. (2007). persister cells, dormancy and infectious disease. nat rev microbiol, 5(1), 48-56. 9. maisonneuve, e., & gerdes, k. (2014). molecular mechanisms underlying bacterial persisters. cell, 157(3), 539-548. 10. mosovsky, k., silva, e., troyer, r., propst-graham, k., & dow, s. (2014). interaction of interferon gamma-induced reactive oxygen species with ceftazidime leads to synergistic killing of intracellular burkholderia pseudomallei. antimicrob agents chemother, 58(10), 5954-5963. 11. nierman, w. c., yu, y., & losada, l. (2015). the in vitro antibiotic tolerant persister population in burkholderia pseudomallei is altered by environmental factors. front microbiol, 6, 1338. 12. propst, k. l., troyer, r. m., kellihan, l. m., schweizer, h. p., & dow, s. w. (2010). immunotherapy markedly increases the effectiveness of antimicrobial therapy for treatment of burkholderia pseudomallei infection. 129pathogens & antimicrobial factors • antimicrob agents chemother, 54(5), 17851792. 13. schweizer, h. p. (2012). mechanisms of antibiotic resistance in burkholderia pseudomallei: implications for treatment of melioidosis. future microbiol, 7(12), 1389-1399. 14. stevens, m. p., & galyov, e. e. (2004). exploitation of host cells by burkholderia pseudomallei. int j med microbiol, 293(7-8), 549-555. 15. sutton, s. (2011). accuracy of plate counts. journal of validation technology, 17(no 3), 5. 16. white, n. j. (2003). melioidosis. lancet, 361(9370), 1715-1722. 17. wiersinga, w. j., van der poll, t., white, n. j., day, n. p., & peacock, s. j. (2006). melioidosis: insights into the pathogenicity of burkholderia pseudomallei. nat rev microbiol, 4(4), 272-282. 18. wood, t. k., knabel, s. j., & kwan, b. w. (2013). bacterial persister cell formation and dormancy. appl environ microbiol, 79(23), 7116-7121. 19. wuthiekanun, v., & peacock, s. j. (2006). management of melioidosis. expert rev anti infect ther, 4(3), 445-455. 20. yi, h., cho, k. h., cho, y. s., kim, k., nierman, w. c., & kim, h. s. (2012). twelve positions in a beta-lactamase that can expand its substrate spectrum with a single amino acid substitution. plos one, 7(5), e37585. copyright 2018, fine focus all rights reserved “faculty perspective kristen picardo, ph.d associate professor, biology director, center for student scholarship, research, and creative work st. john fisher college 159 160 • fine focus, vol. 4 (2) on a recent flight i was seated next to an undergraduate ivy league college student. she was exhausted after having chosen to leave a stint in a military training academy and slept for a solid hour, mouth wide open. when she awoke, we assumed the typical mentorstudent chit-chat even before she realized i was a biology college professor and she an undergraduate biology major grappling to plan her future. she was confused and needed to tell someone about it, and i couldn’t help but be her sounding board, offering free advice – get in the lab and try a research experience! often times, students seek out a welcoming professor to ask about how they can get involved as a student researcher. at my small pui, our doors are open, our classes are small, and so we enjoy plenty of student interaction and time to explore such conversations. we connect our students with our colleagues who have research expertise in the students’ favorite course topic and natural connections occur. at times this serendipitous method isn’t the best approach. if a student is one of many or is not comfortable approaching a professor, it can be helpful if the college or program offers details on a website or through scheduled information sessions. some colleges have formalized offices that serve to reach out to educate students about research opportunities on and off their home campus. attending these sessions can be very helpful and informative and remove the hesitation some students feel about approaching a faculty mentor on their own. a survey at my own institution revealed that most of our students learn about research opportunities through talking with other students. attending research presentations hosted by departments is also a great way to learn about opportunities. when students approach me or when i discuss research projects ongoing in my laboratory, i do two things: 1) get them excited and 2) use language that shows them that my research is accessible, understandable, and not just for senior students. in order to succeed, i’ve learned that students in my lab need to be excellent communicators. i am not with them every hour they spend in the lab, and therefore i ask for meetings and pop-ins as well as electronic updates as students plan experiments and decipher their data. sometimes, i am “busy in meetings” when i know i want my students to struggle a bit and work toward a solution to a problem they are facing in their project. in order for a student to be ready to take on a project with me, they must have the maturity to handle to recursive nature of a sometimes frustrating process. finally, they must have time in their schedules to fully commit. the best way for students to be an attractive candidate for a project is to show their potential mentor that they have time in their schedule to fully engage with the research (e.g. minimum course load not including research, through mentored research experiences, students grow in their ability and confidence to creatively analyze information in new ways often leading to novel solutions to complex problems. they learn practical, transferrable skills and resiliency allowing them to grow from challenging experiences. these experiences prepare students for advanced work in their disciplines, and more importantly ignite their curiosity and strengthen their ability to adapt and utilize core skills and habits of mind that will prime them for their futures. it is no wonder that employers and graduate programs look highly on how these experiences prepare the future workforce. as i told the sleepy student, a research experience will transform and direct one’s future in ways traditional classwork cannot. i look forward to reading her first manuscript someday. that will prime them for their futures. it is no wonder that employers and graduate programs look highly on how these experiences prepare the future workforce. gram-negative, oxidase-positive bacteria in rainwater and wind samples kathryn e. preston and jennifer huddleston* department of biology abilene christian university abilene, tx 79699 manuscript received 2 april 2016; accepted 28 july 2016 copyright 2016, fine focus all rights reserved gram-negative, rod-shaped, oxidase-positive bacteria, such as aeromonas and pseduomonas, are widespread in the environment. aeromonas are emerging human pathogens associated with extraintestinal and opportunistic infections. though there are various biological particles known in the atmosphere, these microbial communities are poorly characterized. bacteria have the ability to remain suspended in the air for prolonged periods of time and can be transmitted through both airborne and droplet means. this study aimed to isolate aeromonas and other similar bacteria from samples from the troposphere in order to learn more about the distribution of these organisms. eleven precipitation and wind samples were aseptically collected in abilene, texas, and plated onto aeromonas blue medium plates with and without ampicillin. the 16s rdna sequences were amplified from 28 gram-negative, oxidase-positive isolates. the analyzed sequences showed that none of the isolates belonged to the genus aeromonas, but did include pseudomonas, sphingomonas, massilia, naxibacter, paracoccus, novosphingobium, and mesorhizobium, giving clues to the distribution of these organisms. furthermore, six isolates appeared to be novel species of bacteria, and several more were uncultured before this study. abstract corresponding author jennifer huddleston jennifer.huddleston@acu.edu keywords • aeromonas • pseudomonas • aerobiology • rainwater • atmosphere • wind • 16s rdna aerobiology is the study of the occurrence, dispersion, and passive transport of airborne biological particles, which could be viable or nonviable, and are distributed in and throughout the air (11). a seasonal difference has been found among the amount of culturable bacteria from air samples; the highest concentrations were shown during the summer months (29). microbial presence in air is an important factor that contributes to the transmission of infectious diseases, as evidence from previous studies has illustrated that pathogenic bacteria in the air have the ability to travel over long distances and come into contact (through inhalation and ingestion) with individuals who had no previous contact with the source of the infectious disease (8). although hundreds of thousands of individual microbial cells can exist in a cubic meter of air, the diversity, interactions, and distribution of these organisms are poorly understood (4). there are constant human-microbe interactions, both pathogenic as well as beneficial (17). various bacteria have the ability to remain suspended in the air for prolonged periods of time and can be introduction 93 • fine focus, vol. 2 transmitted through both aerosolized or airborne and droplet means (8). aerosols in the atmosphere have the ability to influence cloud formation and precipitation development, impacting the earth’s climate, water cycle, and atmospheric reactions (22,26). there is evidence suggesting that bacteria may be a major factor within the biological aerosols found in the atmosphere (33). the atmosphere has been described as “one of the last frontiers of biological exploration on earth,” which further supports the importance to investigate its impact on microbial life (34). the water cycle describes the continuous movement of water around and through the earth as its physical state is altered between liquid, gas, and ice. three percent of the earth’s water is found in the atmosphere (22). the lowest part of earth’s atmosphere, known as the troposphere, contains microbial communities that are poorly characterized at high elevations and in the air masses above the ocean (6). the poor characterization of these microbes is due to the difficulty in acquiring samples of adequate biomass, which leads to further challenges of finding sufficient dna to be used for analysis (6). rainwater harvesting is becoming an increasingly popular drinking water source. however, the consumption of rainwater could be dangerous due to the potential health risks of chemical and microbiological contamination (1). though there have been countless studies performed concerning chemical contamination of rainwater, microbial contamination continues to be somewhat unknown due to the difficulty involved in detecting pathogens (16). however, a small number of recent studies have emerged that do confirm the presence of waterborne pathogens in rainwater and air samples, but there is still much to learn (3,16,20). little is known about the occurrence and movement of particular genera of bacteria, such as aeromonas, in the troposphere. aeromonas cells are gram-negative, oxidase-positive, non-spore forming, rodshaped bacteria that are found in various aquatic habitats. this genus is a pathogen found in fish and other poikilothermic organisms, and is also regarded as an emerging pathogen in humans causing bacteremia, gastroenteritis, cellulitis, and sepsis (21). our first hypothesis was that we would isolate aeromonas from wind, rainwater, and hail samples because of its ubiquity in aquatic and other environments. however, aeromonas was not found, so our second hypothesis was that we would isolate other unique bacteria with characteristics similar to aeromonas. the second hypothesis was supported in the successful isolation of other gram-negative, oxidasepositive bacteria such as pseudomonas, aurantimonas, and paracoccus, which could potentially hold more importance than aeromonas and give insight into the microbial communities of the troposphere. materials and methods sampling, media, and growth conditions rainwater, wind, and hail samples were collected in abilene, texas, from may to june of 2014 and from january to march of 2015. the rainwater and hail were collected using applied & environmental microbiology• 94 sterile sampling jars that were set outside away from runoff of roofs and trees. hail was allowed to melt in the lab at room temperature before processing. the wind was collected using plates of aeromonas blue medium (abm) (14) by standing outside in an open field, without buildings in the way to ensure as pure of a collection as possible, with the plate open against the direction of the wind for five minutes. the temperature, time, wind direction, wind speed, humidity, and dew point were recorded for each day of wind collection. abm was made since it is not available commercially, and consisted of 4.0 g of soluble starch (mallinckrodt, st. louis, mo), 0.25 g of nh4cl (mallinckrodt, paris, ky), 1.0 g of tryptone (sigma-aldrich, st. louis, mo), 0.5 g of yeast extract (sigma-aldrich, st. louis, mo), 0.04 g of bromothymol blue (ward’s natural science, rochester, ny) 15.0 g of agar powder (alfa aesar, ward hill, ma), 1 l of distilled water and was adjusted to a ph of 8.0 using 1m koh. after autoclaving and cooling, 0.1 g of sodium desoxycholate (sigma chemical company, st. louis, mo), 5.0 ml of 0.41% l-tryptophan, and 5.0 ml of 0.99% l-phenylalanine were added and mixed. abm was made with and without 32 µg/ml ampicillin (gbiosciences, st. louis, mo). ampicillin was added to the plates in later collection dates in order to select for aeromonas and against pseudomonas since many aeromonas strains have chromosomally encoded β-lactamases and pseudomonads typically do not (14). after collection of wind-generated samples, the abm plates were incubated aerobically at 30˚c for 24 to 72 hours for colony growth. the rainwater and melted hail were diluted in 0.85% nacl to obtain countable colonies and spread onto abm plates with and without ampicillin and also incubated at 30˚c for 24 to 72 hours, depending on how quickly colony growth occurred. after growth on the abm plates, individual colonies were chosen and subcultured onto 0.2x tryptic soy agar (tsa) plates and incubated at 30˚c for 24 to 48 hours. 0.2x tsa plates consisted of 8.0 g of tsa (carolina, burlington, nc) and an additional 12.0 g agar per liter of medium. cultures were subcultured until pure cultures were obtained. screening and amplification of 16s rdna from selected isolates. each presumptive aeromonad colony was tested for a positive oxidase reaction using oxistrips (hardy diagnostics, santa monica, ca). each colony that was oxidasepositive was kept for further testing, since aeromonas species have cytochrome c oxidase in their electron transport chains. then, they were gram stained. gramnegative, oxidase-positive rods were selected for amplification by colony pcr and sequencing of the 16s rrna gene. the protocol is as follows: 5.0 µl of 10x standard taq reaction buffer (new england biolabs inc.), 1.0 µl of 10 mm dntps, 1.0 µl of 10 µm 27fm forward primer 5’-agagtttgatymtggctcag-3’ (9) (invitrogen, carlsbad, ca), 1.0 µl of 10 µm 1492r reverse primer, 5’-taccttgttacgactt-3’ (9) (invitrogen, carlsbad, ca), 0.25 µl of neb taq dna polymerase (new england biolabs inc.), 41.75 µl of nuclease-free water (invitrogen; grand island, new york) and template in the form of a colony, which totaled to 50 µl per reaction. several of the genes from isolates could not be amplified using the colony pcr protocol, so pure dna was extracted as previously described (30). the thermocycling conditions are as follows: initial denaturation: 95˚c for 5 minutes, 95 • fine focus, vol. 2 30 cycles of 95˚c for 30 seconds, 55˚c for 30 seconds, 68˚c for 1 minute 30 seconds followed by a final extension of 68˚c for 5 minutes. the products were visualized on a 0.8% agarose (invitrogen; carlsbad, ca) gel containing ethidium bromide. products of 1.5 kb were purified using dna clean & concentrator (zymo research corporation, irvine, ca) and sent to dna analysis facility on science hill at yale university (new haven, ct) for sequencing. dna sequence analysis. the national center for biotechnology information (ncbi) blastn tool and ribosomal database project (rdp) seqmatch tool were used to search nucleotide databases and to analyze and interpret the results of each gene that was sequenced. two databases were used in order to maximize the chance of finding accurate species identifications. of the ncbi blastn tools, the following information was recorded for each sequenced sample: genus, species, strength (e-value), and identification number (2). of the rdp seqmatch tool, the following information was also recorded for each sequenced sample: genus, species and the strength (s_ab score) (19). results the rainfall on the days rainwater was collected varied from 0.05 cm to 7.20 cm, with temperatures ranging from 0.6 ºc to 28.3 ºc (table 1). wind samples were collected only in may in winds ranging from 4.8 kph to 22.5 kph in temperatures from 22.8 ºc to 28.3 ºc. wind direction, humidity, and dew point were also recorded and shown in table 2. after isolating, subculturing, testing for oxidase, and gram staining, 28 dna sequences from presumptive aeromonads were analyzed. none of these isolates were obtained from the hail sample, as the sample did not yield any bacteria with the phenotypes of interest. as shown in table 3, of those 28 sequences, no isolates belong to the genus aeromonas. however, date genera isolated low temperature high temperature total rainfall 2014-05-13 pseudomonas 17.2ºc 8.9ºc 0.05 cm 2014-05-23 paracoccus, pseudomonas 28.3ºc 18.9ºc 0.61 cm 2014-05-25 massilia/naxibacter, pseudomonas 23.3ºc 17.2ºc 2.39 cm 2015-01-21 none 0.6ºc 9.4ºc 7.20 cm 2015-01-31 agrobacterium/aurantimonas, mesorhizobium, pseudomonas 6.7ºc 12.2ºc 3.60 cm table 1. weather parameters at the time of rainwater sample collections. a “/” between ncbi and rdp genus names is shown when the two databases gave conflicting identifications. applied & environmental microbiology• 96 date genera isolated time temperature wind direction wind speed humidity dew point 2014-05-20 novosphingobium/ sphingomonas, pseudomonas 11:00 26.1ºc south 22.5 kph 48% 14.4ºc 2014-05-22 none 11:00 26.7ºc south 19.3 kph 56% 16.1ºc 2014-05-23 novosphingobium/ sphingomonas 11:00 22.8ºc south 20.9 kph 68% 16.7ºc 2014-05-26 none 16:00 28.3ºc south southwest 4.8 kph 44% 15.0ºc 2014-05-27 none 11:00 23.3ºc north 4.8 kph 68% 17.2ºc 2014-05-28 none 11:00 24.4ºc north 22.5 kph 58% 16.1ºc table 2. weather parameters at the time of wind sample collections. a “/” between ncbi and rdp genus names is shown when the two databases gave conflicting identifications. table 3 (opposite page). ncbi blast database and rdp database comparison for sequence samples. isolates kep1-kep13 were isolated on aeromonas blue medium without ampicillin and isolates kep24-kep28 were isolated on aeromonas blue medium supplemented with 32 µg/ml ampicillin. all strength (e-value) scores were ≤ 2 × 10-165. isolates with a star are those thought to be novel species. uncultured refers to strains that were previously identified through environmental sequencing methods only. the ncbi blast database resulted in the following number and identities of bacteria: 14 pseudomonas, 6 various bacteria that were previously uncultured and only identified through environmental sequencing methods, and 8 other bacteria: massilia varians, 2 strains; paracoccus ‘mali 27’, 1 strain; novosphingobium clone seqseez199, 1 strain; mesorhizobium opportunistum, 2 strains; agrobacterium h13-3, 1 strain; and paracoccus queba07, 1 strain. as a comparison, analysis of the sample using the rdp database resulted in the following number and identities of bacteria: 16 pseudomonas, 4 various previously uncultured bacteria, and 8 other bacteria: naxibacter 6981, 2 strains; sphingomonas 44/40, 3 strains; aurantimonas ureilytica, 2 strains; and rhizobium gls4, 1 strain. the highest s_ab score for the collection of sequences was 1.00 while the lowest was 0.520 (for isolate 6 and isolate 23). the s_ab signifies that the sequences in this study had unique 7-base oligomers shared between the sample sequence and a given rdp sequence (which was then divided by the lowest number of unique oligonucleotides in either of the two sequences). data were analyzed based on date of sampling, source of isolation, and species identification (fig. 1). pseudomonas species were found both in rainwater and wind and both in may and january. paracocuss and massilia/naxibacter and novosphingobium/ sphingomonas were only isolated in may, while agrobacterium/rhizobium and mesorhizobium/aurantimonas were isolated in january. 97 • fine focus, vol. 2 ncbi blast database rdp database isolate genus and species identity genus and species strength (s_abscore) kep1: wind pseudomonas putida 99% pseudomonas putida 0.969 kep2: wind uncultured novosphingobium clone seqseez199 99% sphingomonas 44/40 0.949 kep3: wind uncultured novosphingobium clone seqseez199 99% sphingomonas 44/40 0.942 kep4: wind uncultured novosphingobium clone seqseez199 99% uncultured sphingomonadaceae 0.942 kep5: rain uncultured pseudomonas 100% pseudomonas fulva 0.98 kep6: rain* pseudomonas plecoglossicida 89% pseudomonas cpa30 0.520 kep7: rain pseudomonas mc83 99% pseudomonas mc83 0.974 kep8: rain massilia varians 97% naxibacter 6981 0.870 kep9: wind pseudomonas jsm 2215099 100% pseudomonas fulva 0.997 kep10: rain pseudomonas fulva 100% pseudomonas 471-1 0.997 kep11: rain pseudomonas 9dlp 99% pseudomonas psb1 0.992 kep12: rain paracoccus queba07 99% uncultured proteobacterium 0.974 kep13: rain paracoccus ‘mali 27’ 99% uncultured proteobacterium 0.962 kep14: wind novosphingobium clone seqseez199 99% sphingomonas 40/40 0.949 kep15: wind pseudomonas putida 99% pseudomonas putida 0.969 kep16: wind uncultured novosphingobium clone seqseez199 99% uncultured sphingomonadaceae 0.942 kep17: rain pseudomonas mc83 99% pseudomonas mc83 0.974 kep18: rain massilia varians 97% naxibacter 6981 0.870 kep19: rain pseudomonas jsm 2215099 100% pseudomonas fulva 0.997 kep20: rain pseudomonas fulva 100% pseudomonas u471-1 0.997 kep21: rain pseudomonas 9dlp 99% pseudomonas psb1 0.992 kep22: rain uncultured pseudomonas 100% pseudomonas fulva 0.983 kep23: rain* pseudomonas plecoglossicida 89% pseudomonas cpa30 0.520 kep24: rain* mesorhizobium opportunistum 94% aurantimonas ureilytica 0.911 kep25: rain* mesorhizobium opportunistum 94% aurantimonas ureilytica 0.916 kep26: rain agrobacterium h13-3 99% rhizobium gls-4 0.972 kep27: rain* pseudomonas syringae 96% pseudomonas fluorescens 0.876 kep28: rain* pseudomonas syringae 93% pseudomonas s1(2011b) 0.552 applied & environmental microbiology• 98 discussion & conclusions there are many reasons why aeromonas may not have been found in the samples: 1) the lack of a proximal lake or stream as an initial source of aeromonas prevents detection of these types of cells, 2) aeromonas may not be able to survive in the atmospheric or wind conditions of the study, 3) aeromonas may be present in the precipitation, but the methods were not sensitive enough to detect them, and finally, 4) the microbe may not be present at all in precipitation. even though we did not find aeromonas as we had hypothesized we would, we did find other interesting strains with a both databases resulted in identification of previously uncultured bacteria. thus, this study resulted in successfully culturing those bacteria. for the ncbi blast database identifications, three of the six total previously uncultured bacteria were novosphingobium (table 3). of the samples that were sequenced, the identities of six of the samples appear to be new species (or at least type strains) of bacteria. according to the ncbi blast database, these presumptive new species ranged from 89% to 96% identity to known species (table 3). these isolates could be representatives of previously unknown species since their species identities are less than 97% identical to known species (9). number of isolates massilia/naxibacter rainwater wind m ay 2014 january 2015 novosphingobium/sphingomonas paracoccus pseudomonas agrobacterium/rhizobium mesorhizobium/aurantimonas pseudomonas g enus and d ate of isolation 0 2 4 6 8 10 12 figure 1. number of isolates with regard to date of sampling, source of isolation, and identification. only isolates with the phenotypes of interest that were further characterized are included in this graph. a “/” between ncbi and rdp genus names is shown when the two databases gave conflicting identifications. 99 • fine focus, vol. 2 diversity of implications. the majority of the isolates found were from the genus pseudomonas, including p. putida (isolate 1), p. plecoglossicida (isolate 6, isolate 23), p. fulva (isolate 10, isolate 19, isolate 20, isolate 22), p. syringae (isolate 27, isolate 28), and p. fluorescens (isolate 27) (table 3). members of the genus pseudomonas are often found in water and soil ecosystems, so their abundance among the samples collected in this study is not surprising (23,24). however, since members of this genus have also been associated with the transmission of diseases in humans, animals, and plants, the possibility of coming into contact with pathogens through rainwater or wind exists. (7). another significant characteristic of pseudomonads is that they are thought to play an important role in the atmosphere. p. syringae is associated with bioprecipitation. bioprecipitation is a feedback cycle in which land plants produce airborne particles or aerosols containing microorganisms that impact the formation of clouds by their ice nucleation ability. this feedback cycle leads to rainfall that benefits both plant and microbial growth (25). the microorganisms that are encapsulated within these airborne particles are found in the center of snow and rain on five continents, with many of these microbes appearing in agricultural regions that are increasingly farmed. it is thought that the bacterium p. syringae is “the most prolific ice nucleator” because it can act within a set of warm atmospheric temperatures and may also cause other nucleators to lose their abilities (5). furthermore, as a plant pathogen, p. syringae damages certain agricultural crops through the utilization of its ice making capabilities (5). as stated before, pseudomonads are found in both soil and water. since wind speeds ranged from 4.8 kph to 22.2 kph when the wind samples were collected (table 2), it is possible for particulates from the ground to be swept up and included in the samples. many of the sequenced isolates include genera often found in soil environments. some of these isolates were identified as genera previously known to fix nitrogen, such as a pseudomonas (12). another bacterial isolate found that is also important in the nitrogen cycle was paracoccus, which has previously been reported to reduce nitrate (13). as mentioned before, there were several bacteria whose closest sequence matches were previously identified through environmental sequencing but were not cultured until this study. interestingly, two of these strains have previously been reported to fix nitrogen, a member of the order rhizobiales and novosphingobium (15,28). sphingomonas was also found and is a genus known for its diversity of nitrogen-fixing capabilities as well as its association with plants and their roots (27). other soil bacteria of the genus naxibacter and massilia were also found among the isolates (35). members of the massilia genus are not only closely related to members of the naxibacter genus, but both genera have been isolated from air samples in another study (32). it was also no surprise that aurantimonas was also found, since species within this genus have been isolated from air samples collected in the republic of korea (31). finding aurantimonas, naxibacter, and massilia suggests that there may be an important ecological role of these genera in air. other isolates not usually isolated from air that were found in our study belong to the genus sphingomonas. members of this genus form biofilms within rainwater harvesting tanks that act as a bio-control agent and natural filter by removing applied & environmental microbiology• 100 contaminants and bacteria from rainwater (18). these explanations illustrate that the isolates discovered in this study have unique capabilities and can survive amongst various environmental pressures. expanding both frequencies of collection and diversity of seasons will improve future research in this area. wind samples were only taken in the summer. with samples taken throughout the year, this could increase the diversity of microbes found. as there are different forms of precipitation such as ice, snow, hail, sleet, and fog, collecting these forms of water will improve future research and allow for comparison between the microbes found in each state of water. this study revealed the presence of interesting bacterial strains present in the troposphere. we found organisms important in bioprecipitation as well as those that have the potential to be important in the nitrogen cycle that are typically found in soil. in this study, we easily isolated previously uncultured bacterial strains as well as up to six novel species. this indicates that there is much to discover about the troposphere and how microbes are moved from environment to environment via transpiration and through updrafts of particulate matter from the terrestrial environment. acknowledgments we would like to thank abilene christian university mcnair scholars program for the support of k. e. preston for the duration of this project. we would also like to thank sally hays who carried out preliminary work on this project. 1. ahmed w, huygens f, goonetilleke a, gardner t. 2008. real-time pcr detection of pathogenic microorganisms in roof-harvested rainwater in southeast queensland, australia. appl. environ. microbiol. 74:5490–96. 2. altschul sf, gish w, miller w, myers ew, lipman dj. 1990. basic local alignment search tool. j. mol. biol. 215:403–10. 3. an hr, mainelis g, white l. 2006. development and calibration of realtime pcr for quantification of airborne microorganisms in air samples. atmos. environ. 40:7924–39. 4. burrows sm, 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indiana manuscript received 6 june 2016; accepted 17 november 2016 copyright 2017, fine focus. all rights reserved. 54 • fine focus, vol. 3 (1) the gram-positive bacterium staphylococcus aureus is well known for its ability to develop resistance to various antimicrobial substances. methicillin-resistant s. aureus (mrsa), a cause of nosocomial infections worldwide, is becoming increasingly resistant to the glycopeptide antibiotic vancomycin, one of few antibiotics used to treat serious multiple-antibiotic-resistant staphylococcal infections. with the increase in the number of mrsa strains showing reduced sensitivity to vancomycin, it has become important to investigate alternative treatment options. in this study, we examined the effects of five metals: silver, copper, arsenate, zinc, and cadmium on the growth of a clinical mrsa strain mm66 demonstrating heterogeneous intermediatelevel resistance to vancomycin. disc diffusion and gradient plate experiments were used to compare the metal susceptibility levels of strain mm66 to that of the methicillin-resistant s. aureus laboratory control strain. mm66 grew less successfully when exposed to metals, and showed an overall increased level of susceptibility to metals compared to the laboratory control strain. of the metals tested, silver exerted the highest inhibitory effect on the growth of mm66. abstract corresponding author reena lamichhane-khadka* rlamichh@saintmarys.edu keywords • methicillin-resistance • staphylococcus • vancomycin • metals methicillin-resistant staphylococcus aureus (mrsa) is a major cause of nosocomial and community-acquired infections worldwide (18, 2, 29, 4). since its first appearance in the early 1960s (15), mrsa has been the primary cause of nosocomial skin and bone infections as well as bacteremia (24, 6, 19, 13). mrsa infections account for about one-third of all s. aureus infections in the united states annually (13). in 2005, mrsa infections caused a higher mortality rate than hiv, killing an estimated 6.3 out of every 100,000 individuals. vancomycin was introduced for the treatment of s. aureus infections, including mrsa (24, 13). by 1997, more than 50% of introduction syaphylococcus aureus metal susceptibility • 55 mrsa nosocomial infections responded only to vancomycin (24). however, mrsa strains showing reduced sensitivity to vancomycin have appeared and are now a prevalent problem (24, 2, 19). since the isolation of the first vancomycinresistant s. aureus in 1995 (30), isolates with different levels of resistance have been recognized. high-level resistance to vancomycin is attributed to the vana gene, which was detected in all three of the first vancomycin-resistant (vrsa) isolates (30). intermediate resistance to vancomycin or vancomycin-intermediate s. aureus (visa) is defined by a minimum inhibitory concentration (mic) of greater than 3 mg/l of vancomycin (delgado et al., 2007). however, visa can show an mic up to 16 mg/l, and vancomycin-resistant s. aureus (vrsa) can show an mic of 32 mg/l or even more (30). hetero-vancomycin intermediate s. aureus (hvisa), a subtype of visa, initially show lower vancomycin mic (less than 2 mg/l) but grow successfully when exposed to higher levels of vancomycin, and are likely a precursor to visa (14). antibiotic-resistant s. aureus also demonstrate reduced sensitivity to other antimicrobial substances such as household disinfectants (16) and various metals (3). metals such as zinc, copper, silver, cobalt, and cadmium are well-known for their antimicrobial properties, and have been used in medicine, animal husbandry, and agriculture. metals such as copper and zinc are common in feed supplements for agricultural animals (3). cadmium and arsenic are common pollutants in soil and water, exposing environmental bacteria to their oligodynamic action (28, 34). copper and silver have been used more specifically for their antimicrobial activity. both are used in making water vessels and food containers to keep the contents disinfected (17, 27, 7). copper sulfate and other copper salts have also commonly been used as organic biocides and fungicides as well as in the medical setting as astringents (17). copper salts along with other metals such as mercury salts and tellurium, magnesium, and arsenic oxides have also been used to treat leprosy, tuberculosis, gonorrhea, and syphilis (17). however, many bacteria including s. aureus are becoming increasingly resistant to several metals. resistance of s. aureus to various metals has been reported in clinical cases (23), and has been linked with the widespread use of metal-containing compounds such as feed supplements and biocides. a possible link between antibiotic resistance and crossresistance to metals and biocides has been suggested in several bacteria including s. aureus (21, 16). several studies have suggested that with the development of antibiotic resistance, s. aureus strains may also develop decreased sensitivity to metals that were previously toxic at small concentrations (25, 11, 3, 1). resistance to metals in mrsa has also been associated with methicillin-resistance, being displayed in the same strains (3, 25). in this study, we examined the susceptibility of a clinical mrsa strain mm66 demonstrating heterogeneous intermediatelevel resistance to vancomycin (6) to five metals: silver; copper; arsenic; zinc; and cadmium. two laboratory control strains of s. aureus – a fully antibiotic-sensitive strain atcc 25923, and a mrsa control strain atcc 43300 were used to determine the relative susceptibility of mm66 to the metals tested. based on the available data from studies involving clinical strains of methicillinresistant s. aureus, we hypothesized that mm66 would demonstrate a comparatively reduced susceptibility than atcc 44330 to the metals at concentrations previously tested on similar strains. in contrast, this study revealed that mm66 has lower levels of resistance to copper, zinc, arsenic, and cadmium compared to atcc 43300. growth of mm66, atcc 43300 and atcc 25923 was completely inhibited by silver (2.5 mm). 56 • fine focus, vol. 3 (1) strains, growth conditions, and reagents the clinical hvisa strain mm66 was obtained from prof. john gustafson’s laboratory at oklahoma state university, stillwater, oklahoma. this strain was first isolated in las cruces, new mexico by delgado et al. (2007). two controls were used in this study; american type culture collection (atcc) 25923, a fully sensitive laboratory strain of s. aureus; and atcc 43300, a laboratory mrsa strain sensitive to vancomycin. each strain was streak-plated onto luria bertani agar plate from stock cultures and maintained at 4oc. metal solutions were prepared from metal salts (zinc chloride, copper sulfate, cadmium acetate, silver nitrate, and sodium arsenate) at a concentration of 200 mg/l and filter-sterilized. aliquots of 1 ml each were made at 1 mg/ml, 5 mg/ml, and 10 mg/ml to be used for disc preparation. all stock solutions were stored (at 4°c) in the refrigerator. mueller hinton broth (mhb), mueller hinton agar (mha), luria bertani broth (lb) and luria bertani agar (lba) were prepared per instructions provided on powder containers. overnight cultures were prepared by inoculating 5ml of lb with one isolated colony of bacteria from the stock plates and incubating at 37°c at 200 rates per minute (rpm) in the shaking incubator (new brunswick scientific, i2500 series, enfield, ct). all experiments were performed in triplicates starting with triplicate overnight cultures in lb. all media used in this study were obtained from bbl difco (franklin lakes, nj), and all chemicals were obtained from sigma (saint louis, mo) unless indicated otherwise. the strains used in this study are listed in table 1. disc diffusion assay metal susceptibility was examined by disc diffusion similarly to the method described by poston and saw hee (1991). for the first trial, a 50 microliter volume of overnight culture was used to inoculate 5 ml of sterile mhb. for the second trial, this amount was decreased to 20 microliters. tubes were incubated at 37°c, 200 rpm for approximately three to four hours until they visually matched the mcfarland turbidity standard of 0.5. meanwhile, discs were prepared by adding calculated volumes of sterile stock solution of each metal to sterile paper discs of 4 mm diameter. total liquid volume added to each disc was adjusted to 20 microliters with sterile distilled water to ensure a uniform distribution of the metal salt on the disc. after incubation, if suspensions were less turbid than the standard, they were placed back in the incubator so that further growth could take place. if they were more turbid, they were diluted with sterile mhb. once all tubes matched the mcfarland standard, culture was inoculated onto the entire agar surface of mha plates with a sterile cotton swab. prepared discs were placed on the plates with sterile forceps and gently pressed to ensure that entire disc surface contacted the agar. the plates could sit for one minute before being inverted and incubated at 37°c in a stagnant incubator overnight. zones of inhibition were measured after 18, 20, 24, 48, and 72 hours. metal gradient plate assay gradient plate was performed as described by price et al., 1999. square plates (90mm x 90mm) were obtained from fisher scientific (pittsburgh, pa) and placed at a 14° angle so that bottom layer of agar would solidify at methods and materials syaphylococcus aureus metal susceptibility • 57 the same angle. to make the bottom layer, 40 ml of freshly prepared mha was poured and allowed to solidify. the next day, the top layer mha was prepared by adding calculated volumes of 200 mg/ml stock to the mha before again pouring 40 ml of mha, but this time pouring with the plate flat so that the top layer formed a flat surface. twenty microliters of overnight culture were transferred to 5 ml of sterile mhb and incubated at 37°c, 200 rpm. after incubation, the cultures were diluted to an optical density (od) of 0.1 at 600 nm as measured by a spectrophotometer (thermo scientific, waltham, ma). when od of 0.1 was reached, culture was inoculated onto gradient plate in a line streak from lowest concentration to highest concentration with a sterile cotton swab by streaking the same line three times to ensure equal distribution of the culture along the plate. the plates were then inverted and incubated at 37°c and confluent growth was measured after 18, 20, 24, 48, and 72 hours. strain relevant background source/reference mm66 clinical hvisa; vancomycin mic 3.0 μg/ml delgado et al., 2007 atcc 25923 lab s. aureus strain; sensitive to antibiotics atcc atcc 43300 lab mrsa strain; sensitive to vancomycin atcc table 1. strains used in this study. hvisa: hetero-vancomycin-intermediate s. aureus mrsa: methicillin-resistant s. auerus results disc diffusion assay exposure to 10, 30, 35, and 40 micrograms of zinc chloride, copper sulfate, and sodium arsenate did not have any effect on the growth of the s. aureus isolates. when exposed to 60 micrograms of zinc chloride, atcc 25923 displayed small, faint zones with visible reduction of growth but atcc 43300 and mm66 strains remained unaffected. none of the strains showed any zone of inhibition with 60 micrograms of copper sulfate and sodium arsenate. when exposed to cadmium acetate, the strains showed some zones of decreased growth, herein referred to as ‘zones of inhibition’; however, these zones varied in size across the strain types. after 20 hours of incubation around a 40-microgram disc, mm66 displayed the largest zone of effect (35.3 mm in diameter) whereas atcc 25923 and atcc 43300 displayed smaller zones of inhibition (12.3 mm and 15 mm, respectively) (figure 1). mm66 also displayed clear zones of inhibition at lower concentrations of cadmium acetate (30 and 10 micrograms); no zone of inhibition was observed in the control strains at these concentrations. 58 • fine focus, vol. 3 (1) figure 1. comparison of the zones of inhibition of 40 μg cadmium acetate disks on s. aureus strains atcc 25923 (black bars), atcc 43300 (gray bars) and mm66 (white bars). error bars represent standard deviation (n=3, p<0.05). significantly greater zones of inhibition (connoted by *) were observed in mm66. hours of incubation z on e of in hi bi tio n (m m ) 0 18 5 10 30 15 35 20 40 25 20 24 48 72 atcc 25923 atcc 43300 mm66 clear zones of inhibition were observed in all three strains at all concentrations of silver nitrate. the zone sizes of all strains were similar (figure 2). the results were consistent in a second disc diffusion experiment performed on triplicates of each strain. metal gradient plate assay on the 5mm copper sulfate gradient plates, mm66 displayed reduced susceptibility compared to atcc 25923 and atcc 43300. atcc 25923 grew up to 83.33±9.87 mm, atcc 43300 grew with average of 80.667±2.08 mm, and mm66 grew through the entire length of the gradient (90±0 mm) (table 2). however, when a higher concentration gradient (20 mm) was used, growth of mm66 was more reduced compared to the other strains. atcc 25923 grew an average of 19.67±1.53 mm, atcc 43300 grew an average of 16±1 mm, and mm66 grew an average of 6.33±5.01 mm. (table 3). at 2.5 mm concentration gradient of sodium arsenate, both atcc 25923 and atcc 43300 grew through the length of the plate, measuring an average of 90±0 mm of growth. mm66 grew an average of 39.833±4.26 mm which is significantly less than the growth of the other two syaphylococcus aureus metal susceptibility • 59 figure 2. comparison of the zones of inhibition of 40 μg silver nitrate disks on s. aureus strains atcc 25923 (black bars), atcc 43300 (gray bars) and mm66 (white bars). error bars represent standard deviation (n=3, p=0.05). no significant differences were observed in zone size. hours of incubation z on e of in hi bi tio n (m m ) 0 18 2 4 12 6 14 8 16 10 20 24 48 72 atcc 25923 atcc 43300 mm66 strains (table 2). at 5 mm concentration gradient, atcc 25923 still grew the full length of the gradient, measuring 90±0 mm. however, atcc 43300 grew an average of 85.33±3.21 mm and mm66 did not grow at all. in a third experiment using 20 mm sodium arsenate gradients, mm66 did not demonstrate any growth, atcc 25923 grew an average of 40±1 mm and atcc 43300 grew an average of 23.667±5.68 mm. atcc 43300 grew significantly less than atcc 25923 (data not shown in table). on cadmium acetate, mm66 failed to grow on a gradient of 2.5 mm; atcc 25923 grew to an average of 30±1 mm at 2.5 mm gradient and failed to grow on 5 mm; and atcc 43300 grew to an average of 72.667±5.51 mm on a 2.5 mm gradient of cadmium acetate and 16.667±4.51 mm on 5 mm gradient (tables 2 and 3). the growth of both controls was significantly reduced at a higher gradient of cadmium acetate (10 mm); atcc 25923 grew to an average of 10.667±9.29 mm and atcc 43300 grew to an average of 16.333±1.15 mm, and growth of mm66 was completely inhibited. none of the strains grew on 2.5 mm, 5 mm, and 10 mm gradients of silver nitrate. this result was consistent with all the triplicates. comparative results of gradient plate assay for all strains are presented in tables 2 and 3. 60 • fine focus, vol. 3 (1) the results of the disc diffusion test suggested a lack of sensitivity in both the laboratory and clinical mrsa strains to zinc, copper, and arsenate. compared to the laboratory mrsa strain atcc 43300, mm66 showed increased sensitivity to cadmium acetate. although the growth of all strains was affected by cadmium acetate, growth of mm66 was the most inhibited; the control strains had smaller zones of inhibition surrounding the cadmium acetate discs. clear zones of inhibition were only present on mm66 plates, indicating an increased sensitivity of this strain to cadmium. distinct zones of inhibition were observed around the silver nitrate discs at all concentrations used. this suggested that the mrsa strains are highly susceptible to silver. overall, similar effects of the metals on the growth of the strains were observed by both the disc diffusion and gradient plate methods. therefore, the findings of both experiments supported one another. the gradient plate experiments provided a more comprehensive comparison of susceptibility among the strains. unlike disc diffusion experiments in which discs with specific concentrations of the metals were used, the gradient plate assays discussion strain metal gradient zn 0g2.5 mm cu 0g5 mm cd 0g2.5 mm ag 0g2.5 mm asa 0g2.5 mm atcc 25923 32 ± 1.0 83.33±9.8 30±1.0 0 90±0 atcc 43300 79.3±4.2 80.7±2.1 72.7±5.5 0 90±0 mm66 38.67±2.3* 90±0* 0* 0 49.3±4.5* table 2. metal gradient plate experiment results (at lower concentrations of metals). numbers represent mm grown on 90 mm gradient plates and standard deviations (n = 3). *connotes significant difference compared to mrsa control strain (p < 0.05). strain metal gradient zn 0g5 mm cu 0g10 mm cd 0g5 mm ag 0g5 mm asa 0g5 mm atcc 25923 17 ± 1.7 90±0 0 0 90±0 atcc 43300 36.3±1.5 90±0 16.7±4.5 0 90±0 mm66 14.5±1.6* 64.2±0* 0* 0 0* table 3. metal gradient plate experiment results (at higher concentrations of metals). numbers represent mm grown on 90 mm gradient plates and standard deviations (n = 3). *connotes significant difference compared to mrsa control strain (p < 0.05). syaphylococcus aureus metal susceptibility • 61 used continuous concentration gradients of metals. therefore, bacterial growth starting from the lowest concentration to the highest concentration of the metal could be observed. in the gradient plate experiments, the hvisa strain mm66 showed reduced growth in presence of zinc compared to the lab mrsa strain atcc 43300. zinc is required by the bacteria at a low concentration to carry out various metabolic reactions as catalysts, structural stabilizers, and as coenzymes or cofactors (1). therefore, growth of the strains at the zero end of the concentration gradient was expected. as the concentration becomes higher, the cells cease to grow due to the toxic effects of zinc. in our experiments, atcc 43300 could grow at higher levels of zinc compared to mm66, suggesting that the clinical hvisa strain is probably compromised in its ability to resist higher levels of zinc. the strains showed highly reduced susceptibility to copper sulfate; all three strains grew to almost the entire length of the gradient plates at 5 mm copper sulfate concentration gradients. strain mm66 displayed the longest distance of growth, suggesting a more reduced susceptibility of this strain to copper compared to the mrsa control strain. interestingly, in higher concentration gradients of copper (10 mm and 20 mm), mm66 displayed shorter distances of growth compared to its control counterparts. copper, like zinc, is necessary in trace amounts for the metabolic functions of the bacteria (1). it is possible that mm66 grows more successfully at a lower concentration of copper sulfate, but is affected more rapidly when the concentration gets higher, as seen in the 10 mm and 20 mm gradients. further research investigating the molecular basis of copper resistance in these strains may provide more information in this aspect. when exposed to 5 mm arsenate, mm66 grew to almost half the length of the concentration gradient. however, this growth was significantly reduced when compared to the growth of control strains, indicating that mm66 has reduced susceptibility to arsenate compared to the control mrsa strain. this pattern was consistently observed in other concentrations of arsenate (table 3). despite the toxicity of arsenic, resistance to high levels of arsenic has been observed in isolates of s. aureus (1, 34, 33). bacteria that are resistant to arsenate compounds contain efflux systems that expel the arsenates out of the cells. arsenates are analogs of phosphates which are essential for cellular functions, and bacteria may take the arsenates in by phosphate transport mechanisms. once in the cells, they are converted to arsenites and pumped back out of the cell via efflux pumps (33). the growth of the strains in arsenate gradients indicates that a similar mechanism may be present in these strains. further research may be necessary to determine the arsenite efflux capabilities of these bacterial strains. the results of the cadmium gradient plate assay were consistent with the results of the disc diffusion test. in 10 mm gradient of cadmium acetate, the growth of mm66 was significantly reduced compared to that of the control strains. cadmium is a highly toxic pollutant found in the environment. this may limit its therapeutic use despite its ability to significantly inhibit the growth of mm66. cadmium kills s. aureus by generating oxidative stress and inhibiting thiol metabolism (28). s. aureus isolates that are resistant to cadmium possess the czrc gene and the cad operons that enable them to resist the negative effects of cadmium (3, 5, 8). methicillin-resistance in s. aureus has been linked with cadmium-resistance and the presence of the czrc gene in many mrsa isolates (3). the increased susceptibility to cadmium in mm66 indicates the presence of a previously unidentified mechanism in the hvisa strain. this suggests the need of further studies to understand the genetic basis of this phenomenon in mm66. 62 • fine focus, vol. 3 (1) acknowledgements the authors acknowledge the saint mary’s college sistar award for providing funding for this research. the authors also thank professor john e. gustafson, oklahoma state university, stillwater, ok, for kindly providing the s. aureus strain mm66. silver gradients, like the silver discs, completely inhibited the growth of all three strains of s. aureus at the tested concentrations. the oligodynamic action of silver compounds is a known phenomenon, and silver compounds are commonly used as antimicrobial agents, such as 1 % silver nitrate to prevent opthalmia neonatorum and as silver sulfadiazine to treat infected burn wounds (27, 9). therefore, reduced growth of the s. aureus strains in disc diffusion and gradient plate assays was not completely unexpected. what was unexpected was that the growth of all strains, including the clinical mrsa strain, was completely inhibited even at lower concentrations. even though no differential growth among the strains was observed and hence no data on the relative susceptibility of the mm66 strain could be obtained, it can be said that the clinical hvisa strain mm66 appears to be susceptible to even low concentrations of silver. silver inactivates bacterial enzymes and damages dna, and helps in the accumulation of reactive oxygen species in the cells, leading to the death of bacteria (20). our results are in favor of the proposition that silver compounds could be considered as alternatives to traditional topical antibiotics for the treatment of infections caused by antibioticresistant bacteria (27). since s. aureus is implicated in a variety of skin infections, using silver compounds could be a viable option for treating skin infections. therefore, the possibility of using silver as an effective alternative to antimicrobials in treating mrsa infections should be further investigated. in conclusion, this research provides interesting new information about the metal susceptibility characteristics of a clinical mrsa strain that was reported as a hetero-vancomycin-intermediate s. aureus (6). despite the high occurrence of crossresistance to metals in methicillin-resistant s. aureus isolates, mm66 does not seem to show these tendencies. our results suggest an overall reduced metal susceptibility in the clinical hvisa strain, with silver exerting the highest inhibitory effect on growth. the findings of this research underline the need of further investigation on the use of metals as therapeutic options in treating drug-resistant s. aureus infections. references 1. abd, a.-k. h., abu-raghif, a. r., & samir, r. a. 2011. a study on heavy metals and antibiotic resistance of staphylococcus aureus isolated from clinical specimens. j. med. sci. 9:357-363. 2. applebaum, p. c. 2006. the emergence of vancomycinintermediate and vancomycin-resistant staphylococcus aureus. clin. microbiol. infec. 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(2006). beyond mrsa: visa and vrsa. american j. nurs. 106: 28-30. 31. tsai, k.-j., hsu, c.-m., & rosen, b. p. 1997. efflux mechanisms of resistance to cadmium, arsenic, and antimony in prokaryotes and eukaryotes. zoo. studies 36:1-16 32. willey, j., sherwood, l., & woolverton, c. 2013. prescott’s microbiology. 9th edition. new york: mcgraw hill. 1014 p. 33. yang, h.-c., fu, h.-l., lin, y.-f., & rosen, b. p. 2012. pathways of arsenic uptake and efflux. current topics in membranes, 69:325-358. 34. zelibor, j. l. jr., doughten, m. w., grimes, d. j., & colwell, r. r. 1987. testing for bacterial resistance to arsenic in monitoring well water by the direct viable counting method. appl. environ. microbiol. 53:2929-2934. bactericidal effects of low-irradiance low level light therapy on methicillin-resistant staphylococcus aureus in vitro patrick j. mcmullan1, jakob krzyston2, robert osgood,1 1 department of biomedical sciences, rochester institute of technology, rochester, ny 2 department of biomedical engineering, rochester institute of technology, rochester, ny manuscript received 14 march 2016; accepted 28 may 2016 copyright 2016, fine focus all rights reserved low level light therapy (lllt) within the visible blue spectrum (400-470 nanometers) is a well-documented therapeutic alternative to combat multidrug resistant organism infections through the generation of reactive oxygen species (ros). however, one shortcoming of lllt is that many studies deliver therapy through high powered lasers and lamps. high powered light sources not only require specialized staff to operate, but they also deliver the total light dose (fluence) at an exceptionally high intensity, or irradiance, which could consequently deplete the oxygen supplies required to promote lllt’s bactericidal properties. to overcome these faults, lowirradiance lllt, or delivering the same total fluence of lllt over an extended period of time with decreased irradiance was evaluated in vitro. to further explore this alternative approach, the bactericidal effects of lowirradiance (10.44 mw/cm2) lllt using wavelengths of 405-nm, 422-nm and 470-nm were studied on methicillin-resistant staphylococcus aureus (mrsa) cultures. among these wavelengths, it was determined that 405-nm lllt provided the most effective reduction of bacterial load at the lowest total fluence (75 j/cm2) (94.50% reduction). the bactericidal effects of 405-nm low-irradiance lllt were then further studied by treating mrsa cultures to 75 j/cm2 lllt while using irradiances of 5.22 mw/cm2 and 3.48 mw/cm2. it was concluded that there was a greater reduction of mrsa bacterial load when samples were exposed to irradiances of 5.22 mw/cm2 (95.71% reduction) and 3.48 mw/cm2 (99.63% reduction). this study validates the bactericidal properties of low-irradiance lllt on mrsa, and subsequent studies should be completed to optimize its full therapeutic potential. abstract corresponding author patrick j. mcmullan pjm4302@rit.edu keywords • photoinactivation • bactericidal • mrsa • low level light therapy the plight of antibiotic resistance, partially due to the medical community’s overreliance of antibiotics, has led to efforts by healthcare systems to determine alternative strategies for antimicrobial treatments. in the united states, there are more than 2 million cases of hospital-acquired infections (hais) annually, resulting in annual costs of $20 billion and nearly 100,000 deaths (16,26). methicillin-resistant staphylococcus aureus introduction 105 • fine focus, vol. 2 pathogens and antimicrobial factors • 106 (mrsa) is one of the prominent pathogens involved with both hais and community acquired infections (cais). the prevalence of an individual within the intensive care unit who develops either a hai or cai due to mrsa are greater than 55% and 59%, respectively (10). mrsa has been able to ascend to its present status as a predominant infective agent due to its ability to develop high levels of resistance to several classes of antibiotics through different pathways, including mutation, conjugation, transduction, and transformation (2). there are two distinct sources of mrsa: (1) hospital-associated mrsa (ha-mrsa), which are limited to clinical settings, and (2) community-associated mrsa (camrsa), which are found in a multitude of environments and settings, including beaches, computer keyboards, schools, gyms, athletic fields, and locker rooms. (57). mrsa, an opportunistic pathogen, is not only responsible for localized trauma or postoperative infections, but can also be lifethreatening, causing bacteremia, endocarditis, sepsis or toxic-shock syndrome (11,13,17). research also suggests mrsa is one of the primary pathogens responsible for failure of surgical implants or prosthetics due to biofilm formation at the soft-tissue-implant interface, which compromises patients’ activities of daily living (adls), as well as causes reimplantation to eliminate the bacterial load (28). a long-term solution to better manage multidrug-resistant organism infections is paramount. low level light therapy (lllt), also referred to as “biostimulation,” is the process of illuminating tissues with a precise wavelength of light over a specific period of time. more specifically, the use of high energy (lower wavelength) waves in the ultraviolet (uv) and visible blue spectra for lllt have shown more consistent results when it comes to bactericidal effects and reduction of bioburden. the capabilities of uv (100-400nm) lllt to sterilize wound surfaces has been welldocumented (3,8-9,15). dai et al. investigated the potential of prophylactic uv (200280nm) light treatment for infections developing in superficial cutaneous mouse wounds contaminated with both mrsa and pseudomonas aeruginosa (9). for both bacterial infections, uv lllt significantly reduced the bacterial burden in comparison to untreated wounds, while also increasing the survival rate of p. aeruginosa infected mice (58%) and wound-healing rate of mrsa infected mice (31%) (9). however, a limitation to uv lllt is that it is toxic and carcinogenic in tissues with extended exposure (3,8-9,15). in order to overcome the collateral damage associated with uv lllt, the use of wavelengths in the visible blue spectrum (400-470-nm) have been proven to be efficacious for both their bactericidal and wound healing capabilities in vitro and in vivo (1,10,19-20,23,27). while uv lllt’s bactericidal properties are induced through dna damage, the mechanism of visible blue light involves the photoexcitation of endogenous porphyrins within bacterial species, which is much less detrimental to healthy mammalian cells (1,10,19-20,23, 27). subsequently, this excitation initiates the formation of reactive oxygen species (ros), which are toxic to bacterial cells and biofilms, without compromising the dna of adjacent cells. maclean et al. investigated the bactericidal effects of visible (405-nm) light using a high-intensity xenon lamp, and illustrated that the 405-nm array had a phototoxic effect on a variety of bacteria that are highly prevalent in cais and hais, including gram-positive bacteria: mrsa, staphylococcus epidermidis, streptococcus pyogenes, clostridium perfringens, and 107 • fine focus, vol. 2 gram-negative bacteria: acinetobacter baumannii, pseudomonas aeruginosa, escherichia coli, proteus vulgaris and klebsiella pneumonia (19). the first underlying objective of this study was to compare the bactericidal efficacy of lllt on cultures of mrsa using three specific wavelengths (405-nm, 422-nm and 470-nm) within the visible blue spectrum. additionally, the objective of this study was to evaluate the efficacy of low-irradiance lllt, or the concept of delivering the same total light dose, or fluence, of lllt at a lower intensity, or irradiance, over an extended period of time. this concept contradicts standard lllt where therapy is administered at a considerably high irradiance (>40 mw/ cm2) and delivered over a period of seconds to 15 minutes (10, 14). based on these objectives, this in vitro study was conducted in two separate phases. in phase 1, the bactericidal properties of 405-nm, 422-nm and 470-nm lllt were evaluated to determine which wavelength provided the greatest bacterial load reduction at the lowest possible fluence. mrsa samples were exposed to these three wavelengths at an irradiance of 10.44 mw/cm2, which is substantially lower than precedent studies (10, 14). after determining the optimal wavelength and the precise fluence, phase 2 was conducted by evaluating the efficacy of low-irradiance lllt at the optimal wavelength, but at irradiances of 5.22mw/ cm2 and 3.48 mw/cm2, and durations of 4 and 6 hours. given the recent advancement of implantable and portable lllt devices (4, 25), the process of administering lllt over a period of multiple hours has the potential to be a cost-effective yet clinically feasible alternative to conventional lllt. despite the fact lllt’s bactericidal effects have been well documented within all wavelengths of the visible blue spectrum, it is hypothesized that 405-nm lllt will provide the greatest bactericidal response, due to the higher energy levels associated with the shorter wavelength photons. furthermore, it is expected that low-irradiance lllt will provide a greater bactericidal rate when compared to delivering the same lllt fluence at a higher-irradiance, because of the fact that visible blue lllt is dependent upon oxygen supplies for ros formation. consequently, low-irradiance lllt will not deplete oxygen supplies as rapidly and therefore provide greater treatment efficacy. materials and methods study design: during phase 1, cultures of mrsa were exposed to three separate wavelengths of visible blue light (405-nm, 422-nm and 470-nm) at an irradiance of 10.44 mw/ cm2. the cultures were exposed to lllt for a total of 8 hours. however, aliquots from each of the exposed cultures were taken in two-hour intervals (t=2,4,6,8 hours), which was the equivalent of lllt doses of 75 j/ cm2. these aliquots were then quantified to determine the number of colony forming units per milliliter (cfu/ml) through a standard plate counting technique using a whitley automated spiral plater (wasp). table 1 outlines the total fluence of lllt each culture received at each of the twohour intervals. pathogens and antimicrobial factors • 108 upon completion of phase 1, a prospective analysis was conducted to determine which wavelength would provide the greatest bacterial load reduction at the lowest possible fluence. after determining these conditions, phase 2 was initiated to evaluate the effects of maintaining the aforementioned optimal fluence, but further reducing the irradiance and increasing the total exposure time of lllt to 5.22 mw/cm2 (4 hour exposure) and 3.48 mw/cm2 (6 hour exposure). bacterial isolates: the mrsa strain that was used in this procedure was atcc(r) baa-1761 (atcc, manassas, va). a loopful of frozen inoculum from -80°c frozen stocks was used to quadrant streak onto the surface of a tryptic soy agar (tsa) plate. each plate was then incubated at 37°c and 5% co2 for 24 hours to produce isolated and pure cultures of mrsa for this study. biosafety precautions: throughout the course of the procedure outlined within this experimental study, adequate biosafety measures were taken to comply with the fact that mrsa is classified under the biosafety level 2 risk group. in addition to wearing laboratory coats, gloves and eye protection, all facets of this procedure were conducted using aseptic technique within a class ii laminar flow biosafety cabinet. light emitting device: the led light sources used during phase 1 of this study were leds at 405+5-nm, 422+5-nm and 470+5-nm wavelengths (visual communications company, poway, ca). the light-emitting device was constructed by assembling leds onto an eic-108 3220 tie-points solderless breadboard (eic laboratories inc., norwood, ma). a labnet power station 300 power supply (labnet international, edison, nj) was connected to provide the desired voltages to emit the necessary irradiance. 24 leds were assembled onto each breadboard, and were then positioned directly beneath one well on a 24-well plate, so each well would be illuminated by a single led. to ensure that each well was receiving identical irradiances of blue light, the overall irradiance that each well was illuminated by was measured using a thorlabs optical power meter (thor laboratories, newton, nj). photoinactivation procedure: mrsa was grown for 24 hours at 37°c and 5% co2 in tryptic soy broth (tsb). after isolating colonies of mrsa, a single colony of mrsa was suspended in tsb to grow for 24 hours. following incubation, aliquots of the cultures were serially diluted in sterile saline to a final dilution factor of 1:10,000 (tsb suspended mrsa culture: saline). this dilution was completed in order to quantify the bacteria within the constraints of the whitely automatic spiral plating system outlined below. the concentration of mrsa in the diluted cultures was confirmed through monitoring their optical density (od600) at 600-nm. 275.0 µl of the diluted mrsa cultures were loaded into each well of a corning® costar® 24-well flat bottom cell culture plate with a total well volume of 3.4 ml (corning inc, corning, ny) to receive light exposure. throughout the course of this experiment, there were four separate trials conducted to ensure consistency, and to evaluate both the intraplate and interplate bactericidal effects of the low-irradiance lllt. during each individual trial in phase 1, there were four experimental cohorts where mrsa cultures received either: 1) no light exposure (control); 2) 405-nm lllt; 3) 422-nm lllt; or 4) 470-nm lllt. for each cohort that received 109 • fine focus, vol. 2 lllt, the light was administered at a fixed irradiance of 10.44 mw/cm2. during phase 2, there were also found experimental cohorts where mrsa cultures received either: 1) no light exposure (control); 2) lllt at 10.44 mw/cm2 over 2 hours; 3) lllt at 5.22 mw/cm2 over 4 hours; or 4) lllt at 3.48 mw/cm2 over 6 hours. during phase 2, cultures that received lllt were treated with the optimal wavelength determined during phase 1. with the exception of the light wavelengths from the led source used on the experimental groups, each trial was completed at room temperature in total darkness to eliminate confounding variables from external light sources. additionally, in order to minimize the risk for contamination, the well plates remained covered for the duration of the light treatment. throughout the course of each trial, fifty-microliter aliquots were taken from four randomly selected wells using aseptic technique, although no well was repeatedly sampled, in each well plate during increments of 2 hours of light exposure. these aliquots were then plated onto tsa using the whitley automated spiral plating system. standard plate count procedure: a whitley automated spiral plater (wasp) (don whitley scientific limited, west yorkshire, uk) was used to spiral plate 50 µl of each aliquot onto the surface of a tryptic soy agar plate in an archimedes spiral. the plates were incubated for 24 hours at 37°c in 5% co2. colony counts were manually performed three times per plate, and the mean value was subsequently used to calculate the bacterial load in units of cfu/ml. statistical analysis data throughout the course of this study was analyzed post-hoc using oneway anova and student t-tests. each illumination condition was compared to their control, as well as wavelength differences between groups. p values < 0.05 were considered to be statistically significant. results as illustrated in table 1 and fig. 1, 405nm low-irradiance lllt provided the greatest total bacterial load reduction of mrsa cultures (99.06% reduction [p < 0.05]) in phase 1, when compared to 422nm (98.84%) and 470-nm (98.71%) lllt. furthermore, 405-nm lllt was the only wavelength that successfully provided a 2.0-log bacterial load reduction (> 99.0% kill rate). while 405-nm lllt provided the greatest cumulative bacterial load reduction among the eight-hour light exposure, table and fig. 1 also suggest there was a greater bacterial load reduction seen from a fluence of 75 j/cm2 across the first two hours of 405-nm lllt treatment (94.50% [p < 0.0001]) than what was seen from a fluence of 150 j/cm2 across the first four hours of 422-nm lllt (83.47%), as well as 225 j/cm2 across the first six hours of 470-nm lllt (93.95%), respectively. based on these observations, phase 2 of this study was conducted using the optimal lllt wavelength of 405-nm at a fluence of 75 j/cm2. pathogens and antimicrobial factors• 110 9.5 9.0 8.5 8.0 7.5 7.0 6.0 4.0 2.0 0.0 0 75 150 225 300 fluence (j/cm2) lo g (c fu /m l) **** **** figure 1. mrsa colony forming units (cfus) post-low level light therapy this figure is a graphical depiction of mean bacterial load reductions of mrsa following 405-nm, 422-nm and 470-nm lllt relative to the total lllt fluence received. each experiment was completed at a fixed irradiance of 10.44 mw/cm2. mrsa samples were collected in increments of 75 j/cm2 (every 2 hours of the 8-hour treatment) of lllt. error bars represent standard error of collected samples. asterisks (****) signify p-value < 0.0001 following anova to analyze significant differences between experimental wavelengths. fluence (j/cm2) 405-nm 422-nm 470-nm log (± sem) percent change log (± sem) percent change log (± sem) percent change 75 8.06 (± 0.03) [p < 0.0001] 94.50% 8.93 (± 0.02) 54.32% 9.10 (± 0.02) 36.87% 150 7.67 (± 0.02) [p < 0.0001] 97.53% 8.46 (± 0.02) 83.47% 8.93 (± 0.03) 53.42% 225 7.31 (± 0.02) [p <0.021] 98.93% 7.54 (± 0.02) 96.92% 8.15 (± 0.02) 93.95% 300 7.26 (± 0.02) [p <0.05] 99.06% 7.36 (± 0.02) 98.84% 7.39 (± 0.02) 98.71% table 1. bacterial load reduction of mrsa samples following 10.44 mw/cm2 lllt: this table indicates the mean bacterial load reductions log(cfu/ml)(± sem) of mrsa following low-irradiance lllt at wavelengths of 405-nm, 422-nm and 470-nm. all samples were exposed to light at a constant irradiance of 10.44 mw/cm2, but received lllt at varying exposure times and total fluences (j/cm2). a total of 16 replicates (4 from each experimental trial) of each data point were collected to determine mean bacterial load reductions. p-values signify level of significance differences among the experimental wavelengths using anova. p-values < 0.05 are considered significant. 111 • fine focus, vol. 2 upon determining the optimal fluence and wavelength of lllt in phase 1, phase 2 was conducted to analyze 405-nm lllt when delivering the same total fluence of 75 j/cm2, but through irradiances of 5.22 mw/cm2 and 3.48 mw/cm2. by decreasing the irradiances, the duration of each lllt cycle to receive 75 j/cm2 was increased from the initial two-hour treatment at 10.44 mw/cm2 to 4 hours at 5.22 mw/cm2 and 6 hours at 3.48 mw/cm2. table 2 indicates that there was a correlation associated with a greater bacterial load reduction when an identical fluence of 75 j/cm2 405-nm lllt was administered at a lower irradiance and subsequently an increased exposure time. an increased bacterial load reduction was observed when the irradiance was decreased to 5.22 mw/ cm2 (95.71% reduction [p < 0.023]) and 3.48 mw/cm2 (99.63% reduction [p < 0.0001]). irradiance (mw/cm2) lllt exposure period mean log (± standard error) mean bacterial load reduction p-value 10.44 2 hours 8.06 (± 0.03) 94.50% 5.22 4 hours 7.91 (± 0.04) 95.71% < 0.023 3.48 6 hours 6.85 (± 0.03) 99.63% < 0.0001 table 2: bacterial load reduction of mrsa following irradiance-manipulated 405-nm lllt: this table indicates the mean bacterial load reductions (cfu/ml) of mrsa following 75 j/cm2 of 405-nm low-irradiance lllt. while all samples received a constant fluence of 75 j/cm2, the irradiance and exposure times were manipulated from 10.44 mw/cm2 to 5.22 mw/cm2 and 3.48 mw/cm2. a total of 16 replicates (4 from each experimental trial) of each data point were collected to determine mean bacterial load reductions. p-values signify level of significance differences among the experimental irradiances using anova. p-values < 0.05 are considered significant. discussion one significant limitation of conventional lllt is that the high-intensity light sources that are commonly used to administer therapy are relatively expensive, and require a specialized medical facility and staff to operate and maintain. consequently, these centers require frequent travel for patients that are looking to receive lllt, and may not be readily accessible. in order to facilitate introducing lllt into mainstream medicine, recent studies have analyzed the use of low-power light emitting diodes (leds) as an alternative illumination source to the high-powered lasers that are currently used (1, 20, 27). this in vitro study validates the proof of concept of the bactericidal effects of low-irradiance lllt using visible blue low-powered leds on cultures of mrsa. photoinactivation of mrsa was achieved through lllt at wavelengths of 405-nm, 422-nm and 470-nm, as seen in precedent pathogens and antimicrobial factors• 112 literature (1,10,19-20,23,27).). however, it was observed that the optimal wavelength for visible blue lllt was 405-nm (p < 0.0001).. the results of this study are consistent with the findings of bumah et al., which illustrated that 405-nm photoinactivation resulted in a more efficient colony count reduction as opposed to other wavelengths within the visible blue spectrum (6). while multiple studies have confirmed the efficacy of lllt at one discrete wavelength or fluence, this study intended to evaluate the photoinactivation rates of multiple wavelengths to determine the optimal wavelength and fluence of lllt within the visible blue spectrum. as seen in table 1 and fig. 1, 405-nm photoinactivation effectively illustrated a more immediate bactericidal effect compared to its 422-nm and 470-nm counterparts. despite achieving similar bacterial load reductions at each wavelength following the entire 300 j/cm2 light treatment, 405-nm lllt resulted in a 94.50% load reduction following its initial 75 j/cm2 (2 hours) of irradiation. based on the parameters of this phase 1 study, 75 j/cm2 of 405-nm blue light was determined to be the optimal blue light dosage, and was therefore held constant throughout phase 2. following phase 1, the irradiance (mw/ cm2) at which the light was delivered to the mrsa cultures was manipulated during phase 2 to determine if a lower, more continuous light exposure could result in a maximum formation of reactive oxygen species (ros) through a specific fluence. as illustrated in table 2, low-irradiance lllt provided a statistically significant improvement [p < 0.0001] in reducing the bacterial load of mrsa, and should be considered as a viable, therapeutic alternative to high intensity lllt, which conversely deliversthe total fluence over short durations, ranging from 60 seconds to 10 minutes. based on this principle, lllt illumination using a high irradiance light source could potentially deplete oxygen supplies more rapidly than low-irradiance lllt, and therefore compromise the efficacy of the intervention (4, 38). future studies using this low-irradiance strategy will add merit to the growing body of evidence of the efficacy and safety of lllt. one of the limitations of this study was the inability of the led apparatus to administer a uniform irradiance of less than 3.48 mw/cm2 to each of the 24-wells due to a constraint of the power supply used in this experimental setup. based upon the parameters of this study, subsequent studies should be completed to evaluate the bactericidal effects of lllt using irradiances lower than the 3.48 mw/cm2 to determine if there is a minimum threshold to low-irradiance that would consequently compromise efficacy of treatment. another limitation of the current study was that the bactericidal effects of low-irradiance lllt were only evaluated on one strain of mrsa. studies completed by grinholc et al. (13) determined after analyzing over 40 distinct strains of mrsa that the bactericidal effect of lllt was strain-dependent, with bacterial load reduction counts ranging from 0% >99.9% (3 log-reduction) (13). while this study did analyze the effects of lllt using a light source in the visible red (624-nm) spectrum, subsequent analyses of the bactericidal effects of 405-nm lowirradiance lllt should be evaluated among various gram-positive and gram-negative organisms, and their respective strains. in addition to subsequent studies strictly analyzing the efficacy of 405-nm lowirradiance lllt for its bactericidal and wound healing capabilities, there is a growing body of evidence that lllt using multiple wavelengths has provided 113 • fine focus, vol. 2 synergistic effects that benefit the overall therapeutic outcomes when compared to using solely monochromatic lllt (12, 14, 18, 21-22). studies from goldberg et al. (12) and lee et al. (18) observed the efficacy of visible blue (415+5 nm) and red (633+6 nm) combination light for the treatment of acne vulgaris in a 27 patient study, and determined that phototherapy using multiple wavelengths was an effective strategy to reduce bacterial load as well as accelerate skin healing (12, 18). translating the efficacy of combination wavelength treatments to other areas of medicine could possibly reduce the potential of oral tissue infection following oral surgery while simultaneously promoting faster healing of gingival tissue or additional soft tissues within the oral cavity. furthermore, combination wavelength treatments show promise in dermatologic, surgical, and military environments where infection management, tissue rescue, and tissue healing are directly connected to survival or other favorable outcomes. this research received no specific grant from any funding agency in the public, commercial, or not-for-profit sectors. the authors listed in this paper do not hold any financial or nonfinancial competing interests in regards to the research completed in this study. the authors declare that they have no competing interests. we would like to thank the rochester institute of technology’s college and health sciences and technology stock room (rochester ny) and the entire osgood laboratory for their assistance throughout all facets of this study. funding information competing interests acknowledgments pathogens and antimicrobial factors• 114 references 1. adamskaya, n., dungel, p., mittermayr, r., hartinger, j., feichtinger, g., wassermann, k., & griensven, v. 2011. light therapy by blue led improves wound healing in excision model in rats. injury 42:917–921. 2. al-masaudi, s. b., day, m. j., & russell, a. d. 1991. antimicrobial resistance and gene transfer in staphylococcus aureus. j. appl. bacteriol. 70:279–90. 3. aleem, n. a., aslam, m., zahid, m. f., rahman, a. j., & rehman, f. u. 2013. treatment of burn wound infection using ultraviolet light: a case 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with led light modulates healing processes in an in vitro scratchwound model using 3 different cell types. dermatol. surg. 41:261–8. 28. vasso, m., schiavone panni, a., de martino, i., & gasparini, g. 2016. prosthetic knee infection by resistant bacteria: the worst-case scenario. knee surgery, sports traumatology, arthroscopy xylanolytic psychotrophs from andosolic sedge fens and moss heaths in iceland olivia j. rickman1, m. auður sigurbjörnsdóttir2, oddur vilhelmsson 2,3* 1university of reading school of biological sciences, rg6 6ur, england. present address: the university exeter medical school, exeter 2university of akureyri faculty of natural resource sciences, akureyri, iceland. 3university of iceland biomedical center, reykjavík, iceland. copyright 2018, fine focus all rights reserved manuscript received 11 october 2017; accepted 15 may 2018 171 nine xylanolytic bacterial strains were isolated from fen and heath soils in northern iceland. they were found by 16s rrna gene sequencing to belong to the genera paenibacillus, bacillus, pseudomonas, and stenotrophomonas. using a simple, plate-based semiquantitative assay with azo-crosslinked xylan as the substrate, it was determined that although isolated from cold environments, most of the strains displayed greater xylanolytic activity under mesophilic conditions, with only the paenibacilli displaying markedly cold-active xylanolytic activity. indeed, for one isolate, paenibacillus castaneae ov2122, xylanolytic activity was only detected at 15°c and below under the conditions tested. of the nine strains, paenibacillus amylolyticus ov2121 displayed the greatest activity at 5°c. glycohydrolase family-specific pcr indicated that the paenibacilli produced multiple xylanases of families 10 and 11, whereas a family 8 xylanase was detected in pseudomonas kilonensis al1515, and a family 11 xylanase in stenotrophomonas rhizophila al1610. abstract corresponding author oddur vilhelmsson oddurv@unak.is keywords • xylanolytic • bacteria • glycohydrolase • moss heaths introduction 172 • fine focus, vol. 4 (2) xylanases, catalyzing the endohydrolysis of 1,4-β-d-xylosidic linkages in the hemocellulosic polymer xylan, are a widespread group of enzymes that play an important role in plant detritus degradation and are of considerable importance in many industrial processes, such as in the paper and food industries (3, 7). unsurprisingly, xylanase-producing microorganisms are typically found on plants or in association with plant material, such as in the phyllosphere and rhizosphere (15, 32). while xylanases have been isolated from various extremophiles, in particular thermophiles (7), research on cold-adapted xylanases is relatively scarce. several xylanases have nevertheless been isolated from psychrophiles (12, 19) and found to possess common features such as a low temperature optimum and poor thermostability due to fewer salt-bridges and less compact hydrophobic packing as compared to their mesophilic and thermophilic counterparts (7). xylanolytic activity is among the most important polymer degradation activities in the phyllosphere and xylanolytic bacteria can thus be expected to be important players in plant detritus turnover. furthermore, xylanases are valuable enzymes that are used in several processes within the food and paper industries. cold active xylanases and psychrotrophic xylanolytic organisms are thus of considerable interest both to biotechnology and cold-climate ecology. ov2121 aib0216c p. taichungensis bcrc 17757t p. xylanilyticus xil14t p. barcinonensis bp-23t p. oceanisediminis l10t p. massiliensis smarlab biomol-2301065t p. panacisoli gsoil 1411t p. kribbensis am49t p. peoriae dsm 8320t p. nicotianae yim h-19t p. hunanensis fel05t p. stellifer dsm 14472t p. borealis dsm 13188t p. odorifer dsm 15391t p. typhae xj7t p. vini lam0504t p. konsidensis lbyt p. relictisesami kb0549t p. azoreducens cm1t p. rhizosphaerae cecap06t p. cineris lmg 18439t p. favisporus gmp01t p. lemnae l7-75t p. turicensis mol722t p. chitinolyticus nbrc 15660t p. gansuensis b518t p. popilliae atcc 14706t p. thiaminolyticus nbrc 15656t p. profundus si 79t p. thailandensis s3-4at p. granivorans a30t p. agaridevorans dsm 1355t p. sepulcri ccm 7311t p. mendelii c/2t p. gorillae g1t p. catalpae d75t p. xinjiangensis b538t bacillus ss015a p. oenotherae dt7-4t p. cellulosilyticus palxil08t p. kobensis dsm 10249t p. tarimensis sa-7-6t p. algorifonticola xj259t 76 p. endophyticus pecae04t 74 97 94 ov2122 p. castaneae ch-32t 100 100 100 94 67 100 100 p. tibetensis ssb001t p. wynnii dsm 18334t 96 57 61 80 90 33 38 94 96 73 80 58 ov2120 p. amylolyticus nrrl nrs-290t p. xylanexedens b22at 100 p. tundrae a10bt aib0216b p. tylopili mk2t 98 77 99 68 97 64 96 45 84 99 98 37 90 42 66 96 86 83 27 67 32 54 34 52 49 35 41 40 12 0.01 p. wulumuqiensis y24t p. shenyangensis a9t p. dauci h9t p. antarcticus lmg 22078t p. glacialis kfc91t 99 99 173 figure 1. 16s rrna gene phylogenetic analysis of the paenibacillus isolates and selected type strains from that genus. phylogenetic relationships were determined using the neighbor-joining method of saitou and nei (33). the optimal tree had a branch length sum of 0.8577. the percentage of replicate trees in which the associated taxa clustered together in the bootstrap test (1000 replicates) are shown next to the branches (10). the tree is drawn to scale, with branch lengths in the same units as those of the evolutionary distances used to infer the phylogenetic tree. the evolutionary distances (number of base substitutions per site) were computed using the maximum composite likelihood method of tamura et al. (35). all positions containing gaps and missing data were eliminated, resulting in a total of 1369 positions in the final dataset. the analysis was conducted in mega6 (36). in this study, we present nine psychrotrophic xylanolytic bacterial strains from three soil habitat types in northern iceland, identified as paenibacilli, pseudomonads, a bacillus and a stenotrophomonas, and discuss the temperature-dependence of their xylanase activity and the likely glycohydrolase family of their respective xylanases. materials and methods isolation of bacteria leptosolic and andosolic sites (table 1) were sampled aseptically with sterile implements into sterile falcon tubes (becton-dickinson, franklin lakes, nj, usa) or whirlpak bags (nasco, fort atkinson, wi, usa). to obtain microbial suspensions, soil samples were placed in a tenfold volume of butterfield‘s buffer (butterfield 1932) at ph 7.2 in a sterile stomacher bag and stomached in a labblender 400 (seward, worthing, uk) for 2 min. the slurry was then serially diluted in butterfield‘s buffer to 10-6. all dilutions were spread-plated in duplicate onto plate count agar (pca), tryptic soy agar (tsa), and actinomycete isolation agar (aia) (becton-dickinson) and plates incubated in the dark at 15°c for four weeks. the plates were examined for colony morphotypes based on color, sheen, convexity and other visible features. representatives of each morphotype were aseptically picked, streaked onto fresh media and incubated under identical conditions as the original isolation plates. in order to obtain pure cultures, the isolates were re-streaked and incubated at least once more. stocks of purified isolates were prepared by suspending a loopful of growth in 0.5 ml 28% (v/v) glycerol and stored at −70 °c. xylan degradation screens and assays degradation of xylan was screened for by the appearance of blue halos on an 0.5 g l-1 suspension of azo-cross-linked birch xylan (megazyme,wicklow, ireland) in a medium that consisted of 4 g nutrient broth (nb) and 15 g l-1 agar (becton-dickinson). when used as a semi-quantitative xylanase assay, halo emergence on equal-volume (20 ml) plates was monitored twice daily and halo diameter measured with a ruler. one-way anova on terminal halo diameters was used to determine effect of temperature on xylan degradation. precultures were incubated in the presence of xylan. phenotypic characterization several standardized biochemical tests were performed using the api 20e test strips (biomériaux) according to the manufacturer‘s instructions. 16s rrna gene sequencing colonies were picked using sterile toothpicks and suspended in 25 µl colony lysis buffer (1% triton x-100 in 0.02 mol l-1 tris / 0.002 mol l-1 edta at ph 8.0). the resulting suspension was heated to 95°c for 10 min and then cooled to 4°c and 1 µl of the suspension used as a template in a standard taq-pcr reaction (35 cycles, annealing at 51°c for 30 sec, extension at 68°c for 90 sec, denaturing at 95°c for 30 sec) in an mjr ptc200 thermocycler (mj research inc., waltham, ma, usa). the primers 8f (5‘-agagtttgatcctggctcag-3‘) and 1522r (5‘-aaggaggtgatccaaccgca-3‘) 174 • fine focus, vol. 4(2) 175 depth (cm) strain no. isolation medium colony morphology site 1. sandabotnaskarð (65.685°n, 16.769°w). brown andosol. sparse vegetation. 10 aib0216b pca white, flat, dry 10 aib0216c pca white, flat, dry 10 aib0228 tsa cream-colored, convex site 2. svartárkot (65.336°n, 17.242°w) brown and histic andosol. moist moss heath. 5 al1515 pca cream-colored, convex 17 al1610 aia tan, convex, glistening, small 17 al1614 pca white, matte, convex, grainey 25 do1702 aia cream-colored, irregular, glistening, slimey site 3. hesteyrarskarð (66.358°n, 22.930°w) histic andolsol. short sedge fen. 5 ov2120 tsa white, flat, dry 5 ov2121 tsa white, flat, dry 5 ov2122 tsa white, flat, dry were used at a final concentration of 0.2 µm in a total volume of 25 µl of pcr mixture containing 0.15 µl of taq dna polymerase (new england biolabs, ipswich, ma, usa). 16s rrna gene sequencing cont. the primers 8f (5‘-agagtttgatcctggctcag-3‘) and 1522r (5‘-aaggaggtgatccaaccgca-3‘) were used at a final concentration of 0.2 µm in a total volume of 25 µl of pcr mixture containing 0.15 µl of taq dna polymerase (new england biolabs, ipswich, ma, usa). amplicons were visualized on an 0.8% agarose gel using sybr safe (life technologies, carlsbad, ca, usa) and cleaned up for sequencing using 20 u µl-l exonuclease i and 5 u µl-1 antarctic phosphatase (new england biolabs) at 37°c for 30 min, followed by deactivation at 90°c for 5 min. partial sequencing of the purified amplicons was performed with a bigdye terminator kit and run on applied biosystems 3130xl dna analyzer (applied biosystems, foster city, usa) at macrogen europe, amsterdam, the netherlands using sequencing primers 519f (5‘-cagcagccgcggtaatac-3‘) and 926r (5‘-ccgtcaattcctttgagttt-3‘). sequences were examined in abi sequence scanner 1.0 (applied biosystems, framingham, ma, usa) and the forward and reverse complement of the reverse sequence manually aligned and merged. phylogenetic analysis the 16s rrna gene sequences were assigned table 1. strains, source samples, and isolation media; plate count agar (pca), tryptic soy agar (tsa) and actinomycete isolation agar (aia). 176 • fine focus, vol. 4(2) to taxa based on the identification of phylogenetic neighbors as determined by a blastn (1) search against a database containing type strains with validly published prokaryotic names and representatives of uncultured phylotypes (18). the top thirty sequences with the highest scores were then selected for the calculation of pairwise sequence similarity using a global alignment algorithm (22), which was implemented at the eztaxon server (18). for further phylogenetic analysis, sequences were multiply aligned using muscle (9) and bootstrapped (10) neighborjoining trees (33) were calculated in mega6 (36) using the maximum composite likelihood model (35). amplification of xylanase gene fragments genomic dna was extracted and purified from cultures grown on nutrient agar (na) plates using mobio microbial dna isolation kit (mobio laboratories, solana beach, ca) according to the manufacturer’s instructions. primers specific to xylanases of families 8 (xyn8a, forward: 5‘-catcctgttcaggaagacagtagtgggg-3‘, reverse: 5‘-cttgataatccggaaattgccactgacatgc-3‘), 10 (xynfa, forward: 5‘-cacackctkgtktggca-3‘, reverse: 5‘-tmgttkacmacrtccca-3‘), and 11 (paxyna, forward: 5‘-gaytaytggcartaytggac-3‘, reverse: 5‘-atrtcrtangtnccnccrtcrct-3‘) were used for amplification. reaction conditions, amplicon clean-up and sequencing were as described for the 16s rrna gene amplification, above. identification of xylanolytic isolates isolated bacterial strains (106) from andosolic wetland and moss heath sites (table 1) were screened for the ability to yield blue halos on azo cross-linked xylan plates at 15°c, and nine positive isolates selected for further study. the isolates were identified by 16s rrna gene sequencing (table 2) and found to comprise paenibacilli (5 isolates), pseudomonads (2 isolates), a stenotrophomonad and a bacillus. a pairwise distance comparison of the muscle multiply aligned 16s rrna gene sequences obtained for all nine strains (table 3) reveals that four of the paenibacilli, strains ov2120, ov2121, aib0216b, and aib0216c are very closely related to one another, with on average less than 2 base substitutions over the 987 nucleotide positions tested. a phylogenetic analysis of the paenibacilli (fig. 1) was performed using 54 paenibacillus type strain reference sequences. the reference sequences were selected based firstly on their similarity to the test strains as determined by the blastn algorithm on the eztaxon server and secondly based on their completeness, enabling us to run the phylogenetic analysis over 1369 nucleotide positions. the analysis revealed that isolates ov2121, ov2120, aib0216b, and aib0216c form a clade with p. amylolyticus, p. xylanexedens, p. tundrae and p. tylopili, supporting the eztaxon assignments of isolates ov2120 and ov2121 as p. amylolyticus, and aib0216b and aib0216c as p. tundrae. the divergence of strain ov2122 from the other four paenibacilli was further demonstrated on the phenotypic level by biochemical characterization using the api 20e strip test, in which ov2122 was distinguished by lack of onpg hydrolysis, presence of arginine dihydrolase and gelatinase, citrate utilization, and lack of sucrose and amygdalin utilization (table 4). results applied & environmental microbiology • 177 presence and family of xylanase genes to independently verify the presence of glycohydrolase family 8, 10 or 11 xylanase genes, pcr reactions using primer pairs xyn8a, xynfa, and paxyna were run on dna extracted from pure cultures of isolated strains, as well as of the az-xylan screen-negative strain do1702 as a negative control (fig. 2). selected product bands were extracted and sequenced (table 5). under the reaction conditions employed, the family 8 primer pair xyn8a yielded apparent primer dimers in all cases, but nevertheless clear product bands at approximately 400 bp were visible for strains al1515, ov2121, and ov2122. the al1515 band was sequenced and found by blastx to contain a 359-bp product most similar to a hypothetical protein of unknown function in the betaproteobacterium andreprevotia chitinilytica, and thus probably constitutes an unspecific amplification of an unknown component of the al1515 genome, or possibly a hitherto unidentified xylanase. the family 10 primer pair xynfa yielded a variety of amplicons: a large (>10 kb) product from strain al1610, an approximately 150 bp product from strain ov2122, an approximately 250 bp product from strain ov2122, three products (3 kb, 1 kb and 700 bp) from each of strains ov2120 and ov2121, and two products (150 and 750 bp) from strain aib0216b. of these, three amplicons were sequenced (both aib0216b products and the aib0228 product), one of which was found to correspond to a paenibacillus xylanase, while the others showed greatest similarity to proteins of unknown function (table 5). the family 11 primer pair paxyna yielded an approximately 200 bp-product from strain aib0216c, and approximately 800-bp products from strains al1610, ov2120, ov2121, and ov2122. the products for al1610, ov2121, and ov2122 were partially sequenced and all displayed greatest similarity to paenibacillus endo-1,4-beta xylanases (table 5). isolate sequence length (nt) genbank accession most similar taxon at eztaxon1 (% similarity) aib0216b 1523 kx349197 paenibacillus tundrae (99.87) aib0216c 1452 kx349198 paenibacillus tundrae (99.79) aib0228 1436 kx349200 pseudomonas baetica (99.30) al1515 998 kx349201 pseudomonas kilonensis (99.50) al1610 1503 kx349202 stenotrophomonas rhizophila (99.73) al1614 1435 kx349199 bacillus subtilis (99.23) ov2120 1459 kx349195 paenibacillus amylolyticus (99.72) ov2121 1451 kx349194 paenibacillus amylolyticus (99.79) ov2122 1457 kx349196 paenibacillus castaneae (99.73) table 2. identification of xylanolytic bacterial isolates by 16s rrna gene sequencing. 1 the isolates were identified using the eztaxon server (http://www.ezbiocloud.net/ eztaxon; kim et al., 2012) on the basis of 16s rrna gene sequence data. 178 • fine focus, vol. 4(2) figure 3. xylanolytic activity as measured by the emergence and diameter of dye halos on azo-crosslinked birch xylan (megazyme) agar media. plates were incubated at 5°c (a), 15°c (b), 22°c (c), and 35°c (d). values are means of measurements of triplicate plates. error bars are omitted for clarity (standard deviations of terminal values can be seen in table 6). solid circles indicate strain aib0216b, open circles strain aib0216c, solid downwards-pointing triangles strain aib0228, open upwards-pointing triangles strain al1515, solid squares strain al1610, open squares strains al1614, open diamonds strain ov2120, solid upwards-pointing triangles strain ov2121, open downwards-pointing triangles strain ov2122. applied & environmental microbiology • 179 temperature-dependence of xylanolytic activity the xylanase screen was adapted to a semi-quantitative assay by monitoring the emergence and diameter of the blue dye haloes on triplicate plates incubated at 5, 15, 22, and 35°c over a period of up to 10 days. the effect of incubation temperature on xylanolytic activity varied substantially among the strains (fig. 3), with ov2121 displaying the most prominent activity at 5°c, whereas at 35°c al1614 and al1515 displayed the most activity. although variation among replicates was in many cases high (table 6), especially at lower temperatures, one-way anova on terminal halo diameters revealed significant differences in xylanase activity among the strains at 35°c (f(7,16) = 8.45; p<0.001) and at 22°c (f(8,16) = 4.45; p=0.005), while at 15°c one-way anova did not reveal a difference (f(7,16) = 1.44; p=0.257) and at 5°c, while evidence against the null hypothesis of there being no difference was found, it was not significant at the 95% level (f(5,12) = 3.01; p=0.055). most of the isolates displayed higher xylanolytic activity at higher temperatures, although isolate ov2122 only displayed activity below 35°c. no activity was detected at 5°c in any of the replicates of isolates al1515, al1610, or al1614. figure 2. agarose gels with putative xylanase gene fragment amplicons. the amplicons were obtained by taq polymerase pcr using primer pairs specific for xylanases of glycohydrolase families 8 (a; xyn8a), 10 (b; xynfa), and 11 (c; paxyna). all gels show a 1kb dna ladder (neb) in the first lane, showing bands from 0.5kb to 10kb. lanes 2 to 11 contain amplicons from strains as follows: 2, aibo216b; 3, aibo216c; 4, aibo228; 5, al1515; 6, al1610; 7, al1614; 8, do1702; 9, ov2120; 10, ov2121; and 11, ov2122. (c) according to the 16s rrna gene sequencing, five of the strains in present study belong to the genus of paenibacillus. the paenibacilli are ubiquitous in nature, having been isolated from environments as diverse as soil, clinical specimens, and hot springs (16). members of the genus are highly diverse, with member species representing psychrophiles, mesophiles, and thermophiles, strict aerobes and facultative anaerobes, and are known for their production of various glycanolytic exoenzymes (28). since its formation from members of bacillus (2), the genus paenibacillus currently has grown to 200 species and 4 subspecies according to the lpsn (27). xylanolytic activity is common among the paenibacilli (6) and several investigators have reported the isolation and characterization of glycohydrolase family 8 (23), family 10 (17, 37) and family 11 (34, 38) xylanases from these organisms. several paenibacilli are psychrotrophic and, hence, cold-active xylanases are found among them (40). much like the paenibacilli, the pseudomonads are very commonly encountered in plantand soil-associated environments, and a number of studies have described xylanolytic activity in, and the isolation and characterization of xylanases from these bacteria (20, 29). the p. fluorescens family 10 xylanase a and family 11 xylanase e have been well studied (13). the identity of xylanolytic isolate al1610 as a stenotrophomonas sp. was more surprising. while the stenotrophomonads are typically plant-associated bacteria and therefore expected in the habitats under study, they are generally, as the genus name implies, quite fastidious with a nutritional spectrum mostly limited to monoand disaccharides (25). very little literature currently exists on xylanolytic activity in these organisms. indeed, according to raj et al. (30), their study on an inducible, thermostable xylanase from stenotrophomonas maltophilia was the first to describe xylanase activity in that species. malfliet et al. (21) also reported s. maltophilia xylanase activity among the consortia present in malting barley. 180 • fine focus, vol. 4(2) discussion ov2121 0.000 ov2120 0.000 0.000 ov2122 0.068 0.068 0.000 aib0216b 0.002 0.002 0.068 0.000 aib0216c 0.002 0.002 0.068 0.000 0.000 al1614 0.140 0.140 0.151 0.140 0.140 0.000 aib0228 0.267 0.267 0.301 0.267 0.267 0.257 0.000 al1515 0.256 0.256 0.288 0.256 0.256 0.247 0.009 0.000 al1610 0.259 0.259 0.302 0.260 0.259 0.267 0.177 0.179 0.000 ov2121 ov2120 ov2122 aib0216b aib0216c al1614 aib0228 al1515 al1610 table 3. pairwise distances between muscle multiply aligned 16s rrna gene sequences. hydrolysis of: onpg + + + + + + arginine dihydrolase + + + + w lysine decarboxylase ornithine decarboxylase citrate utilization + + + + h2s production urease tryptophan deaminase indole production acetoin production + gelatinase activity + + + + + no3 reduction fermentation/oxidation: d-glucose + + + + + + + + + d-mannitol + + + inositol d-sorbitol l-rhamnose + d-sucrose + + + + w + d-melibiose + + + + + + + + + amygdalin + + + + + + + l-arabinose + + + + + + + w pa en ib ac ill us a ib 0 21 6 b pa en ib ac ill us a ib 0 21 6 c pa en ib ac ill us o v 21 20 pa en ib ac ill us o v 21 21 pa en ib ac ill us o v 21 22 b ac ill us a l 16 14 ps eu do m on as a ib 0 22 8 ps eu do m on as a l 15 15 st en ot ro ph om on as a l 16 10 table 4. selected phenotypic characteristics (positive (+), negative (-) or weak (w)) of xylanolytic isolates obtained using api 20e test strips applied & environmental microbiology • 181 bands primer lgth (nt) best blast x hit % (id)aa al1610 261 paenibacillus sp. hy8 endo-xylanase 99 ov2122 263 paenibacillus amylolyticus 1,4-beta-xylanase 99 ov2121 204 paenibacillus sp. pamc 26794 endo-1,4-betaxylanase 95 aib0216b_ b1 xynfa 157 paenibacillus sp. o199 hypothetical protein 100 table 5. blastx of partial nucleotide sequences obtained from amplicons obtained using xylanase-specific pcr primers. the stenotrophomonads have been used in agricultural and environmental biotechnology, such as for bioremediation, in spite of the potential of s. maltophilia as an opportunistic human pathogen (4). xylanolytic activity may further expand the biotechnological potential of these organisms. a pairwise distance comparison of the aligned 16s rrna sequences, obtained for all nine strains, revealed a close relatedness of strains isolated in widely separated parts of the country. these results are striking but not necessarily unexpected, as the sporeforming paenibacilli disperse easily by wind over considerable distances and are thus not subjected to the physical isolation required for diversification (26). the fifth paenibacillus, strain ov2122 was more divergent, displaying on average 67 base substitutions to each of the other paenibacilli over the 987 nucleotide positions tested. the other four strains were more heterogeneous, although the two pseudomonads were fairly similar to each other (9 substitution over the 987 positions). the highest divergence observed was between paenibacillus ov2122 and stenotrophomonas al1610, 298 base substitutions among the 987 nucleotide positions tested. four isolates, ov2121, ov2120, aib0216b and aib0216c, form a clade with p. amylolyticus, p. xylanexedens, p. tundae and p. tylopili (fig. 1). both p. xylanexedens and p. tundrae were originally isolated as xylanolytic paenibacilli from moist tundra soil in alaska (24). however, related strains have been isolated from other environments, such as barley grain in finland (31) and catfish guts in brazil (8). isolate ov2122 forms a clade with p. castaneae and p. endophyticus supporting the eztaxon assignment of this isolate as p. castaneae, a species described from strains originally isolated from a chestnut tree rhizosphere in spain (39). several of the strains yielded amplicons from more than one xylanase primer pair used for screening. while this may be due to less-than-perfect specificity of the primers employed due to the noted diversity of xylanase amino acid sequences (7), more than one xylanase genes may indeed be present in the respective genomes. several microorganisms, including both pseudomonads and bacilli, are known to produce multiple xylanases (11, 41). taken together, these results indicate that the paenibacilli (strains aib0216b, aib216c, ov2120, ov2121, and ov2122) possess at least family 10 and family 11 xylanases, strains ov2121 and ov2122 possibly also a family 8 xylanase. 182 • fine focus, vol. 4(2) strains 5°c 15°c 22°c 35°c aibo216b 1.3 ± 0.4 3.5 ± 0.5 4.0 ± 0.0 4.0 ± 0.0 aibo216b 0.7 ± 0.6 3.2 ± 0.3 4.2 ± 0.3 4.3 ± 0.3 aibo228 0.3 ± 0.6 2.5 ± 0.9 3.8 ± 0.3 3.8 ± 0.3 al1515 0.0 0.0 3.2 ± 0.3 6.3 ± 0.6 al1610 0.0 3.5 ± 0.7 3.6 ± 0.1 5.2 ± 0.3 al1614 0.0 2.0 ± 0.4 3.6 ± 0.8 6.5 ± 1.3 ov2120 0.5 ± 0.5 3.3 ± 0.6 4.3 ± 0.4 4.3 ± 1.0 acknowledgements this work was in part conducted under an erasmus internship exchange programme and was funded by the university of akureyri research fund. the authors would also like to acknowledge robert w. jackson and other coordinators of the university of akureyri and reading university joint arctic microbiology field trip and laboratory course of 2012 within which environmental sampling and initial culturing of the isolates was performed by students árný ingveldur brynjarsdóttir and luke johnson. the two pseudomonads (aib0228 and al1515) display quite divergent results, with al1515 possibly possessing a family 8 xylanase, although in both cases the identity of the pcr amplicons could not be verified by sequencing as belonging to a xylanase gene. based on the 261-bp paxyna amplicon sequence, the stenotrophomonas al1610 xylanase appears most similar to the family 11 xylanase of paenibacillus hy-8 (14). we adapted the xylanase screen to a semi-quantitative assay and monitored the emergence and diameter of the blue dye haloes incubated at different temperature, ranging from 5 – 35°c. the effect of temperature was noticeable most of the tested strains displayed most activity at higher temperature. of the nine tested strains, only paenibacillus amylolyticus ov2121, displayed the most prominent activity at 5°c. we therefore conclude that of the nine xylanolytic strains studied, paenibacillus amylolyticus ov2121 produces the most cold-active xylanase under the conditions employed, while pseudomonas kilonensis al1515 and bacillus subtilis al1614 display higher activity at higher temperatures. paenibacillus castaneae ov2122 only displays xylanolytic activity at lower temperatures. applied & environmental microbiology • 183 table 6. terminal halo diameters on az-xylan test media at different incubation 1. altschul, s. f., madden, t. l., schäffer, a. a., zhang, j., zhang, z., miller, w., lipman, d. j. 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(1989). identification of two distinct bacillus circulans xylanases by molecular cloning of the genes and expression in escherichia coli. appl. environ. microbiol. 55:568–572. 186 • fine focus, vol. 4(2) mycorrhizal fungi • 65 mixtures of mycorrhizal fungi improve growth of lactuca sativa and reduce levels of zinc in contaminated soil john a. haradean1 tyler ralph-epps1 zach whiteacre1 sarah neumann1 donna m. becker1 1 department of biology, northern michigan university, marquette, mi 49855 manuscript recieved 3 may 2019; accepted 6 september 2019 copyright 2019, fine focus all rights reserved abstract corresponding author john a. haradean jharadea@nmu.edu department of biology, northern michigan university marquette, mi, 49855. bioremediation is currently under investigation as a viable way to remove many environmental pollutants and most commonly involves the use of microorganisms to extract organic pollutants or heavy metals from water or soil. one of the most abundant heavy metals found in industrially polluted sites is zinc (zn); it is often found alongside metals like lead (pb), arsenic (as), and mercury (hg). this experiment investigated the potential bioremediation of pasteurized soil contaminated with zinc using different vesicular arbuscular mycorrhizal fungi (vam) species and lettuce plants (lactuca sativa). soil was amended with 0.4 g of zinc chloride (zncl2) per kg of soil. amended and unamended soils were inoculated with two different mixes of vam, bioag vam-endo™ and mycobloom. for each treatment, l. sativa plants (15 pots per treatment) were grown in a greenhouse setting. plant diameter was measured weekly. plants were harvested after 55-days and the wet weight of leaf tissue was measured before the tissue was sent for analysis of zinc levels. roots were assessed for mycorrhizae using a trypan blue staining procedure. the bioag vam-endo™ mix was the most successful at removing zncl2 from the soil. l. sativa inoculated with vam mixes formed mycorrhizae, grew healthier and removed more zinc from the soil than the noninoculated group. we propose further investigation into the use of mycorrhizal fungi paired with other plant species to remove zinc from contaminated sites with harmful levels of zinc. keywords introduction • bioremediation • mycorrhizal fungi • vam • lactuca sativa • zinc bioremediation uses an introduced or naturally present organism to break down environmental pollutants. several subcategories of bioremediation exist, three of which are phytoextraction, rhizosphere degradation, and mycoremediation. phytoextraction uses plants to take up a contaminant to be stored within its tissue and the tissue is then harvested and typically destroyed. rhizosphere degradation takes advantage of the soil microbiome to help breakdown the contaminant. in mycoremediation fungi are used to degrade or capture contaminants (9, 14). phytoextraction, alone, has not been determined to be an effective method of removing heavy metals from contaminated sites, as of yet (29). however, when multiple bioremediation methods are used in concert, contaminant uptake can be increased. when vesicular-arbuscular mycorrhizal fungi (vam) are associated with a plant, zinc uptake and biomass are significantly increased, in lab and field studies (17, 21). many fungi have powerful enzymes that can break down pollutants directly and they also contain heavy metal chelators, making them particularly suited to remediate areas contaminated with heavy metals (4, 14, 30). fungi can bind these metals to chelators anchored within their cell wall, intracellular chelators within the cytosol, or bind them to extracellular chelators. each of these methods render the metals inactive which can ultimately save organisms, like plants, from bioaccumulation of these toxic agents in tissues, or humans from ingestion of food crops containing toxic quantities (16, 30). fungi have successfully been used to remediate areas of concern, but more research needs to be done in this area to make them more effective. glomus intraradices is one such species of fungus that has been studied extensively. g. intraradices is a species of 66 • fine focus, vol 5 2019 vam that associates with plant roots in a symbiotic relationship. a few studies have showed g. intraradices has a higher tolerance for heavy metals when compared to other vam grown under the same conditions (11, 20). heavy metals are released into the environment through a number of processes. they often accumulate, naturally, through erosion of parent material, such as exposed bedrock and other parts of the earth’s crust. human activity has caused a sharp increase in release of heavy metal pollutants within the environment. one example of a heavy metal is zinc, which can be found in toxic concentrations in many anthropogenically contaminated sites (6). in 2005, 985 of the 1,662 hazardous waste sites that were proposed for inclusion on the epa national priorities list contained zinc, although it is unknown how many of those sites were evaluated for toxic quantities of zinc (1). zinc has many important industrial uses (e.g., manufacturing of batteries, use in paint and rubber products, and zinc oxide). another major source of zinc contamination is through mining, which includes leftover tailing piles, by-products of smelting, and hydraulic fracturing (7). hydraulic fracturing, otherwise known as fracking, is a mining technique used to extract oil and natural gas. since natural gas has gained popularity as a transitional fuel in the race to find a sustainable source of energy, hydraulic fracturing has increased in popularity. in 2015, there were 623 reported fracking fluid spills in the united states, according to the colorado oil and gas conservation commission (27). this is down from the highest recorded number of spills in 2014, which is 786, also reported by the colorado oil and gas conservation commission (27). the use of hydraulic fracturing, like any other form of mining, generates waste. in this case, it is the contaminated water expelled from the hydraulic fracturing wells along with the contents of whatever was in the ground (12). this waste often contains high concentrations of zinc brought up from deep within the earth. inevitably when this water is expelled into the area around the well, zinc as well as other pollutants can accumulate in the soil often in the form of zinc chloride and other organic materials (12, 15). in plants, zinc is used as a constituent in many enzymes and proteins and it is vital to the production of chlorophyll, to the development of cold resistance, and to the conversion of starches to sugars (5, 25). the normal concentration of zinc in healthy leaf tissue is measured to be in the range of 10-15 ppm (5, 19). in toxic concentrations of zinc, plants experience stunted growth, chlorosis of the leaves, and changes in root growth (22). high zinc concentrations also interfere with the uptake of other nutrients such as phosphorus, iron, manganese or copper, ultimately leading to their deficiency. if the toxicity is severe enough, partial or whole plant death will occur (5). noticeable symptoms of toxicity generally occur when zinc levels accumulate to greater than 200 ppm (5, 19). the presence of elevated zinc levels in the environment by things like hydraulic fracturing wastewater has an indirect effect on us. contamination can have detrimental effects on many staple crops, as well as native species. zinc accumulation can be detrimental to a multitude of ecosystems and human well-being. knowing that vam often form large multi-species complexes in nature, mixtures were chosen for this current study rather than using only a single species. these mixes were paired with a popular variety of l. sativa. l. sativa is a common crop grown around the world with a fast maturation rate. l. sativa is also susceptible to different types of environmental pollution, making it a good candidate for study on the effects of soil contamination on plants. this experiment used the same variety of l. sativa but with different mixes of vam, one containing species native to the great lakes region (mycobloom) and another sold for commercial agricultural species (bioag vam-endo™). it was hypothesized that the presence of the vam mixes would reduce zinc concentrations within the soil and that different types of vam will have a different impact on the uptake and bioaccumulation of zinc. mycorrhizal fungi • 67 materials and methods soil preparation approximately 155 kg of agricultural soil was gathered from marquette, michigan. this soil was pasteurized, in a single batch at northern michigan university, twice at 75 °c for 1-hour, with a 24hour resting period in between pasteurizations. a soil sample was then sent to a&l great lakes laboratory, inc. to determine the nutrient profile of the soil before amending. soil treatments six treatment groups were created, each consisting of 15 pots with 1.78 kg of soil in each pot (5.5 inch square jumbo dillon greenhouse pots, growers solution, cookeville, tn). this pot size and volume of soil allowed for proper l. sativa plant growth (whitacre and neumann, unpublished). the first treatment group, t1, consisted of unamended pasteurized soil. the second and third treatment groups, t2 and t3, consisted of pasteurized soil amended with the mycorrhizal inoculants. t2 was amended with bioag vam endo-mix™. t3 was amended with mycobloom. bioag vam endo-mix™ consists of several different species of vam, the predominant one being glomus intraradices (65.8% of the species mix). smaller amounts, each comprising 5.7% of the species mix, of glomus claroidium, glomus deserticola, glomus mosseae, glomus etunicatum, gigaspora albida and glomus clarum were also included. mycobloom consists of seven different vam species native to the great lakes region. the species of vam include: claroideoglomus claroideum, funneliformus mosseae, cetraspora pellucida, claroideoglomus lamellosum, acaulospora spinosa, racocetra fulgida and entrophospora infrequens. the other three treatment groups, t4, t5, and t6, used the remaining half, 38.75 kg, of the pasteurized soil. this soil was moved to a separate container where a 1 l solution of water containing 31.14 g of zncl2 was mixed in using a shovel. this was done for approximately 10-minutes to ensure uniformity mixing of the zncl2. this process added approximately 0.4 g of zncl2 per kg of soil. the pasteurized soil with zncl2 added was then split up into the other 45 pots. this amount of zncl2 was chosen due to the results of previous trials consisting of 0.5 g of zncl2 per kg of soil (whitacre and neumann, unpublished), where plants across all zinc amended groups died in significant amounts.t4 consisted of the pasteurized soil amended with 0.4 g zncl2 per kg of soil. t5, in addition to zncl2, was mixed with bioag vam endo-mix™. t6 was mixed with mycobloom™. the mycorrhizal inoculants for all respective treatment groups were mixed in by hand at 5.6 g per pot. each pot contained approximately 1.78 kg of soil or soil with inoculate. all measurements made were made separately on a taylor digital kitchen scale to the nearest 100th decimal place. seed germination and planting once each of the treatment groups were properly amended, the pots were immediately moved to northern michigan university’s greenhouse. l. sativa seeds (black seeded simpson, johnny’s selected seeds co.) were germinated using a wet paper towel placed inside a plastic bag, for a period of 48-hours. once germinated, two seeds were planted in each of the 90 pots to ensure survival of at least one plant. the pots were covered with plastic wrap and left for 10-days until the seedlings were culled to one seedling per pot. after 2-weeks since planting, the plastic wrap was removed. another 2-weeks from this point, the plant diameter was measured every week for 4-weeks. a growth period of 55-days was chosen to ensure full maturity was reached, according to johnny’s selected seeds description. plants were watered as needed throughout the experiment. the computer control system of the greenhouse conditions were set to not exceed 29 °c or fall below 16 °c. however, 3-days of the experiment temperatures had reached 38 °c due to a malfunction of the greenhouse cooling system. the light conditions consisted of 14-hours of sunlight, and 10-hours of dark. soil and tissue sampling after a total growth period of 55-days the l. sativa leaf and stem tissues were harvested. the harvesting was done by cutting the stem just below the basal rosette, at roughly the same place on each plant. wet weight was taken for each plant to create an average, and then all the plants in each group were weighed together for a total group biomass. the soil from each treatment group was then bulked, taking care to keep each treatment group separate. soil samples were then taken for each separate treatment group. 68 • fine focus, vol 5 2019 results the soil and tissue samples were stored overnight in a cold room at 4 °c. the next day the samples were shipped overnight, unrefrigerated to a&l great lakes laboratory (fort wayne, indiana) and tested for their nutrient profile. the plant tissue was tested for total nitrogen using the dumas method using an elementar rapid-n cube, while mineral analysis was conducted using inductively coupled argon plasma using a thermo icap 6500. the soil analysis procedures were done in accordance with “recommended chemical soil test procedures for the north central region.” staining of roots for vam the roots were stained and analyzed for vam colonization. they were first washed of any remaining soil with deionized water (di water) and placed in separate beakers. each group was then placed in 5 % potassium hydroxide (koh) (w/v) and allowed to soak for a period of 2-days, until they appeared translucent and white (or as some would say, looked like “rice noodles”). the roots were rinsed with di water in between soaking periods. the roots were then rinsed with di water again and placed in a 5% hydrochloric acid (hcl) (v/v) solution to soak for 1-minute. a trypan blue stain was then created using 500 ml of glycerol (c3h8o3), 475 ml of deionized water, 25 ml of acetic acid (ch3cooh) and 0.1 g of trypan blue. the roots were allowed to sit in the trypan blue stain (c34h28n6o14s4) for 7-days and were then observed under a microscope. plant growth and biomass data the relationship between treatment groups and mean weekly plant diameter can be seen in figure 1. t4, which contained zinc only (no vam present) had stunted plant growth compared to all other treatments for all 4-weeks of measurements, however these plants had a clear linear growth rate throughout the 4-weeks. with all other treatments (either the control containing no zinc, or treatments with zinc plus vam), plant diameter measurements were similar as shown by the clustering of the data (fig. 1.). t5 had the greatest plant diameter at the end of the experiment, while t4 had the smallest diameter. the final plant diameters of t4 significantly differed from t1 (p<0.005), t2 (p<0.05), t5 (p<0.00005) and t6 (p<0.0005). other treatments that differed significantly from each other, are t5 when compared to t3 (p<0.05), as shown in figure 2. the mean final biomass of l. sativa tissue is shown in figure 3; the final biomass correlates similarly with the results of the mean final diameter. t3 and t4 had statistically significant lower biomass values than all other treatments (p<0.005), but they were not significantly different from each other. all the other treatments (t1, t2, t5, and t6) were not significantly different from each other. zinc concentration comparisons the zinc concentrations in the above ground tissue of l. sativa are shown in figure 4. plant leaves from all 15 pots were bulked for zinc analysis (due to cost constraints) thus no statistical tests could be run. however, the tissues of the t4 samples contained a considerably larger amount of zinc, 952 ppm, than any of the other zinc amended groups. the tissues of the treatments treated with mycorrhizae and zinc, t5 and t6, contained 573 ppm (60.2% of the amount of zinc as t4), and 476 ppm (50% of the amount present in t4) of zinc respectively. the tissues of the treatments that were not amended with zinc, t1-t3, all contained similar small amounts of zinc at the end of the experiment. mycorrhizal fungi • 69 70 • fine focus, vol 5 2019 the concentrations of zinc remaining in the soil are displayed in figure 5. soil from all 15 pots were bulked for zinc analysis (due to cost constraints) thus no statistical tests could be run. the treatment groups that were not amended with zinc all displayed similarly low levels of zinc. t4 clearly had the highest amounts of zinc in the soil. while the zinc concentrations of t5 and t6 had higher levels of zinc than unamended treatments (t1-t3), the levels of zinc are clearly lower than t4 that did not have any vam treatment. to ensure mycorrhizae had formed between the fungi and the roots in treatments t2, t3, t5 and t6 and that t1 and t4 were not colonized by vam, root tissue was stained and observed under microscope. figure 6 serves as a representative image of roots colonized by vam, which were evident in all treatments inoculated with the mycorrhizal fungi mixes. t1 and t4 had an absence of mycorrhizae (data not shown). figure 6. a representative image of l. sativa root tissue colonized by vam. statistical analysis the results of this experiment were analyzed using the tukey’s honest significant difference test, conducted in the program r, to assess the diameters and biomasses of the various test groups. discussion the l. sativa plants, which were treated with only zinc, experienced stunted growth initially and throughout the experiment (fig. 1). but, plants treated with vam (t5, t6) in zinc amended soils had similar weekly plant diameters and biomass as the treatment groups that had no zinc present (fig. 1). taken together, this suggests the presence of the vam aided in plant growth, likely by protecting the plant from growth-inhibitory or toxic levels of zinc. previous studies mentioned plant biomass had significantly increased due to additions of zinc and vam, but those studies applied lower concentrations of amended zinc (2, 16, 20). at the end of this experiment, t4 (treated only with zinc) exhibited significantly lower growth, biomass, and mean diameter compared to every other treatment group, besides t3 (fig. 2 and fig. 3). there were also two plant deaths that occurred in t4. during statistical analysis, the size measurements for the dead plants were counted as values of zero. the values were used in the analysis, because the deaths were believed to be caused by zinc, due to leaf chlorosis. since this occurred, the treatments amended with zinc were on the threshold of zinc toxicity. unexpectedly, without the addition of zinc, the plants treated with mycobloom (t3) at the completion of the study were affected negatively to the point where they had similar biomass and plant diameter data to the plants in soils treated with zinc only (t4) (fig. 2 and fig. 3). this could be due to non-optimal conditions for one or more of the vam species in the mix, perhaps causing a competitive or parasitic relationship to develop with the plants (10). the bioag vam endo-mix™, however, had a positive effect on plant growth, in both the zinc treated and untreated soils, when compared to their respective controls. this supports data found, in earlier experiments, on the ability of various mycorrhizal species to prevent heavy metal stress in l. sativa (13). the fungal species composition and ratios differed between the two vam mixes, which could help explain the different impacts on l. sativa plant growth. excessive heat in the greenhouse may have also played a role, although visible signs of widespread plant heat stress (e.g., chlorosis, wilting) were not evident through the course of the experiment. plant tissue grown in zinc amended soil without vam (t4) had very high levels of zinc (952 ppm) (fig. 4). in contrast, t5 and t6, also grown in zinc amended soil but with inoculated vam mixes, had mycorrhizal fungi • 71 substantially lower levels of zinc compared to t4 (573 ppm and 476 ppm respectively) (fig. 4). this suggests the vam were either sequestering some of the zinc in their fungal tissues, or possibly had chelated the zinc through release of extracellular chelators. although the zinc levels in the soil remained high (according to the standards followed by the a&l great lakes laboratories) (fig. 5), the quantity of zinc in the soil of t5 was substantially lower than in t4, and zinc levels in the t6 soil was also lower than t4 (fig.5) . this suggests that with longer vam treatment times zinc concentrations may be brought back to within a normal range, if the fungi were sequestering the zinc from the soil into their tissues. the soils of t5 and t6 may also be considered nontoxic according to some standards that report a 300 ppm toxic threshold of zinc and the t5 and t6 values are below that 300 ppm (19). while the average value of t5 and t6 did measure below 300 ppm, health hazards may still exist (2). it is possible that treatments of l. sativa only, without the addition of mycorrhizae, could bring the soil to acceptable levels of heavy metal concentrations. however, using mycorrhizae would be preferential, as the lettuce would likely experience less strain from zinc toxicity (3, 13). quantities of zinc remaining in the soil and tissues of t5 and t6 were different from each other (though not dramatically so); this, along with the unusually low plant biomass seen in t3 (which also had vam treatment), suggest that different vam mixes and pairing with proper plant species should be formulated to accommodate specific heavy metal contaminations more appropriately. choosing vam species that readily form mycorrhizae with the desired plants is important for this process. in this study, visual evidence of mycorrhizae formation was confirmed (fig. 6). the results from this study support other published findings that combinations of vam and their symbiotic plant species can be effective at remediating polluted soils (18, 24). further, we believe combined remediation techniques such as using specific plant species, tailored vam mixes, beneficial bacteria, and addition of chelators could be a highly successful strategy for bioremediation of polluted environments (9, 14, 16, 20, 21). acknowledgments we thank dr. alan rebertus (department of biology, northern michigan university) for his expertise with statistical analyses and the department of biology for financial support for supplies. 72 • fine focus, vol 5 2019 references 1. agency for toxic substances and disease registry. 2019. toxicological profile for zinc https:// www.atsdr.cdc.gov/toxprofiles/tp60-c6.pdf 2. 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phytoextraction: where’s the action? journal of geochemical exploration 151:34-40. 30. zhang, y., hu, j., bai, j., wang, j., yin, r., wang, j., & lin, x. 2018. arbuscular mycorrhizal fungi alleviate the heavy metal toxicity on sunflower (helianthus annuus l.) plants cultivated on a heavily contaminated field soil at a weee-recycling site. science of the total environment 628:282-290. 74 • fine focus, vol 5 2019 letter to the editor: genetic editing of secretory pathway of penicillium chrysogenum after observation of increased secretory rates in an increased stress environment (microgravity), a research proposal by high school students in dubai akio shirali 1 ,* , wilson minter huijsmans 2 ,* and matteo sottocornola 3 ,* 1 the cranleigh school, abu dhabi, united arab emirates * authors have contributed equally to this work copyright 2018, fine focus all rights reserved manuscript received 12 december 2017 this piece was received as an unsolicited contribution to xx at asm from a group of high school students in dubai uae. the form is that of a generally written research proposal. we opted to take it and publish it as a letter to the editor here, in order to serve as an example of the type of research-based assignments in stem curricula internationally. please direct all queries or questions to ms. sarah groves, teacher using email provided. 163 in this study, we aim to amplify the secretory pathway of penicillium chrysogenum within the iss or similar simulated microgravity using the minipcr and/or rtq-pcr and then optimizing penicillium chrysogenum function using crispr cas-9 (clustered regularly interspaced short palindromic repeats), a new technology in the genetics which can help in gene alteration for better drug production. the secretory pathway of penicillium chrysogenum is controlled by genes pcbab , pcbc and pende abstract corresponding authors akio shirali student, cranleigh ad, abu dhabi, uae email: shiraliakio@gmail.com ms. sarah groves science teacher, the cranleigh ad, po box 126888 abu dhabi, uae email: sgroves@cranleigh.ae wilson minter huijsmans student, cranleigh ad, abu dhabi, uae email: wilbuoy@gmail.com matteo sottocornola student, cranleigh ad, abu dhabi, uae email: m.sottocornola@icloud.com introduction 164 • fine focus, vol. 4 (2) key words penicillium chrysogenum crispr cas-9 minipcr rtq-pcr microgravity beta – lactam antibiotics pcbab pcbc pende the secretory pathway of microorganisms have shown promising results in regard to increased secretory rates when simulated by microgravity. therefore, in microgravity, this microorganisms can be used to obtain higher amount of microorganisms’ secon`dary metabolites. beta lactam bacteria are of great biotechnological interest due to their ability to secrete bioactive antibiotica as secondary metabolites. secondary metabolites are organic compounds produced through the modification of primary metabolite synthesis. secondary metabolites do not play a role in growth, development, and reproduction like primary metabolites do, because they are typically formed during the end of log phase or near the stationary phase of growth. the secretory pathway of penicillium chrysogenum is controlled by genes pcbab, pcbc and pende. these genes control the production of penem, a type of beta lactam antibiotics. space flight experiments have been suggested to affect cellular processes in microorganisms. for instance, preliminary reports on the effects of spaceflight on secondary letter to the editor • 165 metabolism. in our study, we plan to fill the gap between same quality higher yields versus same quality lower yields of secondary metabolites during space flights. furthermore, penicillium griseofulvum is found to have a penicillin gene cluster similar to that of penicillium chrysogenum. no other species among the studied fungi were found to produce penicillin or to possess the penicillin biosynthetic genes, except p. verrucosum, which contains the pcbab gene but lacks pcbc and pende. hence penicillium chrysogenum is our fungi of choice. the process of regulation of penicillin biosynthesis has been studied for many years. specifically, the improvement of p. chrysogenum strains to obtain higher penicillin yields is a main intense objective in industrial research. to simulate the microgravity environment on earth, several models have been developed and applied to examine the effect of microgravity on secondary metabolism. the purpose of our study is to use penicillium chrysogenum for production of higher penicillin yields in microgravity environment. methods we divided the method of the experiments into two parts: 1freeze and fly: a simple experiment where the penicillium chrysogenum is monitored in a low gravity environment on earth. this acts as a proof of concept experiment. 2further plan: use of data obtained by the freeze and fly experiment to edit the microbe gene structure using crispr technology in order to produce a permanent artificially induced stress condition to increase penicillin production rate by penicillium chrysogenum. 1freeze and fly experiment: the “freeze and fly experiment” will be a proof of concept and a chance to gather information on how the expression rate of genes is different to the ones on plain gravity. genes for the biosynthesis of secondary metabolites are arranged in clusters together with genes for resistance to the toxic action of secondary metabolites on the producer organisms. likewise, mrna templates of the penicillin biosynthesis cluster of p. chrysogenum pcbab, pcbc , and pende will be used on the minipcr. these templates will be prepared and purified on earth. the frozen mrna templates will be sent on the iss to run the minipcr. the freeze and fly will be used with the three genes (pcbab , pcbc and pende) and their primers (from ref 15): pcbab: fwd: gaa gac gtc ata ctt att ctc tg rev: cgg cat cgg ata aag aga tct gg pcbc: fwd: gat tgg cgc tcc tcg ttc acc rev: cca tta ttt ttc tag tcg aca tgg cat cga ttc cca agg cca atg tcc cc pende: fwd: ccc gca gca cat atg ctt cac atc ctc tgt caa ggc rev: atg aca aac atc tca tca ggg how can this help the world? our idea is there to fill any of two voids: 1. the idea can be used to increase the production of stock which then can help 166 • fine focus, vol. 4 (2) sample 1 control: no polymerase sample 5 primers pair: pcbc sample 2 control: no template sample 6 primers pair: pcbc sample 3 primers pair: pcbab sample 7 primers pair: pende table 1: pcr experiment will be run on the genes pcbab, pcbc and pende from penicillium chrysogenum the experiment will be done in a minipcr in duplicate, with 8 sample reactions as following: while this is being done in microgravity there would be another batch from the same strain of penicillium chrysogenum would be monitored on normal gravitational conditions. decrease the problem of a low inventory. this will make sure that in a financial sense, the constant fear of not being able to accommodate the high demand with a low supply can be cancelled or at the least reduced. 2. the increase in the stock of medicine can be help full as any charity buys the medicines at either a lower cost or in a greater quantity at the same cost which will help the underprivileged to help procure a medicinal cure to ailments that they may suffer from. we hope that our work can have a massive impact on the lives of others, as the blessing and happiness of a person is far greater than any sort monetary gain that anyone or we could gain. considering the method that we have tried to introduce here we believe that this will have many implications on the pharmaceutical business as there are many types of medicines procurable from bacterial secondary metabolites. here are some examples: 1. cancersthe chemotherapy drugs come from secondary metabolites of plant. 2. diabetes type 1 humulin, the most successful medicine to treat type 1 diabetes is the recombinant insulin produced in bacteria escherichia coli and yeast. 2further plan experiment: the expression rates of the three genes on earth would be compared to their expression rates in microgravity. this would be done using a reverse transcription method form their rna to create a cdna, a technique known as rt pcr. when this is done, we plan to create a draft plasmid containing an edited version of the dna to account for the changes that have been made in the expression rates in microgravity. this plasmid would then be implanted into a cell which can express the plasmid; like f+ cell. once f+ cell is placed with a group of f cells, the dna as a plasmid can move from the f+ cell to the fcell via the help of the protein relaxosome and relaxase. this is the process of conjugation. once complete the f+ cell could be placed in a colony with massive amounts of fcells. this would then allow the fcells to procure the same traits of the f+ cell, which is an increased secretory rate. letter to the editor • 167 graph 1: the bacterial growth curve 1 4 this is to ensure that the bacterium does not create offspring that are resistant to the stress because they die at the end of the graph due to lack of nutrients and food. why the stationary phase is important for secondary metabolites production? first, we would like to reemphasize the objective of the experiment “genetic editing of secretory pathway of penicillium chrysogenum , after observation of secretory rates in increased stress environment”. this idea is based on multiple facts: 1. stress that is induced on micro bacteria increases the rate of secondary metabolite production 1 . 2. genetic editing is now quite widely used. 3. other studies done on bacterium with similar genomic structures have shown promising results 2 , 4 . to understand the process of secondary metabolite production, it is important to take into account that secondary metabolite production is only temporary as a results of adaptation to stress by bacteria and fungi. this best to compare to puts of charles darwin, one of history’s best biologist, “it is not the strongest that survives, but the species that survives is the one that is able to adapt to and to adjust best to the changing environment in which it finds itself”. to ensure that the fungi cannot adapt to the stress we propose that the edited stress is added to the micro bacteria in their stationary phase ( see graph 1 ). discussion how can this help the world?? our idea is there to fill any of two voids: 1 the idea can be used to increase the production of stock which then can help decrease the problem of a low inventory. this will make sure that in a financial sense, the constant fear of not being able to accommodate the high demand with a low supply can be cancelled or at the least reduced. 168 • fine focus, vol. 4 (2) acknowledgements • dr. wael mohammed osman (khalifa university, abu dhabi, uae) • dr. claudia schafer (mit, boston, usa) • mr. matthew macnaught (the cranleigh school, abu dhabi, uae) • ms. sarah groves (the cranleigh school, abu dhabi, uae) • mr. damien ward (the cranleigh school, abu dhabi, uae) • minipcr (amplyus 1770 massachusetts avenue cambridge ma 02140 usa) about the authors akio shirali hello, my name is akio shirali, i am a 13 year old student at cranleigh abu dhabi. i have always been interested in the sciences and have always excelled in mathematics. one of my main motivations is my grandmother who was a microbiologist. my area of specialty is biology. wilson minter huijsmans my name is wilson minter huijsmans and i am 15 years old. i have always excelled at mathematics and the sciences, specifically physics. i have won/ achieved multiple things in all these areas, with the most recent being a finalist prize in the genes in space uae competition, the motivation for this paper, alongside my science obsessed teammates, the co-authors of this paper. 2 the increase in the stock of medicine can be help full as any charity buys the medicines at either a lower cost or in a greater quantity at the same cost which will help the underprivileged to help procure a medicinal cure to ailments that they may suffer from . we hope that our work can have a massive impact on the lives of others, as the blessing and happiness of a person is far greater than any sort monetary gain that anyone or we could gain. considering the method that we have tried to introduce here we believe that this will have many implications on the pharmaceutical business as there are many types of medicines procurable from bacterial secondary metabolites. here are some examples: 1cancersthe chemotherapy drugs come from secondary metabolites of plant. 2diabetes type 1 humulin, the most successful medicine to treat type 1 diabetes is the recombinant insulin produced in bacteria escherichia coli and yeast. letter to the editor • 169 references 1. ana m. calvo, richard a. wilson, jin woo bok, and nancy p. keller. 2002. relationship between secondary metabolism and fungal development. microbiol mol biol rev; 66(3): 447–459. 2. hong gao, zhiheng liu, lixin zhang. secondary metabolism in simulated microgravity and spaceflight. protein cell 2011. 2(11): 858–861 doi 10.1007/s13238011-1125-z 3. microbiology book series number 2: current research, technology and education topics in applied microbiology and microbial biotechnology (volume 1), publisher: formatex research center, badajoz, editors: a. méndez-villas, pp.577-588. 4. nitin kumar singh, adriana blachowicz, aleksandra checinska, clay wang, kasthuri venkateswaran. draft genome sequences of two aspergillus fumigatus strains, isolated from the international space station. genome announc . 2016 jul-aug; 4(4): e00553-16.published online 2016 jul 14. doi: 10.1128/ genomea.00553-16 5. efimov vp, morris nr. the lis1-related nudf protein of aspergillus nidulans interacts with the coiled-coil domain of the nude/ro11 protein. j cell biol . 2000 aug 7; 150(3): 681–688. 6. marco a van den berg, richard albang, kaj albermann, jonathan h badger et al. 2008. genome sequencing and analysis of the filamentous fungus penicillium chrysogenum. nature biotechnology 26:1161-1168. 7. eric fourest, catherine canal and jean-claude roux. 1994. improvement of heavy metal biosorption by dead biomasses and penicillium ph control and mycelial (rhizopus arrhizus, mucor miehei chrysogenum). fems microbiol. rev. 14(4):325-332. 8. david j. smith, martin k. r. burnham, john h. bull et al. b-lactam antibiotic biosynthetic genes have been conserved in clusters in prokaryotes and eukaryotes. 9. . henry f. chambers, delphine moreau, david yajko et al. 1990. can penicillins and other -lactam antibiotics be used to treat tuberculosis? embo j. 9(3):741-747. matteo sottocornola hello, my name is matteo sottocornola. i’m italian and french and speak fluently three languages. i’m currently studying for my a levels as i am a 16 years old. my main interests lie in the fields of physics and mathematics although i also take interest in subjects such as biology and chemistry. • fine focus, vol. 3 (2) barbara stedman, ph.d. director of national and international scholarships ball state university how to write a good recommendation letter recommendation letter • 187 186 • fine focus, vol. 3 (2) in my work as the director of national and international scholarships at ball state university, i see dozens of letters of recommendation each year, the vast majority of them written by faculty. over the past decade i’ve learned that some faculty members may view writing a letter of recommendation as a burden, as an interruption to their own research agendas or other activities they consider more important than supporting a student’s application. these recommenders may dash off a letter in 15 minutes, using the same trusty boilerplate that they’ve used for a hundred other students. thankfully, though, i’ve learned that most of our faculty view letter-writing as a privilege, as an important way to help advance a student’s academic and professional careers. these letterwriters easily spend an hour or more crafting and editing a letter, and these are typically the instructors who are asked again and again to provide letters for students’ applications for scholarships, internships, and graduate school. from the students’ perspective, asking for letters of recommendation may seem like a crapshoot: letters are the one component of an application that isn’t under their control, and it typically remains unseen, the content unknown. when the competition for a national scholarship is especially fierce, however, a strong letter can be the factor that ultimately determines a student’s chances of success. in the following pages i offer some general advice for writing strong, persuasive letters of recommendation for scholarship applications in general, along with some specific advice for stem-based scholarship programs, particularly the barry m. goldwater scholarship and nsf graduate research fellowship (nsf grf)1, two of the most important and visible scholarships available to stem students in the u.s. 1) tailor each letter according to the specific criteria of a given scholarship. resist the urge to rely on your go-to boilerplate when writing a letter for a major scholarship competition, and don’t simply change the name of the scholarship when recommending the same student for more than one competition. the applicant should provide you with a list of the criteria for a given scholarship competition; if that hasn’t happened, ask the student for the criteria or go to the scholarship program’s website, where you may find overt guidelines and advice for letter-writers. for stem-based scholarships, research experience and future potential in research are often the most important criteria. goldwater scholarship applicants, for example, are explicitly advised to solicit letter-writers who can discuss their “ability, interest, and experience in conducting research.” recommenders should therefore focus much of their attention on past research experiences, whether in the lab or the field, and should also comment on an applicant’s relevant personal traits: “the motivation, creative thought, stamina, and ability to collaborate that are characteristics of those successfully practicing” in stem research. for the nsf graduate research fellowship, research experience and potential – including the feasibility and significance of the applicant’s graduate research plan statement (one of two essays submitted with the application) – should again be focal points in a letter of recommendation. nsf grf letter-writers are also asked to “include comments on the applicant’s potential for contributing to a globally-engaged united states science and engineering workforce” and to take special care to address the nsf merit review criteria – i.e., the intellectual merit and broader impacts criteria. in addition to providing information about the scholarship criteria, applicants should have provided you with reminders of their past accomplishments and perhaps even a request to focus on a particular experience (e.g., “i’d appreciate it if you could discuss my leadership of the human genome project”). they may even want you to discuss an experience or quality that they haven’t had opportunity to elaborate on in their part of the application. and while it’s always a good idea for recommenders to read essays that applicants have written for a scholarship application, doing so is especially important for the nsf grf, as noted above. 2) don’t simply summarize the applicant’s activities and accomplishments; interpret and evaluate them. many letter-writers believe that eloquently paraphrasing a student’s résumé is useful and persuasive. it isn’t. students already will have provided the content of their résumés in the application, so restating that same information in your letter provides no new information for the reviewers. instead, bring your knowledge and experience to bear upon your evaluation of this student, to help the readers understand what the applicant’s activities and accomplishments mean, perhaps in the context of other outstanding students. how does this student stand out from the crowd of other brilliant, visionary, accomplished students you’ve known and worked with? sometimes numbers are useful in making that case. for example, stating that a student is in the top 1% of all undergraduates you’ve taught over a 30-year teaching career conveys a powerful message. or favorably comparing the applicant to a previous student who went on to an illustrious research career can be useful, too. joe schall, author of writing letters of recommendation, states that good letter-writers “invite us to imagine ourselves in the presence of the student” and help us to “know the candidate well, in fact to admire the student.” in creating this “invitation,” consider what kind of impressions the student has made on other faculty in your department or on other students; you may want to quote one or two of them. even while inviting readers to admire the student, however, avoid grandiose hyperbole. some scholarship review committees don’t even like to read the word “perfect” in a letter of recommendation (i.e., “jane smith is a perfect candidate for the ___ scholarship”). readers don’t expect any candidate to be perfect and without flaws – which raises the question of addressing failures and weaknesses in a letter of recommendation. should you include them? if the student has significant weaknesses and unaddressed failures, you may first need to reconsider whether you feel comfortable providing a letter for him or her. in general, however, you should discuss failures if they have led to positive growth or if they help explain a gap or weakness in a student’s record. the goldwater foundation even offers this specific advice to recommenders: “if there are any special circumstances or challenges the student has had to face that have negatively affected the student’s performance or that the student has faced and has successfully addressed and performed well in spite of these challenges, please discuss these in your letter.” i would simply add the caveat that if such challenges are personal in nature, ask the student if you may mention them in your letter. 3) support your points with specific, illustrative details and one or more 1. the barry m. goldwater scholarship is aimed at identifying and supporting sophomores and juniors who will become the next generation of our nation’s top stem researchers. the nsf graduate research fellowship provides three years of generous support to outstanding graduate students (including those who will enter their first year of graduate school) in a wide range of the sciences, including social sciences. both programs are federally sponsored and funded. recommendation letter • 189188 • fine focus, vol. 3 (2) personal anecdotes. don’t leave your readers asking “how?” or “in what way?” if you say a student has tremendous initiative, describe an incident in which he or she demonstrated that initiative in a meaningful way. flesh out evaluative statements – “jane uses both her senses and her intuition well in the lab” – by offering an anecdote in which you saw jane’s senses and intuition at work. help the readers experience that scene through engaging description; convey the surprise and pleasure you experienced when you first saw her talents in action. you should also show readers that you know this student as more than simply a good test-taker or lab worker. identify noteworthy character traits, adopt a warm and personal tone in your writing – there’s no need to refer to the student as “miss” or “mr.” – and be sure to employ memorable details. after the scholarship review committee members have finished reading several dozen application packets, what will they remember from your letter? the more that your voice comes through, the more compelling your letter will be. 4) consider the structure, length, and other formatting details. unless you are instructed otherwise, i recommend the following general guidelines. a) greeting: address the foundation or committee, rather than “dear sirs,” or “to whom it may concern.” b) opening paragraph: • state what you’re recommending student for. • indicate the length of time and circumstances in which you’ve known the student. • if needed, provide a brief statement of your qualifications for evaluating the applicant, but don’t make the letter about you; the review committee will not care how accomplished you are. c) readability: • don’t go smaller – or larger – than 11 or 12 point times new roman, 10 point arial, 11 point calibri, or a comparable font and size. • single-space your letter, with adequate paragraph breaks. • don’t use visual gimmicks. it’s fine to boldface or italicize a few key words, but don’t overuse either formatting tool, and don’t use “splashy” fonts, in an attempt to draw attention. i once had a faculty member put a student’s full name in 14-pt. broadway font throughout his letter, thinking that he would help readers remember the student’s name. yes, the reviewers might have remembered the student’s name, but not for the right reasons! d) length: for most scholarships, aim for 1½ to 2 pages or about 1,000 words. a one-page letter may suggest you have little to say, and three pages may try the patience of committee members who are reading and evaluating dozens of applications. be sure to read the instructions for each specific scholarship program, though, as you may be given a character, word, or page limit. recommenders for the nsf grf are limited to two pages, and those for goldwater scholarship are allowed up to three pages. other formatting guidelines are also specified by each program, too, including font options and the width of margins. e) submission protocol: because the vast majority of scholarship applications are now online, you’ll likely be required to copy and paste your letter into a text box or to upload it as a word or pdf file. when copying and pasting into a text box, you don’t need to worry about letterhead or signature. when uploading a file, though, you’ll usually need to have both letterhead and your signature. for uploaded letters, you may be given the option of submitting a word document, but i recommend going with a pdf instead. if you want to print out your letter and then scan it as a pdf, be sure to set your scanner resolution to 300 dpi or higher; 200 dpi will make the text grainy and potentially annoying to your readers. if you have your letterhead and signature saved as a .gif or .jpg (or other image file), however, you may find it easier to simply insert the letterhead and signature into the word version of your letter and then “save as” a pdf. f) proofread: spell the student’s name and the scholarship’s name correctly. i’ve seen students called the wrong name in letters, have their names spelled two different ways, identified by the wrong pronoun, and recommended for the wrong scholarship! for online submission of letters, too, be sure to preview the letter before hitting “submit,” to check for formatting problems created by copying and pasting from word into a text box. 5) try new strategies for improving your letters of recommendation. if you’re not happy with the letters you’ve been writing, or if you’ve often found yourself at a loss to write a substantive letter for some applicants, these strategies might help: a) meet with the student in person, to discuss the scholarship and the student’s accomplishments and future plans. even if the student sends you detailed written information, a 20-minute conversation may help you come up with convincing anecdotes or help clarify what you want to focus on in your letter. b) think of a letter of recommendation as a rhetorical challenge: you’re building an argument about a student’s intelligence, insight, ambition, and potential to have a significant impact on his or her field of study, profession, and perhaps even society. you’re also building a persuasive argument that this student is a good fit for this scholarship. what do you need to tell the readers to convince them of your broad statements about the student? c) if you simply feel “stuck” and can’t figure out how to write more than half a page about a student, try this strategy: identity three of the student’s qualities that make him or her especially well qualified for the scholarship or otherwise stand out from the crowd. write a paragraph or two about each quality, providing supporting evidence and at least one anecdote to illustrate each point. while the strategy itself may sound formulaic, the results are usually distinctive and convincing, allowing the readers to get a clear picture of that particular applicant. 6) be ethical. never ask a student to draft his or her own letter of recommendation. but do ask the student to give you written information about the scholarship, plus reminders about past papers and projects and other relevant accomplishments that you’re especially well-qualified to discuss. if you can’t write the kind of letter that will help a student be a strong contender for a scholarship – even after trying some of the strategies provided here – please say so from the start. writing an unusually short or unenthusiastic letter only hurts the student. even though your letter is almost always considered confidential by scholarship foundations (you’d be alerted to any rare cases in which that isn’t the case), and certainly letters for the goldwater scholarship and nsf grf are kept confidential , you’re welcome to share your letter with an applicant if you like. in fact, you may want to fact-check the content of your letter or simply let an applicant know how highly he or she is regarded. however, you should never comply with a student who asks you to hand over your letter without first confirming the scholarship application’s protocol. writing a strong letter of recommendation takes time and careful thought, but for scholarship applicants your letter can be the deciding factor for a review committee debating between one or more equally qualified candidates. and even for those students who may not win a scholarship, your letter will likely provide a useful foundation for future applications, whether for other scholarships, internships, or graduate school. the investment of time and energy is worth it, so throw out that boilerplate when recommendations really matter. students will be grateful, and you may help provide them with an opportunity that will open door after door in their academic careers and professional futures in science. recommendation letter • 191 190 aspergillus sclerotiorum fungus is lethal to both western drywood (incisitermes minor) and western subterranean (reticulitermes hesperus) termites. gregory m. hansen, tyler s. laird, erica woertz, daniel ojala, daralynn glanzer, kelly ring, and sarah m. richart* department of biology and chemistry, azusa pacific university, azusa ca copyright 2016, fine focus all rights reserved manuscript received 21 october, 2015; accepted 16 january, 2016 termite control costs $1.5 billion per year in the united states alone, and methods for termite control usually consist of chemical pesticides. however, these methods have their drawbacks, which include the development of resistance, environmental pollution, and toxicity to other organisms. biological termite control, which employs the use of living organisms to combat pests, offers an alternative to chemical pesticides. this study highlights the discovery of a fungus, termed “apu strain,” that was hypothesized to be pathogenic to termites. phylogenetic and morphological analysis showed that the fungus is a strain of aspergillus sclerotiorum, and experiments showed that both western drywood (incisitermes minor) and western subterranean (reticulitermes hesperus) termites die in a dose-dependent manner exposed to fungal spores of a. sclerotiorum apu strain. in addition, exposure to the a. sclerotiorum huber strain elicited death in a similar manner as the apu strain. the mechanism by which the fungus caused termite death is still unknown and warrants further investigation. while these results support that a. sclerotiorum is a termite pathogen, further studies are needed to determine whether the fungal species has potential as a biological control agent. abstract corresponding author *sarah m. richart srichart@apu.edu keywords • entomopathogenic • phylogenetics • biological control • pest management • aspergillus sclerotiorum • reticulitermes hesperus • incisitermes minor • termites termites are a common structural and economic pest worldwide. while estimates vary considerably, termites are responsible for between $500 million and $1 billion of wood damage and cost $1.5 billion in control measures per year in the united states alone (7,32). an array of problems have been associated with the use of chemical pesticides to control pests like termites, such as the development of resistance in target organisms, environmental pollution, and toxicity to non-target organisms, including humans (25). avoidance behavior to such chemicals is also a significant challenge in controlling termite populations (33). because of such complications, biological termite control agents that are natural insect pathogens have been proposed, including parasitoids, viruses, bacteria, protozoa, nematodes, and fungi (7). fungi, especially those that occur naturally in termite habitats, are promising candidates for use as biological control agents because they are well-adapted to survive in such environments (33). entomopathogenic fungal species such as beauveria bassiana, introduction 24 • fine focus, vol. 2 (1) termite collection incisitermes minor and reticulitermes hesperus termites were obtained either from decomposing firewood in arcadia, ca, or from branches found in the foothills of the san gabriel mountains in glendora, ca. termite-containing wood was stored in plastic bins in a cool, dark room until used in experiments. to collect termites, wood was broken apart and termites were picked up with lightweight forceps or paintbrushes, and placed in petri dishes with a brown paper towel as food, with additional moisture for r. hesperus. termites were left two or more days in petri dishes after collection and before the start of an experiment to ensure that healthy, uninjured insects were used. fungal cultures, dry spore collection, and quantification. a strain of fungus believed to have eliminated an i. minor colony at azusa pacific university in 2009 (called “apu strain”) was isolated from a deceased termite and cultured on difco sabouraud dextrose agar (sda) (bd diagnostics, franklin lakes, nj). fungus was repeatedly subcultured on sda plates to ensure a pure culture. aspergillus sclerotiorum strain huber (16892, american type culture collection, rockville, md) was used as a comparison strain. for spores that were used to infect termites, cultures of both strains were grown in the dark on sda plates with penicillin-streptomycin (100 u/ ml penicillin and 100 mg/ml streptomycin, diluted from 100x stock solution, hyclone laboratories, logan, ut) at 25°c for approximately 3 weeks, or until a confluent layer of yellow spores was present. each plate was then either used at 3 weeks or later to infect termites directly. to collect spores, confluent plates were inverted and placed atop a 0.45 mm sterile vacuum filter, then gently and repeatedly tapped to dislodge spores. spores were then left in the filter apparatus under vacuum to dry for approximately 3 hours. the resulting dry spore powder was quantified by resuspending in a solution of 0.1% (v/v) tween-20 and counting in a hemocytometer. live spore quantification was determined by standard plate count on sda in duplicate or triplicate for each dilution, and fungal colonies were counted on days 2-4, depending on the growth. materials and methods paecilomyces fumosoroseus, and metarhizium anisopliae have been suggested as potential termite control agents, with the latter being the best studied and most effective of the three (17,19,28). there are limitations, however, to the effectiveness of fungi as termite control agents in their native habitats, due to specific termite behaviors such as allogrooming (6), spore avoidance (22), and vibrational alarm signals in response to fungal spores (22,29). in addition, naturally occurring antimicrobial compounds in termite feces and alimentary canals have been described (6, 23). in this current study, we hypothesize that a novel isolated fungus is entomopathogenic to termites. the research objectives are to genetically and morphologically identify the apu strain, experimentally infect two different species of termites, incisitermes minor and reticulitermes hesperus, with apu strain, and determine if dose has any effect on termite survival. pathogens and antimicrobial factors • 25 its region and β-tubulin gene polymerase chain reaction (pcr) amplification the species identification of the strain of fungus that was presumed to kill a colony of i. minor termites in 2009 (called “apu strain”) was initially performed by pcr amplification of the internal transcribed spacer region (its) of fungal ribosomal dna isolated using zr fungal/bacterial dna miniprep kit (zymo, irvine, ca) according to the manufacturer’s protocol, with the following additional step: prior to dna extraction, fungal samples suspended in water were frozen at -20°c. the primer pair made for amplification of the fungal its region was its1 and its4 (sequences as previously described) which amplifies a region that includes the entire 5.8s rrna gene (31). pcr was also performed using primers (bt2a and bt2b), designed to amplify aspergillus β-tubulin, as previously described (10). pcr products were purified using the wizard sv gel and pcr clean-up kit (promega, madison, wi), and sequenced at the dna sequencing core lab (city of hope, duarte, ca). phylogenetic analysis of its and β-tubulin gene regions of apu strain the apu strain its sequence was subjected to a megablast search using the fungi refseq its database in blast (national center for biotechnology information). the top eight sequences ranked by maximum score were used. aspergillus tanneri, the 8th sequence, was used as an outgroup, similar to a previous phylogenetic study (27). for its, sequences were aligned along with the apu strain sequence using clustalw in mega6 software. default settings were used for the alignment. under the pairwise alignment and multiple alignment sections, the gap opening penalties were set to 15, and the gap extension penalties were set to 6.66. under the multiple alignment section, the gap opening penalty was set to 15 and the gap extension penalty to 6.66. the dna weight matrix was set to iub. the transition weight was 0.5. the use of a negative matrix was turned off. the delay of divergent cutoff was set to 30%. after alignment, the sequences were trimmed to 562bp, and used to construct maximum likelihood phylogenetic trees using mega6. the tamura 3-parameter model was selected, as well as gamma distributed with invariant sites (g + i), as done previously (27). the number of discrete gamma categories was 5. gaps and missing data were subject to complete deletion. the ml heuristic method was set to nearest-neighbor-interchange, and the initial tree was set to default nj/ bionj. the branch swap filter was set to very strong. the number of threads selected was 1. bootstrap analysis of 1000 replicates was performed. the sequence of a region of the β-tubulin gene, a commonly sequenced gene in fungi, was also subjected to a megablast using blast. the first 14 strains of unique species similar to apu strain were chosen for phylogenetic tree construction. the aspergillus tanneri β-tubulin sequence was acquired from ncbi as an outgroup, as done previously (27). a total of 15 sequences of β-tubulin regions were acquired and aligned along with the apu strain sequence in mega6 using the same parameters as mentioned above for the its region. after alignment, the sequences were trimmed to 579bp. a maximum likelihood phylogenetic tree was constructed in mega6. all selected options were the same as done for the its region as described earlier except that the kimura 2-parameter was used as done 26 • fine focus, vol. 2 (1) previously (27). in addition, the first, second, third, and noncoding sites were selected. bootstrap analysis was performed with 1000 replicates. methods for morphological studies and microscopy for light microscopy analysis, fungal microcultures were set up by inoculating each of the 4 edges of a 1 cm x 1 cm square of sabouraud dextrose agar (sda) on top of a microscope slide, and covering the inoculated medium with a sterile glass microscope cover slip. the slide culture was set on a small platform in a petri dish, and extra water was put in the bottom of the dish below the level of the slide to allow proper moisture. fungi were grown in the dark at 25°c for 3-4 days. to view, cover slips were carefully removed and placed on top of clean microscope slides with lactophenol cotton blue stain (medical chemical corp., torrance, ca) and initial photographs were taken using a leica dm70 microscope. for measuring apu strain features, fungus was inoculated onto a sda square but grown directly on a micrometer, and viewed using a nikon tms microscope. to view macroscopic fungal colony growth, fungi were inoculated onto czapek yeast agar (cya) and malt extract agar (mea) plates and grown in the dark at 25°c for 7 days, as previously described (14). an electron micrograph was taken by directly putting a sample on a conductive double-sided adhesive (pelco image tab™, ted pella, inc, redding, ca) and viewed using a hitachi tm-1000 tabletop scanning electron microscope. i. minor termite infection with apu strain or huber strain an initial study was conducted to assess the lethality of apu strain to i. minor. thirty i. minor termites of differing developmental stages were individually inoculated on the back by inoculation loop with apu strain from an overgrown sda culture plate. thirty control termites were untreated. each group was placed in a petri dish with 90 mm whatman no. 3 paper for food. live termites were counted each day for 7 days. to compare the effect of apu strain to a. sclerotiorum huber strain on i. minor termites, 40 termites per group were placed on a sda plate overgrown with either the apu strain or huber strain and allowed to walk around for 2 minutes, during which they became covered in fungal spores. forty control termites were removed from the same colony as the experimental group and were left untreated. each group of 40 was then placed on a piece of 90 mm whatman no. 3 paper in a petri dish, and live termites were counted daily except on day 5 for the duration of the experiment. dose response of i. minor termites to apu strain the response of i. minor termites to increasing doses of a. sclerotiorum apu strain was determined for 4 different doses (150 mg, 70 mg, 30 mg, and 3 mg) by inoculating n=3 groups containing 25 termites each of differing developmental stages with each dose. the 3 mg doses were prepared by adding 9 parts powdered sugar to 1 part fungal spores, and using 30 mg of the sugar/fungal mixture per petri dish. each dose of fungal spores were uniformly spread on a 90 mm disk of whatman no. 3 filter paper in a petri dish, pathogens and antimicrobial factors • 27 fungus isolate exhibits i. minor termite pathogenicity initially, i. minor termites were exposed to apu strain spores and the results of this experiment showed that the number of live termites inoculated with the apu strain declined throughout one week of observation, beginning with the first inoculated termite death on day 3 (fig. 1a). by day 7, all 30 of the inoculated termites were dead. by contrast, all 30 uninoculated control termites were alive until day 6, and on day 7, there were still 27 live control termites. inoculated termites that died were examined using light microscopy for signs of fungal infection, such as shown in fig. 1b, and upon transfer to fungal media (sabouraud dextrose agar), grew fungus that appeared very morphologically similar (data not shown). scanning electron microscopy of dead inoculated termites indicated fungal growth, seemingly of one type that, due to its unenclosed spores, belonged to the phylum ascomycota (fig. 1c). results where termites were kept throughout the experiment and thus exposed to spores for the duration of the experiment. the 0 mg control groups (n=2) were prepared in the same manner, except spread with 75 mg of powdered sugar instead of fungal spores. the termite groups were kept in the dark, and the number of live individuals was determined daily (approximately every 24 hours) over 9 days. each mg of spores corresponded to 1.07 x 108 spores by hemocytometer estimation. viable counts were not performed. a one way anova followed by a tukeyhsd test (rstudio version 0.99.441) was performed in order to determine the significance of the doses on the number of living termites on days 5 and 9 of the experiment. dose response of r. hesperus termites ten or 25 ml of a 1.5% (w/v) agar solution (bacto-agar, bd difco franklin lakes, new jersey) was first pipetted into glass petri dishes and allowed to solidify to provide enough moisture for subterranean termites and inhibit further fungal growth (33). whatman no. 3 filter paper disks (90 mm) were placed on top of the solidified agar as a food source. various quantities of dry apu strain a. sclerotiorum spores were evenly spread on top of the paper. the following quantities of spores were used: 25, 50, 75, 100 mg. each mg corresponded to 1.05 x 108 total spores as counted by hemocytometer, and 4.86 x 105 viable spores by plate count determination. for the control groups, 75 or 100 mg of powdered sugar was spread on the filter paper so as to mimic the powdery nature of the spores. termites were separated into groups of 35 termites of differing developmental stages. the groups were then introduced into dishes containing either fungal spores or powdered sugar (n=2 for 100 mg spores; n=3 for all other dosages, where data were pooled from two separate experiments). the termites lived in the dishes for the duration of the experiment, and their survival was recorded daily for 9 days. all of the dishes were stored in the dark and only exposed to light when survivability was recorded. a one way anova followed by a tukeyhsd test (rstudio version 0.99.441) was performed in order to determine the significance of the doses on the number of living termites on day 9 of the experiment. 28 • fine focus, vol. 2 (1) identification of apu strain as aspergillus sclerotiorum in order to determine if the apu strain was a known insect or termite pathogen, it was necessary to identify the species of fungus. once the its sequence of the apu strain was determined, and a blast comparison using validated reference sequences (refseq, national centers for biotechnology information) was performed, a phylogenetic tree was constructed consisting of 8 species with the most similar its sequences (fig 2a). a. sclerotiorum had the closest sequence to the apu strain its region, making it the probable identity of the apu strain. identification of apu strain was confirmed to be a. sclerotiorum by an independent lab (accugenix, newark, de) that analyzed a smaller region of its (its2). to corroborate these results, a region of the β-tubulin gene from apu strain was chosen to be sequenced as a means of species identification. similar to the its region, the sequence of the β-tubulin gene showed closest identity to a. sclerotiorum (fig. 2b). since sequence results of both its and β-tubulin regions suggested a. sclerotiorum was the apu strain species, the apu strain was compared microscopically to a known reference strain of a. sclerotiorum (huber strain). microscopic examination of apu n um be r of li ve te rm ite s day 35 30 25 20 15 10 5 0 0 1 2 3 4 5 6 7 control a. b. c. apu strain figure 1: healthy incisitermes minor termites inoculated with apu strain show decreased viability over time. a. survivability of infected termites over time. control termites were unaffected. b. light micrograph of a representative deceased i. minor termite after inoculation with apu strain with characteristic yellow spores visible. c. scanning electron micrograph of fungal fruiting body found on deceased termite (800x magnification). pathogens and antimicrobial factors • 29 figure 2. phylogenetic trees based on its sequences and β-tubulin gene sequences of apu strain indicate identity is aspergillus sclerotiorum. the phylogenetic tree for a. its and for b. β-tubulin genetic regions of apu strain were each constructed using the maximum likelihood method in mega6. bootstrapping percentages are shown near the branch they refer to. a strain of aspergillus tanneri was used as an outgroup for each tree. a. its b. β-tublin aspergillus elegans cbs 102.14 aspergillus steynii cbs 112812 aspergillus affinis atcc mya-4773 apu strain aspergillus sclerotiorum nrrl 415 aspergillus ostianus atcc 16887 aspergillus melleus cbs 546.65 aspergillus ochraceus nrrl 398 aspergillus tanneri atcc mya-4905 80 0.005 99 94 39 55 100 aspergillus steynii partial culture collection cbs:112813 aspergillus elegans partial culture collection imi:345568 aspergillus westerdijkiae strain soussec5 aspergillus melleus partial culture collection cbs:121987 aspergillus ochraceus strain gigg2471 aspergillus ostianus partial culture collection cect:2917 aspergillus subramanianii strain dto 245e4 aspergillus sulphureus strain cbs 55065 apu strain aspergillus sclerotiorum strain cbs 54965 aspergillus persii strain gr131 aspergillus bridgeri isolate nrrl 35081 aspergillus roseoglobulosus isolate nrrl 4565 aspergillus neobridgeri isolate nrrl 13078 aspergillus tanneri isolate nrrl 62426 99 62 99 89 95 98 99 46 52 66 64 0.05 30 • fine focus, vol. 2 (1) and huber strains showed very similar morphological features, such as the shape of the conidial head structures, which displayed similar overall spherical shapes with spherically shaped vesicles, as shown in fig. 3a. additionally, the vesicles of both fungi have biseriate appendages (made up of metulae and phialides) leading to the spherical conidia (spores). various other fungal structures of the apu strain were observed or measured by light microscopy and compared to those of a. sclerotiorum from klitch’s key to identifying aspergillus species (14). all structures observed or measured on the apu strain were found to fall within the range of structures found in a. sclerotiorum (table 1), although they were not unique to a. sclerotiorum. apu and huber strains of fungus were also grown on media to compare colony morphologies. on both czapek yeast agar (cya) and malt extract agar (mea), colonies of apu and huber strains looked nearly identical in size, indicating a similar growth rate, as well as colony shape and texture (fig. 3b). both species displayed white mycelia and had liquid colony exudate. the major difference in colony appearance between the two fungal strains figure 3. microscopic and macroscopic comparison of apu strain and a. sclerotiorum huber strain show similarities. a. microscopic examination of fruiting body structures using white light microscopy. b. fungal colony growth at day 7 on either czapek yeast agar (cya) or malt extract agar (mea) medium, as indicated. a. b. apu strain cya mea aspergillus sclerotiorum huber strain table 1. apu strain morphological features compared to those of aspergillus sclerotiorum aall values for a. sclerotiorum are as previously described (14) fungus metulae phialides vesicle spores shape diameter texture diameter a. sclerotioruma 7-12 µm 6-8 µm pyriform/ spherical 17-35 µm smooth/finely roughened 2.5-3.0 µm apu strain 8 µm 8 µm spherical 23 µm smooth 3.0 µm pathogens and antimicrobial factors • 31 % li ve te rm ite s day 120 100 80 60 40 20 0 0 1 2 3 4 6 7 8 9 atcc strain apu strain figure 4. comparison of survival of incisitermes minor drywood termites over time infected with two different strains of aspergillus sclerotiorum. i. minor termites were exposed to apu strain (“apu strain,” black bars) or huber strain (“atcc strain,” grey bars). control termites were unexposed. each group had 40 individuals from the same colony. data are expressed as mean percent of the uninfected control live termites on each day. figure 5. apu strain aspergillus sclerotiorum affects i. minor and r. hesperus termites in a dosedependent manner. termites were exposed to various total mg quantities of dried fungal spores as indicated. data are expressed as mean percent ± s.d of the uninfected control live termites on each day. control termites were exposed to powdered sugar. a. i. minor termites were placed into groups of 25 and exposed to each mg quantity of fungus (n=3) or 75 mg powdered sugar (n=2). each mg contained 1.07 x 108 total spores by hemacytometer count. viable counts were not performed. different letters on day 5 indicate statistically significant differences (p<0.05) with the control belonging to group a. b. r. hesperus termites were placed into groups of 35 and exposed to each mg quantity of fungus (n=3 or n=2) or 100 or 75 mg powdered sugar (n=3) . each mg contained 1.05 x 108 total spores by hemocytometer count, and 4.86 x 105 viable spores. different letters on day 9 indicate statistically significant differences (p<0.05) with the control belonging to group a. 100 80 60 40 20 0 0 1 2 3 4 5 6 7 8 9 10 3 mga 30 mg 150 mg 70 mg b 75 mg 100 mg 25 mg 50 mg 0 1 2 3 4 5 6 7 8 9 10 100 80 60 40 20 0 32 • fine focus, vol. 2 (1) discussion was the spore color: apu strain spores were bright yellow-orange in color, while the spores of huber strain were beige (visible in fig. 3b). incisitermes minor termites die after exposure to both apu and huber strains of a. sclerotiorum i. minor termites exposed to either apu or huber strains showed very similar survivability kinetics over time, and all exposed termites died by day 9 (fig. 4). initially, for the first several days after fungal exposure, there was little termite death seen. a large decrease in live termites then occurred from day 4 to day 6 in both experimental groups. both i. minor and reticulitermes hesperus termites died after exposure to apu strain in a dose-dependent fashion. since initial experiments involving i. minor termites walking on overgrown plates of apu strain (fig 1a and fig 4) or huber strain (fig 4) resulted in fewer live termites over time compared to controls, larger experiments were conducted with termites exposed to varying doses of fungal spores. the results shown in fig. 5 clearly demonstrate that the numbers of live i. minor and r. hesperus termites decreased over time as the dose of dry fungal spores they were exposed to increased, in the range of 3 to 150 mg for i. minor (fig. 5a), and 25 to 100 mg for r. hesperus (fig. 5b). for i. minor termites, all individuals per group died by day 7 when exposed to 30 mg or higher doses (fig. 5a), but on day 5, a statistical difference in the average percent of live termites (p<0.05) was seen for each dose except for 70 and 150 mg doses. statistical analysis confirmed that there was a significant effect of dose in relation to i. minor termite survival on day 5 (anova, f = 66.64, pr(>f) = 1.1e-06), as well as on day 9 (anova, f= 289.4 , pr(>f) = 1.7e-09). for r. hesperus termites infected with varying mg dose quantities of apu strain, there was a more gradual decline in the numbers of live termites over the 9 day course of the experiment, with no dose resulting in the death of all members in the groups. at day 9, however, 18% of termites treated with the highest dose of the spores survived compared to the controls. 86% of those treated with the lowest dose survived. significantly different groups are shown in fig. 5b (p<0.05), and statistical analysis confirmed that there was a significant effect of dose in relation to r. hesperus termite survival on day 9 (anova, f =36.2, pr=1.49e-05). in 2009, a colony of incisitermes minor western drywood termites housed at azusa pacific university (apu) died mysteriously, and dead termites were inspected and found to be covered in a fungus with yellow spores, which was subsequently isolated. it was possible that either the fungus was feeding off of termites that had died of other causes or it contributed to the termites’ demise. therefore, i. minor termites were initially infected with the purified apu strain, all of which died by day 7. this indicated that the fungus contributed to termite death. pathogens and antimicrobial factors • 33 to see if apu strain was a novel termite pathogen, it was identified by genetic analysis. apu strain was found to be most genetically similar to aspergillus sclerotiorum fungus based on both its internal transcribed spacer (its) region sequence within the ribosomal rna gene loci and its β-tubulin gene sequence (fig. 2). this its region has been shown to be the most accurate “dna bar coding” region of fungal genomes for species identification to date (24) and has the most numerous fungal sequence submissions to genbank (approximately 800,000). external independent corroboration of the apu strain as a. sclerotiorum was also determined by sequence examination of a smaller its region, its2 (accugenix). a second region of the apu strain genome, a portion of the β-tubulin gene, was also sequenced, since this region has been shown to be polymorphic and useful in identifying filamentous fungi within phylum ascomycota (10). the species used to construct the β-tubulin tree (fig. 2b) that were genetically similar to a. sclerotiorum were different from those used in the its region tree (fig. 2a) because the β-tubulin gene sequences were used from the entire genbank database, rather than selected its reference strain sequences in the refseq database. however, this β-tubulin phylogenetic tree shows similar species relationships to a phylogenetic tree previously published based on three dna regions: β-tubulin gene, calmodulin gene, and its region (27). these three genetic analyses all confirm that apu strain belongs to a. sclerotiorum. apu strain also had similar morphological features to a reference strain of a. sclerotiorum huber when examined microscopically (fig. 3a). more extensive observation of its microscopic morphology and measurement of several of its features (table 1) fit within published parameters for a. sclerotiorum (14), although they did not rule out many related aspergillus species that have similar features. this was helpful because it did not contradict the genetic analyses performed. the colony morphologies of apu and huber strains when grown on solid media also appeared similar (fig. 3b). the major difference in appearance between the two strains was the color of the spores, however, it is not unusual for strains of the same aspergillus species to have a range of spore pigmentation (14). taken together, these genetic and morphological approaches to species identification indicated that the identity of apu strain was a. sclerotiorum. this was especially interesting since a. sclerotiorum has not generally been studied for its entomopathogenicity. infection of western drywood termites with two different strains of a. sclerotiorum (apu and huber) resulted in decreased viability of termites over time, and both strains displayed similar kinetics (fig. 4). these similar kinetics further suggest that the two belong to the same species. this was not necessarily an expected result, as different strains of the same species of entomopathogenic fungi may show selective pathogenicity, depending on the source of their isolation (13,30). since the apu strain of a. sclerotiorum was isolated from dead termites, and the huber strain was originally isolated from a decaying apple in 1933, they could have had very different effects on drywood termites (12). interestingly, they were both lethal to termites when termites were exposed to each fungus in the same manner, which suggests an inherent pathogenicity for termites. in addition, to our knowledge, it is the first demonstration of a. sclerotiorum as an i. minor termite pathogen. 34 • fine focus, vol. 2 (1) the apu strain of a. sclerotiorum was not only lethal to western drywood termites (family kalotermitidae), but also to western subterranean termites (family rhinotermitidae). groups of termites of both species infected with a. sclerotiorum had fewer live termites than control termites over time, and this decrease in viability was dose-dependent (fig 5). in this set of experiments, each group of termites as a whole was exposed to a particular fungal dose; this was preferable to individually exposing termites because it has been shown that the drywood termite incisitermes schwarti is more susceptible to death by fungal infections when in isolation, rather than in groups of 10 or 25 (3). additionally, in these experiments, control termites were exposed to powdered sugar, an inert but particulate substance, in order to establish that the fungus was entomopathogenic and not causing or contributing to termite death in some non-specific way, for instance, by physically blocking respiratory structures. the apparent greater lethal effect that the 30 and 70 mg doses of spores had on i. minor compared with the 25 and 75 mg doses with r. hesperus may either be due to a greater susceptibility that i. minor has for the fungus, or that there were more viable spores per mg in the i. minor experiments than in the r. hesperus experiments, although the total amounts of spores per mg were very similar. this second possibility is likely, because the experiments with r. hesperus were done much later than the i. minor experiments with the same preparation of spores, and it very likely lost viability in that time, although it is not possible at this point to rule out the first possibility. low doses of fungal spores resulted in greater termite viability than higher doses, which suggests that both termite species have antifungal defenses to protect them from the lower doses of fungus, although their immune systems are largely uncharacterized. one complicating factor, however, is that the 3 mg dose of spores used to infect i. minor contained powdered sugar. this could possibly have given an immune advantage to the i. minor as has previously been reported in odontotermes formosanus (8), and may explain why this dose was not significantly different from the control uninoculated termites on either day 5 or 9. in any case, this is the first study indicating that a. sclerotiorum is pathogenic to both of these species of termite pests, which are among the five important termite pest species out of 45 total termite species in the united states (26). the mechanism for a. sclerotiorum’s infection of and entomopathogenicity to termites is unknown. spores could be ingested and germinate in the alimentary tract, as seen in termites infected with beauveria bassiana, or they could invade the termite by secreting cuticle-degrading enzymes, as seen in termites infected with metarhizium anisopliae (4,15). a. sclerotiorum may then cause disease by one or more of its known excreted metabolites, such as the insecticidal aspochracin molecules (27). one aspochracin molecule previously isolated from aspergillus ochraceus has been shown to be toxic to silkworm larvae (21). control termites in the dose-dependent experiments in our study (fig. 5) were treated with powdered sugar to try to control for the possibility that spores might be blocking termite respiratory spiracles or interfering with other aspects of their physiology as particles, and not by some fungus-specific mechanism. however, the powdered sugar is not uniformly sized compared to the spores, and so using inert particulates of the same size as the fungal spores, perhaps pollen, would make a better control. finally, it is also possible that a. sclerotiorum pathogenesis is related to the termite’s immune response to the fungus. both the route of infection and the mechanism for pathogenesis of a. sclerotiorum in termites are currently being explored. pathogens and antimicrobial factors • 35 potential use of a. sclerotiorum as a termite biological control agent the use of microbiological agents as biological controls for pests like termites includes the use of the fungi m. anisopliae and b. bassiana. any fungus introduced into an environment to control termites must be horizontally transmissible to other nest mates, since in many termite species, direct treatment of the nest may not be possible (22). whether a. sclerotiorum is transmissible from infected to uninfected termites is currently under investigation, but seems likely since it was found on many individuals from the same colony upon initial isolation. if it proves to be transmissible, then infecting individuals and introducing them into nests or other approximations of termite nests, such as termite planar arenas, would be necessary to determine the feasibility of a. sclerotiorum as a termite control agent (5,6). biological control agents must also be benign to humans and other inhabitants of the area in which they are applied. as a candidate species for termite biological control, a. sclerotiorum shows minimal pathogenicity to humans. there have been only a few documented human cases of a. sclerotiorum disease, including nail infection (onychomycosis) and ear canal infection (otomycosis) (1,9,11). however, like most fungi, it could potentially pose a more serious threat to a person with underlying immunodeficiency. less wellknown are the effects of a. sclerotiorum on other insect species that may occupy similar niches as termites. m. anisopliae, the most extensively studied entomopathogenic fungus, has been shown to be lethal to all species of termites tested which is part of what makes it such a promising control for termites (20). however, m. anisopliae is also lethal to a number of other non-related insects, including locusts and grasshoppers (18), ticks (2), and mosquitoes (25), just to name a few. whether a. sclerotiorum is less harmful to other insects than it is to termites is not well-established; to date, it has only reportedly been tested on mosquitoes. in that study, two species of mosquito larvae (culex quinquefasciatus and aedes fluviatilis) infected with low doses of a. sclerotiorum (4.5 x 105 or 1.75 x 105, respectively) displayed 0% or 18% mortality rates, respectively, by day 10 post-infection, which was lower than many other different species of aspergillus tested in the same study (16). it will be interesting to see if a. sclerotiorum is less pathogenic to other insect types, as well. if it is less pathogenic, it may prove to be a better choice of fungal species for use in termite biological control. 1. amri, m., gorcii, m., essabbah, n., belhajali, h., letscher-bru, v., zili, j., azaiez, r., & babba, h. 2010. aspergillus sclerotiorum: about a case of onychomycosis in tunisia. j. mycol. med. 20:128-132. acknowledgments this work was in part funded by a grant from the faculty research council at azusa pacific university. thanks to jürgen ziesmann for noticing this yellow fungus on his termites in the first place, and for collecting and letting us use so many drywood termites in these experiments. thanks to joshua morris for help with phylogenetic tree construction. 36 • fine focus, vol. 2 (1) 2. benjamin, m. a., zhioua, e., & ostfeld, r. s. 2002. laboratory and field evaluation of the entomopathogenic fungus metarhizium anisopliae (deuteromycetes) for controlling questing adult ixodes scapularis (acari: ixodidae). j. med. 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(1990). amplification and direct sequencing of fungal ribosomal rna genes for phylogenetics. in m. a. innis, d. h. gelfand, j. j. sninsky & t. j. white (eds.), pcr protocols: a guide to methods and applications. (pp. 315-322). san diego: academic press. 32. wright, m. s., osbrink, w. l. a., & lax, a. r. (2004). potential of entomopathogenic fungi as biological control agents against the formosan subterranean termite. in w. m. wilson (ed.), agricultural applications in green chemistry (pp. 173-188). washington, d.c.: american chemical society. 33. zoberi, m. h. 1995. metarhizium anisopliae, a fungal pathogen of reticulitermes flavipes (isoptera, rhinotermitidae). mycologia. 87:354-359. 38 • fine focus, vol. 2 (1) 76 | fine focus avery m. runnebohm1,2, melissa d. evans1,2, adam e. richardson1, samantha m. turk1, james b. olesen1, philip j. smaldino1, and eric m. rubenstein1,3 1. ball state university, department of biology, muncie, in 47306 2. these authors contributed equally to this work. 3. corresponding author author email addresses: avery m. runnebohm: averykirschbaum@yahoo.com melissa d. evans: melissa.evans96@outlook.com adam e. richardson: aerichardso3@bsu.edu samantha m. turk: smturk@bsu.edu james b. olesen: jolesen@bsu.edu philip j. smaldino: pjsmaldino@bsu.edu eric m. rubenstein (corresponding author): emrubenstein@bsu.edu manuscript received 1 october 2019; accepted 7 february 2020. loss of protein quality control gene ubr1 sensitizes saccharomyces cerevisiae to the aminoglycoside hygromycin b avery m. runnebohm, melissa d. evans, adam e. richardson, samantha m. turk, james b. olesen, philip j. smaldino, and eric m. rubenstein volume six | 77 abstract ubr1 is a conserved ubiquitin ligase involved in the degradation of aberrant proteins in eukaryotic cells. the human enzyme is found mutated in patients with johansonblizzard syndrome. we hypothesized that ubr1 is necessary for optimal cellular fitness in conditions associated with elevated abundance of aberrant and misfolded proteins. indeed, we found that loss of ubr1 in the model eukaryotic microorganism saccharomyces cerevisiae strongly sensitizes cells to hygromycin b, which reduces translational fidelity by causing ribosome a site distortion. our results are consistent with a prominent role for ubr1 in protein quality control. we speculate that disease manifestations in patients with johanson-blizzard syndrome are linked, at least in part, to defects in protein quality control caused by loss of ubr1 function. 78 | fine focus introduction the structure and function of the ubr1 ubiquitin ligase are conserved across diverse eukaryotic organisms (8). in humans, mutation of ubr1 causes johansonblizzard syndrome, a disorder characterized by multiorgan dysfunction, physical malformations, and cognitive impairment (19). ubr1 has been extensively investigated in the model unicellular eukaryote saccharomyces cerevisiae (budding yeast), where it contributes to multiple aspects of protein quality control. experiments performed with yeast have been foundational in the discovery of molecular mechanisms of quality control conserved among eukaryotes (3). among other roles, ubr1 promotes turnover of substrates of the n-end rule (17), endoplasmic reticulum-associated degradation (erad) (12, 13), stress-induced homeostatically regulated protein degradation (shred) (14), and cytoplasmic quality control (cytoqc) (7, 11) pathways. yeast lacking ubr1 exhibit divergent responses to pharmacologic interventions expected to increase the abundance of misfolded proteins. for example, ubr1∆ yeast display enhanced sensitivity to the hsp90 inhibitor geldanamycin, while they are resistant to the proline analog l-azetidine-2-carboxylic acid (15). materials all yeast strains used in this study are presented in table 1. these strains have been constructed in previous reports (6, 16). the entire coding sequences of ubr1, hrd1, and doa10 have been replaced with the kanmx4 allele in gene knockout strains (16). yeast were cultured in yeast extract-peptonedextrose medium (1% yeast extract, 2% peptone, 2% glucose, 0.002% adenine, 2% agar) (5) with the indicated concentrations of hygromycin b (corning). methodology and results we tested the hypothesis that ubr1 is necessary for optimal growth in conditions associated with elevated abundance of aberrant proteins. the aminoglycoside hygromycin b reduces translational fidelity by causing ribosome a site distortion and is toxic to yeast at 200 µg/ml (1, 4, 9). we analyzed the growth of yeast lacking ubr1 in the presence of sublethal doses of hygromycin b, which are expected to increase the cellular concentration of misfolded proteins (figure 1). wild type yeast, ubr1∆ yeast, and yeast lacking one or both genes encoding the primary erad ubiquitin ligases (hrd1 and doa10) (10) were subjected to six-fold serial dilution, beginning with an optical density at 600 nm of 0.2. each dilution (4 µl) was spotted onto agar plates containing rich yeast growth medium with no drug or increasing concentrations of hygromycin b. plates were incubated at 30°c and imaged at the indicated times. a detailed explanation of the yeast growth assay procedure can be found in (18). all data were analyzed using prism software (graphpad software inc., san diego, ca). because of highly variable data, values of zero were normalized to 1 to make all data positive and positive data were log-transformed. all means between groups were compared by one-way anova followed by tukey post-hoc analysis. a p value less than 0.05 was designated as statistically significant. in the absence of hygromycin b, all yeast strains exhibited similar growth. consistent with previous results (2), yeast lacking both hrd1 and doa10 exhibited a pronounced growth defect in the presence of 75 µg/ml hygromycin b. individual deletion of hrd1 or doa10 also impaired growth in the presence of hygromycin b, but to a lesser extent than the double mutant. finally, loss of ubr1 impaired growth in the presence of the compound more severely than any of the other mutations tested; this is most evident at 50 µg/ml hygromycin b. this experiment was piloted by undergraduate volume six | 79 figure 1. loss of ubr1 sensitizes yeast to hygromycin b. (a) six-fold serial dilutions of yeast of the indicated genotypes were spotted onto agar plates containing rich medium (no drug) or rich medium containing increasing concentrations of hygromycin b. plates were imaged after 1-2 days (as indicated) of incubation at 30oc. (b) growth in the second column of each plate (dashed rectangles) from three replicate experiments was quantified by densitometry. data were analyzed by one-way anova followed by tukey post-hoc analysis (*, less than wild type; **, less than wild type, hrd1δ, doa10δ, and hrd1δ doa10δ; p < 0.05). error bars represent standard error of the mean. 80 | fine focus students in the methods in cell biology (bio 315) course at ball state university and has been validated by three replicates in the research laboratories of emr and pjs. discussion as we hypothesized, our results indicate ubr1 is crucial for optimal growth of yeast in conditions associated with elevated abundance of aberrant proteins, consistent with ubr1 function in protein quality control. mutations in ubr1 are found in patients with johanson-blizzard syndrome. a previous study demonstrated that homologous mutations also reduce ubr1 function in yeast (8). we speculate that disease phenotypes present in patients with johansonblizzard syndrome harboring mutations in ubr1 are linked, at least in part, to defects in protein quality control. one limitation of the present work is that hygromycin b is expected to trigger the accumulation of a large and heterogeneous population of aberrant proteins. therefore, it is difficult to determine which types of protein aberrancies present the most substantial challenge to cellular health in the absence of ubr1. future biochemical experiments will be necessary to characterize the substrate range of ubr1 in yeast and human cells. such biochemical analyses may also provide insight into the divergent responses of ubr1∆ yeast to different forms of cellular stress. acknowledgements this work was funded by nih grant r15 gm111713 (emr). this project was conceived while emr was supported in part by a ball state university excellence in teaching award. research in the lab of pjs is supported by grant 18-iia-406 from the amyotrophic lateral sclerosis association. we thank the ball state university department of biology (particularly kemuel badger, clare chatot, and susan mcdowell) for material and moral support for the design and implementation of an inquiry-based research course, where this experiment was piloted. we thank jacob price for serving as a teaching assistant in that course. we thank bryce buchanan and courtney broshar for technical assistance. volume six | 81 table 1. yeast strains used in this study. all strains used in this study are congenic with by4741 (16). 82 | fine focus references 1. brodersen, d. e., clemons, w. m., jr., carter, a. p., morgan-warren, r. j., wimberly, b. t., & ramakrishnan, v. 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(2001). systematic genetic analysis with ordered arrays of yeast deletion mutants. science, 294(5550), 23642368. https://science.sciencemag.org/content/294/5550/2364.full 17. varshavsky, a. (2011). the n-end rule pathway and regulation by proteolysis. protein sci, 20(8), 12981345. https://onlinelibrary.wiley.com/doi/full/10.1002/pro.666 18. watts, s. g., crowder, j. j., coffey, s. z., & rubenstein, e. m. (2015). growth-based determination and biochemical confirmation of genetic requirements for protein degradation in saccharomyces cerevisiae. journal of visualized experiments, (96), e52428. https://www.jove.com/video/52428/ growth-based-determination-biochemical-confirmation-genetic 19. zenker, m., mayerle, j., lerch, m. m., tagariello, a., zerres, k., durie, p. r., . . . reis, a. (2005). deficiency of ubr1, a ubiquitin ligase of the n-end rule pathway, causes pancreatic dysfunction, malformations and mental retardation (johanson-blizzard syndrome). nat genet, 37(12), 1345-1350. https://www.nature.com/ articles/ng1681 characterization of prodiginine compounds produced by a vibrio species isolated from salt flat sediment along the florida gulf coast stephanie morgan1, matthew j. thomas2, katherine m. walstrom1, eric c. warrick2, brittany j gasper3* 1division of natural sciences, new college of florida, sarasota, florida 2department of natural sciences, state college of florida, bradenton, florida 3department of biology, florida southern college, lakeland, florida manuscript received 30 september 2016; accepted 29 december 2016 copyright 2017, fine focus. all rights reserved. 34 • fine focus, vol. 3 (1) prodiginines are secondary metabolites produced by several known species of bacteria. these metabolites are known for their bright pigmentation and their potential medicinal uses. biosynthesis of prodiginine compounds, including the well-studied prodigiosin, has been well characterized in serratia marcescens and other bacterial species, including several marine bacteria. in an effort to isolate and identify natural products from marine organisms, an environmental sample was taken from a salt flat along the florida gulf coast and cultured for bacterial growth. a bacterial species that produces a vibrant pink pigment was isolated and identified as a member of the vibrio genus and was named mi-2. whole genome sequencing identified a 13-gene operon with homology to the s. marcescens prodigiosin biosynthetic operon. the pigment produced by mi-2 was hypothesized to be composed of prodigiosin or related prodiginine compounds and was purified by flash column chromatography and identified by mass spectrometry. abstract corresponding author brittany j. gasper* bgasper@flsouthern.edu keywords • prodigiosin • vibrio • antimicrobial • biosynthesis • prodiginine the prodiginine family of bacterial alkaloids includes many vibrantly pigmented compounds, most of which are red, produced as secondary metabolites by a variety of bacterial species. the most notable of these is serratia marcescens from which the best studied prodiginine, prodigiosin, was first isolated in pure form and structurally charcterized (14, 19). interest in the prodiginines comes not only from their strong red pigment but also from their potential medicinal uses. in addition to the antimalarial activity of prodigiosin itself (4), prodigiosin and other prodiginine derivatives have been shown to have immunosuppressive functions introduction with novel mechanisms of action (7, 21, 30, 38, 42) and apoptotic effects in human cancer cells (11, 27, 33, 42). since the discovery of prodigiosin in s. marcescens, prodiginine compounds have been discovered in other bacterial organisms, including streptomyces coelicor a3(2), alteromonas rubra, hahella chejuensis, and vibrio gazogenes (9, 19). biosynthesis of prodigiosin has been best studied in s. marcescens, s. coelicor, and h. chejuensis. in s. marcescens atcc 274, the prodigiosin biosynthesis (pig) cluster consists of 14 genes abbreviated as piga through pign arranged as an operon that are transcribed as a single 14-gene polycistronic mrna (17). h. chejuensis kctc 2396 also contains 14 prodigiosin biosynthetic genes, hapa through hapn, that are similar in their gene layout to that of s. marcescens (22). in s. coelicor a3(2), the red cluster is responsible for prodiginine biosynthesis (5). the genetic arrangement of the red cluster is significantly different from s. marcescens and h. chejuensis, however the presence of 12 homologous genes between it and the pig operon suggests the two biosynthetic pathways are similar (17). several of the known prodiginine producers, including h. chejuensis, v. gazogenes, and a. rubra are marine microorganisms. the isolation of natural and potential medicinal products from diverse marine microorganisms has been described recently, with several new products originating from microorganisms isolated from marine environments (2, 3, 45). we report here the isolation of a prodiginine-producing vibrio species known as marine isolate-2 (mi-2) from a marine salt flat environment along the central gulf coast of florida. originally isolated as a marine antibiotic-producing bacterium, mi-2 was unique in its ability to produce a distinctly pink prodiginine product under appropriate media conditions. whole genome sequencing of mi-2 identified a 13-gene prodiginine biosynthetic gene cluster. the analysis of this prodiginine biosynthetic pathway and structural identification of the purified prodiginine compounds are discussed. prodiginine produced by a marine vibrio species • 35 isolation and genus identification of mi-2 mi-2 was isolated from a salt flat sediment sample aseptically obtained from leffis key in bradenton, fl. the sediment sample was diluted in 0.5 m nacl to 10-5 grams soil/ml and grown on 0.5 m nacl potato dextrose agar (pda) modified with the following contents: tryptone (2 g/l), nacl (0.5 m), glucose (5 mm), and 10x neidhardt mops salts (0.1x final concentration). the neidhardt mops salt components were prepared as described (32). to inhibit fungal growth, 100 μg/l cycloheximide was added to the initial isolation plates. after initial isolation of mi-2, the organism was maintained on 0.5 m nacl modified pda plates and 0.5 m nacl lb agar plates and stored at 25°c. materials and methods 36 • fine focus, vol. 3 (1) identification of the genus of mi-2 was completed by whole colony pcr amplification of the 16s rdna. the 16s primers 63f (5’ cag gcc taa cac atg caa gtc – 3’) and 1387r (5’ ggg cgg wgt gta caa ggc – 3’) obtained from integrated dna technologies (idt) were used to amplify the gene using the mytaqtm mix obtained from bioline (taunton, ma). the whole colony pcr reaction conditions were: 1 cycle of 95°c for 10 minutes; 30 cycles of 95°c for 30 seconds, 58°c for 30 seconds, and 72°c for 1 minute; and 1 cycle of 72°c for 5 minutes. successful pcr amplification was confirmed by agarose gel electrophoresis on a 1% agarose gel in 1x tae buffer and purified using the isolate ii pcr and gel kit from bioline. purified pcr products were sequenced at the dna analysis facility on science hill at yale university. media and growth effect on pigment production the effect of media composition and nacl concentration on the pigment production of mi-2 was determined qualitatively by quadrant streaking mi-2 onto modified pda plates prepared as described previously with supplementation of 2%, 4%, or 6% nacl and lb plates supplemented with 2%, 4%, or 6% nacl. the plates were incubated at 30°c for two days before being photographed. the effect was determined quantitatively by inoculating a colony into modified potato dextrose broth (pdb) and lb liquid cultures at 2%, 4%, and 6% nacl and incubating at 30°c for 48 hours. absorbance of the sample was taken by wavelength scan from 400 to 700 nm at 5 nm intervals. the effect of time on pigment production of mi-2 was determined qualitatively by quadrant streaking mi-2 onto modified pda or lb plates with supplementation of 2% or 4%. the plates were incubated at 30°c for nine days, and photographs were taken after two and nine days. the effect was determined quantitatively by inoculating a colony into pdb and lb liquid cultures at 2% and 4% nacl and incubating at 30°c for 48 hours. absorbance of the sample was taken by wavelength scan from 400 to 700 nm at 5 nm intervals. genomic sequencing and identification of a putative prodigiosin biosynthesis gene cluster genomic dna from mi-2 was extracted from an overnight culture grown in 4% nacl lb liquid media using the phenol chloroform method (44). the extracted dna was checked for integrity by agarose gel electrophoresis and quantified using a nanodrop 2000 (thermo scientific). whole genome sequencing of the extracted dna was conducted using hiseq 2500 technology at purdue university. the resulting genomic sequence was analyzed using the sequence viewer and annotator tool artemis (35). the genes with homology to the pig gene cluster of s. marcescens were identified manually using the national center for biological information (ncbi) protein basic local alignment tool (blastp) (15). prodiginine produced by a marine vibrio species • 37 comparison of putative prodigiosin biosynthesis gene cluster from mi-2 and other organisms comparison of prodigiosin biosynthesis genes from mi-2 and other known prodiginine compound producing organisms serratia marcescens atcc 274, hahella chejuensis kctc 2396, and streptomyces coelicor a3(2) was done by aligning amino acid sequences using clustal omega for each gene within the cluster (37). gene sequences were acquired using the ncbi genbank database and the accession numbers as follows: s. marcescens – ajb33002, h. chejuensis – dq266254, and s. coelicor – al645882. purification of prodigiosin an overnight culture of mi-2 in 4% nacl lb liquid media was centrifuged at 6000 xg for 10 minutes; the supernatant was discarded and the pellet was resuspended in approximately 2 ml of a methanol and 2 n hcl mixture (24:1). the addition of acid was necessary to break down a suspected prodigiosin-associated protein that may sequester the pigment molecule (14, 24). the resuspended pellets were combined in 35 ml scintillation vials wrapped in aluminum foil and left for 12-18 hours on an orbital rotator. extracts were then centrifuged at 6000 xg for 10 minutes to remove any cellular residue. the supernatant was collected, and the solvent was evaporated using a rotary evaporator. hydrophilic impurities were removed by a series of chloroform-water liquidliquid extractions that were repeated on the organic layer until the water layer no longer appeared cloudy (1). the chloroform was evaporated via rotary evaporation, and pigment was redissolved in acetonitrile. flash column chromatography was performed using silica gel as the stationary phase and acetonitrile as the mobile phase. fractions from flash chromatography that corresponded to a pigmented smear at 0.82 to 0.63 retardation factor (rf) regions on silica thin layer chromatography (tlc) plates with acetonitrile as the mobile phase were combined and concentrated by rotary evaporation. uv-vis ph assay the ph of methanol solvents was adjusted to 1.8, 5.0, 7.0, 8.0, 11.1 and 12.0 using solutions of 1 m hcl and 1 m naoh. one hundred μl of purified prodigiosin pigment extract was suspended in each methanol solvent, and absorbance spectra were measured from 350 nm to 700 nm at 0.25 nm intervals. a baseline correction was performed for all samples. mass spectrometry electrospray ionization (esi) tandem mass spectrometry (ms/ms or ms2) was performed using fourier transform mass spectrometry (ftms) mode on an ltqorbitrap with helium used as the collision gas. relative collision energy (rce) ranged from 30-35 in the ion trap component of the instrument. high-resolution mass spectra were obtained with full width half maximum resolving power of 100,000 at 400 m/z in profile mode. 38 • fine focus, vol. 3 (1) figure 1. isolation and colony appearance of mi-2 panel a shows the original isolation of mi-2 from leffis key in bradenton, fl on modified pda media. the red/pink colony of mi-2 selected for pure culture isolation is shown in the black square. panel b shows the pure culture and pink pigment production of mi-2 on modified pda media. b)a) isolation and genus identification of mi-2 the marine bacterium mi-2 was originally cultured from salt flat sediment obtained at leffis key in bradenton, fl on modified pda media supplemented with 0.5 m nacl for marine organisms and tryptone as described in the methods to support growth of fastidious organisms. figure 1 shows the original marine sediment isolation plate in panel a and the pure culture growth of mi-2 in panel b. the purpose of this isolation was to identify antibiotic-producing microorganisms from diverse marine environments. mi-2 was identified as an antibiotic-producer (data not shown), but its ability to produce a bright pink pigment when grown on the modified pda prompted further characterization of this microorganism. sequencing of the 16s rrna gene identified the organism as being a member of the vibrio genus with vibrio ruber being the closest related species (data not shown). results prodiginine produced by a marine vibrio species • 39 media 2% nacl 4% nacl 6% nacl max abs. λ (nm) avg. abs. ( ± se) max abs. λ (nm) avg. abs. ( ± se) max abs. λ (nm) avg. abs. ( ± se) pdb 545 1.70 ± 0.14 545 1.87 ± 0.08 540 1.73 ± 0.08 lb 500 0.72 ± 0.02 500 1.12 ± 0.03 500 1.24 ± 0.05 figure 2. effect of media composition and nacl on mi-2 pigment production mi-2 was quadrant streaked onto modified pda and lb plates at the indicated nacl concentrations and incubated at 30°c for 48 hours. panels a-c show growth on pda at 2%, 4%, and 6% nacl concentrations. panels d-f show growth on lb at 2%, 4%, and 6% nacl concentrations. the shade of the pigment is different when grown on pda compared to lb. this is confirmed by the corresponding table that shows the maximum absorbance wavelength (max abs. λ) and the average absorbance value (avg. abs.) ± standard error (se) for three replicates grown in pdb and lb at 30°c for 48 hours. b)a) d) e) c) f) 2%nacl pda lb 4% 6% 40 • fine focus, vol. 3 (1) 2% nacl pda 4% nacl pda 2% nacl lb 4% nacl lb nacl concentration pdb lb two days five days two days five days max abs. λ (nm) avg. abs. ( ± se) max abs. λ (nm) avg. abs. ( ± se) max abs. λ (nm) avg. abs. ( ± se) max abs. λ (nm) avg. abs. ( ± se) 2% 545 1.70 ± 0.14 545 2.02 ± 0.30 500 0.72 ± 0.02 450 0.781 ± 0.10 4% 545 1.87 ± 0.08 540 2.30 ± 0.01 500 1.12 ± 0.03 450 1.39 ± 0.20 figure 3. effect of time on mi-2 pigment production mi-2 was quadrant streaked onto lb and modified pda with 2% and 4% nacl and incubated at 30°c for nine days. photographs were taken after two days of growth (2d) and after nine days of growth (9d). the corresponding table shows the max abs. λ and avg. abs. ± se for three replicates grown in pdb and lb at 30°c for two days and five days the max abs. λ changed dramatically from two days (500 nm) to five days (450 nm) when grown in lb with 2% and 4% nacl but not in modified pdb at the same salt concentrations. the pigment intensity appeared to be notably decreased after nine days of growth, but this decrease was not reflected quantitatively in the absorbance values. 2d 2d 2d 2d 9d 9d 9d 9d prodiginine produced by a marine vibrio species • 41 media and growth effects on pigment production the effects of media (lb vs. pda) and nacl concentration (2%, 4%, or 6%) on pigment production of mi-2 are shown in figure 2. while the pigment produced by mi-2 appeared bright pink on the 2% nacl pda plate, it was more red on the 2% nacl lb plate. this difference was quantitatively confirmed by measuring the absorbance after growth in liquid samples (tabular data in figure 2). the maximum absorbance wavelength (max abs. λ) was 540-545 for pdb and 500 for lb. there was a slight change in the max abs. λ for pdb at 4% (545 nm) to 6% (540 nm) which may explain the slight change seen in the pigment color from panels b to c. pigment production on both media types visually appeared to show a decrease in intensity as the concentration of nacl in the media increased as seen by comparing panels a to c and d to f, however this decrease was not replicated quantitatively as there is no decreased absorbance seen with higher nacl concentrations. the effect of time on pigment production of mi-2 is shown in figure 3. after nine days of growth, pigment appearance of mi-2 appeared notably altered from where it was after two days of growth. extensive growth on pda appeared to result in decreased pigment content, and altered pigment color from red-pink to red-orange was seen after growth on lb. the change in pigment appearance was more dramatic on both media types at 4% nacl than at 2% nacl. quantitative analysis of this (tabular data in figure 3) confirmed a change in the max. abs. λ from 500 nm to 450 nm when grown in lb while the max. abs. λ of pdb remained relatively unchanged by prolonged growth. average absorbance values do not show the decreased pigment production that is visible in the figure as values for all samples actually increased from two days to five days. identification of putative prodigiosin biosynthesis gene cluster in order to better characterize and identify mi-2, its genome was sequenced. after whole genome sequencing and annotation, the putative prodigiosine biosynthesis gene cluster was identified based on homology to the well characterized 14 gene pig operon found in s. marcescens (17). the mi-2 prodiginine biosynthesis gene cluster is approximately 20.3 kb in length and consists of 13 genes (figure 4). the overall structure of the mi-2 cluster is highly similar to prodiginine biosynthesis clusters in both the arrangement of protein homologs as well as in the relative sizes of proteins and intergenic spacing. six of the open reading frames (orf) overlap with one another, but a significant gap of 177 base pairs was found between the putative pigc and pigd. this is comparable with the structure of the pig cluster in s. marcescens as well as the hap cluster in h. chejuensis (17, 22). the major difference between the structure of the putative pig cluster in mi-2 and s. marcescens is the absence of piga in mi-2. a nucleotide alignment of bases preceding pigb with the coding nucleotide sequence for piga in s. marcescens atc 2744 showed short regions of homology (data not shown). this suggests that a piga homolog may have been present at one time but has since been lost. 42 • fine focus, vol. 3 (1) figure 4. schematic representation of putative prodigiosin biosynthesis gene cluster in mi-2 the cluster is approximately 20.3 kb in length and contains 13 genes. the arrows show the directionality of orfs. letters for orfs correspond to the pigb-n homologs. different arrow patterns indicate the putative role of the enzyme in the bifurcated prodiginine biosynthesis pathway in which pigc condenses the compound 4-methoxy-2,2-bipyrrole-5-carboxyaldehyde resulting from synthesis of the mbc pathway with the compound 2-methyl-3-n-amyl-pyrrole resulting from the synthesis of the map pathway). the corresponding table shows the size of each orf, the nucleotide start position for each orf and the nucleotide end position for each orf. pig homolog orf size (bp) start* end* b 2,070 1 2,070 c 2,288 2,440 4,728 d 2,628 4,905 7,533 e 2.556 7,530 10,086 f 1,010 10,170 11,186 g 204 11,197 11,461 h 1,944 11,464 13,408 i 1,467 13,410 14,877 j 2,471 14,821 17,292 k 320 17,302 17,622 l 814 17,563 18,377 m 1,220 18,210 19,430 n 1,094 19,119 20,213 *nucleotide start and end positions are given relative to the start position of orf b. table rows highlighted in gray indicate an orf that overlaps with the orf preceding it. 1kb mbc pathway map pathway mbc-map condensing enzyme b c d e f g h i j k l m n prodiginine produced by a marine vibrio species • 43 mi-2 s. marcescens h. cheuensis s. coelicor homolog % aai homolog % aai homolog % aai a* piga 55 hapa 54 redw 43 b pigb 57 hapb 38 reds 35 c pigc 69 hapc 54 redh 40 d pigd 73 hapd 51 e pige 80 hape 61 f pigf 73 hapf 58 redi 19 g pigg 64 hapg 45 redo 21 h pigh 72 haph 58 redn 59 i pigi 58 hapi 43 redm 40 j pigj 61 hapj 33 redx k pigk 68 hapk 47 redy 45 l pigl 17 hapl 19 redu 19 m pigm 48 hapm 33 redv 26 n pign 17 hapn 14 redf 15 table 1. aai of prodiginine biosynthetic gene cluster homologs the table shows the aai of mi-2 putative prodigiosin biosynthetic gene cluster homologs compared to known prodiginine biosynthetic proteins from s. marcescens atcc 274, h. chejuensis kctc 2396, and s. coelicor a3(2). aai with mi-2 homologs were calculated using clustal omega. pigd and pige homologs in s. coelicor have not been found. *piga homolog is not found within the mi-2 cluster. protein sequence comparison of the putative prodigiosin biosynthesis cluster the amino acid identities (aai) of the proteins coded for by the putative prodigiosin biosynthesis genes were compared to their homologs of s. marcescens, h. chejuensis, and s. coelicor (table 1). in addition to the structural homology of the gene cluster, there was also consistent homology between genes in the mi-2 cluster and corresponding genes in the prodiginine biosynthesis operon of the other species. the aai between mi-2 and pig homologs typically ranged from 50% to 80%, a comparable range compared with other studies that have shown an aai of only 23% or more between vibrio and serratia species (16, 20, 39). exceptions to this can be seen in pigl and pign homologs, which had very low aai; however, neither of these two proteins are required for prodigiosin production (43). 44 • fine focus, vol. 3 (1) figure 5. uv-vis spectra of mi-2 pigment extract at various phs the graph shows the absorbance from wavelengths ranging from 380 nm to 620 nm of mi-2 pigment extract in aqueous methanol solvents at varying phs. two maxima can be observed at 470 nm and 535 nm. absorbance at 535 nm is greater at acidic ph while absorbance at 470 nm is greater at basic ph. an isosbestic point can be observed at 490 nm. a bs or ba nc e wavelength (nm) 0.24 0.2 0.16 0.12 0.08 0.04 380 400 420 440 460 480 500 520 540 560 580 600 620 ph 2 ph 5 ph7 ph8 ph11 as mentioned previously, there is no homolog for piga found in the mi-2 putative progidiosin biosynthesis gene cluster. however, a different protein found elsewhere in the mi-2 genome did show homology to piga. it is the sequence of this putative protein that is used for piga comparison from mi-2 in table 1. purification and uv-vis spectra of mi-2 purified pigment extract at different ph extraction and purification of the mi-2 pigment was completed by flash column chromatography. the uv-vis spectra of the purified mi-2 pigment extract resuspended in different ph solutions is shown in figure 5. the spectra showed two main peaks that depended on the ph of the solvent. this suggests that the compound exists in either a protonated or nonprotonated form (34). the peak absorbance for what was presumed to be the protonated form occurred at a wavelength of approximately 535 nm and the non-protonated peak absorbance occurred at approximately 470 nm. these results were consistent with the uv-vis spectra for prodigiosin (18). the spectra for the mi-2 pigment extract revealed an isosbestic point at a wavelength of approximately 490 nm. this is the wavelength in which the absorbance remains constant as a conformational change occurs due to the changes in ph of the aqueous methanol. additionally, the data suggested that the ph at prodiginine produced by a marine vibrio species • 45 figure 6. electrospray ionization mass spectra of mi-2 pigment extract the full mass spectra of the purified mi-2 pigment extract is shown. peaks represent ions of a particular mass (x-axis) with a relative abundance (y-axis). the peak at 324 is the most prominent while peaks at 338, 352 and 371 appear as minor components. re la tiv e ab un da nc e m/z 324.2074 338.3422 352.2387 371.1017 100 50 0 which the absorbance between the two peaks would be equal was just above ph 8, which is consistent with the known pka value of 8.25 for prodigiosin in acidified ethanol (18). mass spectra of mi-2 purified pigment extract the full mass spectrum of purified pigment extract from mi-2 is shown in figure 6. this spectrum revealed a mixture of four different compounds in different abundances. a compound with a mass of 324 g/mol was the main component of the mixture, and compounds with masses of 338 g/mol, 352 g/ mol and 371 g/mol were present in the extract as minor components. the tandem mass spectra of each of these four peaks are shown in figure 7. tandem mass spectra of compounds with a mass of 324, 338 and 352 exhibited similar fragmentation patterns that were consistent with prodiginine compounds. specifically, all three contained a peak at 252 m/z, which can be explained by the loss of the alkyl substituent group to yield the 2-methoxy prodiginine core. additionally, all three showed an ion at a mass consistent with the loss of the methyl group from the methoxy. compounds 324, 338 and 352 can be represented by compounds with varying lengths of alkyl chains, as there is a mass difference of 14 that can be represented by the addition of a ch2. the compound with a mass of 271 appeared to be unrelated as it did not contain the prodiginine core 252 peak; however, further analysis would be required to fully elucidate the structure of this compound. the spectrum from this peak is not shown in figure 7. 46 • fine focus, vol. 3 (1) figure 7. tandem mass spectra of peaks from the full mass spectra the tandem mass spectra of the relevant prodiginine peaks from the full mass spectra in figure 6 are shown. peaks represent ions produced as a result of gas-induced dissociation (gid) of the parent molecule. panel a shows the tandem mass spectra of a 324 molecular mass molecule with the proposed structure prodigiosin. the most stable ion produced appears at m/z 309 and is the ion produced by cleavage of the methyl on the methoxy group. the peak at 252 represents an ion created as a result of the alkyl chain being cleaved from the molecule and 292 is the loss of the oxygen. these fragmentation patterns are shown with the dashed arrows. panel b shows the tandem mass spectra of a 338 molecular mass molecule with the proposed structure 2-methyl-3hexyl prodiginine. the most stable ion produced appears at m/z 323 and is the ion produced by cleavage of the methyl on the methoxy group. the peak at 252 represents an ion created as a result of the alkyl chain being cleaved from the molecule, and the peak at 310 is likely loss of an ethene. other peaks represented are not readily explainable given the proposed structure. it is possible this sample that is in low abundance is contaminated. panel c shows the tandem mass spectra of a 352 molecular mass molecule with the proposed structure prodigiosin. the most stable ion produced appears at m/z 337 and is the ion produced by cleavage of the methyl on the methoxy group. the peak at 252 represents an ion created as a result of the alkyl chain being cleaved from the molecule, and 320 is produced after the loss of the oxygen. 0 100 50% 252.1134 292.1812 309.1841 h n hn n o 309 292 252 m/z a) 0 100 50 h n hn n o 337 320 252 % m/z 252.1133 320.2124 337.2151 352.2385 c) 0 100 50% 323 252h n hn n o 203.9438 237.9829 252.1132 303.3048 310.1916 323.1994 m/z b) discussion the biosynthesis of prodigiosin is an interesting metabolic phenomenon as it is a secondary metabolite with no known direct benefit to cellular growth (43). pure non-pigmented strains of s. marcescens show no significant difference in viability from pigmented strains (41). however, in the natural environment, prodigiosin and its derivatives may serve a purpose in bacterial defense as many have antibacterial properties (12, 25). mi-2 is a prodigiosin-producing marine bacterium of the vibrio genus. its species designation appears to be closest to vibrio ruber, a known prodigiosin producer (8), though its biochemical test profile differs from v. ruber in multiple ways, suggesting mi-2 is a strain of v. ruber unique from the wild type (manuscript in preparation). although prodiginine compounds and the organisms that produce them have been studied for a long time, there is limited research on prodigiosin biosynthesis clusters in different genera. to date, prodigiosin biosynthesis clusters have only been examined in s. marcescens atcc 274, serratia spp. 39006, h. chejuensis kctc 2396, and some streptomyces species (5, 17, 22, 36, 43). the addition of another prodiginine biosynthesis cluster in vibrio species will add more information on the potential catalytic mechanism for each enzyme by revealing key residues in conserved regions of each gene. the appearance of the prodigiosin pigment production of mi-2 is affected by the media on which it grows and the length of incubation. the pigments produced by growth in lb and modified pdb showed different max absorbances, verifying the different colored apearance seen on the plates in figure 2. pigment production on both lb and modified pda media types appeared to show a decrease in intensity as the concentration of nacl in the media increased, suggesting an inhibitory effect of excess nacl on pigment production. this phenomenon could not be validated quantitatively in liquid broth (tabular data of figure 2), however it is difficult to draw a comparison in intensities from the absorbance values as differences in the concentration of cells in each sample will affect the level of pigment production and therefore the absorbance values reached. these could be further complicated by the regulation of prodigiosin production by quorum sensing (43). additionally, increasing the length of incubation from two days to nine days resulted in altered coloration, particularly in lb media, and the appearance of decreased pigment intensity in plates, though this occurrence could also not be reproduced quantitatively in liquid (figure 3). the qualitative data suggest the pigment produced by mi-2 may not be stable for extended periods of time in the conditions it was grown (at room temperature and exposed to oxygen) as has been shown for other prodigiosin analogs (40). however further testing with a purified sample of the pigment would need to be performed to confirm this. growth after nine days on pda with 2% nacl showed more red coloration on the right hand side of growth in the third quadrant compared to the rest of the plate. this difference is believed to be caused by the pattern of pigmentation fading in the colonies and not an effect of the media itself. prodiginine produced by a marine vibrio species • 47 48 • fine focus, vol. 3 (1) when comparing the structure of the putative prodigiosin biosynthesis cluster in mi-2 to s. marcescens, the most notable difference is the absence of piga in mi-2. piga functions as a flavoprotein desaturase whose catalytic role in the prodigiosin biosynthetic pathway occurs after pigi but before pigj. an alternative reaction converting the substrate of piga to the correct product without the use of piga has been experimentally confirmed (13), perhaps negating the need for piga in the prodigiosin biosynthetic pathway of mi-2. additionally, as mentioned in the results, a different protein elsewhere in the genome did show homology to piga and may be able to function in its place in prodigiosin biosynthesis. as shown in table 1, the aai between proteins encoded by the putative prodigiosin biosynthetic gene cluster and known prodiginine biosynthetic proteins were typically between 50 to 80%. however, aai comparison is likely not the best method for comparing protein function. conservation of specific domains may be a better determinant as to whether a group of proteins will serve the same function. although h. chejuensis and s. marcescens both produced prodigiosin, the amino acid identity between pig and hap homologs generally ranged from only 30% to 55% (23). despite the seemingly lower amino acid identities between these homologs, these enzymes served the same function in prodiginine biosynthesis (23, 43). a better comparison of protein function can be done by comparing specific amino acid residues that are essential to the function of the protein. according to the ncbi protein basic local alignment tool, the mi-2 homolog of pigc contained a domain belonging to the pyruvate phosphate dikinase (ppdk) superfamily. pigc performs the final reaction of the prodigiosin biosynthesis pathway, which combines the two precursor molecules 4-methoxy-2,2bipyrrole-5-carboxyaldehyde (mbc) and 2-methyl-3-n-amyl-pyrrole (map) into the final prodigiosin structure (29, 43). the mechanism for pigc is similar to that of ppdk enzymes in that they phosphorylate a carbonyl by facilitating the transfer of a phosphoryl group from atp using a histidine residue in a phosphoryl transfer domain (ptd) (6). s. marcescens strains with a pigc knockout completely lost the ability to produce the prodigiosin pigment (43). selective mutagenesis experiments on pigc in serratia spp. 39006 showed that replacement of his840 with an alanine residue completely eliminated activity. similarly, residues predicted to be important for atp binding, glu281 and arg295, showed significant reduction in activity when replaced with alanine residues (6). closer analysis of the amino acid sequence in the mi-2 homolog also revealed residues his841, glu282 and arg296 that are analogous to key residues in pigc (data not shown). given this similarity, it is likely that pigc and the mi-2 protein homolog function identically to one another. investigations to experimentally confirm the function of the putative prodigiosin biosynthetic proteins in mi-2 are underway. the prodiginine compounds isolated and identified in this study have been previously recognized and documented in several other species (1, 22, 26). given the homology between pig, hap, and mi-2 prodiginine gene clusters it is plausible that mi-2 would produce similar prodiginine derivatives to those found in serratia species and h. chejuensis. the mass spectrum of the purified mi-2 pigments contains multiple peaks from prodiginine compounds with different alkyl chain lengths. prodiginine compounds varying in the length of the alkyl chain substituent group on a pyrrole of the structure are well documented and have been produced as minor byproducts in other species (1, 14, 26). the difference in mi-2 appearance on the modified pda and the lb plates may be due to the amount of references 1. alihosseini f., ju k-s., lango j., hammock b. d., and sun g. 2008. antibacterial colorants: characterization of prodiginines and their applications on textile materials. biotechnol. prog. 24:742–47. 2. ameri a. 2014. marine microbial natural products. jundishapur j. nat. pharm. prod. 9:e24716. 3. blunt j. w., copp b. r., keyzers r. a., munro m. h. g., and prinsep m. r. 2015. marine natural products. nat. prod. rep. 32:116–211. 4. castro a. j. 1967. antimalarial activity of prodigiosin. nature. 213:903–4. 5. cerdeño a. m., bibb m. j., and challis g. l. 2001. analysis of the prodiginine biosynthesis gene cluster of streptomyces coelicolor a3(2): new mechanisms for chain initiation and termination in modular multienzymes. chem. biol. 8:817–29. prodiginine produced by a marine vibrio species • 49 overall prodiginine production and the ratio of different prodiginine compounds produced on each medium. this ratio has been previously correlated with color change in v. gazogenes (1). additional pigment spots on the tlc (data not shown) indicated that there may be additional prodiginine compounds produced by mi-2. in other studies, hplc was used with a reversed-phase c-18 column to purify prodiginines (1, 28). preparative tlc has also been used to purify different prodiginines (10, 31). additionally, use of acetonitrile as a solvent for flash chromatography may have impeded separation by not stabilizing a single conformation during separation techniques. prodigiosins prefer the β conformer in acetonitrile and would likely prefer the α conformer in response to interactions with the silica, which is slightly acidic (34). a similar separation was achieved using ratios of chloroform, methanol and 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discovery and development of marine microbial natural products. mar. drugs. 11:700–717. prodiginine produced by a marine vibrio species • 51 the campus ambassador program (cap) was mentioned in this section of our last issue, and continues to be cultivated by our student editorial and marketing teams as a very important stem outreach initiative. last autumn, the effort was led single-handedly by student monica neeb, who had a minor in marketing/management and earned credit for this outcome. the cap idea is meant to extend our network with carefully selected partner institutions. monica started with the university of detroit mercy (udm), and developed a cap contract, formalizing the arrangement with udm biology chairperson dr. stephanie conant. this cap partnership will allow fine focus resources (print journals & promotional materials) to be made available to faculty and students, as well as ensuring students at each partner institution are aware of publishing opportunities for undergraduates in microbiology research and facilitating submission of their manuscripts. overall, the model of the cap is meant to encourage high quality manuscripts from traditionally underrepresented students in the sciences, and to teach them the fundamentals of peer review. at the same time, the cap partner universities will serve as feeder institutions for manuscripts in the long-term. in addition to udm, we have initiated cap arrangements for two international universities: the technical university of dresden (germany), and the university of akureyri (iceland). each fine focus cap site will also have a heavy emphasis on general aspects of professional activities relevant to all areas of stem, not just microbiology. we envision each cap site to be best implemented as a recognized student organization on each campus. perspective“objective lens john l. mckillip, ph.d managing editor, fine focus associate professor of biology, ball state university objective lens • 151 each fine focus cap student organization will establish a constitution and by-laws (using the existing template at ball state university fine focus as a guide), officers, and will be comprised of students from all stem disciplines. although the frequency and specific format for cap meetings and activities will vary slightly at each institution, the activities could include: • cultivate new ideas on how fine focus might work with the american society for microbiology (asm, our existing primary community partner) to promote diversity and inclusion in the sciences • promote the submission of eligible manuscripts to fine focus using print and other promotional materials (pens, business cards, pamphlets, t-shirts, and print journals) sent from ball state university • negotiate the campus library at each institution to carry fine focus in the stacks. • work with student leaders and undergraduate researchers to advocate for resources, events, policies that support the practice of undergraduate research • work with their campus uro/urc to help deliver needed student programming • provide information on best practices in undergraduate research and creative activity to their department and campus leaders • meet with state and federal elected officials to advocate for undergraduate research at professional conferences in their discipline if you are interested in learning more about how your institution could become a potential cap partner, please get in touch with me; i would be happy to discuss additional details. we if you are interested in learning more about how your institution could become a potential cap partner, please get in touch with me; i would be happy to discuss additional details. we always welcome your suggestions and feedback by email at finefocus@bsu.edu jlm 152 • fine focus, vol. 4 (2) undergraduate perspective • 77 the art of the sciences through the lens of undergraduate research hannah fluhler copyright 2019, fine focus all rights reserved introduction i have always been a year younger than my peers, desperate to prove i measured up to them, meaning anything that could serve as a competition—from memorizing the unit circle to mastering a combination of challenging tap dance rhythms—has naturally manifested as such in my brain. academics, of course, were the foremost concern in my life when my twin and i both accelerated past the eighth grade into high school from seventh, and i was eager to challenge myself with tough high school math classes (my favorite) and advanced placement courses. from 8am to 3pm i was a student, working through my lunch breaks and free periods on all homework assignments so that once 4:15pm came around, i could dive head first into my dance classes that would last until nearly 10pm on weeknights. then, i would go to bed and start all over the next morning. this drive to be my best self in all facets and the focus i gained from this rigorous schedule served to propel me through school and allow me to succeed when i put my mind to something. at some point early in my sophomore year of high school, a friend introduced me to music and memory, a student organization at ball state through which burris laboratory school students could also volunteer to provide music therapy to nursing home residents with alzheimer’s and dementia. this was my first exposure to creative arts therapies, and looking back, it was the primary catalyst that propelled me toward the current path i am on now. the use of the arts in aiding the physical and mental healing process of adults was a new idea to me in high school, but once a friend pointed out that i could go on to pursue something similar to this music project called dance/movement therapy, it all clicked for me. coming into ball state as a freshman, i still was not quite sure how to combine my love for dance with my ever-present desire to care for others. i knew i could learn about dance/movement therapy through a master’s program after graduating, but i did not know what that would mean for these next few years. after several major changes and picking up a dance performance minor the day before classes started that fall, i somehow made my way to now, as a double major in nursing and pre-dance/movement therapy (an integrated studies major i created through the honors college—that’s a story for another day). i have been extremely blessed in being a recipient of several life-changing scholarships. i am now a college senior and the inaugural recipient of a named, paid fellowship. through this fellowship, i have applied for and received permission after a full review from the local hospital’s institutional review board to begin a research study in the neonatal intensive care unit. this study is exploring the physiological safety of movement and music using a mamaroo baby swing for newborns suffering from withdrawal due to maternal drug use, known as neonatal abstinence syndrome. in addition, i have presented information about safe sleep and the use of movement and music to help similar infants to mothers in meridian health services’ maternal treatment program alongside a neonatal nurse practitioner, applied for grants to fund the donation of mamaroo swings to these mothers, and attended conferences about neonatal abstinence syndrome and the use of creative arts therapies with this population. in my next phase of life, i will be moving to melbourne, australia, for 10 months to pursue much different research in the genome stability lab at st. vincent’s medical institute for research through a fulbright scholarship. i will be exploring new treatment methods on the biochemical level for a rare disease called fanconi anemia (fa). because the cellular processes of fa are so similar to various cancers, this research offers hope for furthering cancer research, too. needless to say, though i am truly just beginning the process of completing undergraduate research, and in a completely different light than microbiology, i have failed more than i have succeeded and am grateful for the opportunity to pass along what little knowledge i have gained from the hurdles along the way. 78 • fine focus, vol 5 2019 the dos and don’ts of undergraduate research first and foremost, the first and most important step i took to accelerate the research process was to find an excellent—not just great—mentor. a few things specifically ensured i did this: • i reached out to potential mentors whose research interests me. whether current or past pursuits, the wisdom these individuals can pass down through a short, simple face to face conversation is immense. so, send that email to the address you found online, or reach out to that person your nutty professor nudged you toward. the worst thing that could happen is they don’t respond. and, if that happens, show up to their office. be persistent about gaining the best background information on the subject or project you are most interested in—it can only help you be better. • i became comfortable with casually bringing up my research ideas with anyone who would listen. i can’t specifically remember how long it was between the first time i thought about newborns suffering through drug withdrawal and actually engaging my mentor for the first time, but i can recall the most receptive people who i bounced ideas off of in the mean time. not everyone will understand your idea, especially for those in the microbiology field, but you never know who will ask the right questions or spark the “ah-ha” moment when you mention your most specific interest and its applications to broader topics. for me, it was my psychiatric and mental health nursing clinical instructor who stood with me several wednesdays in a row and happened to nod her head at the right times and egg me on when i doubted that my idea of using movement with newborns could be viable as a research topic. that instructor ultimately pointed me toward my eventual mentor in this endeavor. the worst mindset i had during the process of developing an undergraduate research project was that if i worked harder and faster for more hours than i already was, that the whole cycle would magically speed up. this was problematic simply because so many factors were out of my control along the way. from the beginning, i knew that however i went about this, it would require the collaboration and careful leadership of dozens of moving parts, beginning with putting together a research team. although individuals working as lab assistants do not necessarily have to assemble their own team, learning how to navigate these environments with opinions and viewpoints of people vastly different than yourself is beyond necessary. from team members, to research boards approving your work, to advisors and sources of funding, learning how to communicate with and make decisions as a part of something larger than yourself is integral to the success of your project. it could be argued that communication is one of the most important pieces of any endeavor, but when it comes to research, it is everything. communication has a role in applying to be a part of the project, proposing the steps to the project or dissecting the instructions for the research work, and finally, of course, documenting and disseminating the work you have done. while i do not believe there is a one-sizefits-all recipe to effective communication, i do know that patience, conciseness, and knowing your audience can solve most problems faced. mastering the art of science as an undergraduate, i have found power in connecting my interests across the lines of various classes and subject matters. from jazz dance technique class to my pediatric nursing courses, and even throughout my 5-week summer abnormal psychology course, i found that i learned so much more when i became confident enough to inquire about my interests, and to approach my peers or my professors who may help me connect the dots i am seeing. i was first urged to apply for a named, paid fellowship by a friend who recently graduated from bsu who is also a recipient of a scholarship which pays for full tuition, room, and board, and instantly knew i wanted to take advantage of the opportunity by at least applying. for several weeks, i struggled to choose which of my pursuits would best fit this fellowship. it wasn’t until nearly two weeks before the application deadline that i knew what i would write a 10-page proposal about, and it undergraduate perspective • 79 connected almost directly to the major i had created for myself: exploring a very physiological problem through an artistic approach. i know a lot of people who are undergraduates and took years to decide what single major they would pursue. for me, i always knew i would need more than one major, but that it was likely it would be best if i was able to combine several. dozens of my friends had played a sport or had a relatively “artsy” extracurricular activity that took up most of their time in high school, but most of them had all but abandoned this main love of theirs for a more typical career path by the end of our freshman year. even my best friend had told her dad she wanted to be a teacher, but was faced with the statement that it wouldn’t make enough money, so she should choose another path. while this may seem like an either-or decision for her, i was not so willing to give up my biggest pull—dance—to help people as a health care professional. so, i sought out the circumstances necessary to create my own path, which of course came with its own set of challenges. my background as a competitive dancer taught me what i believe a lot of scientists, even undergraduate research lab assistants, should know. pushing yourself out of your comfort zone while remaining open to constructive criticism, and then using that feedback to continue to grow, can be a game-changer in an environment as self-driven as research. whether in a kinesiology, microbiology, or creative arts-based pursuit, being able to continuously make adjustments to your thoughts, actions, and words can be the difference between completing a project with your head barely on straight or reaching the finish line with a successful result and fresh energy to pursue the next set of experiments. shaping yourself to best fit your passions, your vision for your future, and the requirements for a project you desperately want to be a part of is vital to research. i had never completed my own literature review before this research, nor had i presented my ideas in front of a board of community professionals with the hope that they would give me the green light instead of stopping my project in its tracks. these were two of the biggest growing experiences for me. along with the task of forming the research team, these experiences will help me endlessly in my future. if i had not adjusted my mindset going into this project, knowing that i would have to do hard things i had never even tried before, i would not have gained approval. i can imagine that lab work is similar—whether you have executed an experiment before or not, it is nearly guaranteed that you will be a professional at the research task at hand when completed. all it takes is to have the courage and drive to begin, and the passion to continue to see it to the end. finding your focus: through your lens one of my outstanding literature classes in high school taught me that everyone sees life in general, as well as individual conversations, through a certain lens. whether this lens is one of gender, race, class, or even profession, we all have a certain lens through which we automatically view most things. we all also have the potential to switch our lenses to match that of someone else or of a new perspective, theory, or ideology of our choosing. i know there is extreme value in viewing one thing through several different lenses, and that this can offer a new perspective otherwise unseen through our initial lens. i have also learned lately that focusing on your passions and pursuits is impossible without the consistency of a personal lens. for example, a white, snowy background can turn red or blue with different pairs of sunglasses on, but you wouldn’t want to wear a different pair of glasses to attempt to perform different parts of a single experiment on the snow, as this could cause skewed results and inaccurate data collection. for the same reason, i have tried my best to gather all of the information available to me about newborns withdrawing from various drugs and the causes and treatments used for them. i began from day one of my undergraduate career forming my own individual lens through which i have been able to take into consideration the psychological and physiological causes of addiction in the mothers. addiction competes with a mother’s drive to do her best to keep her baby healthy, in order to best empathize with her when asking for her consent to enroll her three-day-old child in a research study. if i were to listen to one physician’s biased opinion that all of these mothers, like his first patient who came in high to visit her baby, are simply addicts who have no hope of providing a good life for their child, i would not see another mother’s sobriety. if i were to listen to one counselor’s viewpoint that every mother with an addiction can be helped with an opioid agonist, i would not hear another mother’s opinion that it did not work for her. if i were to simply believe that the 80 • fine focus, vol 5 2019 current line of treatment for babies with neonatal abstinence syndrome—treating their symptoms with morphine— is the best and only option there will ever be, i would be cheating babies with mild symptoms of a drug-free neonatal period. if i were to take any one biased opinion and take it as truth, use it as my lens, i would not be able to contribute to literature on these topics to hopefully improve the treatment and outcomes of these mothers and their children. i would love to thank kelsey davis for asking me to write this perspective piece and professor john mckillip for fostering my first spurs of interest in undergraduate research during my time in fine focus. many thanks to dr. renee twibell for being the most incredible mentor i could imagine, and to each and every one of my professors and clinical instructors for the constant encouragement and questioning about my impossible quest to graduate with several majors in seemingly unrelated fields. finally, thank you to sweet aria for being the brightest star in the sky and to her mother, rachael, for the continuous inspiration to keep fighting the good fight to improve the lives of the most complex pediatric patients. acknowledgments undergraduate perspective • 81 • fine focus, vol. 3 (2) antibiotic resistant bacteria in an urban freshwater ecosystem in central texas annie kwok, michael c. davis, and sanghoon kang* department of biology, baylor university, waco, tx copyright 2017, fine focus. all rights reserved. antibiotic-resistant bacteria • 113 manuscript received 30 march 2017; accepted 07 june 2017 112 • fine focus, vol. 3 (2) antibiotic resistance is a growing concern for the human population and is becoming prevalent in many environments. for example, increasing occurrences of antibiotic resistance genes (args) in aquatic ecosystems elevates the risk of pathogenic microbes acquiring those resistance genes. there is an urgent need to more closely examine the relationship between antibiotic resistant bacteria (arb) and antibiotic residues in urban freshwater environments. thus, our main objective was to investigate the presence of antibiotic resistance in wastewater treatment plant (wwtp) influent and effluent leading into the brazos river using several commonly used antibiotics: penicillin, ciprofloxacin, erythromycin, trimethoprim, tetracycline, sulfamethoxazole, and gentamicin. an additional aim was to explore possible mechanisms of resistance emergence to these antibiotics using techniques such as replica plating, the luria-delbrück fluctuation test, the newcombe test, and 16s rrna sequencing. four samples of influent and treated effluent wastewater were collected from the wwtp to enumerate resistant bacteria in the community and to investigate whether mutations causing resistance in arb might be induced or spontaneous. we found that penicillin had the highest rate of resistance in all samples and that a similar trend of resistance appeared across all four samples. according to the luria–delbrück fluctuation test and the newcombe test, different antibiotics appear to be associated with different tendencies of resistance emergence, with certain groups of antibiotics producing different results, which raises evolutionary questions about the roles of random mutation and induction. most arb detected from the luria–delbrück fluctuation test belong to the klebsiella, enterobacter, and aeromonas genera. this study provides a baseline understanding of the urban freshwater ecosystem status in central texas and quantitatively examines the degree of resistance emergence. abstract corresponding author sanghoon kang* department of biology baylor university one bear place 97388 waco, texas 76798-7388 usa 1-254-710-2140 sanghoon_kang@baylor.edu keywords • antibiotics • antibiotic resistance • antimicrobial resistance • wastewater since the discovery of penicillin in 1928 (13, 14), antibiotics have played a crucial role in the fight against pathogens and infections, as well as increasing livestock growth and health (41). however, recent concerns have become more pressing due to the increasing number of antibiotic resistant pathogens in medical settings across the world – resulting in the loss of viable treatment methods. in an attempt to stifle the alarming rate at which antibiotic resistance has been occurring, researchers have begun to investigate the role of the environment in the spread of antibiotic resistance to both human and animal populations (23). modern wastewater treatment plants (wwtps) have been found to serve as an important source of antibiotic resistant bacteria (arb) and antibiotics (12, 22). the primary goals of wwtps are to remove solids, reduce numbers of pathogens, and sequester nutrients such as organic carbon, nitrogen, phosphorous, and fatty acids, and are not designed to remove antibiotics and other pharmaceuticals that ultimately pollute receiving bodies of water (15, 24, 44). some of these remaining antibiotics are minimally biodegradable in freshwater ecosystems (1), which can result in antibiotic residues of variable concentrations with unknown selective consequences for environmental bacteria (17). the presence of antibiotics can select for bacteria already carrying antibiotic resistant genes (args), allowing for preferential growth and propagation of their genome through horizontal gene transfer (hgt) and asexual reproduction (3, 28). there have been numerous studies within the last several decades which present data on the exacerbation of antibiotic resistance in microbial communities in various sources of freshwater as a result of improperly treated run-offs from large facilities such as hospitals, pharmaceutical production factories, and livestock farms (4, 9, 33, 36, 37). in addition to the problem of antibiotic and pharmaceutical compounds, many treatment measures used in wwtps may not be effective in removing resistant microbes themselves from sewage influent (18, 25, 29). this allows bacterial contaminants to be flushed into receiving bodies of freshwater (20, 31, 41, 45). these bacterial contaminants increase the likelihood of spreading args by residing in environments with potential for frequent hgt to downstream aquatic microbiota. this cycle produces an accumulation of args and arb, allowing urban water sources to serve as both reservoirs and breeding grounds of resistance (2). urban aquatic ecosystems in brazil have demonstrated communities capable of tolerating antibiotic concentrations up to 600 times higher than levels in clinical usage (11). args in aquatic microbes may be relatively harmless to humans when found in non–pathogens, but they can be transferred to pathogens or human and animal commensals (7, 12). when resistance occurs, especially in opportunistic pathogens, it can serve as a risk to human health when affected freshwater sources are used for human consumption and recreation. infections become increasingly dangerous as readily available and widely used treatments may no longer be effective (6) with the accumulation and spread of antibiotic resistance. many of the aforementioned studies in freshwater arb and arg presence have been conducted in europe and asia, however, there have been no such studies for urban aquatic ecosystems in central texas. this research gap introduction antibiotic-resistant bacteria • 115114 • fine focus, vol. 3 (2) highlights the lack of information on antibiotic presence and resistance in important texas watersheds, and as a result, the severity of arb proliferation needs to be studied to gain a grasp on the current situation. the research objective was to investigate the current status of arb in a central texas urban aquatic ecosystem, and the possibility of antibiotics in wwtp influent and effluent genetically influencing and selecting for arb in an urban freshwater environment. we hypothesized that antibiotic resistance would be present in the samples collected from the wwtp and that mutation tests will show a prominent trend of spontaneous mutation in resistance. methods sampling water samples were collected from the waco metropolitan area regional sewage system (wmarss). wmarss is a joint wastewater treatment plant that serves eight urban cities with an average flow of 37.8 million gallons per day. the effluent from the treatment plant leads into the brazos river. in addition to the wmarss, other local watersheds entering the brazos river near waco include several cattle pastures, other smaller treatment subsets of wmarrs, and an artificial wetland ecosystem constructed by the city of waco north of the brazos river. four 500 ml samples of influent and effluent were collected in nalgene™ lab quality amber hdpe wide mouth bottles. the bottles were machine washed, rinsed 3x with distilled water, treated with a 10% sodium hypochlorite solution, then rinsed thoroughly with molecular-grade purified and filtered water. this was considered adequate to remove not only potential dust-borne or water-borne contaminating bacteria, but extracellular dna is eliminated by the sodium hypochlorite treatment. the samples were placed on ice immediately after collection and transported to the laboratory, kept refrigerated (-4 °c), and were processed within 48 hours. sampling was carried out over four weeks, taken once a week in the afternoon. isolation of antibiotic resistance bacteria two types of media were used: trypticase soy agar (tsa) and eosin methylene blue (emb). tsa is considered a nonselective, general-purpose medium, thus it was used to isolate a wide range of culturable bacteria. emb was used to enumerate coliform growth. 100 μl of these dilutions were spread plated onto one tsa and one emb plate, respectively. since effluent contains far fewer bacteria, 250 ml of each effluent sample was filtered on pall gn-6 metricel® mce membrane disc filters to concentrate the bacteria. the filter was then vortexed for 5 minutes at full speed in conical polypropylene tubes with 10 ml of the respective effluent sample to release the bacteria into the solution. 10 microliters of this solution plated onto one tsa and one emb media plate. these four plates comprised the master plates for each sampling date. all emb plates were incubated for 24 hours at 37oc and all tsa plates for 24 hours at room temperature. tsa plates were incubated at room temperature to more closely approximate the average temperatures encountered by bacteria in effluent and in the brazos river. wastewater effluent and river water temperatures are quite variable, and depend on many factors, including time of year, depth of water, flow rate, shade available, etc. for approximately half of the year, surface temperatures of the brazos river are below 25 oc (data available at https://waterdata.usgs.gov/ nwis/uv?08117300). in order to facilitate the growth of a greater diversity of environmental bacteria, the tsa samples were incubated at room temperature (generally between 20 and 25oc). although identification of resistant potential human pathogenic coliforms in the wwtp and effluent were a primary concern, it was useful to have a non-selective culture for comparison to the coliform-selective emb. in addition, some types of resistance that may be harbored by non-pathogenic environmental bacteria have potential for hgt to human pathogens (23, 44). seven classes of antibiotics were chosen based on common use in medicine and agriculture, and were used to create antibiotic infused tsa and emb media culturing plates. concentration values for each antibiotic (table 1) were referenced from minimum bactericidal concentrations (mbcs) determined by an article that has established mbcs for a common gut microbe (19). for each type of sample master plate – influent-tsa, influentemb, effluent-tsa, effluent-emb – seven antibiotics were placed on sterile tsa and emb media culturing plates and left for several hours to sit and absorb into the agar. using velvet, the master plates were replica plated onto the antibiotic infused plates. a total of 28 antibiotic plates were incubated for 24 hours, at 37oc for emb plates and room temperature for tsa plates. bacterial colony growth on each antibiotic plate were deemed resistant to the specific antibiotic and recorded. the luria–delbrück fluctuation test during the isolation process, each set of antibiotic infused plates were compared to their respective master plates and a colony that was observed to be susceptible to all seven antibiotics was chosen from each master plate. a first round of pure culture was inoculated on a single plate of respective media using this colony, incubated for 24 hours as before on emb and tsa plates. using a random colony from the first round, a second round of pure culture was inoculated on a single plate of respective media and incubated again with the same conditions. this process was repeated, using the previous round to inoculate the next round, until the fifth round. the fifth round was used to inoculate seven antibiotic infused media plates and then incubated. this last procedure was replicated five times to produce 35 plates, and the number of antibiotic resistant colonies were observed and recorded. the luria–delbrück experiment intends to test two possibilities: induced or spontaneous mutation (26, 39). the repeated culturing between each generation provides time for mutation to occur during cell division. if the mechanism for antibiotic resistance in bacteria is induction by antibiotics, the number of colonies between the five sets of antibiotic infused plates from the final step should not vary greatly. however, if antibiotic resistance in bacteria is due to spontaneous mutation, then a mutation can occur at any point in the culturing process – either in earlier generations or later generations. this should produce a large amount of variance in colony number between each set of antibiotic infused plates. levene’s test of equality of variances was used to test the significance of results from the luria-delbrück test. the newcombe test the fifth round from each respective sample and plate was used to additionally inoculate seven respective antibiotic infused plates. the plates were incubated at their respective temperatures for 24 hours and then re-spread before being incubated for another additional 24 hours. if the colonies and bacterial cells had spontaneously mutated prior to exposure to the antibiotics, then the re-spread plate should have a higher number of bacteria present due to the moved bacterial cells forming new colonies of their own (32). analysis of variance (anova) was used to test the significance of antibiotic-resistant bacteria • 117 116 • fine focus, vol. 3 (2) results from the newcombe test. 16s rrna gene sequencing using the qiagen dneasy® blood and tissue kit, dna was extracted from resistant isolates of the final sets of antibiotic infused plates from the luria-delbrück’s experiment cultured in bd difco™ nutrient broth. the extracted dna was run on 1% agarose gel at 85v at 115ma for 45 minutes, stained with gel-red (biotium, inc.) in order to ensure bacterial dna was intact. using the isolated dna, a 25 μl pcr reaction mixture consisting of 200 μm primers (universal primers set 27f-1492r), 1 μl of template dna (~1 ug), 9.5 μl of dna safe water, and 12.5 μl of amresco® hot start pcr-togel taq pcr master mix 2x was made for each sample. the pcr was run on the applied biosystems® veriti ® 96-well thermal cycler, beginning at 96oc for five minutes, then 30 cycles of denaturation at 94oc for 30 seconds, annealing at 57oc for one minute, and extension at 72oc for two minutes. the pcr was finished with a seven-minute final extension at 72oc. products were run on a 1% agarose gel at 85v at 115ma for 45 minutes to ensure presence, with quantity confirmed using nanodrop 2000 (thermo scientific). pcr products were sent for sequencing to macrogen usa (rockville, md), and the sequence results were analyzed through the national center for biotechnology information’s (ncbi) basic local alignment search tool (blast) and the ribosomal database project’s (rdp) seqmatch tool. results isolation of antibiotic resistant bacteria in the replicate-plate antibiotic resistance, total culturable count of arb drastically decreased post-treatment in effluent in comparison to influent (figure 1.a). however, while overall culturable count of the samples had decreased in the effluent by 107 cfu/ ml, percentages of resistant bacterial colonies in comparison to total bacterial colonies on the master plate were comparable (figure 1.b). the penicillin-infused media agar plates had the highest percentage of surviving bacteria grown across all four types of samples when compared to the master plates. resistance to ciprofloxacin produced the second highest percentages among the samples, with the exception of influent-emb. erythromycininfused media agar plates generally had the third highest percentage of surviving bacteria followed loosely by trimethoprim, tetracycline, and sulfamethoxazole. gentamicin-infused media agar plates had the lowest percentages of resistance. the luria–delbrück fluctuation test bacterial colony forming units (cfus) were observed and recorded across all five sets of plates within each sampling group. cfus of respective antibiotic-infused plates from each set were averaged and the deviation of each plate from the mean was calculated. to display the variability in resistant colonies for each antibiotic, the range of bacterial colony count deviation from the mean was used and the significance was tested using levene’s test (figure 2). the range in bacterial cfu among the antibiotic-infused plates did not have a clear trend, with ranges for each antibiotic sometimes varying widely. however, trimethoprim, tetracycline and sulfamethoxazole produced consistently low range values in comparison to other antibiotics. levene’s test showed significant differences in variances between the lower variance (trimethroprim, tetracycline and sulfamethoxazole) and the higher variance group (rest of antibiotics); in which only influent samples on tsa media had marginal significance (p = 0.070) while other set of samples showed much stronger significance (p < 0.001). the newcombe test in order to compare the number of cfus on plates before and after the respreading process, the difference in the number of colonies formed before spreading and after spreading was calculated (figure 3). overall tetracycline, trimethoprim and sulfamethoxazole are among the lowest in cfu difference, but there was no statistical significance between this group from the rest of antibiotics. an anova was not able to reject the null hypothesis for the global figure 1. results for culturable antibiotic resistant bacteria (arb) for (a) total arb count (cfu per unit volume), and (b) percentage of arb in comparison to the master plates from the replicate plate antibiotic resistance assay. error bars indicate standard error of the means. a b 0.000 0.500 1.000 1.500 10 8 cf u/ m l penicillin ciprofloxacin erythromycin trimethoprim tetracycline sulfamethoxazole gentamicin 0.0% 5.0% 10.0% 15.0% 20.0% 25.0% 30.0% 35.0% 40.0% tsa plated influent emb plated influent tsa plated effluent emb plated effluent pe rc en ta ge o f a rb penicillin ciprofloxacin erythromycin trimethoprim tetracycline sulfamethoxazole gentamicin 0.000 0.500 1.000 1.500 cf u/ 10 m l tsa plated influent emb plated influent tsa plated effluent emb plated effluent antibiotic-resistant bacteria • 119 118 • fine focus, vol. 3 (2) penicillin erythromycin trimethoprim ciprofloxacin tetracycline sulfamethoxazole klebsiella enterobacter aeromonas influent plated on tsa effluent plated on tsa influent plated on emb effluent plated on emb test of significance as well as the pairwise comparisons (tukey’s method) at p = 0.05. 16s rrna gene sequencing dna extracted from bacteria was visualized as bands on the gel-red stained agarose gel. using ncbi’s blast and rdp’s seqmatch tool, 16s rrna gene sequencing results from the final round of the luria– delbrück fluctuation test were analyzed. gentamicin infused plates did not produce the growth of any resistant isolates. all identified bacteria fell under the phylum α -proteobacteria, and included the genera klebsiella, enterobacter, and aeromonas (figure 4). several isolates were identified at species level (table 2). discussion we carried out these experiments to investigate the current situation of antibiotic resistance in a central texas urban freshwater ecosystem, particularly in an area of the brazos river where the wmarrs operation releases the effluent. minimal bactericidal concentrations (mbcs) of antibiotics in media were used to produce a selective environment in which resistant bacteria would be able to survive and grow, allowing for the observation of active arb. results demonstrated that antibiotic resistance was clearly exhibited in influent sewage leading into the treatment plant as well as treated effluent water leading into the brazos river, in both general media and coliform-selective media (figure 1). the most notable resistance was to members of common antibiotic classes, including a penicillin, a fluoroquinolone, and a macrolide. a b c d 0 5 10 15 20 25 ra ng e of b ac te ria l c fu 0 5 10 15 20 25 ra ng e of b ac te ria l c fu 0 5 10 15 20 25 ra ng e of b ac te ria l c fu 0 5 10 15 20 25 ra ng e of b ac te ria l c fu f = 3.34 p = 0.070 f = 11.83 p < 0.001 f = 12.25 p < 0.001 f = 19.82 p < 0.001 figure 2. range of individual plate counts across respective antibiotic-infused plates from the luriadelbrück fluctuation test. accompanied statistical results are by levene’s test of equality of variances between lower and higher variance group. (a) tsa plated influent (b) emb plated influent (c) tsa plated effluent (d) emb plated effluent. error bars indicate standard error of the mean. -15 -10 -5 0 5 10 15 co lo ni es re sp re ad – co lo ni es un sp re ad penicillin ciprofloxacin erythromycin trimethoprim tetracycline sulfamethoxazole gentamicin tsa plated influent emb plated influent tsa plated effluent emb plated effluent figure 3. difference between colony counts for re-spread vs. un-spread plates from the newcombe test. a negative value indicates a possibility for induced mutation and a positive value indicates a possibility for spontaneous mutation. error bars indicate standard error of the means. figure 4. distribution of identified antibiotic resistant bacteria (arb) by 16s rrna gene sequencing per samples and media from the luria-delbrück fluctuation test. antibiotic-resistant bacteria • 121120 • fine focus, vol. 3 (2) it is notable that the third most common type of antibiotic resistant bacteria in tsa-plated effluent are resistant to an antibiotic that has been (repeatedly) detected in wmarrs effluent – erythromycin (bryan brooks, personal communication). fluoroquinolone and macrolide were not detected in the effluent, although bacteria resistant to them were among the most abundant. it is possible that there may be temporal variations in antibiotic concentration, and the particular sampling might have missed the overall trend which may be responsible for the high resistant bacteria abundance. further studies including that possibility are being planned. treatment provided by the wwtp was still effective in reducing overall total culturable arb between influent and released effluent. despite being an effective in reducing solid wastes, nutrients, and pathogenic microbes, the wmarrs, as with most wwtps (18, 25, 29), did not completely eliminate arb in the effluent samples. in fact, a higher percentage of arb were found in the effluent compared to the influent in some samples, which has also been reported by previous studies (18, 25, 29), possibly due to the increased opportunity of horizontal gene transfer within the wwtp. this presents an alarming possibility, as accumulation of resistant bacteria in this freshwater ecosystem can potentially create a significant reservoir for the spread and persistence of antibiotic resistance in the environment. as most wwtps are not designed to remove antibiotics, pharmaceuticals, and other personal care products, the ineffectiveness of reducing antibiotics have been noted in other areas in the world (5, 8, 10, 30). the detection of antibiotic residues in the wwtp indicates the possibility that at some point prior to reaching the plant, antibiotic concentrations may be high enough for an increased mutation rate, in addition to selective pressure, increasing the prevalence of antibiotic resistance in the environment. mutation tests, the luria–delbrück fluctuation test and the newcombe test, were used to test this possibility and to serve as a premise for any future experiments to come. in the luria–delbrück fluctuation test, a lower range in deviation from mean colony count would indicate an induction mechanism due to antibiotic selective pressures, whereas a larger range in deviation would indicate spontaneous mutation occurring through replication and cell division. although there was no significant trend for the antibiotic residues tested, some antibiotics such as trimethoprim, tetracycline and sulfamethoxazole may be more predisposed to being induced as they produced consistently similar colony counts (figure 2). additionally, the fact that antibiotic resistance emerged from antibiotics at mbcs with five rounds of incubation indicates realistic possibilities in the aquatic environment in which conditions are not far from what was used in the luria–delbrück fluctuation test. results from the newcombe test were not conclusive (figure 3). after taking the difference between number of colonies on the un-spread plates and the re-spread plates, a positive number indicated that the re-spread plate had a greater number of colony forming units. a higher difference would be indicative of spontaneous mutation, whereas a lower difference would be indicative of induced mutation. again, the lowest numbers in cfu differences were found with trimethoprim, tetracycline and sulfamethoxazole. there was no clear trend between samples and media, which may have been due to the inadequate number of replicates, which points to the need for follow-up experimentation to determine whether the presence of antibiotics and other factors could be altering the mutation rate leading to these resistance genotypes. in the final part of the study, 16s rrna gene sequencing was used to provide a potential list of arb that have emerged quickly under strong antibiotic concentration pressures. the presence of some α-proteobacteria was not surprising, as it is one of the main bacterial phyla present in the human gut microbiome (21, 42). however, both the tsa and emb media may have been more selective than was originally anticipated, resulting in little diversity in the isolated cultures. among the identified species, many were opportunistic and commensal pathogenic microbes, and some 16s sequences matched multidrug resistant strains. influent plated on penicillinand ciprofloxacininfused tsa media, and effluent plated on sulfamethoxazole-infused emb media, grew bacteria that were identified as aeromonas jandaei strain ash05 (genbank accession number ku725738), a multi-drug resistant pathogenic strain isolated post-flood in chennai, india (unpublished). a species of aeromonas identified in influent cultured on trimethoprimand ciprofloxacininfused tsa plates, and in effluent cultured on sulfamethoxazole-infused tsa plates (genbank accession number eu260204), has been referenced in a study examining the antimicrobial resistance in gram-negative bacteria in a lake under heavy anthropogenic influence (34). in effluent cultured on penicillin-infused tsa, an environmental enterobacter species (genbank accession number eu420931) has been cited in a study exploring the incidence of extended spectrum beta-lactamases (esbl), and plasmid-mediated ampc beta-lactamase genes and integrons in a eutrophic bay (unpublished). sequence matches for klebsiella pneumoniae involving multi-drug resistance and nosocomial infections (genbank accession numbers cp019772, cp017985, cp015392) were found from influent bacteria isolated on tetracycline and sulfamethoxazole –infused emb media (35). effluent cultured on ciprofloxacininfused emb media produced matches with identified strains of klebsiella pneumoniae that exhibit antibiotic resistance in the environment (genbank accession numbers kj806466 and kp297443). a wide variety of putatively identified strains isolated in this experiment had resistance profiles which matched antibiotics detected at wmarrs; and with strains known for multiple antibiotic resistance, common presence in wwtps, and pathogenic potential (including nosocomial infections). all of the identified bacteria are in the clinically relevant genera klebsiella, enterobacter, and aeromonas, including sequence matches to species known for potentially lethal virulence factors (16, 37). this supports the idea that the natural environment and wwtps can be a significant reservoir for the exchange and maintenance of antibiotic resistance, through horizontal gene transfer and/or selective pressures (26, 42). despite antibiotic concentrations used in this study being minimum bactericidal concentrations for common gut microbes, numerous microbes were still cultured in effluent samples leading into a freshwater ecosystem. if freshwater sources containing multi-drug resistant pathogens are intended for anthropogenic use, it can serve as an alarming issue. recently, a study has examined drinking water in six states in the united states and identified the presence of ctxm (an extended-spectrum β-lactamase) and oxa-48 (a carbapenemase) genes (40). using culturing techniques, antibiotic resistant bacteria have been characterized at a wwtp in central texas and the freshwater ecosystem receiving its effluent. there are substantial percentages of bacteria discovered to be resistant to most antibiotics, penicillin being the most abundant and gentamicin being the last abundant. the luria–delbrück fluctuation and newcombe tests indicate certain antibiotics having particular mechanisms of mutation and evolution, or that resistance may arise through a mixture of induction and random mutation. the identified resistant isolates that rapidly emerged from the experiment have presented findings that include known pathogens with multi-drug antibiotic-resistant bacteria • 123 122 • fine focus, vol. 3 (2) resistance, which imposes this wwtp and urban aquatic ecosystem in central texas as a potential risk. future work in this area includes more detailed examinations of mutation emergence through more extensive mutation tests, general gene sequencing of samples taken from wmarrs, looking at other freshwater ecosystems affected by anthropogenic activity in central texas, and examining other commonly used antibiotics as selective pressures in resistance reservoirs. the studies and work conducted in this paper were supported by erick lebrun, swastika raut, and abigail antrich in the kang microbial ecology laboratory in baylor university. funding was provided by the jack g. and norma jean folmar research grant, and the undergraduate research and scholarly achievement (ursa) initiative at baylor university. this research is in compliance with institutional policies relating to infectious agents. antibiotic-resistant bacteria • 125 acknowledgements references 1. al-ahmad, a., daschner, f. d., & kümmerer, k. 1999. biodegradability of cefotiam, ciprofloxacin, meropenem, penicillin g, and sulfamethoxazole and inhibition of waste water bacteria. arch environ 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d., & natangelo, m. 2000. presence of therapeutic drugs in the environment. lancet 355:178990. 45. zurfluh, k., hachler, h., nuesch-inderbinen, m., & stephan, r. 2013. characteristics of extendedspectrum β-lactamase and carbapenemase-producing enterobacteriaceae isolates from rivers and lakes in switzerland. appl environ microbiol 79:3021-3026 antibiotic-resistant bacteria • 127126 12 | fine focus noawarat cheeptham* department of biological sciences thompson rivers university kamloops, b.c., canada archana lal labette community college pittsburg, ks *corresponding author, ncheeptham@tru.ca manuscript received 17 june 2019; accepted 28 january 2020. factors affecting learning gains among students in microbiology class: a preliminary study between a u.s. community college and a canadian comprehensive university naowarat cheeptham and archana lal volume six | 13 abstract though in the past, serious concerns have been raised about students’ interest and learning gains in stem courses, not much research has been done to examine the differences in learning science at community colleges and universities. the purpose of this paper is to close this gap. this paper analyzes the influence of students’ demographics, preparedness, major, and attitudes on their learning gains in an introductory microbiology class at a community college vs. a university. student demographics, information about their preparedness level, major, and attitudes were collected in a questionnaire and students’ learning gains were assessed by comparing student performance on a preand post-test on four different topics in microbiology. our results indicate that students’ majors and attitudes such as their willingness to actively participate in the classroom discussions and spend time outside the classroom to learn are major factors that enhance their learning. age and marital status positively impact learning gains while gender, employment status, and citizenship status show no impact on learning gains in students. our results also indicate that students at the community college who had less exposure to science classes in high school or biology classes in college achieved statistically higher learning gains despite having overall lower scores on two of the four post-tests. 1. introduction 14 | fine focus in recent years, there has been a growing need for students trained in the fields of science, technology, engineering, and mathematics (stem) in the u.s., while an increased interest in stem education has steadily been recognized in canada. stem’s crisis is not as obvious in canada, however, stem-related career and job preparedness is more prominent. the canadian government has created and put forward many stem initiatives and educational programs to prepare canadians for stem-related careers and jobs (1). canadian stem graduates continue to demonstrate disparities between gender and minority representation (2). in the 2011 national household survey (nhs), it was found that only 18.6% of adults with a postsecondary certificate, diploma, or degree are qualified in stem fields of study. of those, among younger stem graduates, women held a higher share of university degrees, while men still held the majority of university stem degrees in older demographics. it seems that overall canada is better prepared for stemrelated careers and jobs than the u.s. (3,4). blotnicky et al. demonstrated that overall atlantic canada middle school students (7th to 9th graders) from public schools lacked knowledge about stem career requirements and their findings support the need to create better accessibility of knowledge pertaining to stem career requirement to facilitate students’ understanding of the nature of a stem career (5). at mcgill university, montreal, quebec on november 28, 2015 the canadian science & policy exchange convened a working group composed of stem students and experts representing the perspectives of academic, government, and private sectors to discuss current challenges and opportunities in canadian stem education (6). canadian stem students voiced many challenging issues in regard to the current delivery of their stem education. these challenges include issues in, one, curriculum and structure (large class size, lack of applied learning), two, critical skills development, three, career exposure, and, four, metrics and evaluation. after working with experts during the working groups, several solutions to these issues were recommended from the students. the first four solutions at the top of the list were, one, reducing class sizes to improve student engagement, two, developing interdisciplinary courses to expose stem students to diverse perspectives for problemsolving, three, increasing direct training in the critical skills that employers expect from stem students, and, four, fostering an early awareness of stem careers and encouraging students to be proactive in forming a career plan (6). however, there is less research published on stem education related topics in canada in relation to the u.s. though the number of students pursuing their education in stem fields in the u.s. has increased, it has not been enough to meet the increasing demands in stem (7). there has been a shortage of workers and students proficient in math and science (8). the national math and science initiative (nmsi) (http:// www.nms.org/) states that the united states is losing its competitive edge in stem areas (9) and this shortage is appropriately called “the stem crisis”. in 2012, the president’s council of advisors on science and technology (pcast) issued a report suggesting an increase in the number of stem graduates in the next decade to meet projected employment needs (8). a major reason for “the stem crisis” in the u.s. has been a declining trend in the adequate preparation of volume six | 15 high school students to be successful in college level courses. according to an nmsi estimate (9), in 2009, only 21% of 12th grade students performed at or above proficient level in science; in the high school class of 2012, of all the students who took an advanced placement test, only 19.5% earned a qualifying score; and only 13% of high school graduates in 2013 were ready for college level science. in 2012, u.s. students were ranked 27th in math and 20th in science while china, korea, japan, and canada were all ahead of the u.s. recognizing the stem crisis, in december 2018, the u.s. federal government has adopted a detailed strategy to make the u.s. a global leader in stem literacy, innovation, and employment by pursuing three objectives; one, build a strong foundation for stem literacy, two, increase diversity, equity, and inclusion in stem, and, three, prepare the stem workforce for the future (10). given the seriousness of issues surrounding science education, it is not surprising that several studies have examined the factors that influence post-secondary students’ interest and desire to pursue careers in stem fields. astin and astin (1992), for example, reported a significant positive correlation between institutional traits and background characteristics of students and their interest in studying science and related fields during their post-secondary studies (11). they stated that the level of mathematical preparation in high school is the strongest and most consistent predictor of students’ initial interest as well as continuation of their education in stem fields. several studies have examined the impact of background characteristics such as age (12), gender (13, 14), marital status (12), family income (12), academic success (13), financial situation (15), and previous educational experiences (11) on retention of stem majors into and during post-secondary education. leppel found that age, marriage, and hours employed have a negative impact on college persistence while family income and gpa have a positive impact on both men’s and women’s persistence (12). similarly, cabrera and la nasa showed that factors like family income, parental education, gender, ethnicity, and exposure to at–risk factors in lowest socio-economic status place students at a disadvantage and make their path to college extremely difficult (13). whalen and shelley showed that underrepresented (female and/or minority) students do not perform as well in stem majors as traditional white, male students (14). academic performance of students has always been a topic of interest, discussion, and research among educators in many disciplines. much of the earlier research analyzed the relationship between students’ demographics, experiences in high school, their entry qualifications, type of institutions and their performance in college classes. effects of many different demographic factors on student learning in different fields have been examined, for example, age (16, 17, 18, 19, 20, 21), gender (17, 19, 20, 22, 23, 24), race (24), employment status (13, 17), and citizenship (17, 18). in studies where the effects of age and gender on academic performance were examined, mixed results have been reported. in an introductory biochemistry class, mlambo reported that gender and age did not cause any significant variation in the academic performance (19). similarly, in a study by colorado and eberle, age was 16 | fine focus not a significant factor affecting academic performance of graduate students enrolled in online classes (16). jayanthi et al. also reported no significant impact of age on academic performance of post-secondary students (17). newman-ford et al. reported minor impact of age and gender on educational achievements in first year undergraduates (20). in a study by richardson and king (1998), older students exhibited more desirable approaches to learning in terms of their persistence and attainment (21). in some studies, no link was found between gender or race (except for latinos) and student performance in an introductory college chemistry class (24) and mathematics class (23). in contrast, in other studies, female students were found to outperform male students (17, 22). few studies reported a correlation between nationality and student performance. a study done in singapore reported that international students performed better than domestic students (17). in addition to age, gender, race, and nationality, other factors that influence student achievement in postsecondary education have also been researched. it is widely accepted that well prepared students are more likely to pursue their initial choice of a science major. astin and astin (1992) suggested that it may be possible for more students to pursue science majors and careers if the level of overall academic competency could be increased at the secondary level (11). newman-ford et al. reported a significant impact of prior educational attainment, and attendance on educational achievement of first year undergraduate students in u.k (20). along the same lines, jayanthi et al. reported that intention to pursue higher studies and learning preferences contribute to students’ academic performance significantly (17). on the contrary, in a study in an introductory biochemistry class, it was concluded that learning preferences and entry qualifications do not cause any significant variation in the academic performance of students (19). it is well known that students who have no visible or significant gaps or barriers in learning in high school are better prepared to handle the rigor of college. tai et al. reported a significant correlation between pedagogical experiences of students during high school class and their performance in introductory college chemistry class (24). they also concluded that demographic predictors like parents’ educational level, family income, and affluence of the community were linked to significant differences in how students perform in their college class. despite the complexity and the importance of issues relating to student learning in stem fields, we found very few studies that compared learning gains between twoand four-year educational institutions. the purpose of this paper is to fill this gap. thus, this paper examined learning gains of students enrolled in an introductory microbiology class at a two-year college in the u.s. and at a four-year university in canada. this paper compared the student composition (in terms of demographics, level of preparation, major, and attitudes) at the two educational institutions by means of a questionnaire administered at the beginning of the semester. we also analyzed the results of the same preand post-tests (details are provided in the methods section) taken by students at both the institutions to test whether their learning gains are linked to the nature of institutions, students’ prior exposure to volume six | 17 the subject matter, attitudes towards microbiology, and demographics. further, we have provided several plausible explanations consistent with our findings of differences in students’ learning at two types of institutions. 2. methods this study was conducted at a u.s. community college (uscc) and a canadian university (cu). the microbiology class at uscc was capped at 20 students and there was a mix of traditional (25 or younger) and non-traditional students (above 25 years of age) (72% vs 28%) and the majority of them were pre-nursing majors (88%). at cu, most students were traditional students (98%), majoring in biology/microbiology/ pre-med (67%) and the class size was capped at 80 (one big lecture class divided into 5 lab sections with a maximum of 16 students in each lab section). the lab sections met for microbiology experiments (once a week at cu and twice a week at uscc) for a total of 170 minutes per week. at both the institutions, students worked in a group of 2-4 students during each lab period. though microbiology is a sophomore level class at both the institutions, at uscc it has no prerequisite and almost all the pre-nursing students take it during their freshman year. in contrast, at cu, first year biology and chemistry are the prerequisites to take microbiology, and hence all the students enrolled in microbiology are sophomores. at both the institutions, the traditional lecture discussion format was used in the classroom (lecture followed by discussions from student questions). despite the differences in class sizes and prerequisites, both the instructors had similar expectations from their students in terms of learning. both the courses had the same learning objectives and students at both the institutions were given the same preand post-tests. to minimize differences in teaching styles and content, both instructors communicated on a weekly basis to discuss the lesson plans and the mode of delivery. at the beginning of the semester, the purpose as well as the details of this research study were explained to the students. although, the participation in this study was voluntary and students were asked to sign a consent form, all the students present on day 1 of the semester, chose to participate. students were also explained that their identities would remain anonymous, the information they provided would always remain confidential, and their names would never be released in any form in the study. the data were not analyzed until after the semester was over and the students’ names or identities were not shared between the two instructors (authors of this paper). an irb approval was obtained at cu before the beginning of the study whereas no irb approval was required at uscc. a questionnaire and a test for each of the four selected topics in microbiology were developed (supplemental materials available upon request). the questionnaire consisted of fifteen questions and was administered at the beginning of the semester to collect information on: (a) the demographics of each student, i.e., age, gender, marital status, number of hours working per week, and citizenship status; (b) student preparedness, i.e., number of science courses they completed in high school, other biology courses completed in college before enrolling in microbiology, and their major; (c) 18 | fine focus students’ interest in microbiology and their attitudes, study habits, and behavioral engagement towards learning, i.e., their willingness to actively participate in the classroom, their attitude towards the laboratory participation, and their willingness to spend time learning outside the classroom. all the questions in the tests, administered before (pre-test) and after (posttest) class room discussion, were either selected from the test bank of the microbiology text books (difficulty level 1) or written by the authors at the lower levels of bloom’s taxonomy (knowledge and comprehension). each question was carefully selected to assess students’ basic knowledge and understanding in the following topics: ● introduction to microbiology ● cell structure ● microbial metabolism ● microbial growth we assessed the prior knowledge of basic concepts of students enrolled in microbiology class by administering a test before the topic was discussed in class. after the topic was discussed, we administered the same test as a post-test to determine their learning gains by comparing each student’s score on post-test to their score on pre-test. students were not told that they would be taking an identical test for both pre-test and post-test. each test comprised of a total of 10 questions worth 1 point each, for a maximum of 10 points. the data were collected from both the institutions for statistical analyses after the semester was over. we compared the demographics of students enrolled in the microbiology class at uscc and cu and related them to student learning gains on the post-test versus the pre-test. students’ responses to different questions on the questionnaire were statistically analyzed and the results are presented in fig. 1. to statistically analyze the differences in student demographics and their preparedness, a binary classification was used (table 1). the data for students’ attitudes were collected on a 5-point likert scale, where 1 represents the least value and 5 represents the highest value (for example, regarding interest in microbiology, 1 represents no interest and 5 represents high interest). to analyze differences between variables, a test of equality of means was used. we have used 94% level of confidence to accept or reject the null hypothesis. thus, p-values, based on t-tests, of less than or equal to 0.06 (6%) rejects the null hypothesis of equality of means between variables. eviews software was used for statistical analysis. 3. results at the beginning of the semester, on the first day of classes, a questionnaire was administered to collect the basic information of students enrolled in microbiology class at both the institutions. fig. 1 (a), (b), and (c) summarize the differences between uscc and cu students based on their responses to our questionnaire. fifteen students at uscc and 70 students at cu completed and submitted the questionnaire. based on the analysis of our data, fig. 1 (a) shows that cu had volume six | 19 table 1. binary classification used for the statistical analyses for the variables relating to students’ demographics, preparedness, and major. 20 | fine focus a statistically significant higher proportion of younger (age 25 or below) and single (based on their marital status) students as compared to uscc. however, our results show no statistically significant difference in student composition in terms of gender, employment status, and nationality. as shown in fig. 1 (b), in case of students’ preparedness, a statistically significant higher proportion of cu students had completed three or more science classes during high school and had completed one or more college level biology classes before enrolling in microbiology class as compared to uscc students. thus, the students at cu were academically better prepared to learn microbiology (or any science course) relative to students at uscc. also, a statistically significant higher proportion of students at cu were biology, microbiology or pre-med majors relative to uscc. fig. 1 (c) summarizes the results with respect to students’ interest in microbiology and their attitudes towards learning. our results indicate no significant difference in students’ interest in microbiology or their desire to be an active leader during laboratory exercises at both the institutions. however, it is interesting to note that students at uscc were statistically more interested in participating in classroom discussions and in learning course material outside the classroom relative to students at cu. next, students’ performance in pre-tests and posttests at uscc and cu was analyzed to assess if mean preand post-test scores in the four topics statistically volume six | 21 figure 1a. comparison of student demographics between uscc (n = 15) and cu (n = 70) based on a survey administered on first day of class. binary classification of variables (age = 1, if below 25 years; gender = 1, if male; marital status = 1, if married; employment = 1, if hours worked > 10 hours; citizenship = 1, if domestic student and 0 otherwise) was used to test for equality of means between cu and uscc. results show that age and marital status were statistically significantly different between the students at uscc and cu. * p ≤ 0.06. 22 | fine focus figure 1b. comparison of student demographics between uscc (n = 15) and cu (n = 70) based on a survey administered on first day of class. binary classification (number of science classes completed in high school = 1, if 3 or more; college level biology classes completed before enrolling in microbiology = 1, if 1 or more; major = 1, if biology, microbiology, or pre-med, and 0 otherwise) was used to test for equality of means between cu and uscc. results show that the number of science classes completed in high school, number of college level biology classes completed before enrolling in microbiology, and students’ major were statistically significantly different between the students at uscc and cu. * p ≤ 0.06. volume six | 23 figure 1c. comparison of students’ interest and attitudes towards learning microbiology between uscc (n = 15) and cu (n = 70) based on a survey administered on first day of class. a 5-point likert scale was used to compare students’ interest and other attitudes towards learning, with 1 representing least interest in microbiology and 5 representing high interest in microbiology; 1 representing less than 1 hour to spend outside the classroom and 5 representing over 5 hours to spend outside the classroom; 1 representing not likely to actively participate in the class and 5 representing very likely to actively participate in class; 1 representing do not care about lab participation and 5 representing taking the lead role in the lab group. using test of equality of means, the figure show that hours devoted to learning outside the class and willingness for active class participation were statistically significantly higher amongst uscc students relative to cu students. * p≤ 0.06. 24 | fine focus vary between the students at two institutions. as shown in fig. 2, our results indicate that the student scores in pre-tests for introduction, cell structure, microbial metabolism, and microbial growth were statistically significantly higher (at 6% level) at cu relative to uscc, reflecting better prior knowledge about these topics at cu relative to uscc. however, the knowledge gap was closed as indicated by no statistically significant difference in students’ mean scores in introduction and microbial metabolism posttests between the two institutions. in other two topics, cell structure and microbial growth, students at cu had a statistically significant higher post test scores as compared to uscc students. we further analyzed and compared student performance in preand post-tests in different topics at both the institutions and the results are presented in fig. 3. our results show that classroom instruction led to significantly higher post-test scores (as compared to their pre-test scores) in all four topics at uscc and there was no significant statistical difference between pre-test and post-test scores in any of the four topics at cu. thus, based on our analysis, after students were exposed to the topic in the classroom at uscc they had higher learning gains (as reflected by statistically significant higher post-test scores of uscc students in each of the four topics as compared to their pre-test scores). to analyze learning gains between the students at uscc and cu, the difference in performance of each student before and after a topic was discussed was computed. the means of these differences were compared between the two institutions for each of the four topics. our analysis shows that students at uscc exhibited statistically significant higher learning gains (at 6% level of significance) in three out of four topics (introduction, cell structure, and microbial metabolism) (fig. 4). at cu, the students did not exhibit learning gains in any of the four topics. 4. discussion & conclusions this research was conducted to fill the current gap in the literature regarding learning gains of students in a microbiology class at two different types of institutions, a two-year community college in the u.s. and a fouryear canadian university, and their relationship to the demographics, preparedness, attitudes, study habits, and behavioral engagement towards learning. the results of this paper are based on (a) students’ responses to a questionnaire on their demographics, preparedness for an introductory course in microbiology, major, and their attitudes and study habits towards learning; and (b) students’ test scores in 4 different topics in microbiology, administered before and after the topic was taught in the classroom (preand posttests). further, all students’ responses were compared between 2-year college in the us and 4-year university in canada. our research finds several interesting results: based on our sample results, statistically, students at cu were relatively younger and unmarried when compared to students at uscc. statistically, students at uscc scored better grades in post-tests as compared to their pre-tests in all the topics. it may be mentioned that our volume six | 25 figure 2. comparison of mean performance of students in preand post-tests at uscc relative to cu. results from the test of equality of means show that uscc students’ mean scores were statistically significantly lower in all four pre-tests relative to cu students. however, performance of uscc students was statistically significantly lower in only 2 out of 4 post-tests (cell structure and microbial growth) relative to cu students. * p≤ 0.06. 26 | fine focus figure 3. comparison of means of the preand post-test scores of students at uscc and cu. test of equality of means shows that students at uscc scored statistically significant higher scores in all 4 post-tests relative to pre-tests, reflecting higher learning gains after the topic was discussed in class. however, performance of students at cu was not statistically significantly different between preand post-tests in all four topics. * p≤ 0.06. volume six | 27 figure 4. difference of means between the post-test vs pre-test scores at uscc and cu. test of equality of means shows that there was a statistically significant difference between the post-test versus pre-test scores amongst uscc students relative to cu students in 3 out of 4 topics (introduction, cell structure, and microbial metabolism). * p≤ 0.06. 28 | fine focus results reflect correlations between these variables rather than causality. however, it is interesting to note that richardson and king concluded that older students tend to exhibit more desirable approaches to learning in terms of both their persistence and attainment (21). newman-ford et.al also reported that despite attaining lower results overall, older students achieve a higher proportion of better grades and age had some impact upon educational achievement (20). our results show no statistical difference between cu and uscc with respect to other three demographic factors, gender, citizenship, and number of students working 10 hours or more per week. we found that statistically cu students were academically better prepared for introductory microbiology class relative to students at uscc, when preparedness is measured in terms of number of science courses completed during high school and their prior exposure to college level biology classes. a statistically higher proportion of cu students were biology/microbiology majors and had more exposure to science courses in high school and first year of college before taking microbiology class. our results show that students at cu had statistically significant higher pre-test scores as compared to students at uscc in all four topics studied. despite this, students at uscc scored statistically significant higher grades in all the post-tests as compared to their pre-tests. these results are contradictory to newman-ford et al. who found a strong, statistically significant relationship between prior attainment and subsequent results (20). our results are consistent with mlambo who also reported that entry qualifications do not significantly affect academic performance in an introductory biochemistry class (19). colorado and eberle also reported that academic performance in online learning environments was not significantly affected by the number of educational degrees attained (16). we also found that students at uscc were willing to devote more time outside the classroom to study and to actively participate in class to learn microbiology when compared to students at cu. our results show that students at uscc attained significantly higher posttest scores as compared to their pre-test scores. these results are consistent with the result of a study by harb and el-shaarawi that students who participate in class discussion outperform students who do not (22). we then compared students’ scores on post-test with students’ scores on pre-test at each of the two institutions separately. interestingly, we found that the posttest scores of students in all four topics were statistically significantly higher from their pre-test scores at uscc. no such statistical difference between students’ performance on preand post-tests in the case of cu students was found. it is evident from the questionnaire data that students at uscc were not as well prepared to handle the rigor of college microbiology class (as evident from their lower scores in pre-tests, number of science classes completed at high school, and number of biology courses completed at college before enrolling in microbiology). but after exposure to the topic in the class they achieved statistically higher learning gains as compared to the students at cu. a study by yager et al. showed that students with or without prior high school science work exhibited similar performance on all assessments after course completion simply because volume six | 29 students without high school science program worked extra hard and devoted more time to learning (25). an interesting question is why students’ scores were consistently higher on post–test relative to pre-test in case of uscc and not so in the case of cu. there could be several explanations for this. the class size was much larger at cu relative to uscc. though the same lecture-discussion format was used to discuss a topic in the classroom and both the instructors tried to use the class and lab time in a similar manner, due to the smaller class size at uscc, students might have had the advantage of getting more interaction with the instructor and among themselves as compared to the 4-year university where the class size was big. though, at both the institutions, instructors maintained regular office hours to interact individually with the students and involve them in one-on-one discussion, the students at cu who were mostly biology/microbiology majors with heavier course load and more exposure to college level courses may not have utilized that opportunity. based on students’ responses, our results indicate that students at cu were better prepared to learn microbiology, as statistically a higher proportion of them had completed three or more science classes during high school and were also exposed to college level biology courses. it is also evident from their significantly higher pre-test scores in all the topics. despite the lower pre-test scores of uscc students, statistically they were more willing to spend more time outside the classroom to study and actively participate in the classroom to learn when compared to cu students and it is evident from their higher learning gains. statistically, at uscc, students were older and married and hence, may be more responsible and committed to learning as compared to more traditional students at cu. as reported by newman-ford et al. despite attaining lower results overall, older students achieve a higher proportion of better grades and age had some impact upon educational achievement (20). richardson and king also concluded that older students tend to exhibit more desirable approaches to learning in terms of both their persistence and attainment (21). it must be acknowledged that though students at cu did not have any statistically significant learning gains in any of the four topics in microbiology, they still had higher overall scores on the post tests. however, performance of uscc students was statistically significantly lower in only 2 out of 4 post-tests (cell structure and microbial growth) relative to cu students. in conclusion, our results indicate that if students are willing to spend time to learn, and have the right attitudes towards learning, they can overcome their prior deficiencies and achieve higher learning gains. this study, though preliminary and exploratory, has created a space for the further investigation of learning gains of major and non-major students in a microbiology class at two different types of institutions with different student demographic. it serves as a 30 | fine focus starting point of research in such an area. 5. research challenges this research has few limitations. first, it is limited by the differences in class size at the two institutions. cu had a significantly larger class size as compared to uscc. second, though every effort was made to follow the same type of lecture-discussion format in both the classrooms and both instructors were in frequent communication via phones and email messages about the instructions and worked closely together throughout the project, the individual instructor differences might have caused some differences observed in the results. third, at uscc, students could enroll in microbiology as freshmen and the course has no prerequisite, whereas at cu it is a sophomore level class with a year of biology and chemistry as prerequisites and, hence, students at cu were academically better prepared for the class. 6. suggestions for future research this research was done at one 2-year community college and one 4-year university for one semester. it will be interesting and useful to expand it to include more 2-year and 4-year institutions in the u.s. and canada to increase the sample size and conduct additional statistical investigation to further explain the differences in students’ learning gains at different institutions. acknowledgements the authors are grateful to dr. anil k. lal, professor of economics for his numerous suggestions during statistical analysis of the data. the authors are also grateful to the american society for microbiology’s biology scholars 2016 writing and publishing course, from science education research to publication workshop, and would like to thank the course cohort, dr. marcy kelly, the course instructor, and other mentors for the meaningful discussions and their feedback during the writing phase of this manuscript. our thanks go to drs. karen densky and catharine dishke hondzel and kara loy of the centre for excellence in learning and teaching, brenda smith and shane neifer of library, gabrielle kam and chadabhorn insuk of faculty of science, for their assistance. the authors declare that there are no conflicts of interest. volume six | 31 references 1. the government of canada. 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(1998). adult students in higher education: burden or boon. journal of higher education, 69, 65-88. 22. harb, n., & elshaarawi, a. (2007). factors affecting students’ performance. journal of business education, 82(5), 282-290 23. tahir, i. m., & abu baker, n. m. (2009). influence of demographic factors on students’ beliefs in learning mathematics. international education studies, 2(3), 120-126. 24. tai, r.h., sadler, p. m., & loehr, j. f. (2005). factors influencing success in introductory college chemistry. journal of research in science teaching, 42(9), 987-1012. doi: 10.1002/tea.20082 25. yager, r. e., snider b., & krajcik, j. (1988). relative success in college chemistry for students who experienced a high school course in chemistry and those who had not. journal of research in science teaching, 25(5), 387-396. doi:10.1002/tea.3660250506 erratum: author information has been updated as follows. archana lal1 & naowarat cheeptham2 1corresponding author; labette community college, parsons, ks 67357, usa email: archanal@labette.edu 2department of biological sciences, thompson rivers university, kamloops, b.c., canada mailto:archanal@labette.edu 3318-article text-5931-1-10-20201026 erratum bdellovibrio bacteriovorus protects caenorhabditis elegans from bacterial pathogens elizabeth a. b. emmert, zachary m. haupt, katherine m. pflaum, jennifer l. lasbury, justin p. mcgrath, allison e. collins, and christopher h. briand department of biological sciences, salisbury university, salisbury, maryland copyright 2014, fine focus all rights reserved manuscript received 29 april, 2014; accepted 14 june, 2014 bdellovibrio bacteriovorus is a naturally predatory bacterium that multiplies inside gram negative prey bacteria. there is much interest in using bdellovibrio as a living antibiotic to control infections by gram negative pathogens. in recent years caenorhabditis elegans has proven to be an attractive animal model of bacterial pathogenesis for a range of pathogens. we have used the c. elegans animal pathogenesis model to examine the ability of b. bacteriovorus to protect nematodes from four bacterial pathogens. in all cases, nematodes treated with b. bacteriovorus and the pathogen survived at a significantly higher level than nematodes treated with the pathogen alone. treatment with b. bacteriovorus alone was nontoxic to the worms. we monitored the persistence of e. coli k-12 and e. coli op50 in both b. bacteriovorus treated nematodes and control nematodes. e. coli k-12 levels were significantly lower in b. bacteriovorus treated nematodes than in control nematodes one day after bdellovibrio exposure and e. coli k-12 was eliminated from the worm gut two days faster in b. bacteriovorus treated nematodes. e. coli op50 also demonstrated significantly lower levels in b. bacteriovorus treated nematodes and faster elimination from the worm gut. the successful use of b. bacteriovorus as a therapeutic agent in c. elegans indicates that it may be useful as a living antibiotic in other animal systems. abstract corresponding author elizabeth a. b. emmert eaemmert@salisbury.edu keywords • bdellovibrio bacteriovorus • caenorhabditis elegans • pathogenesis, biocontrol • infection model 52 • fine focus, vol. 1 introduction bdellovibrio bacteria are intriguing because they naturally reproduce inside other gram negative bacteria. the bdellovibrio life cycle involves attachment to and penetration of prey cells, elongation inside the prey periplasm using prey components for growth, fragmentation into multiple cells, and finally, lysis of the prey cell (1). because bdellovibrio lyses prey as it multiplies, and because it cannot infect eukaryotic cells, there is growing interest in using bdellovibrio as a “living antibiotic” (2). numerous researchers have demonstrated in vitro killing of pathogens by bdellovibrio, (3, 4, 5, 6) supporting the idea of using bdellovibrio to control infections. additionally, bdellovibrio has been shown to attack prey within bacterial biofilms and reduce biofilm biomass (7, 8, 9). two studies have put the living antibiotic concept into practice, demonstrating protection against aeromonas hydrophila infection in fish and protection against proteus penneri infection in shrimp through the use of bdellovibrio bacterial host interactions • 53 (10, 11). fish and shrimp mortality was significantly lower when the animals swam in water containing both the pathogen and bdellovibrio as compared to animals in water containing only the pathogen. however, it was not determined whether the mechanism of bdellovibrio protection was simply a reduction of the pathogen level in the water, the killing of the pathogen within the animal, or a combination of the two. until recently, the use of bdellovibrio as an in vivo treatment for infection has been an intriguing, but theoretical option. in 2011 atterbury et al. demonstrated bdellovibrio could be used therapeutically to control salmonella infection in chickens without negative effects on the birds (12). this was the first study to demonstrate in vivo efficacy of bdellovibrio as a treatment for bacterial infection. here we continue the use of bdellovibrio as an in vivo therapeutic agent, but in the c. elegans bacterial pathogenesis model. in 1999 tan et al. first reported the use of the nematode c. elegans as an animal model for bacterial pathogenesis (13). since then numerous researchers have demonstrated that this system can be used for multiple bacterial pathogens including pseudomonas aeruginosa, salmonella enterica, serratia marcescens, and staphylococcus aureus (14, 15). genes identified in c. elegans as important in pathogenesis have been confirmed in mouse models of pathogenesis, validating the use of c. elegans as a pathogenesis model (16). using c. elegans as an animal model for pathogenesis is attractive for numerous reasons such as low cost, short generation time, complete genome sequence and ease of genetic manipulation (17). when c. elegans are maintained in the laboratory they are grown on petri plates containing lawns of nonpathogenic e. coli op50 as their food source and the worms typically live two weeks (18). when grown on a pathogen instead of op50, worm survival is greatly reduced (16). our lab has taken advantage of the wellstudied c. elegans bacterial pathogenesis model system to examine the use of bdellovibrio to protect c. elegans from bacterial infection. in this study, we first established an infection in the nematode and then examined the curative effect of a brief exposure to bdellovibrio. we show that worms treated with both bdellovibrio and a pathogen live significantly longer than worms treated with the pathogen alone. we also demonstrate that bacterial levels are lower and cleared faster in bdellovibrio treated worms than control worms. this work demonstrates that bdellovibrio can be used as a therapeutic treatment for bacterial infections in a welldefined animal model. materials and methods nematode and bacterial strains wild type c. elegans n2 worms were used in all nematode assays. worms and nonpathogenic e. coli op50 were supplied by the caenorhabditis genetics center (minneapolis, mn). worms were grown on nematode growth medium (ngm) with e. coli op50 as the food source (18). pathogens tested were e. coli k-12, enterobacter aerogenes atcc 13048, pantoea agglomerans ls005, and salmonella enterica serovar typhimurium lt2 (19). b. bacteriovorus hd100 was used for all biocontrol assays (20). e. coli hb101 was used as the nonpathogenic control in the biocontrol assays since our early work in this system used b. bacteriovorus 109j, which does not infect e. coli op50, but does infect e. coli hb101. 54 • fine focus, vol. 1 however, all the experiments described here used b. bacteriovorus hd100, which does infect both e. coli op50 and e. coli hb101. b. bacteriovorus hd100 was cultured using e. coli k-12 as prey according to standard protocols (21). b. bacteriovorus prey lysates were checked microscopically for active, motile b. bacteriovorus cells and an absence of prey cells. prey lysates contained approximately 6 x 108 b. bacteriovorus cells per ml. the persistence assays utilized kanamycin-resistant e. coli k-12 derivative strain jw1863-1 (22), supplied by the e. coli genetic stock center (new haven, ct) and ampicillin-resistant e. coli op50-gfp strain db15, kindly supplied by j. ewbank (centre d’immunologie de marseille-luminy, marseille, france). pathogenicity assay bacteria were grown overnight in lb broth and 50 μl culture was spread on 60 mm diameter ngm plates. plates were incubated for two days at 25°c to establish bacterial lawns. c. elegans were reared on ngm with lawns of e. coli op50 as the food source. one-day old adult worms were placed on ngm plates containing lawns of bacteria. worm survival was monitored daily for the next nine days. worms were considered dead when they did not respond to gentle prodding with a platinum wire. surviving adult worms were transferred daily to fresh bacterial lawn plates to separate them from newly hatched juvenile worms. each trial measured the survival of 30 worms per treatment. biocontrol assay bacteria were grown overnight in lb broth and 50 μl culture was spread on ngm plates. plates were incubated for two days at 25°c to establish bacterial lawns. c. elegans were reared on ngm with lawns of e. coli op50 as the food source. one day old adult worms were placed on ngm plates containing lawns of a pathogen or nonpathogenic e. coli hb101. after exposing the worms to the pathogen or hb101 for 48 hours (32 hours for e. coli k-12), worms were washed three times in ca/hepes buffer (21) to remove external bacteria. e. coli k-12 treated worms were exposed to e. coli for 32 hours instead of 48 hours because a 48 hour exposure to e. coli k-12 was too toxic and killed the majority of the worms. washed worms were suspended in 1 ml of an active b. bacteriovorus prey lysate or 1ml of ca/hepes buffer for 15 minutes. a 15 minute exposure to b. bacteriovorus was chosen because this is the time required for b. bacteriovorus to attach to prey cells (2). then the worms were pelleted and placed on ngm plates containing lawns of the nonpathogenic e. coli hb101. worms were transferred to new e. coli hb101 plates daily and worm survival was monitored daily for the next seven days. each trial measured the survival of 40-50 worms per treatment. e. coli persistence in c. elegans nematodes were exposed to an antibioticresistant strain of e. coli (32 hour exposure for kanamycin-resistant e. coli k-12 derivative jw1863-1 or 48 hour exposure for ampicillin-resistant e. coli op50-gfp strain db15) followed by three washes in ca/hepes buffer. the washed worms were suspended for 15 minutes in either 1 ml of an active b. bacteriovorus prey lysate or 1 ml of ca/hepes buffer, then pelleted and placed on ngm plates with e. coli hb101 lawns. worms were transferred daily on to fresh e. coli hb101 plates as described above for the biocontrol assays. numbers of internal bacteria persisting in the nematodes after b. bacteriovorus or buffer exposure were determined daily using the protocol of garsin et al. (23) with the following modifications. briefly, 5 worms were placed on a lb agar plate containing the appropriate antibiotic (50 μg/ml) and washed twice with 4 μl results pathogenicity assay we tested the pathogenicity of four species of bacteria, comparing them to the standard, nonpathogenic e. coli op50 routinely used to maintain c. elegans. all four species tested were pathogenic when compared to e. coli op50, greatly reducing worm survival (fig. 1). the pairwise comparisons examining worm survival between the four pathogens indicated that all four pathogens were similar in pathogenicity (p=0.9926). we also tested e. coli hb101 and found it to be nonpathogenic. worm survival on e. coli hb101 was not significantly different from worm survival on e. coli op50 (p=0.5482). worms grown on all four pathogens survived significantly less than worms grown on e. coli op50 (p<0.001) and worms grown on all four pathogens survived significantly less than worms grown on e. coli hb101 (p<0.001). we proceeded to use e. coli hb101 as the c. elegans food source when monitoring worm survival in our biocontrol assays rather than e. coli op50 since our early work in this system used b. bacteriovorus strain bacterial host interactions • 55 figure 1. survival curves for c. elegans exposed to e. coli op50 ( ), e. coli hb101 ( ), e. coli k-12 ( ), e. aerogenes ( ), p. agglomerans ( ), and s. enterica ( ). data are from one trial representative of two independent trials. 100 90 80 70 60 50 40 30 20 10 0 0 1 2 3 4 5 6 7 8 9 10 pe rc en t s ur vi va l time (days) m9 medium to remove surface bacteria. washed worms were suspended in 20 μl m9 medium and ground with a pestle. 30 μl of m9 medium was added to the worm solution to bring the total volume up to 50 μl; the solution was diluted in ca/hepes buffer and plated on lb agar containing the appropriate antibiotic (50 μg/ml) for bacterial enumeration. statistics kaplan-meir survival analysis followed by pairwise logrank tests (24, 25, 26) was used to analyze c. elegans survival over time. the mann whitney test was used to analyze e. coli persistence data. data analyses were performed using graphpad prism® 4 (27). the significance level for all statistical analyses was set at a = 0.05. 56 • fine focus, vol. 1 pe rc en t s ur vi va l time (days) 100 90 80 70 60 50 40 0 2 4 6 8 pe rc en t s ur vi va l time (days) 100 90 80 70 60 50 40 30 0 2 4 6 8 0 1 2 3 4 5 6 7 8 30 40 50 60 70 80 90 100 time (days) pe rc en t s ur vi va l 0 2 4 6 8 40 50 60 70 80 90 100 time (days) pe rc en t s ur vi va l a. b. d. c. time (days) time (days) time (days) time (days) pe rc en t s ur vi va l pe rc en t s ur vi va l pe rc en t s ur vi va l pe rc en t s ur vi va l fig. 2. survival curves for c. elegans exposed to (a) e. coli k-12 (b) e. aerogenes (c) p. agglomerans and (d) s. enterica. worms were treated with nonpathogenic e. coli hb101 ( ), hb101 and bdellovibrio ( ), pathogen ( ), or pathogen and bdellovibrio ( ). worms were exposed to bdellovibrio or control buffer on day one. data are from three independent trials for each pathogen. p values for pairwise comparisons in the biocontrol assay survival curves.. pathogen comparison hb101 vs. hb101 + bda hb101 vs. pathogen hb101 vs. pathogen + bd hb101 + bd vs. pathogen hb101 + bd vs. pathogen + bd pathogen vs. pathogen + bd e. coli k-12 0.4958 <0.0001 0.0047 <0.0001 0.0412 <0.0001 e. aerogenes 0.4402 <0.0001 <0.0001 <0.0001 <0.0001 <0.0001 0.7376 <0.0001 0.0207 <0.0001 0.0098 <0.0001 s. enterica 0.7318 <0.0001 0.1901 <0.0001 0.3292 <0.0001 a bd indicates p. agglomerans table 1 abd indicates bdellovibrio p values for pairwise comparisons in the biocontrol assay survival curves. bacterial host interactions • 57 109j, which did not prey on e. coli op50. biocontrol assay to determine whether b. bacteriovorus could protect nematodes from bacterial pathogens, we established infections in the nematodes, briefly treated infected worms with b. bacteriovorus, placed worms on non-pathogenic e. coli hb101, and monitored worm survival for seven days. for all four pathogens tested, worm survival was significantly improved when worms were treated with b. bacteriovorus as compared to the pathogen alone (fig. 2). for each pathogen, the pairwise comparison between worms treated with the pathogen alone and worms treated with both the pathogen and bdellovibrio was highly significant (table 1). worm survival was unaffected by b. bacteriovorus treatment when worms were grown on nonpathogenic e. coli hb101 (table 1), demonstrating that b. bacteriovorus is nontoxic to worms. bdellovibrio and pathogen treated worms had significantly longer survival than worms treated with the pathogen alone. however, for three of the four pathogens, bdellovibrio treatment was unable to restore the same level of worm survival as with the nonpathogenic e. coli hb101 control, and there were still significant survival differences between control worms and pathogen plus bdellovibrio treated worms. s. enterica infection was the only one completely rescued by bdellovibrio with no significant difference in survival curves between control worms and s. enterica plus bdellovibrio treated worms (table 1). e. coli persistence in c. elegans we also monitored the persistence of one of the four pathogens (a kanamycinresistant derivative of e. coli k-12) as well as ampicillin-resistant e. coli op50 in both 1 2 3 4 5 6 7 0 1 2 3 4 time (days) lo g cf u pe r 5 w or m s a a a a a a b b * * * fig. 3. persistence of antibiotic-resistant derivatives of (a) e. coli k-12 and (b) e. coli op50 within c. elegans treated with bdellovibrio (black bars) or control buffer (grey bars). worms were treated with bdellovibrio or control buffer on day zero. values with the same letter for a single time are not significantly different (p≤0.05). asterisks indicate values with zero variance and thus these days were excluded from analysis. log transformed data are from four independent trials and error bars indicate standard error. 1 2 3 4 5 0 1 2 3 time (days) lo g cf u pe r 5 w or m s b a a a * * time (days) time (days) a. b. lo g c fu p er 5 w or m s lo g c fu p er 5 w or m s discussion while many have used c. elegans as a model for bacterial pathogenesis, we have extended that model to investigate control of four bacterial pathogens by bdellovibrio. the non-vertebrate c. elegans has many advantages as an animal model for bdellovibrio infection control studies including short life span, ease of manipulation, low cost, consumption of bacteria as food, and absence of ethical concerns. our work in c. elegans supports and extends earlier work using bdellovibrio as a therapeutic agent to control bacterial infections in chickens (12). interestingly, the one log reduction in s. enterica by bdellovibrio in chickens is similar to the reduction in e. coli k-12 levels we demonstrated in c. elegans (fig. 3a). in agreement with the chicken study, our work demonstrated improved animal health with a single, discrete dose of bdellovibrio. using bdellovibrio to control infection is often compared to bacteriophage therapy with bdellovibrio having the advantage of a wider prey range than phage (2). indeed, similar to our results, one group has demonstrated the ability of phage to protect c. elegans from salmonella infection (28) confirming the robustness of the c. elegans model. our pathogenicity assay results demonstrate a clear difference in nematode survival between the four pathogens tested and the two non-pathogenic e. coli strains (fig. 1). this highly significant survival difference is also reflected in the biocontrol assay comparing the hb101 treated worms with the pathogen treated worms (fig. 2). although e. coli k-12 is typically considered to be nonpathogenic in animal models and our referring to e. coli k-12 as a pathogen may seem inaccurate, others have also demonstrated that e. coli k-12 is pathogenic in c. elegans (29). e. coli op50 is the strain typically used as a nonpathogenic food source for c. elegans; however we have demonstrated that e. coli strain hb101 is also nonpathogenic. similar nematode survival curves between op50 and hb101 have also been demonstrated by researchers examining the effect of bacterial nutrition on c. elegans lifespan (30). interestingly, when survival is examined beyond ten days, worms live longer on hb101 compared to survival on op50 (30). although bdellovibrio provided intermediate protection from most pathogens, the significant improvement in survival along with the complete protection of salmonella 58 • fine focus, vol. 1 bdellovibrio treated and control worms. one day after exposure to bdellovibrio or a control buffer, e. coli k-12 levels were significantly lower in worms treated with bdellovibrio compared to control worms (fig. 3a). levels of pathogenic e. coli k-12 decreased to undetectable levels in worms three days after bdellovibrio treatment, while it took five days for pathogenic e. coli to drop below detectable levels in control worms. e. coli op50 showed a similar trend in that bacterial levels were lower in bdellovibrio treated worms, although a significant difference between bdellovibrio treated and control worms was not detected until three days after bdellovibrio treatment (fig. 3b). e. coli op50 was also cleared to undetectable levels faster in bdellovibrio treated worms and e. coli op50, unlike e. coli k-12, persisted in the control worms for the entire seven day experiment. the limit of pathogen detection was five cfu per five worms. treated worms clearly demonstrates the protective ability of bdellovibrio in this system (fig. 2 and table 1). the variation in bdellovibrio protection of c. elegans from pathogens may be due to the difference in bacterial colonization of the worms. s. enterica serovar typhimurium kills worms through a persistent intestinal colonization while e. coli kills through a non-persistent intestinal colonization (16). the ability of s. enterica to multiply within and distend the worm intestinal lumen, establishing a persistent infection after the worms are no longer being fed s. enterica cells (31), may provide a more concentrated source of pathogen cells to support increased bdellovibrio growth and predation, leading to complete recovery from infection. interestingly, these data suggest that the more numerous the pathogen cells are in the host, the more effective bdellovibrio treatment may be for resolving the infection. we followed the persistence of two e. coli strains in this system using antibioticresistant derivatives of e. coli k-12 and e. coli op50 to examine the effect of bdellovibrio on e. coli clearance from the worm. pathogenic e. coli k-12 levels were significantly lower in bdellovibrio treated worms one day after treatment and e. coli k-12 was cleared from the worms two days quicker in bdellovibrio treated worms (fig. 3a). this marked reduction in pathogenic e. coli levels by bdellovibrio was enough to significantly improve worm survival, but not enough to restore worm survival back to the level seen in non-pathogen treated control worms (table 1). our results are based on a single, 15 minute exposure of the worms to bdellovibrio and increased survival may occur with longer or repeated exposures of the worms to bdellovibrio. we chose a 15 minute exposure to allow time for bdellovibrio to attach to prey cells and begin invasion of the prey cell (2). even without bdellovibrio treatment, e. coli k-12 was cleared from the worms, in agreement with earlier research demonstrating that pathogenic e. coli does not establish a persistent infection in worms (16). levels of nonpathogenic e. coli op50 were also significantly lower and cleared faster in bdellovibrio treated worms (fig. 3b). however, unlike e. coli k-12, nonpathogenic e. coli op50 was able to persist in the control worms for seven days. the levels of e. coli op50 we detected in control worms on day one agree closely with those found by others investigating viable e. coli op50 counts in c. elegans lysates (30), validating our work in this system. c. elegans appears to be an ideal model system for refining and exploring the use of bdellovibrio as a therapeutic agent. since c. elegans is a bacteriovore, exposure of the worms to pathogenic bacteria is simple and easy. the lower growth temperatures favored by c. elegans (20-25°c) compared to birds and mammals coupled with bdellovibrio’s optimal growth temperature of 28°c makes c. elegans an attractive animal system to investigate the use of bdellovibrio as a biocontrol agent. we administered bdellovibrio as a liquid treatment for precise, controlled dosing, but worms could also be treated with bdellovibrio through placement on plaque plates (17) containing both the pathogen and bdellovibrio. our work prepares the way for future experiments with c. elegans and bdellovibrio to examine additional pathogens, dosage and frequency of bdellovibrio treatment, persistence of bdellovibrio in worms, effect (if any) of bdellovibrio on worm morphology, as well as other variables. while an intriguing hypothesis, the use of bdellovibrio as a feasible therapeutic agent has only been demonstrated in vivo in chickens against salmonella (12). here we extend bacterial host interactions • 59 this work was funded by the richard a. henson school of science and technology at salisbury university. thanks to rebecca pinekenstein for her work in the early stages of this research. thanks to jonathan ewbank for supplying the e. coli op50-gfp strain db15. thanks to j. martin bland, dept. of health sciences, university of york, york, uk for information on the logrank tests. references acknowledgements 1. sockett, r. e. 2009. predatory lifestyle of bdellovibrio bacteriovorus. annu. rev. microbiol. 63:523-539. 2. sockett, r. e. & lambert, c. 2004. bdellovibrio as therapeutic agents: a predatory renaissance? nat. rev. microbiol. 2:669-675. 3. fratamico, p. m. & cooke, p. h. 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graphpad software inc. 28. santander, j. & robeson, j. 2004. bacteriophage prophylaxis against salmonella enteritidis and salmonella pullorum using caenorhabditis elegans as an assay system. electron. j. biotechnol. 7:206-209. 29. browning, d. f., wells, t. j., franca, f. l. s., morris, f. c., sevastsyanovich, y. r., bryant, j. a., johnson, m. d., lund, p. a., cunningham, a. f., hobman, j. l., may, r. c., webber, m. a., & henderson, i. r. 2013. laboratory adapted escherichia coli k-12 becomes a pathogen of caenorhabditis elegans upon restoration of o antigen biosynthesis. mol. microbiol. 87:939950. 30. so, s., tokumaru, t., miyahara, k., & ohshima, y. 2011. control of lifespan by food bacteria, nutrient limitation and pathogenicity of food in c. elegans. mech. ageing dev. 132:210-212. 31. aballay, a., yorgey, p., & ausubel, f. m. 2000. salmonella typhimurium proliferates and establishes a persistent infection in the intestine of caenorhabditis elegans. curr biol. 10:1539-1542. 32. iebba, v., santangelo, f., totino, v., nicoletti, m., gagliardi, a., debaise, r. v., cucchiara, s., nencioni, l., conte, m. p., & schippa, s. 2013. higher prevalence and abundance of bdellovibrio bacteriovorus in the human gut of healthy subjects. plos one 8:e61608. bacterial host interactions • 61 6 | fine focus objective lens addressing the global antibiotic resistance crisis through a microbiology undergraduate course-based research experience (cure) dr. john mckillip lead editor and professor of biology, ball state university, muncie, in volume six | 7 fine focus has, since our first issue back in 2014, showcased undergraduate research efforts globally. we have grown in many ways and have developed our review team and marketing team activities to continually evolve and stay relevant. through our recent partnership with the small world initiative (www.smallworldinitiative.org), fine focus would like to be a venue to publish novel findings on new antibiotic discovery. last year, ball state university became one of the newest partner institutions with the swi, and i’d like to share with you here how we are in the process of redesigning our majors microbiology course into a format that will allow for crowdsourcing new antibiotic discovery, all the while leading eventually to original research manuscripts publishable in fine focus. i invite and welcome other puis to do the same and would be happy to answer questions on how your cure could submit a manuscript to our journal and showcase the work of your undergraduates, or to collaborate with us. statement of the problem: if no new antibiotics are isolated and made available by 2050, the cdc estimates that 10 million annual deaths will occur globally as a result of this inaction. yet, surprisingly few have even heard of antibioticresistant bacteria or understand the implications for global health. in fact, no new classes of antibiotics have been developed to treat microbial infections in well over 30 years, as pharmaceutical companies have instead pursued research and development of more lucrative drugs for non-infectious diseases. since this trend is likely to continue into the foreseeable future, this crisis must be addressed using alternative creative approaches. we propose to change our undergraduate microbiology class laboratory’s focus from a traditional model to a course-based undergraduate research experience (cure) design. specifically, this effort at ball state university to become a cure can and must involve collaborations with colleagues in the bsu department of chemistry to isolate and identify novel antibiotics from soilborne bacteria and fungi as well as a variety of dairy-relevant natural environments (raw milk, silage, manure, aged cheese, and feedlot samples). these natural environments have not been widely explored in this context before, and yet we will be able to rapidly screen hundreds of isolated bacteria and fungi using standard microbiological methods and well-established chemistry separation protocols used routinely in undergraduate teaching labs. using resources, expertise, and protocols modified from the small world initiative (swi), our goal is to isolate and identify multiple novel compounds from bacteria. transforming the undergraduate microbiology curriculum in this way is completely new teaching and research direction for the pi, and will blend pedagogy and original research into an entirely different direction than at present. description of cure development: increasing bacterial resistance spanning over 70 years, combined with a cessation in the discovery of new antibiotics for over a generation has resulted in a global healthcare catastrophe. crowdsourcing antibiotic discovery is a viable option to meet the challenge of this crisis. the american society for microbiology has been instrumental in supporting the small world initiative, swi, (www.smallworldinitiative. org), an international not-for-profit consortium of research and teaching universities in over 17 countries. swi began in 2012 at yale university, and now has partner universities, community colleges, and high school partner institutions in 43 u.s. states. the swi provides a wide range of expertise and physical resources for faculty to transform their research agenda and/or laboratory teaching curricula to systematic discovery of new antibiotics from natural environmental samples, largely soil. this effort will overview our plans to retool our undergraduate microbiology course laboratory’s focus to directly 8 | fine focus align with the swi mission in order to identify and isolate new types of antibiotics with promise in the global healthcare sector. we will do this by rewriting the class lab to become a course-based undergraduate research experience (cure). our hypothesis is that through the cure model, we will be able to isolate multiple species of naturally occurring bacteria and fungi from raw dairy milk (cow and goat), silage, manure, aged cheese, and feedlot samples, and that at least one novel antimicrobial compound (antibiotic) will be isolated and purified from the host microorganism on a larger scale for promise as a chemotherapeutic agent.  goal and objectives: our research goal is reflected in our hypothesis. within this broad goal, we have two smaller objectives that will be addressed over the course of two semesters using a total of 150 undergraduate microbiology students (~75 each semester) in the cure structure: using standard microbiological methods, we will isolate and identify multiple bacteria and/or fungi, at least one of which will produce a secreted compound demonstrating measurable antibacterial activity. secondly, we will use traditional chemistry separation and analytical methods to purify (on a larger scale) and determine the structure and specific activity of each antibiotic quantified in the first objective. using a modified course syllabus and student learning outcomes suggested by the american society for microbiology (asm) swi, we will be able to pilot the cure for two semesters in order to generate the necessary research results and preliminary assessment data essential to show proof of concept and submit a larger, more collaborative proposal to nsf for external funding in early fall 2021. assessment: a combination of both formative and summative assessments will be used and applied throughout the semester, including but not limited to weekly written quizzes, materials and methods oral selfquizzes (given among the student working groups), and an end-of-the semester capstone poster for each group presented at the spring university student research symposium. vigorous consultation will occur regularly throughout both semesters with our two science education research faculty, and assessment experts available through the swi faculty network to ensure measureable data are being collected from these assessments to include in the nsf proposal upon submission in 2021. 83applied & environmental microbiology • chytrid fungi associated with pollen decomposition in crater lake, oregon kathleen a. page* and meghan k. flannery manuscript received 25 october 2017; accepted 27 january 2018 copyright 2018, fine focus. all rights reserved. department of biology, southern oregon university, ashland, or usa 84 • fine focus, vol. 4(1) we identified chytrid fungi that were attached to pine pollen on the surface of crater lake. fungi were identified by large subunit (lsu) rrna gene sequencing of lake pollen extracts and by isolation of a chytrid fungus that was present on the pollen. lsu rrna pcr products were cloned, sequenced and identified. the majority of eukaryotic lsu rrna sequences associated with pollen were found to be members of the chytrid order rhizophyidiales. a fungal isolate was characterized culturally, morphologically, and by dna sequencing and was identified as a member of the genus paranamyces, in the order rhizophydiales. in addition, protist lsu rrna sequences from the phylum ciliophora were found. the concentrations of dissolved organic matter, nitrogen, and phosphate in surface water that had visible pollen rafts increased according to the concentration of pollen in the water. each of these nutrients was detected at several fold higher levels in water with pollen rafts as compared to surface water lacking pollen rafts. these results provide evidence for the role of chytrid fungi in nutrient release from pollen deposited on crater lake. abstract kathleen a. page page@sou.edu keywords • chytrid • pollen • crater lake ecosystem • food webs • fungal aquatic ecology the occurrence of pollen in crater lake: seasonal pine pollen deposition is a source of allochthonous organic matter for crater lake, oregon. crater lake is a high-elevation, collapsed volcanic caldera lake and the deepest lake in the united states. it has an average depth of 350 m and a maximum depth of 594 m. it is a nitrogen limited, oligotrophic lake that supports a relatively low density of phytoplankton, heterotrophic bacteria, fungi, zooplankton, and fish (12). during spring and early summer, coniferous forests in the vicinity of crater lake produce massive amounts of wind-dispersed pollen, introduction corresponding author 85applied & environmental microbiology • primarily from pine trees including pinus contora and pinus ponderorsa (35). pine pollen grains have air bladders that allow them to float on water and form yellow aggregates known as pollen rafts. the appearance of pollen rafts on crater lake and on many northern forest lakes is a yearly occurrence (7, 16, 33, 44). terrestrial organic matter does not flow readily into crater lake; it is fed by rain, snowmelt, and groundwater. other than a few small springs on the steep caldera walls, there are no stream inputs and sources of allochthonous organic matter are presumed to be scarce. crater lake has seasonal stratification of the water column and deep-water mixing events every 2 to 5 years. pollen dispersal occurs during summer stratification when warmer water floats on top of colder, denser water. this effect provides stability within the photic zone and would be expected to favor growth of microbial communities associated with buoyant pollen. however, pollen grains are highly refractory nutrient sources. when not colonized by microorganisms, they eventually sink and can be preserved intact in sediments for millennia (34). because pollen had not yet been studied as a nutrient source in crater lake, we set out to discover if the pollen is subject to frequent microbial colonization and what role, if any, fungi play in its decomposition. release of nutrients from pollen in support of aquatic food webs: pollen is rich in fatty acids and supplies organic and mineral nutrients when broken down by microorganisms (28, 32). conifer pollen inputs have been shown to increase nutrient levels, primary production and zooplankton biomass in oligotrophic lakes (16). chytrid fungi are often observed attached to pollen grains that float on lake surfaces. unlike most aquatic microbes, chytrids have the ability to break down the outer exine wall of pollen to obtain nutrients (22, 38, 41). chytrids that grow on pollen grains can complete their life cycle attached to pollen (15) and are considered to be major pollen decomposers along with bacteria (33). once the exine wall is breached, a variety of other organisms can participate in pollen decomposition. organic matter released from pollen serves as a growth substrate for heterotrophic organisms. mineralized phosphate and nitrogen from organic matter decomposition supports the growth of phytoplankton (4). the size of pollen grains and their buoyancy make them susceptible to zooplankton grazing. partial microbial digestion of pollen greatly increases the nutrient quality of pollen for zooplankton because zooplankton lack the ability to break down the outer pollen wall (32). in addition, chytrid zoospores are consumed by zooplankton and have been shown to promote their growth (22). thus, pollen infection by chytrid fungi initiates food webs that nourish primary producers, heterotrophic microbes and larger consumers such as zooplankton. the role of chytrid fungi in lentic food webs has received little attention, perhaps because of its seasonal pollen dependence and difficulty in fungal identification (20). because of the crucial role that chytrid fungi play in nutrient release from pollen, we sought to identify and culture pollen-associated chytrids from crater lake and compare dissolved nutrient 86 • fine focus, vol. 4(1) levels in pollen rafts relative to lake water lacking pollen. aquatic food webs were initially imagined as linear food chains from phytoplanktonproduced organic matter to zooplankton and animals such as fish. the current view is more complex and includes the consideration of allochthonous nutrients and the roles of heterotrophic bacteria and fungi (36, 37). heterotrophic microbes thrive in most aquatic environments, including crater lake, and participate in food webs and microbial loops (9, 43). most of the available organic matter in aquatic systems is not readily digestible by organisms other than heterotrophic bacteria and fungi (6, 8). heterotrophic microbes effectively consume organic matter and serve as food sources for larger microbes and zooplankton. in addition, they are able to mineralize and release phosphate and nitrate that are often limiting nutrients in aquatic ecosystems (9). phytoplankton and heterotrophic microbes may be in competition for inorganic nutrients, but on the whole, heterotrophs are considered to be essential for production of mineral nutrients that support phytoplankton (4, 38). until now, pollen has not been considered as an allonchthonous nutrient in crater lake. pollen decomposition by chytrid fungi in crater lake would support food webs by releasing the nutrients present in pollen and by promoting chytrid zoospore production. chytrid sporangia and zoospores serve as food for larger organisms including zooplankton and invertebrates (22, 23, 33). characteristics of chytrid fungi and their growth on pollen: chytridiomycota is a phylum of fungi that reproduces with flagellated spores known as zoospores. these organisms, commonly called chytrids or zoosporotic fungi, are genetically diverse and have been shown to occupy a basal position in fungal phylogeny (10). there are five taxonomic orders within chytridiomycota, with rhizophydiales having the majority of described genera and species (30). two other newly recognized phyla, blastocladiomycota and neocallimastigomycota, are similarly comprised of zoosporotic fungi although these phyla have relatively few species represented within them (19). the morphologically distinct features of chytrids are their sporangia and their flagellated zoospores, which are produced within the sporangia. in the case of eucarpic sporangia, filamentous rhizoids are produced, which are attached to the sporangia. zoospores develop into germlings, which are the precursors of sporangia. zoospores and/ or germlings typically attach to a nutritive substrate such as pollen or phytoplankton and develop either as epibionts or as endobionts. these morphologic and life cycle features are used to classify zoosporotic fungi (2, 41). in addition, genetic sequences, particularly large subunit ribosomal rna sequences, are a valuable aid to identification (2, 42). chytrids are found in aquatic and terrestrial environments and are often parasites of phytoplankton or saprophytes with the ability to break down refractory nutrient sources such as pollen, chitin, and cellulose (2, 18, 20). lake pollen is considered to be a prime nutrient for many aquatic chytrids (15, 33). we hypothesize that crater lake pollen is subject to colonization and decomposition 87applied & environmental microbiology • materials and methods sample collection and processing: a crater lake research and sample collection permit number crla-2016-sci-0005 was obtained from the national park service. surface water from crater lake was collected in 0.5 liter bottles from the shore at a rocky location (42°58’36.5”n , 122°5’16.8”w) where the dropoff into the water was steep. the sampling site was selected based on the presence of a thick pollen raft in early june, 2016. approximately 0.4 liters of water (with or without pollen raft) were collected on june 3, june 20, july 27, and august 31 of 2016. aseptic techniques included use of sterilized sample containers, careful skimming of surface water during collections to avoid contamination, and aseptic laboratory techniques. the volume of buoyant pollen raft that was collected above surface water was measured relative to total sample volume using the graduations on the sample bottles. water was kept at ambient temperatures for up to 12 h until it could be stored in a 4°c refrigerator. upon arrival at the laboratory, all samples were analyzed by microscopy to determine the concentration of pollen grains and observe associated microorganisms. pollen concentrations were determined by counting the number of pollen grains in a fixed volume using a hemocytometer and microscope. in addition, 150 ml of each by chytrid fungi, and that this leads to an increase in nutrient availability. food webs at several trophic levels would be supported sample was processed by pre-filtration through a 100-micron filter (to prevent clogging) and subsequent filtration through a 0.2 micron pore size filter to separate particulates from dissolved nutrients. the filtered water was stored frozen for later chemical analysis. buoyant pollen raft from the june 20 water sample was separated from the water beneath it by aspiration, transferred to a 100-micron sieve, rinsed with sterile water and used for isolation of chytrid fungi and analysis of eukaryotic rrna gene sequences. water chemistry: nutrient analysis, including measurements of dissolved organic carbon (doc), phosphate, ammonia nitrogen, and nitrite plus nitrate nitrogen was performed by the cooperative chemical analytical laboratory (ccal, corvallis, oregon) operated by oregon state university college of forestry and the u.s. department of agriculture forest service. ccal’s methodology and quality assurance program conforms with american public health association (apha) and us environmental protection agency (epa) surface water chemistry criteria. ccal tested random duplicate samples for each parameter to ensure that duplicate measurements of identical samples were within their quality by a general increase in nutrients given that crater lake is a nutrient-limited ecosystem. 88 • fine focus, vol. 4(1) assurance limit of ten percent. nitrogen concentration from ammonia was measured using apha protocol 4500-nh3 g (salicylate method), nitrogen concentration from nitrite plus nitrate was measured using apha protocol 4500-no3 f (cadmium reduction method), ortho phosphate was measured using apha protocol 4500-p f (ascorbic acid method), and dissolved organic carbon was measured using apha protocol 5310 b (1). genetic analysis: pollen from water collected on june 20, 2016 was retained in a 100-micron sieve and rinsed with sterile deionized h2o. chitinase from streptomyces (sigma aldrich, st. louis, missouri) was dissolved at a concentration of 1 mg per ml in phosphate buffered saline, ph 6.0. 200 µl of pollen suspension was mixed with 200 µl of chitinase and incubated for 1h at room temperature to break down fungal cell walls. similarly, an isolated colony from a cultured crater lake chytrid was digested with chitinase at ph 6.0 at room temperature for 1h. reagents and methodology from a dnaeasy kit (promega, fitchburg, wisconsin) were used to prepare dna extracts from the chitinase digested lake pollen and fungal isolate. these extracts were used as templates during polymerase chain reaction (pcr) amplification of partial 5.8s rrna, intragenic spacer (its) region, and 28s rrna gene sequences. the primers used for pcr amplification were 5.8sr (5’-tcgatgaagaacgcagcg-3’) and l7 (5’-tactaccaccaagatct-3’). pcr reactions were catalyzed by promega taq polymerase at these thermal cycler settings: 94°c for five minutes followed by 35 cycles of denaturation at 92°c for 30 seconds, annealing at 50°c for one minute, and extension at 72°c for one minute. pcr products were purified after agarose gel electrophoresis using qiaquick gel extraction and spin column kit reagents (promega). amplicons were cloned into pgem t-easy (promega). cloned dna was sequenced using bigdye™ terminator v3.1 cycle sequencing technology and a 310 genetic analyzer (applied biosystems, foster city, california). primers for dna sequencing were 5.8sr (5’-tcgatgaagaacgcagcg-3’) and l7 (5’-tactaccaccaagatct-3’). dna sequences were analyzed by blast to determine percent sequence similarities. a phylogentic tree was prepared using a neighbor joining distance method based on pairwise alignments (39) and sequence similarity identified by blast analysis (https://blast.ncbi.nlm.nih.gov/). pollen clone dna sequences were submitted to genbank under accession numbers mg132640mg132663. crater lake chytrid isolate paranamyces sp. cl sequence was submitted under accession number mg195571. culture techniques and microscopy: pollen from water collected on june 20, 2016 was transferred to a sterile sieve and rinsed with sterile deionized h2o. the pollen was suspended in sterile water and counted. chytrid mpmtg agar was supplemented with 120 mg/l penicillin and 200 mg/l streptomycin to inhibit bacterial growth (11). chytrid agar plates were inoculated by spreading with approximately 50 pollen 89applied & environmental microbiology • grains in a volume of 0.5 mls of sterile water. the cultures were incubated at 15°c for three weeks until chytrid colonies appeared. zoosporotic fungal colonies were purified by three rounds of streak plating on mpmtg agar. one of these isolates was characterized extensively by microscopy and its/lsu rrna gene sequencing. liquid cultures of the chytrid isolate were maintained in mpmtg broth or sterile distilled water with 6 mg/l penicillin, 10 mg/l streptomycin, and 2 x 104 ponderosa pine pollen grains/ml. a leica dm1000 microscope with digital mc100 camera was used for microscopy of stained and unstained samples. samples were observed by phase contrast microscopy and by staining with lactophenol cotton blue followed by bright field microscopy. cooperative chemical analytical laboratory performed measurements of doc in pollen and chytrid culture supernatants after filtration through 0.2 micron membranes. results chemical and microscopic analysis of water samples: climatic conditions during late spring of 2016 resulted in the appearance of substantial rafts of aggregated pollen on crater lake, visible from the caldera rim. the yellow pollen rafts stood out in stark contrast to the water of this lake that is renown for its deep blue color. on june 3 and june 20, pollen rafts were observed floating in swirled patterns on the surface of the lake in various locations, especially near the shoreline. a wind-blown pollen raft accumulated as particularly thick layer near cleetwood cove on june 3. a sample of surface water from this location on june 3 consisted of buoyant, aggregated pollen (20% of sample volume) and optically clear water beneath the pollen raft. on june 20, a pollen raft at cleetwood cove constituted 5% of the surface water sample volume. later in the summer, on july 27 and august 31, pollen rafts were absent from the lake, but pollen grains were suspended in surface water. pollen concentrations and chemical analysis of filtered water samples are shown in table 1. dissolved organic carbon (doc), phosphate and mineralized nitrogen were present at higher concentrations in surface water associated with pollen rafts. in the month of june, pollen raft waters had 5-160 times more doc than nonraft associated water during late july and august. phosphate and mineralized nitrogen levels (nitrite plus nitrate) were also greatly elevated (3-330 fold) by the presence of pollen rafts. pollen rafts on crater lake were concentrated at particular locations, usually near the shore. it is important to note that the pollen raft collected on june 3 was unusually thick. the vast majority of crater lake’s surface was free of pollen rafts throughout 2016. microscopic observations of crater lake water samples revealed the presence of zoosporotic fungi, bacteria and ciliates attached to and/or grazing on pollen in water samples from june 3, 20, and july 27. 90 • fine focus, vol. 4(1) in the water sample from june 3 there were an average of two zoosporotic fungal sporangia attached to each pollen grain. a representative photograph of chytrid colonization of pollen grains is provided as figure 1. figure 1. phase contrast micrograph of crater lake pollen with attached fungal sporangia and bacteria. table 1 91applied & environmental microbiology • genetic analysis of pollen associated organisms: dna was extracted from pollen water collected on june 20, 2016 and lsu rrna sequences were amplified by pcr and cloned. twenty-four lsu rrna clones were sequenced and identified by blast and by phylogenetic analysis of fungal sequences (table 2 and figure 2). fourteen lsu clones were identified as members of the chytrid order rhizophydiales. among these, 12 clones were 98-100% identical to each other, forming a distinct clade most closely related to chytrid genera alphamyces and kappamyces. the two other rhizophydiales clones, pollen428 and pollen429, did not fall within the tight clade of clones but clustered with known rhizophydiales genera in the neighbor joining tree shown in figure 2. the closest blast matches to clones pollen428 and pollen429 were rhizophylictis (96% sequence similarity) and entophylictis (91% sequence similarity), respectively. clone pollen416 had a relatively novel lsu rrna sequence that was most similar to species in the chytrid order blastocladiales and phylum blastocladiomycota. nine of the lsu clones were identified as members of the protist phylum ciliophora including three clones that were 99% -100% similar to sterkiella histriomuscorum, and one clone that was 99% similar to oxytricha longa. five of the protist clones had lsu rrna sequences that could not be definitively identified table 2. classification of cloned pollen lsu rrna gene sequences based on blast analysis and neighbor joining analysis. the range of percent dna sequence similarity to the closest matches in genbank are shown. 92 • fine focus, vol. 4(1) figure 2. neighbor joining phylogenetic analysis of cloned pollen lsu rrna gene sequences identified as fungal, crater lake chytrid isolate sequence, and closely related sequences from genbank. scale bar indicates percent dna sequence difference. beyond the phylum ciliophora, but were most likely members of the orders sessilida, haptorida, and endogenida. dna sequence results were consistent with microscopic observations of zoosporotic fungi attached to crater lake pollen and protozoa grazing on pollen. cultivation and characterization of a fungus from crater lake pollen: a chytrid isolate was cultured from crater lake pollen and identified by its and lsu rrna gene sequencing, morphology and cultural characteristics. the dna sequences had 99.8% sequence identity to paranamyces uniporus (812/814 nucleotides), a chytrid in the family halomycetae and order rhizophydiales (31). dna sequence similarity between paranamyces uniporus, the crater lake pollen isolate, and other rhizophydiales is shown in figure 2. the crater lake chytrid isolate grew in mpmtg broth and on pollen grains suspended in distilled water, and completed its life cycle in six days at 15°c. it generated zoospores that were 3-4 microns in diameter, spherical to slightly oval, with a single flagellum 8-15 microns long. zoospores developed into spherical 93applied & environmental microbiology • figure 3. crater lake chytrid developmental stages. a) zoospores stained with lactophenol cotton blue. b) germlings stained with lactophenol cotton blue. c) sporangia stained with lactophenol cotton blue. d) unstained lodgepole pine pollen after four days of chytrid growth. germlings with branched rhizoids on single axis. mature thalli were spherical sporangia ranging from 20 to 80 microns in diameter. sporangia were monocentric and eucarpic, inoperculate with fine rhizoids tightly aggregated on a single axis. exit pores were not apparent. the morphologic features of the crater lake isolate were consistent with its dna sequence identification as paranamyces (31). photographs of its various developmental stages are shown in figure 3. in an experiment designed to determine if organic carbon was released during chytrid growth on pollen, the crater lake isolate was mixed with ponderosa pine pollen in distilled water, penicillin and streptomycin. after four days at 15°c, the pollen grains were heavily colonized with developing sporangia. the culture supernatant contained 469 mg/l doc, whereas control samples of incubated pollen alone or fungi alone had 178 mg/l and 84 mg/l doc, respectively. the growth experiment provides further evidence that chytrid fungi are able to thrive on pollen and participate in nutrient release. 94 • fine focus, vol. 4(1) we used microscopy, rrna gene sequence analysis, and isolation of a chytrid fungus to demonstrate the presence and growth of chytrids on crater lake pollen. similar to other published results (21, 29, 45), we found that the majority of eukaryotic rrna gene sequences associated with lake pollen were chytridiomycota in the order rhizophydiales. the abundant presence of chytrid fungi on pollen in crater lake undoubtedly contributes to the release of nutrients for consumption by other aquatic microorganisms and improves the digestibility of pollen for zooplankton (32). zoospores produced during chytrid growth on pollen can also nourish zooplankton directly (22). we showed that lake water associated with pollen rafts had increased concentrations of doc, phosphate, and mineralized nitrogen. we measured nutrients in surface water near shore, whereas previous researchers reported dissolved nutrient levels in crater lake surface water miles from shore, over the deepest part of the lake. for example, larson et al (26) reported finding 0.012 mg/l phosphate, 0.003 mg/l nitrogen from ammonia, and 0.001mg/l nitrogen from nitrite plus nitrate. these values are similar to our late summer water sample that lacked pollen. in 1999, hargreaves et al detected doc values of 0.05 to 0.15 mg/l in crater lake surface water over the deepest part of the lake (17). we found higher levels of doc, perhaps because pollen and other allochthonous macronutrient inputs are likely to be higher near shore. our results are consistent with results from other lake systems where the addition of pollen increased dissolved nutrients (16). isolation of a paranamyces chytrid from crater lake pollen adds to the list of zoosporotic fungi available for laboratory studies. we anticipate that this isolate will be useful for studies of pollen decomposition and metabolic activities of chytrid fungi. fungi have been identified as important decomposers in aquatic and terrestrial ecosystems but the dominant research focus has been on woody and leafy detritus found in streams, soils, shallow lakes and wetlands (23, 24). filamentous, hyphomycete fungi are commonly observed in these systems. however, hyphomycete growth is limited by their non-motile spores and their reliance on hyphal networks (45). in a deep-water, oligotrophic lake such as crater lake, leafy and woody detritus are rare, whereas the seasonal pulse of pollen is predictable. chytrid fungi produce motile zoospores that are able to chemotax toward nutritive substrates such as pollen (14) and chytrids are well adapted to nutrient extraction from pollen. this study detected a diversity of chytrids in association with pollen from crater lake, with representatives from chytridiomycota and blastocladiomycota. our results align well with previous reports of chytrid diversity within aquatic dicussion 95applied & environmental microbiology • ecosystems (21, 29, 40, 45). high trophic transfer efficiencies have been reported from pollen to chytrid zoospores as well as from chytrid zoospores to daphnid zooplankton establishing a direct connection between pollen and higher trophic levels within aquatic ecosystems (13, 22). daphnia and 11 other species of zooplankton with similar feeding patterns are present in crater lake (4, 12, 27). previous research on zooplankton within crater lake found significant seasonal and annual variability in zooplankton density but could not identify a mechanism for that variability (27). because pollen may represent a large fraction of the macronutrients that enter crater lake, its degradation and nutrient uptake by chytrid fungi likely contributes to zooplankton abundance. we speculate that variation in pollen abundance contributes to variations in zooplankton abundance in crater lake. daphnia are present within crater lake and are a species of interest for the crater lake long-term limnological monitoring program because of their important relationship with fish species (3, 12). based on analysis of stomach contents, the diet of crater lake’s kokanee salmon consists almost entirely of daphnia (3). increases in fish populations were correlated with increases in daphnia abundance over a period of 28 years (12). allochthonous nutrients such as pollen that support daphnia would therefore be predicted to support salmon in crater lake. crater lake is oligotrophic with low concentrations of minerals and doc. thermal stratification and nutrient upwelling from water below 200m to productive water above 120m are important crater lake processes that regulate ecosystem dynamics (5). the depth and frequency of vertical water column mixing control the extent that deeper, nutrient-rich water mixes with relatively nutrient-poor epilimnion and metalimnion. thermal stratification occurs in crater lake at the same time as pollen input. during this time, a warmer epilimnion floats on colder deep water, and prevents deep water mixing. the upper 200m of crater is mixed by wind (5, 25). nutrients derived from pollen rafts will diffuse and be assimilated quickly in the epilimnion and/or leach slowly into deep water. wintertime mixing to the very bottom of the lake occurs every 2-5 years in crater lake, due to a reversal of thermal stratification. during reverse stratification, colder surface water is forced downward, causing nutrient upwelling (12). if chytrid fungi were absent from crater lake, undecomposed pollen grains would likely sink to the lake’s bottom and be effectively removed from the aquatic food web. pollen decomposition by chytrids and other microorganisms in the summer-stratified epilimnion ensures that much of the organic and inorganic nutrients from pollen remain in the upper water column, supporting the growth of phytoplankton, heterotrophic microorganisms and larger organisms. pollen-derived nutrients that leach into water below 200 meters are removed from the food web until an upwelling event brings them back up to the more productive epilimnion and metalimnion. 96 • fine focus, vol. 4(1) organisms that live in crater lake contend with a nutrient poor environment. low levels of nitrate in the euphotic zone limit the growth of phytoplankton and food is scarce for organisms at higher trophic levels (26, 43). low levels of primary production increase the impact of allochthonous nutrients on food webs. the seasonal pulse of pollen input supports crater lake organisms at several trophic levels. release of mineral nutrients and doc from pollen supports microorganisms at the base of food webs. pollen particles with decomposed exine walls are high quality food for we thank mark buktenica and scott girdner, along with other aquatic biologists and rangers at crater lake national park for their invaluable assistance with pollen collection and discussions about crater lake aquatic zooplankton, as are chytrid zoospores that are generated during chytrid growth on pollen (22). atmospheric deposition of particulate nutrients such as pollen has not been quantified for crater lake (25). to fully understand nutrient dynamics in crater lake it will be important to estimate the amount of pollen entering the lake. longterm studies that track pollen abundance and daphnia abundance might reveal a correlation between them. additionally, studies that trace flow of carbon and nitrogen from pollen through aquatic food webs should be considered. ecology. peter letcher kindly provided information about paranamyces and evaluated micrographs. acknowledgements 97applied & environmental microbiology • references 1. apha 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(2014). importance of saprotrophic freshwater fungi for pollen degradation. public library of science one 9(4): 1-12. 64 | fine focus isolation and identification of dermatophytes from collegiate runners l ga a kalnina1,3, stephanie m guzelak2, dpm and maryann ab herman, phd1. 1st. john fisher college, 3690 east avenue, department of biology, rochester, ny 14618 2our lady of lourdes memorial hospital, 169 riverside drive, binghamton, ny 13905 3cornell university, 630 west north street, department of plant pathology and plant-microbe interactions, geneva, ny 14456 corresponding author: dr. maryann ab herman, associate professor of biology at st. john fisher college, rochester, ny 14618. mherman@sjfc.edu manuscript received 30 january, 2021; accepted 24 august 2021 keywords: dermatophytes, collegiate runners, mycology, fusarium species complex, fungal molecular identification volume seven | 65 abstract competitive runners experience various risk factors that render them more susceptible to superficial cutaneous fungal infections, including the use of occlusive footwear, shared locker rooms, submission of feet to constant maceration, trauma, sweating, and having depressed immune function. the goal of this work was to assess the prevalence of athlete’s foot fungi in cross country runners at st. john fisher college. toe webs of 16 collegiate runners were sampled and volunteers surveyed about their shoe habits, foot hygiene, and average miles run per week. lack of tinea pedis-causing fungi in asymptomatic crosscountry runners shifted the study to investigate the identities of fungi morphologically similar to athlete’s foot and look for correlations with volunteers’ running habits and hygiene. thirty-five distinct fungal cultures were isolated and compared to a known trichophyton rubrum strain both microscopically and macroscopically. four samples were preliminarily identified as tinea pedis-causing fungi and sequenced to confirm molecular identification. fungal dna was isolated, purified, and pcr amplified using primers for the internal transcribed spacer region, d1/d2 region of the 28s subunit, and β-tubulin gene. three of the four isolates were identified as fusarium equiseti, a soil-borne plant pathogen with rare human pathogenicity reported. the fourth isolate was beauveria bassiana, a common soil-borne pathogen that can infect immunocompromised individuals. correct dermatophytic identification and understanding of the interplay between species is important to provide correct treatment, prevent spread among athletes and within facilities, and determine how opportunistic pathogens might play a role in people with immune suppressed function, which includes runners. 66 | fine focus introduction athletes, particularly long-distance runners, are at high risk for tinea pedis infection as they train in occlusive footwear, use shared locker rooms, frequently endure minor foot trauma, and have periods of decreased immune function (7,18,20,22,23,29). tinea pedis, or athlete’s foot, is a common superficial fungal infection caused by several dermatophytes, most commonly trichophyton, microsporum, and epidermophyton species. with the increase in superficial fungal infections over the past 30 years, correct identification of tinea pedis-causing fungi is critical for epidemiological purposes, recognizing potential infection sources, and providing proper treatment, such as topical and/ or oral antifungals (1,5,9,29). many symptoms can mimic the symptoms of tinea pedis, which further indicates the need for proper identification, as incorrect treatment can lead to secondary fungal and bacterial infections (9,11). infection by nondermatophytic molds can also result in tinea pedis. little is known about the impact of multiple fungal species on morbidity (3). sports-related infections can be a major cause of disease in individuals, as well as the whole team, due to close contact and sharing of equipment and facilities (20). dermatophyte infections are difficult to cure completely and frequently recur at the initial site of infection (7,29). clinical identification in practice is typically based on symptoms rather than morphological or molecular identification. further morphological identification involves assessing growth rate, colony pigmentation, hyphal structure, size and shape of conidia, and examining stained foot scrapings for hyphae or pseudo hyphae (1,3,10). these methods are imprecise and can lead to incorrect identification of other fungi inhabiting the foot, as the foot hosts a plethora of other organisms. gram positive bacteria, such as corynebacterium minutissimum, and candida species can cause scaling and maceration that mimic tinea pedis symptoms (10), while other soil-borne species such as microsporum gypseum, can live on skin and cause acute infections (30). accurate identification of asymptomatic tinea pedis and other potential opportunistic pathogens needs to be coupled to fungal morphology with a dna sequence-based approach. molecular targets such as ribosomal dna (rdna), beta tubulin, and mitochondrial dna (mdna) have been successful in identifying dermatophytes to a species level (1). many fungi can be identified by comparing the internal transcribed spacer (its) region of ribosomal dna; though closely related sister taxa, significant sequence variation, and imprecise typification of species dictate the use of additional conserved sequence regions (26). the its region is the most commonly used target for fungal sequencing due to sensitive detection by pcr. multiple copies of the ribosomal gene are present in all organisms and provide an optimal target for developing specific pcr primers that discriminate among closely related species (31). additional targets, such as the d1/d2 region of the 28s subunit and the β-tubulin gene, provide a more robust molecular picture for dermatophyte identification (11). the goal of this research was to investigate the prevalence of tinea pedis-causing fungi on asymptomatic cross-country runners at st. john fisher college and look for correlations with their running habits and hygiene. it was predicted that approximately a quarter of the runners would be carriers of athlete’s foot-causing fungi based on a previous survey of asymptomatic marathon runners (14). morphological and molecular techniques were used to identify potential tinea pedis isolates to determine prevalence of specific dermatophytes among runners. results of this work provide basic information on soil-borne fungi that are morphologically similar to tinea pedis but not typically associated with human disease complexes. further understanding of the prevalence and interactions of these fungi with dermatophytic pathogens can help develop recommendations to reduce spread among teammates and within facilities and improve proper diagnosis and treatment. volume seven | 67 materials and methods in the fall of 2014, male and female crosscountry runners at st. john fisher college were invited to participate in this study. a document describing the purpose, methods, and risks were explained, presented to, and signed by each participant. the voluntary nature of participation and procedures for ensuring confidentiality of participants and their samples were ensured. the study was approved by st. john fisher college institutional review board (irb) on october 2nd, 2014 (irb file no: 3370 091814 07). each participant completed an eight-question foot condition survey addressing their individual amounts of running, footwear habits, and foot hygiene. sample collection, culture identification, and maintenance the stratum corneum of the third and fourth web spaces of both feet of 16 volunteers was sampled using a sterile swab (6). samples were isolated on bbl sabouraud dextrose agar (sba) amended with 0.2 mg/ml chloramphenicol and incubated at 27℃ for 6 days, checked daily for visible growth, then subcultured until pure (22). for spore visualization, samples were grown on potato dextrose agar (pda) at room temperature for 7 days until a visible ring pattern emerged. dna extraction, sequencing and analysis samples for dna extraction were isolated from pure fungal colonies and grown in sabouraud dextrose broth for 7 days at room temperature using a cel-grotissue culture rotator (thermofisher scientific, waltham, ma). fungal dna was isolated using the powersoil dna isolation kit (mo bio laboratories, inc., carlsbad, ca). the internal transcribed spacer region (its), ribosomal large subunit d1/d2, and β-tubulin genes were amplified via polymerase chain reaction (pcr) using the following primers: its1/ its4 (31), its5/its4 (31), nl-1/nl-4 (13,25), bt2a/ t1 (9,17) using a biorad t100 thermal-cycler. reactions for pcr amplifications (50 µl) consisted of: 35 to 50 ng of template dna, primers (0.2 µm each), 1x amplitaq table 1: participant survey of running habits and hygiene. 68 | fine focus gold master mix containing 1.25 u amplitaq gold dna polymerase, 0.2 mm dntps, 2.5 mm mgcl2, and geneamp pcr gold buffer (applied biosystems, waltham, ma). cycle parameters for its primer combinations were an initial denaturation at 94℃ for 5 min, followed by 35 cycles consisting of denaturation for 1 min, annealing at 56℃ for 1 min, extension at 72℃ for 1 min, and a final extension at 72℃ for 10 min (17,18). cycle parameters for nl-1/nl-4 bt2a/t1 primer sets were an initial denaturation at 95℃ for 5 min, followed by 35 cycles consisting of denaturation for 45 sec, annealing at 52℃ for 90 sec, extension at 72℃ for 90 sec, and a final extension at 72℃ for 10 min (17,18). pcr products were visualized on a 0.8% agarose gel, purified using the e.z.n.a. cycle pure kit (omega bio-tek, norcross, ga), and submitted for sanger dna sequencing (acgt, inc. wheeling, il). sequences were compared with sequence entries in genbank database using the basic local alignment search tool for nucleotides (blastn) (20). using the calculated percent identity score, specimens were assigned to a genus and species with a minimum average of > 93% homology across the primer sets used. results sixteen collegiate cross-country runners, 3 female and 13 male, participated in this study. (table 1). runners sampled in this study ran between 35-60 miles per week and none had active tinea pedis infections. survey results did not yield any consistent patterns in frequency and temperature of washing running shoes, whether running shoes were worn in the locker room or used for outside of training, or if shower shoes were worn in the locker room. all but two volunteers always wore socks with their athletic footwear. foot washing habits varied among participants; most runners washed their feet daily with soap and water while a few did this periodically or not at all. sampling both feet of each volunteer resulted in 35 distinct fungal cultures (from a total of 24 of the samples) that varied in size, shape, and color (fig. 1). four isolates (3r, 4r, 11r, 13l) were tentatively identified as tinea pedis-causing fungi based on their macroscopic and microscopic morphology when grown on sba. isolates 3r, 4r, and 13l were initially classified as trichophyton rubrum as they exhibited chains of round macroconidia of varying shape and size and fluffy to cottony white colonies. isolate 11r appeared similar to trichophyton mentagrophytes as it grew numerous small and circular spores and exhibited small, starshaped, white colonies (table 2). the four isolates morphologically similar to tinea pediscausing fungi came from male runners who ran between 40-50 miles per week, none wore their shoes without socks, and all used soap and water to scrub their feet. from the 3 runners where f. equiseti was isolated none wore shower shoes; all wore their training shoes for other activities. shoe washing habits varied as one runner didn’t wash their running shoes, one washed with cold water, and the third washed with figure 1: macroscopic diversity of dermatophyte fungi isolated from collegiate runners. picture (a) shows an original sample, pictures (b-i) are a representation of morphological diversity of isolated subcultures. volume seven | 69 warm water. only one of the three wore shoes in the locker room. the runner with b. bassiana wore shoes in the locker room, didn’t wear their running shoes outside of training, and infrequently washed their shoes in hot water. when isolates were grown on pda, colonies of 3r, 4r, and 13l exhibited a pink/ orange pigmentation on the underside of the colony while 11r still appeared white, with one large colony. isolates 3r, 4r, and 13l also produced sickleshaped, septate macroconidia with aerial conidiogenous cells. macroconidia, typical of fusarium species, are highlighted with arrows in microscopic morphology of isolates 4r and 13l in table 2. colony 11r grew globus, hyaline conidia on aerial hyphae. pcr products for all samples using primer sets its-1/its4, nl-1/nl-4, and t1/bt2b were in the 500-700 bp range, as expected. the its-5/its-4 primer set did not amplify products under the conditions tested. top sequencing results showed 92.89-99.83% identity to the unknown isolates (table 3). isolates 4r and 13l were identified as fusarium equiseti and 11r as beauveria bassiana as the sequencing produced the same species for each of the six primers tested. isolate 3r is likely fusarium equiseti as it was the top result for four of the primers, while the d1/ d2 region of isolate 3r came back as the closely related species fusarium incarnatum discussion a range of fungi were isolated from the feet of collegiate crosscountry runners, four of which initially resembled tinea pedis species on sba and chosen for further identification. this preliminary morphological identification is the most common method of identification of tinea pedis in clinical settings as it is accurate (particularly when an individual is symptomatic) and inexpensive (1,3). dna sequencing can be costly but has the advantages of increased sensitivity and reproducibility, minimal sample handling, and speed, compared to culturebased diagnosis (1,26). asymptomatic individuals, variation in fungal morphology, and inconsistent morphological results have created a need for more straightforward and reliable identification methods. runner hygiene was variable across the across the sixteen volunteers. inconsistent habits of shoe, sock, and foot hygiene can influence the spread and survival of fungi, yet there is no standard protocol for hygiene in collegiate runners (8). consideration of community spread of dermatophytes has led to recommendations of not wearing running shoes in the locker room, implementation of additional sanitation measures for shoes and socks, and increasing washing temperature for clothing and shoes to 60˚c in order to kill fungal spores (8). table 2: macroscopic and microscopic morphology of tentative tinea pedis-causing fungal isolates. arrows highlight macroconidia. 70 | fine focus four isolates appeared to be tinea pediscausing species based on their morphology on sba media, commonly used to culture dermatophytic fungi (22). isolation of these fungi from 25% of runners surveyed was consistent with lacroix et al.’s findings where 27% of european marathon runners were asymptomatic carriers of trichophyton spp. (14). using a more general growth medium (pda), cultures demonstrated inconsistent morphologies to those of athlete’s foot-causing fungi. isolates 3r, 4r, and 13l resembled trichophyton when grown on sba; however, once placed on pda, they produced curved, septate macroconidia characteristic of fusarium species. isolate 11r grew as a fluffy white colony on sba similar to athlete’s foot fungi, though microscopic morphology was consistent with beauveria bassiana on both growth media. sba media did not hinder spore formation for the species as it did for fusarium species complex isolates. dna sequence analysis indicated that none of the isolates were typical species that cause athlete’s foot. it was not surprising that no fungal dna was amplified using the its5/its4 primers as it was previously documented that some fungal isolates only work with its5/its4 or its1/ its4 (18). all primer set identities agreed except for isolate 3r. this isolate was identified as fusarium equiseti (β-tubulin and its) and fusarium incarnatum (d1/d2). inconsistencies about species identification and recognition exist in the fusarium incarnatum-equiseti complex due to significant genetic variability and need for further research on differences among species (2,26,30). the translation elongation factor 1-alpha and partial rna polymerase second largest subunit genes have distinguished over 20 species within the fusarium incarnatum-equiseti species complex; use of both primer sets could identify isolate 3r to species level (2,29). blast scores in the low 90% range can be explained by limited fungal data in databases and sequencing phred scores between 20 to 40 (99.0-99.99% accuracy). asymptomatic infections of tinea pedis, referred to as occult tinea pedis, are common among athletes and complicate the spread and diagnosis of tinea pedis (10). while not previously isolated from runners, both b. bassiana and members of the fusarium incarnatum-equiseti species complex are opportunistic human pathogens that can result in superficial dermal lesions to deep tissue, systemic infections (28,29). long distance runners have depressed immune function and are especially prone to traumatic and environmental dermatoses due to repeated physical stress on their feet and exposure to soil. b. bassiana and fusarium species are ubiquitous in soil and track and field athletes may come in contact while running through soil during practice and outdoor track meets, which then gets tracked into locker rooms. limited washing of running sneakers can contribute to repeated exposure to soil fungi directly or through shared surfaces. table 3: sequencing results and percent identity for its, d1/d2, and β-tubulin regions for dermatophytic isolates 3r, 4r,11r and 13l. volume seven | 71 molecular identification tools, such as sequencing of conserved dna regions, can improve the consistency and accuracy of identification of dermatophytic fungi (10). this study demonstrated morphological identification could eliminate 75% of the isolates cultured from runners’ feet from being tinea pedis-causing fungi. four isolates resembled trichophyton when grown on sba media, but exhibited atypical morphology when pda media was used. dna sequencing was required to determine no isolates were tinea pedis-causing fungi. the presence of opportunistic human pathogens in areas of common tinea pedis infection highlights the need to identify microbes inhabiting asymptomatic and healthy runners. more diagnostic techniques are being developed for dermatophyte identification, such as matrixassisted laser desorption/ionizationtime of flight mass spectrometry, and these tools will continue to improve fungal identification (26). greater understanding of dermatophytic fungal interactions and their role in disease development can further inform methods of prevention, identification, and treatment. acknowledgments this work was funded by a council on undergraduate research (cur) biology division research supply grant, st. john fisher biology department research funds, and the st. john fisher college center for student research & creative work. we would like to thank dr. jonelle mattiacio for critically reading and providing feedback on the manuscript. citations 1. ahmadi, b., mirhendi, h., shidfar, m.r., nouripoursisaakht, s., jalaziland, n., geramishoar, m., & shokoohi, g.r. 2014. a comparative study on morphological versus molecular identification of dermatophyte isolates. journal de mycologie medicale. 25:29-35. 2. avila, c.f., moreira, g.m., nicolli, c.p., gomes, l.b., abreu, l.m., pfenning l.h., haidukowski, m., moretti, a., logrieco, a., & del ponte, e. m. 2019. fusarium incarnatum-equiseti species complex associated with brazilian rice: phylogeny, morphology and toxigenic potential. international journal of food microbiology 306:1-8. 3. canavan, t.n., & elewski, b.e. 2015. identifying 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(pp. 307-328). national center for biotechnology information. 22. purim, k.s.m., & leite, n. 2014.sports related dermatoses among road runners in southern brazil. anais brasileiros de dermatologia 89: 457-592. 23. robert, r., & pihet, m. 2008. conventional methods for diagnosis of dermatophytes. mycopathologia 166:295-306. 24. sabadin, c.s., benvegnu, a.a., da fontoura, m.m.c., saggin, l.mf., tomimori, j., & tischman, o. 2011. onychomycosis and tinea pedis in athletes from the state of rio grande do sul (brazil): a cross-sectional study. mycopathologia 171:183-189. 25. sasagawa, y. 2019. internal environment of footwear is a risk factor for tinea pedis. the journal of dermatology 46:940946. 26. sting, r., eisenberg, t., & hrubenja, m. 2019. rapid and reasonable molecular identification of bacteria and fungi in microbiological diagnostics using rapid real time pcr and sanger sequencing. journal of microbiological methods 159:148-156. 27. suh, s-o, grosso, k. m., carrison, m. 2018. multilocus phylogeny of the trichophyton mentagrophytes species complex and the application of matrix-assisted laser desorption/ionizationtime-flight (maldi-tof) mass spectrometry for the rapid identification of dermatophytes. mycologia 110:118-130. 28. taschdjian, c. 1955. fountain pen ink as an aid in mycologic technic. journal of investigative dermatology 24:77-80. 29. tucker, d.l., beresford, c.h., sigler, l., & rogers, k. 2004. disseminated beauveria bassiana infection in a patient with acute lymphoblastic leukemia. journal of clinical microbiology 42:5412-5414. 30. wang, m.m., chen, q., diao, y.z., duan, w.j., & cai, l. 2019. fusarium incarnatum-equiseti complex from china. persoonia. 43:70-89 31. white, t.c., findley, k., et all. 2014. fungi on the skin: dermatophytes and malassezia. cold spring harbor perspectives in medicine. 32. white, t.j., bruns, t.d., & leach, l.d. 1990. pcr protocols: a guide to methods and applications. in m.a. innis, d.h. gefland & j.j. sninsky (eds.), amplification and direct sequencing of fungal ribosomal rna genes for phylogenetics (pp. 315-322). academic press. 74 | fine focus undergraduate student perspective undergraduate research student interview airhenvbahihea edionwe received a b.s. from ball state university 2021 in pre-med studies interviewed by sammie campaniello received a b.s. from ball state university 2021 in science education tell me a little about your research, and the broad goal. title: bacteriophage control of salmonella enterica in artificially contaminated 1%, pasteurized milk the genus salmonella is made up of over 2,500 serovars between two species: salmonella enterica and salmonella bongori. salmonella spp. are the number one causative agent of food-borne illnesses in the united states. consuming food that is contaminated with salmonella can cause a condition known as salmonellosis. salmonellosis, also known as food poisoning, occurs when salmonella spp. enter the intestinal tract and invade the epithelial cells of our small and large intestines. it is characterized by vomiting, diarrhea, fever, and abdominal cramps. usually this infection will only last up to 48 hours but in the immunocompromised community, this can be life threatening. if a person’s immune system is not able to fight off the salmonella infection, then the bacteria will enter the bloodstream and cause a systemic infection which can be fatal. an infection of the intestinal tract cannot be treated with antibiotics. treatment with antibiotics can leave the patient vulnerable to more infections of the gi tract. however, if antibiotics were a treatment option, they would be ineffective against a salmonella infection. salmonella spp. are resistant to multiple antibiotics leaving only a few that can be used to treat these infections. alternatives to antibiotics are needed to treat infections where the bacteria are resistant to all antibiotics. bacteriophages are known as the “viruses of bacteria”. bacteriophage, also known as phage, insert their genetic material into their hosts, hijacking their dna synthesis machinery in order to produce more phage inside the host cell. there are two forms of bacteriophage, lytic and lysogenic. both phages hijack their hosts in the same way, but how long it takes to kill the host is where these two differ. lytic phage get their name by causing the host cell to lyse after producing more phage. lysogenic phage will incorporate their genome into the hosts and not cause lysis until a switch is made. there is no set time for how long a lysogenic phage will keep the host cell alive but once the switch is made, the host cell will produce more phage eventually causing itself to lyse. bacteriophages have been considered an alternative to antibiotics because of this lytic ability to kill their hosts. the goal of this project is to isolate and characterize bacteriophage volume seven | 75 from a raw milk environment to use as a biological control of salmonella enterica contamination of food products. we expect to see that the bacteriophage are able to control salmonella contamination at a low density but as the density increases, the phage becomes less effective. we also expect that the bacteriophage will have a higher affinity to the s1 (unknown salmonella species we isolated from the farm) bacteria than it does to the lab grown salmonella enterica . can you give a brief overview of some of your daily activities in the lab? each day i go to to the lab and subculture set of s1 and s. enterica with tsb tubes. if we are out of material we need, i usually make sets of tsa plates and tsb tubes. each day’s task depends on what we have going on in the lab at a particular time. how has working in the lab helped to better prepare you for your future? from just in the lab this semester i have gained some confidence on what it takes to be a microbiology student and work in a microbiology lab. also, working in the lab i feel has gotten me ready for my ms program and what it takes to be a scientist and also with having the experience in the lab will increase my chances of getting into medical school. what are some limitations that you seem to be facing in this pandemic regarding your research? some limitations we faced during this pandemic are noting having in materials and phages we need for experiment not getting delivered time due to the pandemic, also due to the pandemic the amount of time we are allowed in lab is limited in other to accommodate all students doing research to help reduce the numbers of students allowed in the lab at a given time. do you foresee these limitations changing anytime soon or do you think research has forever been changed due to covid-19? i think the limitations we are currently facing could change. we have a vaccine for this virus and the pandemic is over. with that being said i do not think research is forever changed due to covid-19 because i believe a good scientist/ researchers should be able to accommodate changes and be able to work around these unplanned changes but change is the only constant time in life. what is some advice that you would give a new undergraduate researcher that would have helped you be better prepared for your research experience? • do not wait till your last year of your undergraduate degree to start research. • failure is part of being a researcher • don’t be afraid to ask professors to work in their labs from your first of your undergraduate career. • take initiative and work hard. • don’t be afraid to ask questions if unsure. do you have any further comments about fine focus or undergraduate research in general? i would encourage any science/biology major student to sign up for a research immersive learning class because it will help prepare them for plans after their undergraduate degrees such as graduate school, phd program. also it will also look good on your resume and most importantly makes you a better scientist. 36 | fine focus real-time screening of foods using repetitive element pcr reveals a dna marker characteristic for enterotoxigenic bacillus species breanna r. brenneman1, kyla l. adamson2, matthew r. beer3, yenling ho4, kiev s. gracias5, chelsea m. priest6, erika n. biernbaum7, and john l. mckillip* department of biology, ball state university, muncie, in 47306 1current address: university of virginia, charlottesville, va usa 2current address: elanco, clinton, in usa 3current address: cooper genomics, houston, tx usa 4cdc foundation, indianapolis, in usa 5department of biology, oakland city university, oakland city, in 47660 6current address: community hospital, anderson, in usa 7current address: food safety and enteric pathogens research unit, national animal disease center, agricultural research service, u.s. department of agriculture, ames, ia, usa volume seven | 37 manuscript received 1 september 2020; accepted 9 december, 2020 key words: bacillus, enterotoxigenic, detection, rep-pcr, dna fingerprinting abstract bacillus cereus is traditionally thought to be the only member of its genus accepted as a pathogen in foods like grains, fruits, vegetables, and milk due to the presence of the nonhemolytic (nhe) operon. however, many other bacillus spp. may also harbor the nhe operon and be pathogenic, including not just food-associated gastrointestinal toxicoinfections, but human endophthalmitis as well. real-time pcr targeted the nhea gene in 37 samples obtained from food, soil, and reference cultures by analyzing the standard deviations of melt peaks. repetitive element pcr was used to compare the banding patterns of each sample against b. cereus atcc 14579 and three b. thuringiensis strains to “fingerprint” each isolate. of the original 43 isolated tested, 37 were gram-positive rods. the remaining six samples were gram-positive cocci. twenty-five of the 37 gram-positive bacillus spp. were nhea positive, while twelve were negative. many of the nhea positive strains were species not previously known to contain nhe and were capable of causing gastroenteritis in consumers. 38 | fine focus introduction bacillus spp. are gram-positive endospore-forming rods ubiquitous in soil worldwide and are primarily aerobic to facultatively anaerobic saprophytes (3). over 148 distinct bacillus species have been described. this large number of individual species reflects a high degree of genetic diversity. taxonomical identification of species within the bacillus genus has changed over time as differentiation methods have improved (17). currently, bacillus species are divided into two groups the b. subtilis and b. cereus divisions. the b. cereus group includes b. cereus, b. mycoides, b. anthracis, b. thuringiensis, and b. weihenstephanensis. these species are also grouped under the name b. cereus sensu lato (51). phylogenetically, b. cereus is quite closely related to the entomopathogen b. thuringiensis and the human pathogen b. anthracis, a fact that has led to vigorous discussion on shared virulence properties, dna sequence conservation among strains, and prevalence in the environment (19, 39, 48). along with b. weihenstephanensis, these species constitute a single genetic subgroup, a rather arbitrary classification designation that brings into question how a species is even defined in this family of bacteria. for example, it seems clear that at least for many bacillaceae other than b. cereus, presence and expression of enterotoxin genes is not uncommon (7, 11, 18, 20); nor is it atypical to identify strains of b. cereus lacking detectable enterotoxin genes (5, 48). moreover, b. cereus may harbor virulence genes on plasmids more commonly associated with b. anthracis (49). many pathogenic strains of bacillus spp. are primary isolates from clinical, food, and environmental sources. naturally, many published studies on the b. cereus group mention the difficulty in selecting features for reliable identification of these species. the involvement of species in the b. cereus group in foodborne illness, as a leading cause of ocular infections (endophthalmitis), and as an indicator of water quality (4, 25), begs the question of how such a closely related set of species and strains could manifest itself so differently in varied environments. accordingly, a reassessment of identification strategies is in order as even more reports appear in the literature of bacillus spp. involvement in novel ecological niches. the search for a reliable dna-based typing approach for bacillaceae has explored several technologies in recent table 1. table 1: foods screened for the presence of bacillus spp. by food type as described in materials and methods. volume seven | 39 years, including repetitive element pcr (rep-pcr) (34), next generation sequencing (ngs) of whole genomes to identify polymorphic regions (12), and multilocus sequence typing (mlst) (6, 21, 22), which relies on the pcrbased amplification of 400600bp internal fragments of housekeeping genes. however, these conserved gene targets are frequently not adequate to effectively resolve bacillus species or strains for identification. clearly, a repertoire of both phenotypic and novel genotypic-based methods must be utilized for the ever-increasing number of strains appearing in the literature. this trend reflects a growing interest in this group of bacteria (4). the objective and hypothesis of this research is that one may develop a genotypic screening method to reliably detect enterotoxigenic bacillus spp. from contaminated food without the need for culture-based methods. use of dna typing/fingerprinting compared to positive control enterotoxigenic (nhe-producing) bacillus, we show the potential in rep-pcr as a rapid and highthroughput screening tool for a variety of contaminated foods. materials and methods bacillus spp. isolation from soil and food. using a previously described method (46), soil was collected (at 4-inch depths) from multiple locations around the ball state university campus in muncie, in. a total of 41 varieties of flavoring/ powder, seasonings, milk, coffee creamers, cheese, snacks, spreads, and drink additives were obtained at local retailers in order to isolate bacillus spp. from these naturally contaminated foods (table 1). each sample (5g) was added to 100 ml of brain heart infusion broth (bhib; bd diagnostic systems, franklin lakes, nj). after mixing, the solution was incubated at 32°c while shaking at 160 rpm for 72h. samples were heat-treated at 80°c for 30 min on a hot plate using a water jacketed vessel and constant shaking, after which the suspension (1 ml) was pipetted onto quadruplicate tryptic soy agar (tsa; alpha biosciences, baltimore, md) plates and incubated overnight at 37°c. streak plates were performed from initial growth and incubated at 37°c overnight to obtain pure cultures, confirmed by gram and endospore staining. reference strains (table 2) were obtained from presque isle cultures (erie, pa usa) and dr. james mitchell (ball state university, muncie, in) and subcultured table 2. table 2: bacillus reference strains used in this study. 40 | fine focus onto tsa slants. all cultures were refrigerated at 4°c until dna extraction and real-time pcr analysis. dna extraction. each isolate, including reference strains, was separately cultured in tryptic soy broth (tsb, weber scientific, hamilton, nj usa) grown aerobically by shaking for 24h as previously described (31). bacteria were pelleted by centrifugation at 9,000 x g for 3 min at 4°c and the pellets were resuspended in 300µl te buffer (amresco, solon, oh) containing 30 µl of 10% sds (promega, madison, wi), and 20 µl of 20 mg/ml proteinase k (ambion, austin, tx). following a 37°c 30 min. incubation, an equal volume of phenol:chloroform:isoamyl alcohol (25:24:1) solution (amresco) was added, vortexed, and centrifuged for 10 min. at 10,000 x g and 4°c. the aqueous phase was carefully transferred into clean microcentrifuge tubes and mixed with 0.1 volume of cold 3m sodium acetate (fisher scientific, pittsburgh, pa) and one volume of cold 95% isopropanol (greenfield ethanol co., brookfield, ct). the microcentrifuge tubes were inverted to mix and centrifuged for 30 min at 16,000 x g at 4°c. the supernatant was discarded, and the dried dna pellets were quantified spectrophotometrically to assess yield and purity. uniplex pcr. all dna samples were normalized to a concentration of 1 µg/ µl in sterile water. all reactions were performed in triplicate. real-time pcr was used initially to target the nhea gene (32). primer sequences used for this and other experiments are shown in table 3. pcr was performed as previously described (18) with minor modifications. the annealing temperature was changed from 55°c to 52°c to better support annealing of the nheaf primer to template dna, and a melting curve was used to resolve and validate amplicon identity. each pcr reaction consisted of 1x iq sybr green supermix (bio rad, hercules, ca), 100 pmol nheaf and nhear primers (integrated dna technologies, coralville, ia), and 0.5 µg of template dna. nuclease-free water (promega) was added for a final volume of 25 µl in 0.2 ml pcr tubes (corbett research, concord, nsw). a positive control was included in each set of reactions, consisting of template dna from b. cereus atcc 14579, (american type culture collection, manassas, va), previously shown to harbor the nheabc operon (41). pcr reactions were performed in a rotor gene rg-3000 thermocycler (corbett research) using an initial 94°c 120s denaturation step followed by 35 cycles of 94°c for 20s, 52°c for 60s, and 72°c for 60s. a final 72°c 6 min. extension step preceded melting curve analysis (40°c to 95°c in 0.7°c per second increments). amplicon melting peaks were plotted using rotor gene 6 software and melt peak data were exported into microsoft excel for analysis. only melt peaks within 1 standard deviation of the average melt peak of positive control b. cereus atcc 14579 were considered as positive for the presence of nhea. rep-pcr. dna templates from all strains analyzed (table 1) were subjected to repetitive elementpcr (rep-pcr) using a diversilab kit (bacterial barcodes, athens, ga) specific for fingerprinting bacillus spp. dna of each sample previously isolated for real-time pcr was re-standardized to 50 ng/ µl. primers for repetitive elements within bacillaceae were included in the diversilab kit and are shown in table 3 (24). all reactions were completed in triplicate and consisted of 18 µl rep-pcr mm1 buffer, 2.5 µl of geneamp® 10x pcr buffer, 2.0 µl of primer mix, and 0.5 µl of taq dna polymerase (5 prime, gaithersburg, md) and 100 ng of template dna. positive kit controls were included with each set of replicates, as were no template controls (ntc). all reactions were performed using a rotor gene instrument and consisted of an initial 94°c 2 min. denaturation followed by 35 cycles of 94°c for 30s, 55°c for 30s, and 70°c for 90s. following a final extension step for 3 min at 70°c, a subset of reactions was subjected to melting curve analysis as described earlier, while others were analyzed using agarose gel electrophoresis. for the latter samples, 5µl of each pcr product was loaded into a 1.5% (w/v) agarose gel (bioexpress, kaysville, ut) containing 0.625 μg/μl ethidium bromide volume seven | 41 (invitrogen, carlsbad, ca) (43) and the gel electrophoresed for 1.5h at 70v (constant). the gel was visualized on a gel doc xr (bio-rad, hercules, ca) using uv light. the resulting banding patterns were recorded in microsoft excel as a virtual gel (table 4). banding patterns of nhea positive and nhea negative were compared against the b. cereus reference strain and three b. thuringiensis strains, representing additional members of the b. cereus genetic subgroup. sample bands identical to each reference strain (b. cereus, b. thuringiensis var. kurstaki, b. thuringiensis var. japanensis, and b. thuringiensis var. israelensis) were divided by the number of total bands in each reference strain. the resulting number was multiplied by 100 to determine the percent each sample was identical to b. cereus, b. thuringiensis var. kurstaki, b. thuringiensis var. japanensis, and b. thuringiensis var. israelensis. sequencing of diagnostic rep-pcr product. the 1,230bp diagnostic band (11) earlier found to be unique to enterotoxigenic bacillus spp. was identified in real-time melting curve plots of b. cereus atcc 14579 and other strains. results samples excluding reference microbes purchased from presque isle cultures, a total of 45 food and soil samples were screened for the presence of bacillus spp. of these, 21 isolates (48.9%) were found to contain no detectable bacillaceae. twenty isolates (44.4%) were gram-positive, spore-forming rods after heat-treatment and subsequent streak-plating on tsa. these included: basil seasoning, nutmeg seasoning, tazo tea powder, a beef taco from taco bell, lesaffre yeast, prairie farms whole milk, food club quick oats, ann’s house healthy energy blend nuts, peter pan peanut butter, great value peanut butter, dannon yogurt, chevre fresh goat cheese, saputo stella gorgonzola cheese, black creek extra sharp cheddar cheese, pilgrim’s choice blue stilton cheese, cooper science building soil, lucina building soil, christy woods soil, and ball gymnasium soil. an additional six isolates, three of which were isolated from jiffy corn muffin mix, were gram positive cocci. aside from jiffy corn muffin mix, grampositive cocci were isolated from mustard seasoning and mix n’ drink powdered skim milk. these gram-positive cocci accounted for a total of 6.7% of the entire sample pool. seventeen bacillus spp. reference strains were purchased from presque isle cultures for subsequent real-time pcr analysis. overall, a total of 37 samples were either pure-type cultures or gram-positive rods that were subsequently subjected to dna extraction in preparation for real-time and rep-pcr. real-time pcr b. cereus atcc 14579 was used as a positive control to test for the presence of nhea and had an average melt peak of table 3. table 3: primer sequences, melting temperatures, and guanine and cytosine content for the nhea gene used in uniplex pcr and repetitive element (rep-pcr) palindromic sequences in bacillus spp. 42 | fine focus table 4. table 4: banding patterns of all nhea positive samples, excluding samples 9, 10, 22, 27, 31, 39, and 43. red cells represent the length in base pairs of each dna ladder band. yellow cells represent the column of positive control b. cereus atcc 14579. green cells represent banding of b. thuringiensis var. kurstaki (#15), b. thuringiensis var. japanensis (#16), and b. thuringiensis var. israelensis (#17). 81.96°c over three runs. as shown in table 5, fourteen test samples (37.84% = green highlighted) were consistently within 1 standard deviation (sd) of the positive control over three separate realtime pcr runs (sd determined using sigmastat for windows). standard deviations of samples positive all of three replications for nhea are included in table 6, part b. these samples included reference strains b. macerans, b. brevis, b. cereus, b. thuringiensis var. kurstaki, b thuringiensis var. japanensis, and b. thuringiensis var. israelensis. food samples consistently within one sd of the positive control originated from prairie farms whole milk, ball gymnasium soil, lucina hall soil, cooper science soil, christy woods soil, basil seasoning powder, “clean” peter pan peanut butter, and great value peanut butter. five samples (13.51% = yellow highlighted had two melt peaks within 1 sd of the positive control strain. these included the pure strain b. laterosporos and food samples from a beef soft taco from taco bell, nutmeg powder, chevre fresh goat cheese, and saputo stella gorgonzola cheese. volume seven | 43 six samples (16.22% = orange highlighted) had one melt peak within 1 sd of the positive control train. these included pure strains b. polymyxa and b. coagulans. in addition, food samples with only 1 of 3 melt peaks consistent with b. cereus positive control were from food club quick oats, pilgrim’s choice blue stilton cheese, ann’s house energy blend nuts, and a jar of peter pan peanut butter involved in a food recall that may have contained salmonella. twelve of the 37 samples (32.43% = red highlighted) had either no melt peaks or melt peaks greater or less than one sd of the positive control strain. these included: pure strains b. subtilis globigii, b. stearothermophilus, geobacillus stearothermophilus, b. spaericus, b. megaterium, g. pumulis, b. circulans, and b. subtilis. foods negative for nhea included black creek extra sharp cheddar, tazo tea powder, dannon yogurt, and lesaffre yeast. as shown in table 6, part a, there were 4 of 16 food samples that tested positive for nhea for three melt peaks. additionally, 4 food samples displayed two positive melt peaks, while 4 more displayed one melt peak. four food samples displayed zero melt peaks. all soil samples were positive for nhea with three melt peaks. six reference strains displayed three positive melt peaks for nhea, while only one strain had two melt peaks consistent with the positive control. two strains had one melt peak in line with the positive control, while 8 strains were completely negative for the presence of nhea. table 5. table 5: samples were positive or negative for the presence of nhea 3/3, 2/3, 1/3, or 0/3 times. green samples indicate positive detection of nhea in real-time pcr over three separate runs. yellow samples indicate nhea positive samples in two of three real-time pcr runs. orange samples indicate nhea positive samples in one of three real-time pcr runs. red samples indicate negative nhea detection in real-time pcr. 44 | fine focus range of the dna ladder, as shown in table 4. these bands corresponded to lengths of 1500 bp, 1200 bp, 1100 bp, 1025 bp, 950 bp, 900 bp, 825 bp, 750 bp, 700 bp, 625 bp, 575 bp, 550 bp, 450 bp, 375 bp, 300 bp, and 250 bp. table 7 includes all banding patterns for all nhea positive samples. all other banding patterns were compared against b. cereus reference strain (#14) and three b. thuringiensis spp. reference strains (#15-17). all samples were compared against b. cereus, b. thuringiensis var. kurstaki, b. thuringiensis var. japonensis, and b. thuringiensis var. israelensis. the resulting percent identities of the banding patterns to each reference strain of each sample are recorded in table 7. b. cereus (sample #14) samples 17 and 25 were 6% identical to the banding pattern of sample 14. samples 6, 15, 18, 24, and 32 were 13% identical to sample 14. samples 8, 13, 26, and 28 were 19% identical to sample 14 banding. samples 16 and 42 were 25% identical to sample 14, while samples 33 and 35 were 31% identical. samples 36 and 37 were 44% identical to sample 14. no samples were more than 44% identical to sample 14. nine nhea negative samples were analyzed using rep-pcr, and include samples 1-5, 7, 9, 11, and 12 (data not shown). samples 2, 3, 5, and 7 were 6% identical to the banding table 6. table 6: a: number of samples with nhea positive melt peaks three, two, one, and zero times in divisions of food, soil, and reference strains. b: samples with corresponding sds < 1 when compared against the positive control strain b. cereus atcc 14579 during real-time pcr. after real-time pcr, sd were calculated for sample melt peaks to compare against b. cereus atcc 14579. samples 6, 8, 14, 15, 16, 17, 18, 24, 25, 26, 28, 32, 37, and 42 resulted in standard deviations < 1 when compared against the 82°c average positive control melt peak. any sample with a sd < 1 indicated a positive detection for the nhea gene. data are shown in table 6, part b. rep-pcr repetitive element pcr was utilized on b. cereus atcc 14579, which was labeled as sample 14 for real-time and rep-pcr. this strain was subsequently used as the standard against which all other nhea positive samples in rep-pcr were compared. sample 14 displayed 16 bands within the volume seven | 45 pattern of sample 14. samples 1, 4, and 12 were 19% identical to sample 14. sample 11 was 44% identical to the banding pattern from sample 14. b. thuringiensis var. kurstaki (sample #15) when compared against b. thuringiensis var. kurstaki, samples 17, 24, 25, and 35 shared no identical banding. samples 8, 16, 18, 26, 32, 33, and 36 were 20% identical to b. thuringiensis var. kurstaki. samples 6, 13, 14, 28, and 42 were 40% identical, while sample 37 was 80% identical to b. thuringiensis var. kurstaki. b. thuringiensis var. japanensis (sample #16) samples 6, 17, and 35 shared no identical banding with b. thuringiensis var. japanensis, while samples 15 and 18 were 8% identical. samples 28 and 32 were 17% identical to b. thuringiensis var. japanensis, but samples 8, 13, 26, and 37 were 25% identical. samples 14 and 42 were 33% identical to b. thuringiensis var. japanensis. samples 24 and 33 were 42% identical to b. thuringiensis var. japanensis banding, while sample 36 was 50% identical. table 7. table 7: comparison of all rep-pcr banding patterns with b. cereus (sample #14), b. thuringiensis var. kurstaki (sample #15), b. thuringiensis var. japanensis (sample #16), and b. thuringiensis var. israelensis (sample #17). yellow cells denote the specific sample all other banding patterns were compared against. blue cells represent banding patterns 20% to 29% identical to each yellow reference strain. green cells represent banding patterns 30% to 39% identical to each yellow reference strain. red cells represent banding patterns identical banding that was 40% and above to each yellow reference strain. no samples were more than 44% identical to b. cereus. sample 37 was 80% identical to b. thuringiensis var. kurstaki. sample 36 was 50% identical to b. thuringiensis var. japonensis. samples 13, 35, and 42 were 50% identical to b. thuringiensis var. israelensis, while samples 8 and 18 were 75% identical. 46 | fine focus b. thuringiensis var. israelensis (sample #17) when compared against b. thuringiensis var. israelensis, samples 15, 16, 24, and 25 were 0% identical. samples 6, 14, 26, 28, 32, 33, 36, and 37 were 25% identical to b. thuringiensis var. israelensis. samples 13, 35, and 42 were 50% identical to b. thuringiensis var. israelensis, while samples 8 and 18 were 75% identical. as shown in table 5, samples 9, 10, 22, 39, and 43 had one nhea positive amplicon during real-time pcr. samples 27 and 31 had two nhea positive amplicons late in analysis after initially appearing to only contain one positive melt peak. consequently, these samples were not subjected to rep-pcr, as the real-time results were inconsistent. rep-pcr efforts were instead directed at samples that had either three melt peaks or two melt peaks early in analysis within 1 sd of the positive control. discussion the debate over the ideal method for identification of bacillus isolates has raged for over 50 years (42). recent public awareness of potential bioterrorism using the anthrax toxin produced by b. anthracis has led government agencies to fund multiple studies aimed at rapidly differentiating b. anthracis from other closely related bacillus species, such as b. cereus and b. thuringiensis. b. anthracis produces the anthrax toxin encoded by two plasmid-based operons, pxo1 and pxo2 (3, 17, 42). the anthrax toxin primarily kills herbivore mammals but can also kill humans (42, 48). not to be underestimated, b. cereus can cause severe food poisoning through its production of emetic and diarrheal toxins (3, 19). while heavily used as an insecticidal agent in crops with its cry crystalline toxins, b. thuringiensis has also recently been demonstrated to cause food poisoning symptoms in humans similar to b. cereus (3, 42). ironically, species like bacillus coagulans, which was found to contain nhea at least once in this study, are readily used as probiotics in human health (30). it should be noted that the nhe genes have been among the most common reference virulence genes targeted in pcr-based assays performed in foods, including dairy foods (37). nhea/nhea has thus been widely accepted as an indicator of virulence potential in bacillus spp. senso lato. these strains were originally differentiated into species at a time when biologists did not possess the molecular tools to delve deeper than biochemical tests and phenotypical observations (3, 17, 42). while this strategy worked well for other genera, 16s rrna analysis of differences among b. cereus, b. thuringiensis, and b. anthracis have shown these species to have a nucleotide sequence difference of < 1% (48). thus, the emerging “holy grail” of bacillus research would be to accurately differentiate these species. recent advances in molecular biology have allowed scientists to scrutinize the genetic properties of these three “species” (42). after exhaustive studies using dnadna hybridization, 16s and 23s rrna comparative analyses, multilocus sequence typing (mlst), fluorescent amplified fragment length polymorphism analysis, rep-pcr, and small nucleotide polymorphism (snp) analyses, scientists have been unable to reliably differentiate these three bacillus species. while many methods have been pursued, most results have suggested that b. cereus, b. thuringiensis, and b. anthracis should be considered the same species due to highly conserved nucleoidal genetic sequences (3, 17, 39, 43, 48). due to the easily identifiable symptoms of b. anthracis and b. cereus, there is recent concern among biologists that the “b. anthracis” species may in fact be an oversampled subset of b. cereus (42). other scientists speculate that b. anthracis may have only recently evolved to the point to be considered distinct from b. cereus (20). either way, a separate study confirmed that enough of a difference exists between the genome of b. anthracis when compared against b. cereus or b. thuringiensis to consider b. anthracis as identifiable using pulsed-field gel electrophoresis (50). of the 45 total food and soil samples in this study, 20 bacillus isolates were obtained (44.4%). twenty-one samples (48.9%) were not found to contain bacillus isolates. three isolates were plated from jiffy corn muffin mix along with two other samples for a total of 6.7% after heattreatment but were gram-positive cocci. because this research examined bacillus spp., any non-gram-positive rod specimens were not analyzed further. volume seven | 47 bacillus spp. are ubiquitous in nature and form endospores that readily transfer to foods (3, 16, 17). initially for the bacillus isolation approach, nutrient rich bhib incubation overnight at 32°c did not allow for endospore formation. endospores optimally form when the bacteria are stressed and require 1 to 2 days for full development (3, 36). while most samples had already been screened for bacillus presence, the remaining few were instead shaken for three days at the same conditions to allow sufficient time for endospore formation. consequently, endospores were better isolated after this change. it is likely that bacillus spp. endospores were present in many samples that lacked detectable bacillus isolates initially, like nestle nesquik, given their general ubiquity (17). these samples were then subjected to real-time pcr analysis. there are three nhe genes that are encoded on the nheabc operon (3) and have been shown to remain conserved as a cluster during genetic recombination (19). it can reasonably be assumed that the presence of the most proximal subunit of nhe indicates the presence of the other two genes. in the literature, all genes encoding the nhe and hbl enterotoxins have been readily located downstream in both b. cereus and b. thuringiensis (35). of 616 bacillus isolates tested, none were found to harbor only a single or two of the genes for each operon. over three separate real-time pcr runs, all 4 soil samples had three melt peaks within 1 sd of nhea positive b. cereus atcc 14579. thus, they were also positive for the presence of the nhea gene. by extension, these strains were also positive for the presence of the nheabc operon and could be considered pathogenic. samples with three melt peaks consistent with the b. cereus positive control also resulted in standard deviations much less than 1, as shown in table 6b. these melt peaks were extremely similar to each other and to the positive control, meaning that the amplified product was, in fact, nhea. of 16 total food isolates, four displayed three nhea positive melt peaks, while four displayed two nhea positive melt peaks. additionally, 4 food isolates displayed only one nhea positive melt peak, while four were found to contain no identifiable nhea genes. over three real-time pcr runs, samples with three melt peaks within 1 sd of the nhea positive control strain were also considered positive for the presence of nhea. samples with two of three melt peaks within 1 sd of the positive control strain were also considered to be positive for the presence of nhea, even with an erroneous third melt peak. while real-time pcr is an accurate assay for gene detection, it is still sensitive to pipette error as well as pcr inhibitors (29). thus, it is likely that user error prevented a third melt peak within 1 sd of the positive control. samples with one of three melt peaks within 1 sd of the positive control were treated as potentially positive for the presence of nhea. however, further research of these strains needs to be performed for a definitive answer. one positive melt peak was not determined to be strong enough evidence to ignore two negative results. after real-time pcr analysis, reference cultures b. macerans, b. brevis, b. cereus, b. thuringiensis var. kurstaki, b. thuringiensis var. japanensis, and b. thuringiensis var. israelensis also displayed three melt peaks within 1 sd of nhea positive b. cereus over three runs. additionally, b. laterosporos displayed two of three total melt peaks consistent with the positive control strain, and by extension contained the nheabc operon. the b. thuringiensis and b. cereus sample results were expected and confirm earlier work indicating that both are pathogenic (33, 39, 48). while the reference strains b. circulans and b. megaterium were not positive for the presence of nhea in this study, they were found to harbor each hbl gene in a separate study (44). it is very possible these strains contained a polymorphic version of nhea. to the best of our knowledge these samples, minus b. cereus and b. thuringiensis, are novel findings that are not usually associated with food pathogenicity (3, 16, 17, 18). however, it is an unsurprising find that bacillus isolates harboring the nhea gene were identified in food, at least in b. thuringiensis and b. cereus. b. cereus and b. thuringiensis are arguably the same species (3) and have been demonstrated to be pathogenic in food (18). there is a general consensus among biologists that most, if not all, bacillus isolates undergo horizontal gene transfer (17). 48 | fine focus one study determined that of the b. cereus and b. thuringiensis isolates obtained from rice, 84.3% and 100% of them produced the nhe enterotoxin, respectively (1). sixtyone percent and 100% of these same isolates produced the hbl enterotoxin, respectively. a separate study found that of 136 b. cereus isolates obtained from milk, over half were toxic against hela cells (10). additionally, 73.2% were toxic against hel cells. a third study noted that of emetic strains identified, 77.5% of b. cereus strains also produced nhe (27). yet another study found that the nheabc operon was present in every b. thuringiensis strain tested (35). the presence of the nheabc operon does not necessarily indicate a virulent strain, but has a very high likelihood of expressing these genes in a host environment or in food under permissive conditions (3, 41). thus, future work to determine the pathogenicity of nhea positive samples would include the use of a tecra via immunoassay kit to detect enterotoxin proteins (3, 17, 18, 27). without this step, the virulence of nhea positive samples cannot be definitively determined. these data suggest that at least 8 of the 16 isolates from food were positive for the presence of the nheabc operon. an additional four food isolates may also be enterotoxigenic, meaning that there is a 75% chance of any food isolate consumed being potentially enterotoxigenic. additionally, three reference strains were identified that have not been previously known to harbor enterotoxigenic genes. a large degree of genetic variation exists in nhe sequences among bacillus spp. (18), giving rise to false negative results in pcrbased detection assays. strains negative for nhea in real-time pcr have been found to produce the enterotoxin nhe as determined using a tecra via kit. it is very possible that some of the nhea negative strains from real-time pcr may still be enterotoxigenic due to polymorphism (15). after real-time pcr analysis, it was necessary to determine how similar the unidentified bacillus food and soil isolates were to the reference strains b. cereus, b. thuringiensis var. kurstaki, b. thuringiensis var. japanensis, and b. thuringiensis var. israelensis using reppcr. if banding patterns of the unidentified isolates were very similar to rep-pcr banding patterns of reference strains, then this research would not have identified new strains harboring enterotoxigenic genes. within bacillus, most virulence factors are encoded on plasmids (42), which have been demonstrated to readily transfer between differing species (3, 19). indeed, a recent study indicated that the virulence genes associated with b. cereus infection undergo frequent rearrangement both within the bacterial nucleoid and between species (26). thus, a better method than traditional biochemical tests to detect pathogenic bacillus strains is to screen for virulence operons present in plasmids or in nucleoidal dna (28, 42). bacillus genomes that have been sequenced display a high level of genetic synteny in their gene order. two genes that encode for bacterial ribosomes, 16s and 23s rdna, contain genetic sequences that are < 1% different when compared between b. cereus, b. thuringiensis, and b. anthracis (48). a dissimilarity of 3% between 16s or 23s rdna sequences is the minimal “cut off ” between two strains to be considered as distinct species. additionally, the gyrb gene sequence shared among these species is very homologous (38). because these genes are shared among different species within the bacillus genus, they cannot be used to differentiate species (42, 48). however, 16s and 23s rrna can be used to differentiate between different strains of b. anthracis (13). ultimately, the many attempts at differentiating b. cereus, b. thuringiensis, and b. anthracis have led to complete genomic sequencing of 16 strains of these three species (42). this large data pool has allowed bacillus to serve as a good model for genetic conservation and to allow thorough study of virulence gene transfer. additionally, the abundance of sequencing information on bacillus genomes has allowed scientists to statistically differentiate sequencing error from actual polymorphisms. interestingly, there are a number of mechanisms that facilitate the movement of genes between different members of the bacillus genus. one such mechanism is through the natural action of bacteriophage. after lysing its host cell, the bacteriophage will insert its genes into bacillus genomes. while normally either lytic or lysogenic, it is possible for prophage to undergo random mutation, which renders it unable to enter the lysogenic cycle. in this way, genes from one species of bacteria can be transferred to bacillus spp. as previously mentioned, bacillus operons may be on conjugative plasmids. additionally, bacillus spp. are naturally competent, allowing these microbes to naturally take up random dna in their vicinity (19). volume seven | 49 the virulence genes for nhe are present in more strains of bacillus than is currently accepted within the scientific community. this research identified several “species” of bacillus that were not previously known to harbor the nhe enterotoxigenic operon. given that a debate is currently underway about the very identity of b. cereus and other strains, it is improper for food safety experts to screen food products only for b. cereus. phenotypic-based classification techniques have failed to accurately differentiate bacillus species. additionally, no molecular-based approach can accurately differentiate bacillus (42). the bottom line is the determination of species within bacillus does not even matter when concerned with food safety. molecular techniques should instead screen for virulence determinants in microbes instead of identifying said microbes (47). since endospore formation enables bacillus spp. to be ubiquitous in the environment and on 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environmental biology. 73: 3446-49. 51. zwick, m.e., j. sandeep, x. didelot, p.e. chen, k.a. bishop-lilly, a.c. stewart, k. willner, s. lentz, 52. n. nolan, m.k. thomason, s. sozhammannan, a.j. mateczun, l. du, and t.d. read. 2012. genomic characterization of the bacillus cereus sensu lato species: backdrop to the evolution of bacillus anthracis. genome res. doi: 10.1101/ gr.134437.111. 84 | fine focus enrichment of microbes potentially degrading polyethylene using a microcosm approach ana maria barral and ben stenson national university, san diego, ca national university 11255 north torrey pines road, la jolla, ca 92037 contact email: abarral@nu.edu manuscript received 12 may 2020; accepted 11 june 2020. volume six | 85 abstract plastic pollution is a worldwide phenomenon with concerning effects on the biosphere and particularly on the marine environment. biodegradation is considered an environmentally friendly alternative to combat the increasing quantities of plastic pollutants where different microbial sources are tested for plastic degradation potential. in this project, a microcosm approach was used as an enrichment method for marine microbes degrading polyethylene. pieces of low-density polyethylene (ldpe) and highdensity polyethylene (hdpe) previously deployed in ocean water have been explored as a source of microbial biomass. this source plastic was added to a synthetic medium containing sterilized pieces of ldpe and hdpe as the sole carbon source and were incubated for extended periods (32-86 days) in the laboratory to promote growth of microbes that can degrade plastic. biodegradation of polyethylene was confirmed by dry weight measurements and fourier transform infra-red (ftir) spectroscopy. for both ldpe and hdpe a significant reduction in dry weight was observed. ftir analysis showed peaks suggesting oxidative changes in polyethylene’s chemical composition. in summary, the microcosm approach can be considered a viable approach for enrichment of plastic-degrading marine microbial populations. 86 | fine focus introduction few topics span so many disciplines and touch the public interest as much as the issue of plastic pollution. plastic is an essential element of our everyday lives, and its introduction in multiple spheres of human activity brought economically and scientifically sensible solutions and innovations. plastic can be defined as long-chain synthetic polymers created through polymerization of monomers obtained from oil or gas with chemical additives (1). plastic material is used to package almost every commodity, and it is estimated that one-third of plastic manufactured is used in disposable one-time use products (2). due to its non-degradable nature, plastic has been accumulating in the environment at exponentially increasing rates since mass production began in the 1940s (1, 3). some of the plastic accumulating in the environment inevitably makes its way into the ocean environment. rain, wind, tsunamis, streams, and other phenomena carry plastics into the ocean, where they fragment into microscopic pieces. additionally, microplastics may enter the marine environment through cleaning scrubbers, abrasive beads for cleaning boats, and deterioration of larger pieces of plastic (4). plastic litter and contaminants can be found on remote beaches throughout the world constituting not only an eyesore, but also a hazard for the environment. dangers of plastic include harm to animals (5), ability to transport contaminants around the ocean (6), as well as release of toxic chemicals upon degradation (4, 7, 8). diverse abiotic and biotic processes can degrade plastic in marine environments (9–11). degradation can be defined as a process that leads to a decline of polymerlike properties through multiple steps. weathering is the physical deterioration of plastic, which occurs in the ocean through wind and waves. photodegradation is the leading cause for degradation of plastic, as uv-b radiation from sunlight breaks the c-h bonds on the polymer backbone, creating a carbon free radical that most often reacts with oxygen creating a peroxy radical. olefins, aldehydes, and ketones are the most common products, but c-c crosslinking can also occur (11). photo-initiated oxidative degradation is the rate determining step of degradation in the marine environment. biotic degradation is not as well documented as abiotic processes (12, 13) and it is almost always preceded by abiotic degradation (14). microbes can excrete extracellular enzymes, which break down and utilize the carbon backbone of most plastics. in the marine environment, conditions are not optimal, so both abiotic and biotic degradation occur slowly on a scale of decades. potential microbial candidates for biodegradation of plastic have been previously described, including a penicillum species from a landfill (15), pseudomonas (16, 17) bacillus (18) and rhodococcus (19) among others. recent reviews list a number of marine microbial groups with described or potential plastic degradation ability (20–22). polyethylene (pe) is the most common plastic and can be found in two varieties: high-density pe (hdpe) and low-density pe (ldpe). pe represents 64% of all plastic produced and it is the most common floating plastic as well as the most common plastic found on beaches (23). due to its light weight and hydrophobicity, pe floats in ocean water. its backbone is composed solely of carbon atoms, which can be the target of microbial activity originating at any terminal methyl group. previous studies have described the “plastisphere,” a unique microbial ecosystem living on and in floating plastic, containing organisms potentially able to use plastic polymers as a food source (12, 13, 24, 25). an ongoing study exploring the bacterial populations volume six | 87 attaching to floating plastic in coastal waters (26) has produced plastic with a rich biomass. we hypothesized that this biomass could be a suitable source for the identification of potentially plastic-degrading microbes. the microcosm approach, first described in the 19th century (27) has been a debated model to recreate complex ecological systems in a laboratory environment (28–30). a number of authors have used this approach to isolate and identify microbes with the ability to degrade plastic from diverse complex sources (31–33) in this study, we tested a microcosm approach to enrich for marine microbes with the ability to degrade pe. we used pe previously exposed to ocean water as microbial source, and uv-irradiated pe as the sole carbon source in the growth medium. plastic degradation was tested using chemical methods such as fourier transform infrared spectroscopy (ftir) and dry weight measurements. our results indicate modest degradation by decrease in weight and observed oxidative changes. materials and methods microcosm figure 1 shows the overall setup of the microcosm experiment to select plastic degrading microbes. for microbial source, pe that had been previously exposed to ocean water (either in the field or in a laboratory setting, see section below) was added to a synthetic medium containing no carbon. as a carbon source, sterilized pe was used. sterile mason jars were used to contain the medium. microbial source as source of microbial biomass, pe samples previously incubated in ocean water were employed. these samples are part of a project studying microbial colonization in coastal waters (26). briefly, plastic squares about 5cm x 5cm were sterilized and placed in sampling cages, which were either attached to the scripps oceanography pier in san diego, ca (gps coordinates 32°52’00.8”n 117°15’28.1”w, approximately 5m under the water surface), or placed in 5l carboys containing ocean water in the laboratory incubator set at 16 oc. microbial biomass was collected on three different occasions, after 33 days (pier), 153 days (lab), and 70 days (lab) incubations. collection times were variable due to both weather conditions and a related educational field trip experience (34). samples were collected using gloves and sterilized instruments, and placed in sterile pouches. the collected plastic samples with the attached biomass were used as microbial sources for three sets of microcosm experiments. a 3cm x 3cm piece of pe (either hdpe or ldpe), cut with sterile scissors using aseptic technique was added to the microcosms. control cultures with no microbial source were set up in parallel for the first microcosm experiment. source of pe sources of high-density pe (hdpe) and low-density pe (ldpe) were clean produce and grocery bags, respectively. the same batches of bags collected from sprouts and amazon packaging were used for all experiments. the chemical nature of each polymer was confirmed through ftir (see section below). pretreatment of pe 88 | fine focus figure 1. microcosm setup. pe pieces previously incubated in the ocean were used as source of microbes. the synthetic medium imitated ocean salinity but contained no carbon source. carbon was provided as sterilized pe fragments (5 per culture). volume six | 89 prior to placement in medium, the pe used as carbon source was sterilized through serial washes with shaking (40 min in sds 2%, 15 min in deionized water, 30 min in 70% ethanol), using a method adapted from das & kumar (18) . after the last wash, the pe pieces were handled using aseptic technique, stored in sterilized pouches, and left to dry overnight at 60°c. to simulate photo-initiated degradation (naturally occurring in the marine environment by solar radiation), pe samples were incubated under a uvp uv emitter (model tm-10e, upland, california, usa). plastic was cut into 1cm x 1cm squares using aseptic technique and exposed to uv light at 253.7nm for 48 total hours. five squares per sample were added to each microcosm experiment. culture conditions for the microcosm experiment, cultures were kept in a previously described synthetic medium (14), at 16°c in the dark. the medium mimicked ocean salinity while containing no carbon sources other than the added plastic. it contained: 1g nh4no3, 1g k2hpo4, 0.15g kcl, 0.2g mgso4, 0.1g cacl2, 0.1g yeast extract, 1mg mnso4, 1mg feso4, and 1mg znso4 per 1l distilled water. cultures were set up in 250 ml sterile mason jars. hundred ml of the medium were supplemented with 5 pieces of 1cm x1cm pe squares and one piece of previously incubated pe as microbial source. paraffin oil, a non-ionic surfactant, was added at a final concentration of 0.05% v/v to the second and third experimental sets to enhance colonization (17). three sets of microcosm experiments were completed for a length of 32, 70, and 86 days. determination of dry weight weight of the supplemented pe was recorded after the plastic had been sterilized and left to dry overnight at 60°c, but before it was transferred to the synthetic medium (“before”). after the incubation time concluded, plastic was treated with the same sterilization and drying process and weighed again (“after”) on a mettler toledo scale (model xs603s, switzerland). the scale was previously calibrated according to the manufacturer’s protocol and had a sensitivity of ±0.01 mg. dry weight measurements were compared using a t-test. ftir analysis ftir studies were performed on the individual pe pieces at the end of the incubation after the dry weight was recorded. as controls pe fragments sterilized and uv-treated but not incubated were used. ftir was performed on a nicolet iz10 spectrometer and spectra analyzed using the omnic anywhere cloud computing system (thermo scientific, waltham, md). the double bond index (dbi) was calculated based on the relative intensity of the double bond band at 1,650 cm−1 to that of the methylene scissoring band at 1,460 cm−1 (35). the dbi of the control and the experimental samples was compared using a one-way anova. for statistical analyses the graphpad prism version 8.0.0 for mac was used (graphpad software, san diego, ca). results microcosm experiments table 1 shows the details of the microbial sources and incubation times for the three microcosm experiments. pier and laboratory incubations had been set up in parallel to guarantee samples for an educational project, using the same sampling system and as similar conditions as possible. laboratory experiments used ocean water from the pier location. water temperatures at the scripps pier were averaged based on the daily readings from the incubation 90 | fine focus *this set also included a negative control (no microbial source added). **data provided by the shore stations program, with current funding provided by the california department of parks and recreation, division of boating and waterways, award# c1670003. data are collected by staff aquarists and volunteers with the birch aquarium at scripps. (°c) volume six | 91 figure 2. a: a ldpe microcosm at the beginning of incubation. b: the same microcosm at the end of a 70-day incubation. 92 | fine focus period, while the temperature was set at 16 oc for the laboratory incubations. figure 2 shows a ldpe sample from the set #2 microcosm experiment at day 0 and day 70, respectively. increased turbidity can be observed in the medium. overall, microcosm samples consistently showed microbial growth as evidenced by increased turbidity. uneven loss of volume due to evaporation was observed, preventing measurement of turbidity as a reflection of growth. dry weight dry weight of the plastic specimens was measured before and after incubation. due to the extensive manipulation required for the sterilization and drying of the pe samples, some hdpe samples were lost during the process. the negative controls (pe incubated in the microcosm without a microbial source) from set #1 did not show reduction in weight. weight loss was recorded in 69% of samples while no change was recorded in 31% of samples. there was no clear relationship between weight loss and length of incubation tested or source of microbial culture. figure 3 shows the dry weight change in all hdpe and ldpe samples tested before and after the incubation. the average weight for ldpe samples decreased from 11.4 mg (sd=1.35) to 10.1 mg (sd=1.37). similarly, hdpe weight decreased from 5.5 mg (sd=2.9) to 4.5 mg (sd=2.17). the decrease of dry weight was statistically significant for both ldpe (p=.0133, t=3.074, df=9) and hdpe (p=.0409, t=2.739, df=5). ftir when comparing the ftir spectra of control (pretreated but not included in the microcosm) pe with the microcosm ldpe and hdpe samples, peaks indicative of oxidative processes could be observed. figure 3 shows the ftir spectra corresponding to representative ldpe and hdpe samples after 70 days. compared to the control, both ldpe and hdpe show additional peaks corresponding to the range of alkynes (2,200 2,300 cm-1) as well as a robust peak corresponding to double bonds such as the carbonyl group (1,640 1,680cm-1). figure 5. shows the double-bond index (dbi) calculated for each microcosm set. there is no significant difference between the control and the day 30 samples for either ldpe and hdpe. while there seems to be an overall increase in the dbi for both ldpe and hdpe by day 70, only in hdpe was the increase significant from m=0.200 (sd=.0956) to m=0.403 (sd=.07319) (p=.0063). the day 86 samples were not significantly different from the control. discussion the term “great plate count anomaly” was coined by staley and konopka in 1985, which described the discrepancy between total microbial counts from a natural environment and colonies isolated by traditional microbiology methods (36). it is estimated that the latter can only recover 1% or less of the bacterial diversity in most environmental samples (37). therefore it has been a continuing challenge for microbial ecologists to describe complex microbial populations and the key interactions between their components. culture-independent methodologies, based on dna analysis using metagenomic approaches are often used to characterize complex populations (38). such analyses have helped the identification of microbes whose genomes contain volume six | 93 figure 3. average dry weight significantly decreased for both ldpe (m=11.4 mg, sd=1.35 to m=10.1 mg, sd=1.37) and hdpe m=5.5 mg, sd= 2.89 to m=4.5 mg, sd= 2.17) after the microcosm incubation. as controls pe fragments sterilized and uv-treated but not incubated were used. the * indicates significant differences of p=.0133 and p=.0409 for ldpe and hdpe, respectively. 94 | fine focus figure 4. representative ftir profiles of ldpe (a-before, c-after) and hdpe (b-before, d-after). distinct peaks corresponding to alkynes and double bonds are highlighted with arrows. volume six | 95 figure 5. double bond index values calculated from the samples of the three microcosm experiments using ftir. 96 | fine focus genes for enzymes that may be involved in plastic degradation (12). however, interest for the isolation and characterization of microbes using culturedependent approaches remains, particularly for marine organisms (39). a microcosm is a miniature, controlled environment (40) that can act as proxy of complex populations (30, 41). in this study, it served as a “low-tech” approach to select microbial entities with the potential to degrade plastic from a complex biomass. results showed presence of microbial populations capable of degrading ldpe and hdpe as assessed by decrease in dry weight and chemical changes detected with ftir. previous studies have shown chemical signs of degradation in pe and polypropylene samples deployed in coastal waters (26, 42–44). the microcosm approach of this article was developed to promote the potential biodegradation process in a controlled environment while limiting abiotic processes such as weathering and photodegradation. the setup was conducive to microbial growth, which could be observed as increased turbidity, especially after the addition of paraffin oil to the synthetic medium. paraffin oil has been described to increase microbial attachment and formation of a biofilm, which ultimately results in enhanced degradation (17). due to evaporation of culture media in some samples, measurement of absorbance of the culture was deemed not reliable, an aspect that should be improved in future experiments. field experiments are notorious for the influence of factors outside the control of researchers. the first set of the microcosm experiments had a microbial source resulting from an incubation in the ocean for 33 days, and itself had a duration of 32 days. based on the literature and our data showing minimal changes in the ftir spectra, a decision was made to test microbial sources resulting from laboratory incubations that could be kept for longer times. these incubations were completed at controlled temperatures in large carboys containing ocean water. sets #2 and #3 of the microcosm experiments, using the laboratory microbial sources, exhibited more growth and stronger signs of degradation according to ftir, which seems related to the length of the original incubation. it has been described previously that plastic biodegradation is an extremely slow process (9), as also evidenced by our results. while clear ftir peaks indicative of oxidation were observed after more than 2 months incubation, ldpe and hdpe lost only an average of 11% and 18% of weight, respectively. ftir spectroscopy is a commonly used analytical tool to detect changes in chemical composition. formation of new functional groups as well as the disappearance of others are indicative of significant chemical changes (18). our results are in agreement with previous authors’ findings that show signs of pe oxidation as a result of biodegradation. interestingly, we did not observe a defined carbonyl stretch peak at 1712 cm-1, a typical finding in uv-treated pe, which is often used as a measure of photodegradation (45). however, the presence of double bonds increased, as shown by both a larger peak in the range of 1640-1680 cm-1 and the concurrent changes in double bond index, particularly for hdpe at day 70. this could be explained by microbial activity attacking the carbonyl groups generated by abiotic factors, resulting in unsaturated chains (35). the fact that only hdpe from the second (70-day) set showed a significant difference compared to the control, while the 86-day third set hdpe did not, is intriguing. however, the second set’s microbial source originated from a 153 day incubation, more volume six | 97 than double the length of the third set experiment’s source (70 days). enrichment of pe-degrading microbes may have already taken place in the source material of the second set. previous ftir analyses of ocean-incubated samples have shown that signs of degradation appear first for hdpe and later for ldpe (manuscript in preparation). if this is due to chemical differences in hdpe and ldpe, or to different physical characteristics is not yet known. the ftir results highlight some of the limitations of this study, which are standardization and repeatability. as a side project to a field-based educational project that is highly dependent on weather conditions and logistical issues, these initial samples had microbial sources with variable incubation conditions. however, overall the results seem to suggest that 1) laboratory samples to generate microbial sources are as effective as field samples, and 2) length of the original incubation is a paramount factor. metagenomic characterization of the biomass and surrounding water is ongoing. preliminary results indicate an extensive overlap between plastic and water, which shifts over time; as well as a high prevalence of unknown taxa (dr. r. simmons, personal communication). another ongoing study uses culture-dependent methods by swabbing media plates with the biomass to characterize the resulting colonies via 16s pcr (26). future experiments will continue the microcosm approach to enrich in plastic-degrading bacteria using longer incubation times to achieve even higher degradation rates. to isolate and characterize the microbes responsible for degrading pe, standard microbiology dilution techniques combined with selective media will be used, similar to the approach used to isolate a pet-degrading bacterium (46). we plan to use metagenomic sequencing of the original microbial sources and the microcosm cultures to characterize changes in populations. in addition, whole genome sequencing of the population may provide clues to the enzymes involved in the degradation pathway. acknowledgments the authors would like to thank dr. jeff bowman at scripps institution of oceanography (ucsd), dr. emilia deforce of thermo fisher scientific, and dr. rachel simmons of national university for making this project possible. we are grateful to 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a, echevarría f, gonzález-gordillo j, irigoien x, ubeda b, hernández-león s, eriksen m… yamaji h. 2016. microbes on a bottle: substrate, season and geography influence community composition of microbes colonizing marine plastic debris. plos one 11:e0159289. 44. erni-cassola g, wright rj, gibson mi, christie-oleza ja. 2020. early colonization of weathered polyethylene by distinct bacteria in marine coastal seawater. microb ecol 79:517–526. 45. dussud c, hudec c, george m, fabre p, higgs p, bruzaud s, delort am, eyheraguibel b, meistertzheim al, jacquin j, cheng j, callac n, odobel c, rabouille s, ghiglione jf. 2018. colonization of nonbiodegradable and biodegradable plastics by marine microorganisms. front microbiol 9:1571. 46. yoshida s, hiraga k, takehana t, taniguchi i, yamaji h, maeda y, toyohara k, miyamoto k, kimura y, oda k. 2016. a bacterium that degrades and assimilates poly(ethylene terephthalate). science 351:1196–9. volume seven | 9 biogenesis of lipoproteins in gram-negative bacteria: 50 years of progress james c. kuldell* (jkuldell1@pride.hofstra.edu) harshani luknauth* (hluknauth1@pride.hofstra.edu) anthony e. ricigliano* (aricigliano1@pride.hofstra.edu) nathan w. rigel† (nathan.w.rigel@hofstra.edu) department of biology, hofstra university *all authors contributed equally †corresponding author manuscript received 31 january 2020; accepted 20 november 2020. department of biology hofstra university 318a gittleson hall hempstead, ny 11549 nathan.w.rigel@hofstra.edu phone: 516-463-6542 fax: 516-463-5112 keywords: acylation, pre-prolipoprotein, prolipoprotein, apo-lipoprotein, cell envelope 10 | fine focus abstract the outer membrane is the defining characteristic of gram-negative bacteria and is crucial for the maintenance of cellular integrity. lipoproteins are an essential component of this outer membrane and regulate broad cellular functions ranging from efflux, cellular physiology, antibiotic resistance, and pathogenicity. in the canonical model of lipoprotein biogenesis, lipoprotein precursors are first synthesized in the cytoplasm prior to extensive modifications by the consecutive action of three key enzymes: diacylglyceryl transferase (lgt), lipoprotein signal peptidase a (lspa), and apolipoprotein n-acyltransferase (lnt). this enzymatic process modifies lipoprotein precursors for subsequent trafficking by the lol pathway. the function of these three enzymes were originally thought to be essential, however, in some gram-negative bacteria, namely acinetobacter baylyi, the third enzyme lnt is dispensable. here we review the function and significance of lgt, lspa, and lnt in outer membrane biogenesis and how noncanonical models of lipoprotein processing in acinetobacter spp. can enhance our understanding of lipoprotein modifications and trafficking. volume seven | 11 introduction the general structure of gram-negative bacteria such as escherichia coli and acinetobacter baylyi consists of the inner membrane (im) and outer membrane (om) separated by an aqueous periplasm containing a thin peptidoglycan layer (1). the outer membrane is the defining characteristic of gram-negative bacteria and is essential for the maintenance of cellular integrity. the om is composed of phospholipids and a lipopolysaccharide asymmetric bilayer containing β-barrel om proteins (omps) (2). in order for correct biogenesis of the om to occur, lipopolysaccharides must be delivered to the om by the lpt machinery while the beta-barrel assembly machinery (bam) complex assembles omps into the om (3). proteins destined for the om are synthesized in the cytoplasm with a signal peptide used for translocation to the im by the general secretion (sec) pathway for unfolded proteins or through the twin-arginine translocation (tat) pathway for fully folded proteins (4–6). deviations or loss of function in the assembly machinery needed to synthesize the om results in profound defects causing morphological defects such as antibiotic and temperature sensitivity (7). outer membrane lipoproteins have vital functions including aiding in lipopolysaccharide (lps) insertion in the om and forming secretion systems (8, 9). lps is found in the outer leaflet of the om in most gram-negative bacteria and is thus essential because it interacts directly with the outside environment of the bacterium and maintains the impermeability of the om (10–13). the proteins, lptd and lpte are components of the lpt machinery which function to transport the essential lps to the om (8, 9). lpte and lptd form a complex in which lpte is buried inside lptd and functions as a plug to prevent the passage of molecules through lptd into the bacteria (14– 18). there are also surface-exposed lipoproteins such as lpp and pal which function to increase cell wall stability by attaching the om to the cell wall (19–21). other surfaceexposed lipoproteins are part of stress responses including rcsf which is the sensory component of the rcs envelope stress response (22, 23). this stress response can be activated by lps stress and osmotic stress (24–27). lipoproteins are also essential in assembly of the om and regulating the traffic of molecules inside and outside of the gram-negative cell (28). these om lipoproteins can release virulence factors to the surrounding environment resulting in infection (29, 30). they also can release toxins or hazardous chemicals for defense (31). another assembly of om proteins is the bam complex which functions to facilitate the formation of betabarrels in the om (32). blocking om lipoprotein trafficking or inhibiting their function can result in cell death (33). if a lipoprotein is not trafficked, the cell cannot regulate the efflux of molecules across the membrane, depriving it of resources and preventing the cell from expelling hazardous chemicals. therefore, the lipoprotein sorting pathway is integral to gram-negative bacteria because lipoproteins in the om are essential to maintaining the cell envelope. synthesis of lipoproteins begins in the cytoplasm. lipoproteins are synthesized as precursor pre-prolipoproteins in the cytoplasm prior to being trafficked through the abc transporter localization of lipoproteins (lol) pathway (34). once unmodified prelipoproteins destined for the om are translated, they enter the im through the sec or tat pathway (35) (fig. 1a). the signal peptide embeds the protein to the im and allows for recognition for the first step of modification (36). diacylglyceryl transferase (lgt) is the first protein in the pathway and transfers di-acylglycerol to the cysteine residue in the lipobox of the prolipoprotein (37). lgt has been thought to be essential in all gram-negative bacteria because it is the first step of lipoprotein maturation which is required to be recognized and translocated using the lol system (37, 38). however, insertion mutations have been created in the lgt gene of acinetobacter baumannii which have yielded viable cultures (39). the second step of lipoprotein maturation is done by prolipoprotein signal peptidase (lspa) which cleaves the signal peptide leaving the lipobox at the n-terminus. lspa is also essential due to its ability to cleave the anchor peptide from the im. the heavily hydrophobic region is what allows and maintains the protein in the im, therefore its release is integral for lipoprotein movement to the om as well as function and folding. the third and final step in lipoprotein maturation is the addition of one more acyl 12 | fine focus group to the cysteine residue by apolipoprotein transacetylase (lnt) (fig. 1a). together, these processes define the canonical lipoprotein modification pathway in gram-negative bacteria (34). classically it was thought that lnt-mediated tri-acylation was an essential requirement for recognition by the lol system and subsequent lipoprotein trafficking. however, lnt was found to be nonessential in some gram-negative bacteria including francisella, neisseria, and acinetobacter (40,41). the loss of lnt is not without detriment, as the absence of a third acylation interrupts lipoprotein trafficking resulting in visible growth defects such as increased om permeability in acinetobacter spp. (42). the viability of acinetobacter spp. after lnt deletion suggests a non-canonical function of the existing lipoprotein modification and trafficking pathway (fig. 1b). if an apo-lipoprotein is successfully tri-acylated and cleaved, to become a mature lipoprotein and reach the om, it must be translocated there by the lol system (34). the lolcde proteins exist in the im where it receives the tri-acylated lipoproteins, as studied in e. coli (43). if destined for the om the periplasmic chaperone lola then shuttles the lipoproteins to the om receptor lolb for inclusion into the om (38, 44–46). in francisella spp., a fusion protein resembling e. coli lolc/lole exists that shares high homology among functional domains of the transport system (41). this high homology of the fusion protein and subsequent pathway probing identified this protein as lolf, a distinct protein among certain gramnegative bacteria namely neisseria, francisella, and acinetobacter (42, 47). lolf was shown to accept di-acylated lipoproteins as compared to the rejection of di-acylated lipoproteins by lolcde (41). the lolf arrangement and successful trafficking of di-acylated lipoproteins in lnt-deficient a. baylyi suggests the presence of a non-canonical function of the lipoprotein modification and trafficking pathway (fig. 1b). in the process of exploring the non-canonical function of this pathway, it is essential to review the known structures, functions, and mechanisms of the canonical pathway. here we compile research spanning 50 years for the creation of a concise and accessible resource figure 1. lipoprotein biogenesis and trafficking in gram-negative bacteria. (a) in the canonical lipoprotein biogenesis and trafficking pathway, pre-prolipoproteins from the cytoplasm are transported to the periplasm by the sec or tat pathway where lgt adds two acyl chains (green) to the conserved cysteine residue of the lipoprotein precursor. here, lspa cleaves the signal sequence (blue) and the third and final acyl chain is added to the amino terminus by lnt. the im bound lolcde proteins then receive tri-acylated lipoproteins and can be transported by the periplasmic chaperone lola to shuttle the lipoprotein to the om receptor, lolb, for inclusion into the om. (b) in the non-canonical lipoprotein trafficking pathway of gram-negative bacteria with loldf fusion proteins, di-acylated lipoproteins are still capable of transportation to the om by an unknown mechanism or alternative pathway. created with biorender.com. figure 1. volume seven | 13 regarding lipoprotein processing and associated enzymes within gram-negative bacteria. diacylglyceryl transferase (lgt). before lipoproteins are translocated to the om through the lol system, they are first modified by lgt in the im (37). this im protein di-acylates the prolipoprotein which is the first step to becoming a mature lipoprotein. all lipoproteins that have been identified have n-acyldiacylglyceryl-cysteine as their n-terminal amino acid (48). the lgt gene was first discovered in temperature sensitive mutants of s. typhimurium in which there was an accumulation of unmodified prolipoproteins at 42°c (49). the prolipoproteins in these temperature sensitive mutants did not have any glyceryl modification. furthermore, a complementation test with a cloned insert of this deletion restored glyceryl modification activity. this cloned gene was determined to code for diacylglyceryl transferase (lgt). in e. coli, the umpa gene was determined to code for lgt after demonstrating high levels of similarity to the lgt gene in s. typhimurium (50). lgt is generally essential in gram-negative bacteria when compared to gram-positive bacteria. lipoprotein modification has been studied more extensively in gramnegative bacteria compared to gram-positive bacteria. it has been determined that gram-negative bacteria in which lgt is essential include e. coli and s. typhimurium (49, 51, 52). however, there are some gram-negative bacteria such as a. baumannii in which insertion mutations are present due to transposons in the lgt gene, yet there viable cells persist (39). furthermore, lgt has been determined to not be essential in gram-positive organisms such as bacillus subtilis and streptococcus pneumoniae (53, 54). b. subtilis and s. pneumoniae cultures were still viable after lgt deletion (53). however, lgt was deemed essential for virulence in s. pneumoniae (54). it has been hypothesized that the function of lgt in gram-positive bacteria may be analogous to substrate-specific sorting enzymes which translocate wallanchored proteins (55, 56). lgt consists of 291 amino acids and is comprised of seven transmembrane helices. the crystal structure of lgt was identified in e. coli where it is composed of 291 amino acids (33 kda) (57). there are seven transmembrane helices which form the core of the protein and there are two phosphatidylglycerol binding sites, r143 and r239 (fig. 2a). these two binding sites were determined to be critical through the use of complementation tests with lgt knockout cells and different mutant variants. there are six beta strands and four short helices. one of the critical catalytic sites, r143, which faces towards the periplasm, directly binds the substrate phosphatidylglycerol for transfer. this site is positively charged which makes it a prime residue for binding the negatively charged phosphatidylglycerol. the other phosphatidylglycerol binding site is e151. the residue r239 was also determined to be essential for diacylglyceryl transfer and functions by forming a hydrogen bond with the c3 of diacylglycerol. these two key residues are the catalytic sites which transfer di-acylglyceryl to the pre-prolipoprotein. diacylation of the prolipoprotein begins by transferring a non-acylated glyceryl of phosphatidylglycerol to the sulfhydryl group followed by o-acyltransferase catalyzed acylation of the glyceryl moiety. to determine the mechanism of action for this diacyl modification, braun’s lipoprotein was used in e. coli (25, 27, 28). braun’s lipoprotein is a murein lipoprotein which has the same composition as previously identified lipoproteins with a glyceryl cysteine (s-propane-2’,3’-diol)-3-thio-2 aminopropanic acid) at the peptide end attached to two fatty acids with another fatty acid bound at the n-terminal (48, 58). to become a mature lipoprotein, the first step of modification is done by lgt which di-acylates the prolipoprotein (37). the mechanism of di-acylating the prolipoprotein was proposed to begin by transferring a nonacylated glyceryl of phosphatidylglycerol to the sulfhydryl group of the cysteine residue at the n-terminal of the prolipoprotein (48, 60). the sn-2 and sn-3 hydroxyls of the glyceryl moiety are then acylated by o-acyltransferase enzymes to form the di-acylglycerylated lipoprotein. lipoprotein signal peptidase (lspa) signal ii peptidase encoded by lspa in gram-negative bacteria cleaves the signal peptide from the pre-prolipoprotein at the lipobox residue consensus sequence lagc (l−3a−2g−1c+1) during the intermediate step of prolipoprotein processing 14 | fine focus (61). the cleavage event results in an invariable cys residue becoming the n-terminal +1 residue, allowing for the prolipoprotein to proceed to the final acylation step by lnt(62). lspa is ubiquitous among all known gram-negative bacteria and homologs have been found in select grampositive bacteria. lspa is ubiquitously conserved among all known gram-negative bacteria and homologs have been found in select gram-positive bacteria (e.g. staphylococcus aureus) (63). structurally, among both gram-positive and gram-negative eubacteria lspa contains one conserved residue essential for stability, asp-14, and five conserved residues important for catalytic function; asn99, asp-102, asn-126, ala-128, and asp129 (64) (fig. 2b). functionally, in gram-negative bacteria lspa is considered essential under standard laboratory conditions while in grampositive bacteria lspa is considered conditionally essential for virulence (65, 66). there are no known homologs in the domain eukaryota (63). lspa consists of 169 amino acids and is comprised of two main domains containing four transmembrane helices. from the crystal structure of lspa isolated from pseudomonas aeruginosa, the 169 amino acid long (18 kda) inner membrane protein is comprised of two main domains (67). the first domain consists of four transmembrane helices culminating in n and c termini located in the cytoplasm (68) and the second domain consists of a periplasmic domain further separated into two subdomains (67) (fig. 2b). the larger subdomain is a β-cradle that rests on the membrane extending away from the protein’s helical core presenting its polar surface to the periplasm. the smaller and second subdomain contains a periplasmic helix which extends perpendicularly from the β-cradle into the periplasm (67). figure 2. proposed model of peptide topology for lgt (blue), lspa (grey), and lnt (green) in the inner membrane. roman numerals indicate transmembrane domains. (a) lgt contains seven transmembrane domains with six beta strands. pg molecules are bound to the arg143 and arg239 residues in transmembrane domains iv and vi respectively, with glu151 in transmembrane iv essential to the acyl transfer. the loop between transmembrane vi and vii is a gate for the entrance of the pre-prolipoprotein. after the acyl transfer, another pg molecule docks on the protein and the process repeats. adapted from pailler et al., 2012. (b) lspa in addition to the transmembrane domains, has a β-cradle in the first periplasmic exposed domain that retains the majority of the lipoprotein during proteolytic cleavage. lspa also contains a periplasmic helix in the second periplasmic domain. the signal peptide of the prolipoprotein is wedged between transmembrane ii, iii, and iv from recognition sites asp111, asp129, and asn99. adapted from muñoa et al., 1991 and tjalsma et al., 1999. (c) lnt exists as a thioester acyl intermediate with acyl group attached to c387. lnt has a beta-barrel-like structure with a catalytic cavity. the apolipoprotein enters the cavity laterally, stabilized by the loop between transmembrane domains iva and ivb. adapted from gélis‐ jeanvoine et al., 2015. created with biorender.com. figure 2. volume seven | 15 lspa shares a mechanism of action similar to that of the aspartic protease family. based on structural context and the functional domain homology to the family of aspartic proteases, lspa is proposed to form a catalytic dyad at residues asp-102 and asp-129 while residues asn-45, asn99, asp-111, asn-126, and ala-128 create the geometry and recognition site for the lipobox of prolipoproteins (64). the proposed mechanism of lspa mediated signal peptide cleavage is as follows. upon binding to a lipid-modified precursor, the carbonyl carbon of the scissile peptide bond is hydrated creating a tetrahedral intermediate (64). at this point a proton is transferred by means of a lytic water molecule on the initial protonated aspartic acid residue to another aspartic acid residue. the tetrahedral intermediate then donates a proton from one hydroxyl group to the recently charged aspartic acid residue (64). simultaneously the nitrogen atom of the scissile peptide bond receives a proton from the catalytic aspartic acid residue resulting in the peptide bond cleavage of the signal peptide from the lipobox motif of the prolipoprotein. this proposed mechanism of lspa enzymatic action is further supported by lspa ability to function in the absence of metal ions suggesting lspa does not use classical catalytic mechanisms of metalloproteases (69). lspa presents novel targets for drug intervention of bacterial infection. lspa is of particular significance to the development of antibiotics in response to the increasing epidemic of antibiotic resistant bacteria (70). since lspa is broadly conserved and essential in gram-negative bacteria and some gram-positive bacteria it presents as a suitable target for broad-spectrum antibiotic development (63). additionally the correct synthesis of lipoproteins by lspa-mediated enzymatic processes has been implicated in pathogenicity even in bacteria where lspa is not considered essential (e.g. mycobacterium tuberculosis) (71). lspa is also absent in all eukaryotic cells avoiding the possibility of off-target effects on host organisms (63). in comparison to the chronological development of other antibiotics and their respective targets, lspa as a target for antibiotic development is relatively new decreasing the risk associated with “legacy” antibiotics and acquired resistance (70). in early antibiotic discovery trials, a cyclic peptide antibiotic globomycin was isolated from streptomyces spp. and administered to a panel of bacteria to determine antibiotic sensitivities (72). in e. coli and other gram-negative bacteria, growth was severely inhibited by this molecule and resulted in the formation of spheroplasts indicating profound membrane assembly defects (72). further studies in e. coli showed globomycin treatment resulted in the bioaccumulation of di-acylated prolipoproteins in the cytoplasmic membrane and subsequent death of the affected bacterial cell (73). in vitro incubation of lspa enzyme and prolipoprotein substrates in the presence of globomycin showed an inhibition of lspa enzymatic activity on the cleavage of the signal peptide present on the prolipoprotein (74). decades later, the mechanism by which globomycin prevented lspa enzymatic activity was determined by the crystal structure of globomycin bound to lspa from p. aeruginosa (67). globomycin was found to infiltrate the lspa binding pocket consisting of conserved residues, typically specific for prolipoprotein substrates, and tightly bind both aspartic residues implicated in cleavage as described above (67). the occupation of both active enzymatic residues prevents typical enzymatic function resulting in the observed om defects and subsequent death of the bacterium. globomycin is an efficient inhibitor of gram-negative bacterial growth and affords the benefit of targeting lspa, which has no eukaryotic homolog. therefore, the development of globomycin analogs which can more efficiently mimic prolipoproteins and bind lspa is considered a promising avenue of research in the development of new antimicrobials (67). another bacterial secondary metabolite derived antibiotic, myxovirescin, was found to inhibit gram-negative bacteria prolipoprotein processing and subsequent growth in a mechanism similar to that of globomycin despite having a unique molecular structure (75). even though operating on convergent mechanisms of lspa inhibition, myxovirescin was shown to be rapidly bactericidal by a magnitude of almost 10-fold compared to globomycin (75). the advent of another lspa targeted antibiotic with even less incidence of spontaneous resistance compared to globomycin reinforces the importance of lspa as a viable target for antibiotic discovery (63). 16 | fine focus apolipoprotein n-acyltransferase (lnt) the last post-translational modification in the lipoprotein sorting pathway is performed by apolipoprotein n-acyltransferase (lnt), an im bound protein. lnt acquires an acyl group from a glycerophospholipid and transfers to the +1 cysteine, the same residue previously bonded to the now cleaved signal peptide, via an amide bond (52, 76). this final modification acts as a conformational structure for lol system translocation to the om (77). lnt is conserved among gram-negative bacteria but has been found in mycobacterium spp. lnt is extremely well conserved among gram-negative bacteria but has also been found in mycobacterium (78, 79). while lgt and lspa are universally prominent among both gram-negative and gram-positive bacteria, lnt is not. the purpose of the third acylation by lnt is thought to be for recognition for translocation to the om, therefore lnt does not have a function in gram-positives. interestingly, lnt homologs have been identified from blastp in mycobacterium smegmatis and mycobacterium tuberculosis as ppm1. ppm1 has also been confirmed to transfer an acetyl group to the n-terminus, similar to the lnt mechanism (78). the tri-acylated lipoproteins are suspected to contribute to m. tuberculosis virulence factors (71). glycerophospholipids are the prominent substrates for lnt. lnt transfers an acyl group from 3 different glycerophospholipids. phosphatidylethanolamine (pe), the most abundant phospholipid, constituting around 70% of the cellular lipid content, is the prominent substrate for lnt, but not essential for lnt function (80). phosphatidylglycerol (pg), the second most abundant phospholipid, is a substrate for lnt as well, but not used as efficiently as pe (81, 82). interestingly, in mutants lacking pe, pg was used for final acylation without defects. phosphatidic acids (pa) is one of the least abundant membrane lipids but can be used as a substitute for pg efficiently. lnt consists of 512 amino acids and is comprised of six transmembrane segments. lnt has six transmembrane (tms) segments with both the carboxy-terminus and amino-terminus exposed towards the cytoplasm (83). a large, 79 amino acid long, cytoplasmic loop (cl-2) is present between tms-iv and tms-v and contains two hydrophobic segments (tms-iva and tms-ivb) (83). these hydrophobic segments are not fully integrated into the membrane and are partially exposed to the cytoplasm (fig. 2c). similar morphology to a reentrant loop motif, these segments may be used for channeling in substrates, but cl-2 lacks key secondary structure for a reentrant loop (84). it is hypothesized that instead, cl-2 forms a titled fold similar to an intramembrane protease glpg in e. coli (85, 86). lnt also contains a periplasmic exposed nitrilase domain between tsm-v and tsm-vi (83). it is proposed that cl-2, tsmiva, and tsm-ivb are surrounded by the six tms forming a beta-barrel-like structure (85). lnt contains a catalytic cavity inside the beta-barrel-like structure, but the lipoprotein substrate enters the structure laterally, a mechanism similar to integral membrane proteins (87), lnt also exists as a thioester acyl intermediate to allow for higher processivity and acylation for essential om-bound lipoproteins (52). the mechanism for acylation transfer is a two-step process comprised of auto-acylation followed by acyl transfer. the mechanism for acylation transfer is most likely a two-step process, auto-acylation followed by acyl transfer (52, 81). due to the massive amount of lipoproteins transported to the om and the low abundance of the enzyme, lnt existing as an acylated intermediate allows for higher processivity of the acyl transfer (52, 81). the active site of lnt is in the nitrilase domain exposed to the periplasm. for auto-acylation, c387 sulfhydryl group initiates with a nucleophilic attack on the alpha carbonyl of a phospholipid. the resonance from e267 attacks the hydrogen on c387 sulfhydryl group allowing the fatty acid chain to remain on c387, yielding a thioester acyl lnt (52). for the acyl transfer, the newly exposed nitrogen on the diacylated lipoprotein then attacks the alpha-carbon of the acyl group on c387. the next step requires c387, k335, and e267 on the nitrilase domain, w237 on the β1/α1 loop, f358 and m362 on β5/ β6 loop, and r139 and p147 on cl-2 (85). lnt is essential in e. coli containing the lolcde pathways, but not other gram-negative bacteria which have lolfd pathways. in e. coli, lnt is essential. without the final acyl volume seven | 17 modification, om destined lipoproteins are mislocalized (77). this results in envelope stress and an inability to produce functional channel proteins (73). however, in acinetobacter baylyi, acinetobacter baumannii, francisella tularensis, and neisseria gonorrhoeae, lnt is not essential (88). it is unclear if these gram-negative bacteria can translocate di-acylated lipoproteins or another enzyme performs the same function. francisella and acinetobacter produce a novel lol complex, loldf, as opposed to the lolcde characterized in e. coli and many other gram-negative bacteria (41) (fig. 1b). in transcriptomic studies of lnt deficient a. baylyi, lola is upregulated significantly (88) but it is improbable that this upregulation can solely account for a. baylyi survival in lnt deficient states. data from the same transcriptome set shows a twenty-fold increase in hslj, gene expression in lnt deficient a. baylyi (88). additionally, a crystal structures of e. coli lola (89) and putative structure of e. coli hslj (np_415897.1, embl-ebi) are very similar in structure. due to the highly specific mouth-to-mouth transfer mechanism (90) that lola and lolb interact with one another to transfer their inner lipoprotein cargo, homology in structure among lola and hslj is a reasonable cause to investigate hslj as a potential chaperone suppressing the deleterious effects of lnt deficiency. concluding remarks the lipoprotein sorting pathway is more complex than initially thought, the complexity and conditionally essential nature of the genes involved in the processing and transportation pathways provides opportunities to develop additional antimicrobial compounds for both clinical and small molecule pathway-probing applications. though it was initially assumed that the action of lgt, lspa, and lnt were needed for lipoprotein biogenesis in all gram-negative bacteria, this now seems to be an over-simplification. it is certainly true that most gram-negatives require functional lgt, lspa, and lnt to be viable (50, 52, 83). however, it is now clear that there are many exceptions to this rule. a. baylyi is viable without lnt and requires no other genetic manipulations to grow (88). in this species, the potentially more promiscuous lolcde analog loldf is used and a di-acylated lipoprotein is able to be recognized for transport instead of one that is tri-acylated by a mechanism that is still unknown (fig. 1b) (88). by exploiting the ability of a. baylyi to survive in lnt-deficient cellular environments we can use a. baylyi as a model to explore novel lipoprotein processing and transportation constituents. uncovering the mechanism of which a. baylyi can overcome lnt deficiency is essential and in doing so, we open new avenues of non-canonical om biogenesis pathways and chemical interventions. acknowledgements this work was supported by the national science foundation under grant no. 1615822 to nwr 18 | fine focus glossary of terms β-barrel assembly machine (bam): five-protein complex that assembles β-barrel proteins into the outer membrane general secretion (sec) pathway: system for exporting unfolded proteins from the cytoplasm into the inner membrane twin arginine translocation (tat) pathway: system for exporting folded proteins from the cytoplasm into the inner membrane lipopolysaccharide transport (lpt) machinery: transporter system to shuttle lipopolysaccharides across the periplasm to the outer membrane localization of lipoprotein (lol) pathway: pathway responsible for trafficking mature lipoproteins from the inner membrane to the outer membrane pre-prolipoprotein: precursor lipoproteins exported from the cytoplasm prior to acylation by lgt prolipoprotein: precursor lipoprotein with two acyl chains from the action of lgt and with a still-intact signal peptide apo-lipoprotein: di-acylated precursor lipoprotein with cleaved signal peptide prior to final acylation from lnt volume seven | 19 references 1. grabowicz m, silhavy tj. 2017. redefining the essential trafficking pathway for outer membrane lipoproteins. proc natl acad sci 114:4769–4774. 2. okuda s, sherman dj, silhavy tj, ruiz n, kahne d. 2016. lipopolysaccharide transport and assembly at the outer membrane: the pez model. nat rev microbiol 14:337–345. 3. hagan cl, silhavy tj, kahne d. 2011. β-barrel membrane 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factors, lola and lolb. embo j 22:3199–3209. 89. okuda s, tokuda h. 2009. model of mouth-to-mouth transfer of bacterial lipoproteins through inner membrane lolc, periplasmic lola, and outer membrane lolb. proc natl acad sci 106:5877– 5882. 90. pailler j, aucher w, pires m, buddelmeijer n. 2012. phosphatidylglycerol::prolipoprotein diacylglyceryl transferase (lgt) of escherichia coli has seven transmembrane segments, and its essential residues are embedded in the membrane. j bacteriol 194:2142–2151. naowarat (ann) cheeptham, ph.d department of biological sciences, faculty of science, thompson rivers university, bc, canada copyright 2015, fine focus all rights reserved perspective“come dine with microbes: where microbiology, food, and culture meet community outreach and student engagement finaljournal.indd 159 9/25/15 11:38 am introduction thompson rivers university (tru) is located in the interior of the province of british columbia in canada. at tru, we offer a cellular, molecular and microbial biology (cmmb) program alongside other biology programs. students can select a three-credit elective upper level course, biol 4490, entitled “selected topics in biology” which features an advanced seminar in a topic of interest. one of the biol 4490 courses was on microbial applications in industry (industrial microbiology) for the 2014 winter semester and it highlighted the unlimited biochemical capabilities of microorganisms as well as a great variety of microbial fermented products. principles of fermentation technology were introduced and various factors that have a great impact on the biochemical and physiological basis of industrial fermentation processes were discussed. different fermented products and the microbes associated with their production were chosen and the students investigated them as case studies. the general learning objectives of this course were to: 1. recognize the role of microorganisms in industrial microbiology. 2. understand the range of fermentation processes available for different products and the practical benefits and limitations of them. 3. develop an awareness of the importance of microorganisms in industrial processes. 4. learn the biochemical strategies used by microorganisms to produce metabolites that we can use to our benefit. 5. explore the versatility of microorganisms, their diverse metabolic activities and their products. 6. learn about industrial microorganisms and the technology required for large scale cultivation and isolation of fermentation products. 7. learn to read and critically evaluate scientific literature. with the intention of enhancing student engagement and community outreach with topics taught in class, the “come dine with microbes” project was introduced. i was 160 • fine focus, vol. 1 (2) table 1. student evaluation for the biol 4490 microbial applications in industry prior to 2014 winter semester in-class participation (10%) field trip report (10%) field trip participation (10%) individual presentation (10%) term paper (20%) group presentation (10%) final exam (30%) winter 2014 semester in-class participation (10%) field trip report (10%) field trip participation (10%) individual presentation (10%) group presentation (10%) come dine with microbes (50%) finaljournal.indd 160 9/25/15 11:38 am inspired by a laboratory menu passed on to me years ago by a retired faculty member, carolynne fardy (fig. 1). when i saw the menu again prior to the semester, i had a spark and i thought i should ask the class whether they were open to the unorthodox idea of putting on a dinner/fundraiser based on microbial fermented products instead of writing a final exam and a term paper for the 50% of the marks (table 1). with this dinner, we, as microbiologists, could also help raise public awareness of the fact that not all bacteria are bad (fewer than one per cent of all bacteria cause human illness). the majority of microbes are good or neutral to us and in fact we cannot live without them. by introducing this initiative, i hoped that the experience would engage student learning and that at the completion of this event the students would be able to discuss the role of microorganisms in industrial microbiology and the range of fermentation processes available to produce different products with their practical benefits and limitations. i wanted the tru students to communicate their knowledge at a layman’s level by interacting with the norkam secondary school students and dinner guests. furthermore, i hoped that they would communicate to this wider audience the importance of microorganisms in our daily life, the versatility of microorganisms, and their diverse metabolic activities resulting in products for our society. the dinner had the added benefit of intercultural learning through sampling different ethnic fermented foods. i would like to think that i am a part of creating the next generation of global minded scientists; one small class at a time! in september 2014, we had discussed this idea with a local school, “norkam secondary school”, and their culinary arts, foods and home economics program. the instructors, chefs scott roberts and jo-anna allen agreed to help us put on the event on april 11, 2014. in collaboration, we were asked to dedicate two class-times prior to the dinner to go to the secondary school classes and demonstrate to the high school students the impact of microorganisms on food safety so the students can appreciate hand washing and no-touching-face/hairduring-cooking policy. once decided, we used some of our own class times to organize the event. students designed and chose the menu (fig. 2). we worked together to locate sponsors for the event. once the tickets were ready (fig. 3), we had them sold in a very short amount of time. we also met with chefs roberts and allen a few times to go through aspects of getting the kitchen and materials ready for the dinner. our tru students were the main kitchen workforce with the norkam students helping. communications were maintained through email messages. prior to the dinner, the tru students spent two class-times talking to and introducing the norkam students to the wonderful world of microbes with special emphasis on the relationships of microbes with food and food ““ with this dinner, we, as microbiologists, could also help raise public awareness of the fact that not all bacteria are bad (fewer than one per cent of all bacteria cause human illness). perspective • 161 finaljournal.indd 161 9/25/15 11:38 am fig. 1. the menu passed on to the author that sparked the initiative (source: unknown) 162 • fine focus, vol. 1 (2) finaljournal.indd 162 9/25/15 11:38 am safety (fig. 4). tru students were marked on their presentations and interactions with the norkam students. we spent a few hours of the day before the event purchasing ingredients and putting them in the norkam kitchen (fig. 5). the actual day of the event, all of us arrived at norkam early to prepare the foods and the venue. we had a number of volunteers to help us as well. at the dinner, we arranged a non-traditional buffet style by having food stations (for each chosen fermented food) and having tru microbiology students and norkam students man each station (fig. 5). this facilitated communication with the guests when they came up to sample various foods. we successfully hosted 80 guests (approximately 100 people in total, including students and volunteers). guests were very happy to taste delicious and healthy foods while learning about the production of microbially fermented foods through posters (fig. 6) and communication with students. some were pleased to win our raffle baskets filled with different fermented products donated and sponsored by local merchants (fig. 5). a number of guests expressed how much fun it was and that they would like to see more events such as this. at the end, we raised $1,000 that was split between norkam and tru microbiology teaching and research activity. all in all, i think that we achieved what we set out to do given the success of the event. tru students were engaged and showed their learning by teaching the high school students and dinner guests about the role of microbes in production of the various foods that were served. high school students and teachers responded enthusiastically to the microbiology exercises they performed and the food that they helped to prepare. dinner guests were equally enthusiastic about the meal and the information provided by the students. the tru students did very well explaining the role of microorganisms in industrial processes and food production in language that was easily understood by high school students and members of the public at the dinner. the students also demonstrated communication skills through posters (fig. 6), brochures (fig. 7), as well as informal verbal presentations at the dinner. they conveyed to a wide audience the importance of microorganisms in our daily life, the versatility of microorganisms, and their diverse metabolic activities resulting in products that can be used in our society. on february 12, 2014 i was very happy to see an article entitled “chefs and microbiologists break bread anew” published in the asm-microbes volume 9 (1) pp.13-17 (http://www.microbemagazine.org/index. php?option=com_content&view=article&i d=6567:chefs-and-microbiologists-breakbread-anew&catid=1295&itemid=1565). what perfect timing for our class project! my idea was not that far-fetched after all and i was glad that my students were on board and were much more enthusiastic than any class i ever seen before. throughout the course, i found continuous enthusiasm and positive learning energy from my students even though they had to work harder than previous classes preparing for the dinner. this showed how engaged the students were through planning and executing the event. in my 13 years of teaching at tru, i have never felt such a high level of ownership and pride demonstrated by students. perspective • 163 finaljournal.indd 163 9/25/15 11:38 am comments and impressions from students and dinner guests comment #1: “as for my experience, come dine with microbes was the most rewarding project of my undergrad as it provided me with an opportunity to participate in a community outreach project and to network with the public, something our faculty does not emphasize. i not only valued the opportunity to be creative and learn the ropes of hosting a public event, but i also enjoyed that the theme we created which allowed us to educate the public on a scientific topic through the universal language of food! having the opportunity to connect with the public provided me with an opportunity to learn how to communicate microbiology in the “real world” and in a way that left a lasting impression on the students we lectured at norkam, as well as the gracious guests that attended our event!” comment #2: “i would say that overall the dinner was a success for the entire class as well as the community at large. planning for this dinner allowed my classmates and i to discover just how many uses microbes actually have in the industrial world and in the culinary arts (i had no idea that chocolate is the result of fermentation). furthermore, the presentations that we gave to the students at norkam secondary as well as the guests at the dinner allowed us to pass on what we had learned to the community. in the end, i think that the class as well as our audience members understood that most microbes are not bad; many are beneficial and necessary for the little luxuries we often take for granted. i had a great time in this course and would recommend that a similar approach is repeated for future classes.” comment #3: “this class introduced me to the potential health benefits and industrial applications of microorganisms by giving me hands-on real life experience.” comment #4: “this started out as a fun way to spread the word about the importance of microbial fermented food in a healthy diet, and ended up being an incredible learning experience. not only did we discover fermented food products from all over the world and get to make several of them ourselves, we learned how to plan, organize, and carry out a fundraising event! because this was our event and our food, everyone wanted to see it a success and i have never known an entire class to collectively put that much effort into an assignment before!” comment #5: “come dine with microbes was an educational and fun event with lots of good food! the students did a fantastic job of presenting and serving the food. i thoroughly enjoyed meeting new people and old acquaintances as well.” comment #6: “my spouse and i attended the “come dine with microbes” event in april 2014. dr. cheepthams’ passion and desire to promote microbiology concepts applicable to daily life events (i.e. food) was most evident in the enthusiasm and energy her students exhibited throughout the evening. each menu item was delicious and accompanied by a narrative with regard to the microbes utilized in the dish. the event was well organized and educational. in my value judgment, a meaningful dining and learning experience was had by all participants.” comment #7: “i found ‘dinner with the microbes’ to be such a unique and creative learning experience for me and my children (age 4 and 7), in addition to the students who worked so hard to bring it to fruition. i was not sure what to expect with respect to a dining experience, but was intrigued by the concept and believed in the educational merit. to my surprise the food was amazing and my children and i learned a great deal 164 • fine focus, vol. 1 (2) finaljournal.indd 164 9/25/15 11:38 am fig. 2. the menu that students selected to represent at our dinner fig. 3. the ticket for the event come dine with microbes menu starter salad entrees dessert nonalcoholic beverages soup kimchi crostini pickled root vegetable salad tempeh sliders chocolate dipped fruits ginger beer coffee tea sausage and sauerkraut miso soup perspective • 165 finaljournal.indd 165 9/25/15 11:38 am about microorganisms in a way that was engaging and memorable. it was evident that the students who participated in this event were inspired by the innovative teaching techniques offered through this event. i would not hesitate to attend such a function again in the future and could see an event like this growing with time as it was received so well by those who attended.” comment #8: “my wife, 7 year old daughter, and i all attended the “come dine with microbes” dinner at norkam secondary. this event was really enjoyable. first, as we walked through the food buffet, we enjoyed talking with both the tru microbiology students and the norkam science students about how the food served were representative of different microbial and fermentation processes. it was clear the students were enthusiastic about sharing their scientific knowledge in this practical way. moreover, it was a very interesting experience for my family to gain a greater appreciation for the different production processes of the food at the different buffet stations! also, while sitting and eating my family and i also enjoyed talking with other guests and students. everyone at the event agreed how we had gained a greater fig. 4. tru student presentation on the wonderful world of microbes to norkam’s culinary arts, foods and home economics program students prior to the dinner event: a) class presentation; b) demonstration of bacteria cultured on the plates from swabs that norkam students did; c) chef scott roberts and the norkam students interacted with the tru students; d) glow germ hand washing exercise. a b c d 166 • fine focus, vol. 1 (2) finaljournal.indd 166 9/25/15 11:38 am appreciation, and understanding of our food through this event. i really believe events like this contribute to increasing our awareness and knowledge of scientific areas such as microbiology.” comment #9: “i was intrigued when i heard there were tickets being sold for “come dine with microbes” so decided to attend. i found the whole experience very delicious and interesting. the students did a wonderful job of presenting the dishes and were ready to answer our questions on the food and the microbes that created them.” comment #10: “come dine with microbes was a wonderful, unique and engaging way to bring a more positive perspective to the community about the often hidden value of microbes in our lives. my husband and i thoroughly enjoyed the microbial based foods prepared by the enthusiastic students and came away with a better understanding of the health benefits of fermented foods.” comment #11: “the dining with microbes dinner was an amazing opportunity to show just how essential microbes are to life. as students we were able to present a delicious array of food that was possible due to the actions of microorganisms. i think everyone at the dinner had a great time eating the food, winning various prizes all while being educated about the many jobs of microorganisms. the dinner was a great success and i believe our class and the participants had a blast!” comment #12: “i was thrilled with the dinner with microbes’ event last year. it was really beyond my expectations for everything. the students had done a great job providing interesting information about their particular food and they presented it well. the food was delicious, well-prepared and the tempeh sliders were my favorite! i will not hesitate to attend another event such as this.” comment #13: “the event was one of the most interesting things that i have done at tru. it really encouraged me to work well with my fellow students in a way that a regular structured class does not provide. i helped out mostly with decorating the hall, design of posters, media things such as music and the tending to the miso soup station. throughout the entire process i feel like i got to know my instructor and fellow classmates on a more intimate level. many times classes have you sitting and staring at a projector screen trying to absorb somewhat boring words or material. this class and the dinner event focused on the community aspects more, which is something that i really enjoy. the dinner also engaged the public into a world of microbes that maybe they were never aware of existed in these foods. some of the dishes also provided the public and students with a greater idea of how to make/prepare and enjoy these foods in our own home. this dinner was a great ‘awareness tool’ that showed the public that not all bacteria are harmful and having a regular inflow of new microbes into our systems can actually “ “ this dinner was a great ‘awareness tool’ that showed the public that not all bacteria are harmful and having a regular inflow of new microbes into our systems can actually be beneficial and healthy. perspective • 167 finaljournal.indd 167 9/25/15 11:38 am a e b c h d i f g 168 • fine focus, vol. 1 (2) finaljournal.indd 168 9/25/15 11:38 am fig. 5. april 11, 2014 the event: a) both tru and norkam students were briefed by chef scott roberts; b to g) students were having fun on food preparation and setting up the venue; j) raffle ticket drawing and announcement; k) the team; l to o) our happy dinner guests with their raffle baskets; p) cleaning crew! j k l n m o p be beneficial and healthy. overall, it was an awesome experience and it taught me a lot about microbes and how they interact with our foods.” comment #14: “i think there is a lot of unfortunate misinformation about microbes spread by the media. at the very least, the focus is kept on the negative aspects of pathogenic bacteria, which obviously represents only a small portion of the known microorganisms and are actually exceptionally rare considering how many we encounter on a daily (or even hourly) perspective • 169 finaljournal.indd 169 9/25/15 11:39 am basis. i understand why these negative aspects are considered more “newsworthy” -a story about contaminated food killing two people in toronto is more shocking than a species of bacteria living in our digestive tracts helping us to break down our food and preventing illness. however, i think if the mainstream media isn’t going to promote these ideas then somebody should. i know far too may people who constantly disinfect everything they may touch and can’t go an hour without using hand sanitizer. these actions represent someone terrified of “bugs” and “germs” because they see them as purely negative, able to infect you at any moment and cause life-threatening illnesses! of course, we know that this isn’t the case and we understand that having communities of microbes inside your body is a requirement of a healthy individual, but i can understand why the average person gets frightened. caution is completely fine (nobody wants food poisoning!) but i think people take it to a new level. that’s why i think projects like ours are important. everyone knows yogurt is made using microbes but many don’t realize the other food products we gain or the importance of microbes in the process. after the dinner was held, my aunt (an attendee) made one of the dishes we served for easter dinner and spread the word about the fermentation process to the rest of my family because she thought it was interesting. i think the dinner is a good format to spread the word because (if it’s successful) it’s something that people remember easily because they had fun and learned some new information! the same can be said for my own personal engagement. i will always remember it because i had fun and learned new information. i had no idea most of the foods we served were fermented. it was a very memorable learning opportunity! as for how i think we could make it better in the future... (if you do decide to do this with your classes in the future). i think everything worked out in the end which is awesome, but the process of organizing the dinner could also probably be streamlined. i think it would be more beneficial if, instead of people taking on certain individual jobs, if we had people (or small groups of people, 1-3) taking on general roles instead: decorations (table cloths, table placement, centerpieces, screens/ screen placement, music); signage (posters, pamphlets, advertising); tickets (designing them, coordinating the selling of them, ideas for selling outside the university); food (getting donations, figuring out amounts for the final diner (scaling up recipes), being in charge of menus); raffle/donations (getting donations, putting together the raffle baskets, coordinating sponsor logos for the tickets/ pamphlet); secretary (going to all the norkam meetings & taking notes, taking notes at in-class discussions, sharing the notes with the class, making a game plan for how everything will go during dinner ““ i think the dinner is a good format to spread the word because (if it’s successful) it’s something that people remember easily because they had fun and learned some new information! 170 • fine focus, vol. 1 (2) finaljournal.indd 170 9/25/15 11:39 am fig. 6. examples of posters describing microorganisms and fermentation processes involved in the food served. perspective • 171 finaljournal.indd 171 9/25/15 11:39 am fig. 7. examples of student-generated brochures 172 • fine focus, vol. 1 (2) finaljournal.indd 172 9/25/15 11:39 am preparation, etc.). those are six roles and as i assume you generally have approximately 12 people in your class during a semester maybe two people could be assigned to each role? it worked out for us that everyone got all the work done but only because everyone tried really hard and we managed to have a lot of invested people! it may be beneficial to assign roles to make sure everyone does their part in the future. also, this would reduce the amount of classes you would have to hold to discuss things -two people would be responsible for one aspect and could then share ideas with the class and move on. on that subject, i think you could also reduce the amount of class time spent discussing the project by having a plan for each class -what we want to discuss and have a set amount of time for each topic so we don’t run overboard. this would be far easier now that we have a general timeline and some general ideas. i also think we should attempt to sell tickets to people in the community beyond those we know personally. perhaps setting up a table at the mall? or putting posters/flyers out around town? either way i think it would really help the community outreach aspect of it! as a final suggestion, it may be beneficial to try and hold the dinner in the culinary arts building in the future (aka make plans around their schedule) as i think it would reduce a lot of the work and it’s a nicer building! acknowledgements i would like to personally express my sincere thanks to the open-minded students of the winter 2014 microbial applications in industry class for allowing me to be innovative in my teaching and for working tirelessly with a fun attitude to put this event together successfully. you guys rock: jasmeen dhaliwal, rupert heyes, kayla holtslag, jillian lane, arjun randhawa, jordan robinson, mercy rungi, quinton sirianni, laura smylie, dewald van der merwe, and cohord mason. thanks to all the dinner guests who attended and believed in us! this event would have not been made possible without the help of the norkam secondary school’s chefs scott roberts and jo-anna allen and the culinary arts, foods and home economics program students for letting us use their kitchen and space. we had numerous generous sponsors: pipe genie, cheng kwong grocery, crannog ale, nature’s fare market; the village cheese company, harper’s trail, the noble pig brewhouse and restaurant, the old town farm market, and spatial resource services. our sincere thanks to dr. charles hays of tru for his generosity in providing great homemade tempeh to be used in our main dish and for his enthusiasm for tempeh production; it was contagious. thanks to brittany van der merwe, brian tong, solenn vogel, kayla byzick, christopher herbert, and hamza hasan for your assistance in many capacities to make this event a success. my sincere gratitude also goes towards drs. charles hays, ken wagner and mairi mackay for their continued support and their assistance in editing this manuscript. perspective • 173 finaljournal.indd 173 9/25/15 11:39 am volume six | 9 faculty perspective the benefits of mentoring undergraduate research students douglas h. roossien, ph.d. assistant professor of biology, ball state university 10 | fine focus science or history? i initially chose to pursue a degree in cell and molecular biology as an undergraduate because i loved the subject. i loved learning about cells, how they are shaped, how they function. i loved learning about how organized molecules formed the basis of heredity and inheritance. but always at the back of my mind was the question of “how do we know these things?” i was thus inspired by classic experiments by following the logic presented in impeccable experimental design. the cleverness of scientists like thomas hunt morgan, louis pasteur or hershey and chase made me appreciate the human endeavor to understand the world around us. these moments of inspiration were always interrupted by a pang of longing, as i perceived the stream of facts being presented as a complete and static collection of knowledge. a history lesson, if you will. the work was done, we knew how the natural world worked. so much intimate detail about life, how could there possibly be more to learn? thankfully this misconception was shattered through a stint in undergraduate research. my breakthrough what i did not realize when i initially chose my degree program was that it was designed for students interested in pursuing graduate school and/or careers in research. accordingly, the program had strict requirements for participating in undergraduate research. i therefore found myself entering a research laboratory with little to no idea of what to expect. the first small project i was assigned was to clone and sequence a gene involved in organizing microtubules in plant cells. the goal was to understand the evolution of these microtubule organizing centers in the arabidopsis family of plants. my mentor explained it: “there is this oddity in plant cells, they lack the organelles that organize microtubules. yet they clearly get along just fine. the question we’re asking is how have plants evolved independently to accomplish this feat without these seemingly fundamental structures?”. there for the first time i was presented with the unknown. it was not only the question that caught me, but the existence of the question itself. i quickly realized that what we know about the natural world is but a mere beginning to a comprehensive understanding. perhaps even more excited was then being presented with the opportunity to help discover the unknown. mentorship is the key i had amazing research mentors as an undergraduate student: dr. regina mcclinton at grand valley state university and dr. aikseng ooi at the van andel research institute. they both took a keen interest in fostering the excitement and curiosity i brought to the lab. they not only trained me to be technically proficient, but really spent time to train me as a scientist. in hindsight this went beyond the nuts and bolts of scientific inquiry. i learned other fundamental skills: perseverance, critical thinking, foresight, and imagination just to name a few. this took patience and dedication on their part. their investments in me paid off when i realized that i was better prepared for graduate school success than peers whose previous research experience was more task-oriented. i excelled in graduate school where others struggled. i attributed this not to my own intellect or ability, but to the mentorship i received as an undergraduate research student. volume six | 11 the payoff i have had the pleasure of mentoring many undergraduates throughout my career, and the biggest lesson i have learned is that there is not one catch-all approach. students are motivated and inspired by different ideas or different aspects of the scientific process. one of my primary jobs as a mentor therefore is to listen and be attentive. when asked if they think a proposed experiment is exciting or useful, most will respond “yes” to be polite, but body language and mannerisms often tell otherwise. while there will always be unenjoyable tasks in research, my experience has shown that if these are not punctuated with tasks that excite the student (or myself) will not be doing their best work. i strive to perceive the real experience students are having in the research lab and guide them accordingly. it is important to me that i bring undergraduates into the scientific process. my ideal goal for each student entering my research lab is for them to achieve a certain degree of independence with the process of scientific inquiry. i view the student as a researcher in the laboratory, not as a producer of data. to truly engage a student in my research requires exposing my thought processes and rationales rather than simply delivering ideas and goals. this is where the real time commitment comes in, but it is also the source of the big payoff for both myself and the student. as students gain experience in research i try to start asking rather than answering the questions: “what do you think we should do next?”, “what do these data mean to you?”, “how would you test that hypothesis?” etc. watching students develop the skills to critically think about and answer these questions is truly one of the best parts of my career. but also, reality these are idealistic circumstances of course. in reality, not every student that participates in our research will be inspired. most of them may not even pursue a career in research. occasionally a student realizes quite quickly they want nothing to do with research. nonetheless, undergraduate research experiences are still valuable. to give a student a glimpse at the reality of research can provide the fundamental understanding that science is a process and not a collection of facts. how science goes far beyond the dense content we present in our courses, how the work of the pioneers i admired is not complete, how scientific knowledge is not static, but dynamic and under constant revision. this seems imperative now more than ever, as i currently sit writing this in my makeshift home office as a result of the ongoing covid-19 threat. the public response has illuminated many misconceptions about science, both its benefits and limitations. the more students we can expose to the realities of science, the better off we will be. • fine focus, vol. 3 (2) organization of the multigene families of african swine fever virus jacob imbery & chris upton * biochemistry and microbiology, university of victoria, victoria, bc v8w 2y2, canada manuscript received 18 april 2017; accepted 13 june 2017 copyright 2017, fine focus. all rights reserved. african swine fever virus • 155154 • fine focus, vol. 3 (2) african swine fever virus is a complex dna virus that infects swine and is spread by ticks. mortality rates in domestic pigs are very high and the virus is a significant threat to pork farming. the genomes of 16 viruses have been sequenced completely, but these represent only a few of the 23 genotypes. the viral genome is unusual in that it contains 5 multigene families, each of which contain 3-19 duplicated copies (paralogs). there is significant sequence divergence between the paralogs in a single virus and between the orthologs in the different viral genomes. this, together with the fact that in most of the multigene families there are numerous gene indels that create truncations and fusions, makes annotation of these regions very difficult; it has led to inconsistent annotation of the 16 viral genomes. in this project, we have created multiple sequence alignments for each of the multigene families and have produced gene maps to help researchers more easily understand the organization of the multigene families among the different viruses. these gene maps will help researchers ascertain which members of the multigene families are present in each of the viruses. this is critical because some of the multigene families are known to be associated with virus virulence. abstract corresponding author chris upton * biochemistry and microbiology, university of victoria, victoria, bc v8w 2y2, canada keywords • african swine fever virus • multigene family • annotation • bioinformatics • genomics african swine fever virus (asfv) is a large dsdna virus in the family asfarviridae; 23 genotypes have been characterized by sequencing of the p72 gene (1, 12, 13). the virus is endemic in many regions of africa where it infects primarily warthogs and is spread via the bites of soft ticks (9). although asfv causes mild symptoms in warthogs and produces no symptoms while replicating in the ticks, it causes very serious haemorrhagic disease in domestic pigs and wild boar. in these animals, the mortality rate approaches 100% for some asfv strains (18). the relatively recent (2014) but extensive spread of asfv through africa to parts of central europe takes a significant toll on both small and large-scale pig farming operations in these regions, putting a large strain on the global pig trade (5). to date, most successful viral prevention introduction methods rely on routine degenerate pcr screening of wild pig and tick populations together with a rapid and competent diagnosis program when an outbreak is suspected. in addition, strict sanitary control procedures must be implemented to reduce the possibility of infected wild hosts interacting with domestic pigs (7). when outbreaks occur, currently, the only effective response is culling of an infected herd and the imposition of a ban on the movement of adjacent herds (2). this produces serious economic problems for the farmers and may incentivize noncompliance. clearly, an unhindered pork trade would be very beneficial and benefit a large proportion of the population. between 2014 and 2015, close to $55 million was spent on asfv prevention in the baltic states alone, which was considered to have prevented us$4.5 billion in potential losses (7). sixteen full asfv genomes have been sequenced to date, and more than 100 will be sequenced in the next 2-3 years (e. okoth, personal communication). the availability of these genome sequences is important because comparative genomics analyses will allow researchers to better correlate gene content and amino acid sequence variation with virulence and antigenic variation. however, all asfv isolates have at least 5 multigene families (mgf) that are made up of sets of paralogs, which are frequently but not always arranged in tandem. not only do the different viruses have different numbers of these paralogs, but they frequently have indels that remove multiple genes and partial genes resulting in some gene fusions (4,6,14). consequently, when these viral genomes are aligned by software tools these regions are not aligned correctly. the problem is made more complicated by the fact that the individual mgfs have sometimes been mis-annotated due to failure to identify the correct ortholog groups within the sets of paralogs. correct identification of the members of these mgfs is especially important because these genes have been linked with virus virulence (6). the challenge of developing an effective vaccine stems, in part, from the high antigenic diversity distributed among the different strains of asfv and therefore from the genomic variation. although it is possible to induce immunity in pigs that protects from challenge with a homologous genotype, the african swine fever virus • 157 figure 1. maximum likelihood phylogenetic tree of mgf 505 dna sequences was created with raxml using the gtrgamma base substitution model. sequences were aligned with mafft and trees visualized using mega7. 156 • fine focus, vol. 3 (2) generation of protection against a heterologous genotype has proved unreliable (15). in fact, vaccination does not always adequately protect against viruses of the same genotype (19). here we describe the reannotation of the asfv mgfs using a common nomenclature that will facilitate future asfv genome comparisons and provide clarity for the discussion of the differences between viral gene sets. methods data set genomes of the following asfv isolates were used, genbank accession numbers are given in parentheses: asfv-benin_97_1 (am712239); asfv-l60 (km262844); asfv-e75 (fn557520); asfv-ourt_88_3 (am712240); asfv-nhv (km262845); asfv-mkuzi_1979 (ay261362); asfv-ba71v (u18466); asfv-georgia_2007/1 (fr682468); asfv-pretorisuskop_96_4 (ay261363); asfvwarmbaths (ay261365); asfv-warthog (ay261366); asfv-tengani62 (ay261364); asfv-mwi_lil_20_1_1983 (ay261361); asfv-ken05_tk1 (km111294); asfv-ken06 (km111295); asfv-ken_1950 (ay261360). phylogenetic tree and dotplot construction a multiple sequence alignment (msa) of the 16 complete asfv genomes was generated using mafft (10). base-by-base (bbb; (8)) was used to visualize the msa and highlight the differences between the genomes. maximum-likelihood phylogenetic trees were constructed using raxml (16) under the gtrgamma base substitution model using 1000 bootstrap replicates. mega7 (11) was used to visualize and manipulate the phylogenetic tree output. since alignment tools such as mafft treat genomes as linear syntenic sequences, they are unable to accurately display any sequence transpositions. similarly, it can be difficult to assess small differences in the quality of the various possible alignments for the asfv mgfs from a msa. therefore dotplots, which provide a 2-dimensional visualization of all nucleotides-against-all nucleotides were used to supplement genome alignments (jdotter; (3)). a dotplot was created for each individual mgf gene compared against a full length asfv reference genome. the series of matrix alignments across the dotplots created a unique “barcode” describing the relationship of the gene to all the paralogs in the mgfs. the dotplots were especially useful for determining the breakpoints between fused paralogs. mgf paralogs size (bp) conservation mis-annotations mgf 100 3 375-440 high 1 mgf 110 13 315-875 low 27 mgf 300 3 315-800 high 5 mgf 360 19 960-1100 moderate 17 mgf 505 10 1500-1630 moderate 12 table 1. summary of the 5 mgfs for the 16 asfv strains. the relative conservation between the paralogs inversely correlates with the number of paralogs. the “mis-annotations” column indicates the number of figure 2. visual summary from bbb of mgf 110-1l/-2l/-3l paralogs from 9 asfv genomes. thin vertical dark blue bars, red and green blocks represent snps and insertions and deletions with respect to the topmost sequence. light blue blocks represent orfs. gene labels, positioned on the orfs, indicate previous annotations (note inconsistencies) as well as gene number. boxed gene labels indicate new nomenclature. figure 3. visual summary displaying fragmentation of mgf 110-11l orthologs. gene labels indicate the previously annotated orthologs. thin vertical dark blue bars, red and green blocks represent snps and insertions and deletions with respect to the topmost sequence. the mgf 110-10l is not represented in this diagram due to annotation only in warmbaths strain that is more likely a truncated mgf 110-11l ortholog. nucleotide positions are mapped at the bottom of the figure. african swine fever virus • 159158 • fine focus, vol. 3 (2) construction of mgf maps the mgf maps were created as vector graphics with omnigraffle (omni group, seattle) on imac computers. these diagrams can be fully edited to incorporate new genomes and new mgf orthologs as they are discovered. results the goal of this analysis was to create an accurate reference map of the distribution of mgf members throughout the 16 sequenced asfv genomes. since the mgf members are not simply present or absent, an annotation scheme was also required to describe the various gene fragmentation/truncation/ fusion patterns that exist in the different virus strains. since we do not yet know the functional consequences of these multiple rearrangements on the biology of the viruses, the purpose of the map is primarily to flag the various differences that exist between the asfv mgfs. in addition, due to the extremely complex nature of the indels in the mgf regions, which compound when msas are generated, we opted to illustrate general variations in the open reading frame (orf) patterns rather than try to capture every single difference. our results are sufficient to flag differences between the paralogs so that a detailed dna sequence alignment of the region can be performed if more information is required for a particular study. there are currently 5 known mgf series observed in asfv. paralogs within an mgf series are numbered chronologically as they appear in the asfv genome and are classified as “r” or “l” indicating that this gene is either transcribed on the forward or reverse strand respectively. our first step in reviewing the relationships between the paralogs/orthologs of each mgf was to create a phylogenetic tree. the msa was generated using mafft with the dna sequences and the phylogenetic trees were constructed with raxml. figure 1 illustrates the value of the trees by showing a visual representation of the relationship between the paralogs of the mgf-505 series (mgf average size 505 amino acids). for example, the tree shows that paralogs mgf-505-6r and mgf-505-7r result from a relatively recent duplication. however, it must be appreciated that phylogenetic trees also hide the raw data, which may have valuable information about the sequences. for example, recombination events and deletions that fuse two paralogs are likely to be lost if only the tree is viewed. therefore, to ensure that the tree generation step was not flawed by faulty input data, we checked the msas with bbb, a msa editor that also provides highlighting of the differences between pairs of sequences in the msas. this helps the researcher recognize msa regions that have inconsistent similarity levels and may be the result of gene fusion events. in addition to displaying sequence alignments, bbb is capable of generating a “summary view” of sequence alignments, that captures the positions of snps and indel information to allow large msas to be shown on a single page. figures 2 and 3 illustrate the use of bbb to view parts of mgf-110. a truncation of asfv-geo mgf-110-1l is shown together with previous alternate naming of asfv mgf-110-3l orthologs (figure 2). in mgf-110, we propose 13 paralogs whereas 14 had been previously annotated among the 16 genomes because fragments of a single gene had been annotated separately. figure 3 shows several of the difficulties faced figure 4. dotplot of mgf-360 genes. the horizontal sequence contains asfv-mkuzi mgf-360-1l and mgf-360-2l genes (blue arrows). the vertical axis represents 3 sequences; the top 2 panels are the asfvmkuzi mgf-360-1l and mgf-360-2l genes as controls and the bottom panel represents the asfv-e75 mgf-360-1l/2l fusion (red arrow). the dashed line in the bottom panel separates the 5’ half of 360-2l and the 3’ half of 360-1l. self-plots of genes generate a solid diagonal black line whereas plots of paralogs produce fainter intermittent lines. figure 5. example of summaries showing organization of mgf-505. the asfv strain is given in the red boxes at the left. paralog names are shown in the boxes at the top of the diagram along with the size of the ortholog from the reference genome, mkuzi. the annotation of each gene (arrow head indicates direction of transcription) is in two parts: the paralog group followed by the gene number of the virus strain. if the paralog annotation is incorrect, it is labelled in red. the gene box size represents the relative gene size of orthologs, but is not to scale, nor does it reflect the size relationship between paralogs. genes connected by grey boxes indicate the fusion of two orthologs. african swine fever virus • 161160 • fine focus, vol. 3 (2) in trying to annotate the mgfs consistently: 1) mgf-110-11l is fragmented in several viruses, 2) a large deletion in asfv-mwi creates a fusion of mgf-110-9l/11l, and 3) large indels (red and green blocks, which illustrate insertion or deletion with respect to the reference) create orthologs of significantly different sizes (mgf110-12l). since the orfs are displayed by bbb across gapped alignments, they are not accurate representations of their true size. although msas do show raw data (the actual aligned dna sequences), because they display a one-dimensional representation of the alignment they are of less use when regions of sequences may have been rearranged. in such situations, the twodimensional presentation of global sequence comparisons from a dotplot can better show rearrangements. for example, figure 4 shows the comparison of the asfv-e75 mgf-3601l/2l fusion with the 2 parental orthologs. it also shows the results of paralog comparison (1l and 2l for asfv mkuzi) and ortholog comparison (1l for asfv-e75 and asfvmkuzi). after reviewing data from phylogenetic trees, msas and dotplots, we constructed a summary diagram for each mgf. these are presented as supplementary figures 1-6, which are provided at a large scale to present much greater detail . for each summary diagram, there is also an “information sheet” that explains the representations of the mgfs (supplementary figures 7-12). these figures are also available from the viral bioinformatics resource center, in the asfv section (https:// virology.uvic.ca/organisms/dsdna-viruses/ asfarviridae/). figure 5 is an example of one of the summary diagrams and table 1 shows the varying complexity of the individual mgfs. although many of the mgfs are similar among the different asfv strains, there are some differences that are specific to particular clades of the asfvs. examples of these are shown in figure 6 with a full genome phylogenetic tree provided in figure 7. from these figures, it is clear that the mgfs are relatively fluid, with differences appearing even between asfv-e75 and –l60, which are very similar. however, some of this variation is expected given the overall variation between the asfv strains. interestingly, although these viruses are all denoted as asfv strains, there is significant divergence between them. a comparison of the asfv b602l, p32, p54, and p72 genes (as concatenated amino acid sequences) of the 16 asfvs revealed that there are several viruses that have diverged to be 94 95 % identical (aa). in contrast, we found that poxviruses, which belong to a different family of large dna viruses, that are classified as separate species within the capripox or orthopox genera may be 97 99 % identitical (aa) in pairwise alignments. thus, asfvs that are currently classified as different genotypes within a single species may well be classified into different species if taxonomic standards that are used with poxviruses were applied to asfv. discussion the genomic regions that encode the 5 mgfs of asfv presented here are highly variable and are hotspots for indels making both sequence alignment and accurate annotation difficult. this has resulted in inconsistent annotation among the 16 asfv genomes for the identification of paralogs and especially the naming of gene fusions. since figure 6. display of all mgf paralogs as they appear, left to right, in the 16 asfv strains. columns of boxes represent individual mgf orthologs as in figure 5. location of the different mgf series is annotated above the diagram. the arrangement of mgf orthologs separates the genomes (coloured rows) into 4 groups that correlate well with the phylogenetic tree. it is notable that the mgf compilation can be split into 2 regions, between mgf 300-1l and mgf 300-2r, reflecting significantly more variation in region1 than 2. nucleotide positions are given at the bottom of the figure. figure 7. phylogenetic tree for the 16 asfv strains. genotypes are shown in parentheses. boxes indicate groupings defined in figure 6. mafft was used to align the concatenated amino acid sequences of phosphoprotein p32, structural proteins p72 and p54, and the chaperone protein b602l. the tree was created with raxml under a gtrgamma base substitution model using 1000 bootstrap replicates and visualized using mega7. this phylogenetic tree places asfv strains into clades that replicate the groupings generated by overall mgf gene characteristics. african swine fever virus • 163162 • fine focus, vol. 3 (2) a large number of asfv genomes will be sequenced in the near future, we decided that standardizing the annotation and presentation of the asfv mgfs would greatly simplify genome annotation in the future. with a better reference system for the asfv mgfs available, we envision a 3-part process in the annotation process for asfv genomes sequenced in the future. first, there would be a basic sequence similarity search with a set of 10 conserved genes to identify the most similar reference genome to be used with the genome annotation transfer utility tool (gatu; (17)). second, a dotplot would be used to confirm co-linearity between the proposed reference genome and the newly sequenced target genome. third, gatu would be used to transfer as many annotations as possible (> 95 %) from the reference genome to the target, with the use of a full genome alignment of the reference and target in bbb to confirm the positions and numbering of the members of the mgfs. as the number of sequenced and annotated genomes increases, fewer differences will be found between the new target genomes and their references. thus, gatu will become more efficient and less annotator intervention will be required to annotate those few orfs that gatu leaves unfinished. in conclusion, we believe that our figures are an intuitive visualization of the arrangement of genes within the mgfs, especially when there is a need to compare the mgfs of different viruses. it is envisioned that the maps of the asfv mgfs will be living documents, updated, by a volunteer curator from the research community, with any new paralogs that may be discovered in newly sequenced genomes. this is likely to be required since genomes have yet to be sequenced from a large proportion of the asfv genotypes and the mgfs are the most variable parts of the genomes. to this end, the diagrams can be easily updated when new mgf paralogs are discovered by the addition of new columns. although new asfv genomes can be added to the diagrams by simply copying the most similar existing row and editing the sizes of the gene boxes and labels, as the number of genomes grows, space could be saved by showing a single representative if multiple viruses have identical mgfs. these mgf maps will speed up the annotation process and simplify the comparison of new asfv genomes. with more accurate genome alignments, researchers will also be better able to correlate genomic features with virulence levels of the various asfv isolates. african swine fever virus • 165 supplemental figures supp. figure 1. diagram of mgf 100 organization. see supp. figure 7 for additional information describing this mgf. supp. figure 3. diagram of mgf 300 organization. see supp. figure 9 for additional information describing this mgf. supp. figure 4. diagram of mgf 360 organization. see supp. figure 10 for additional information describing this mgf. supp. figure 2. diagram of mgf 110 organization. see supp. figure 8 for additional information describing this mgf. 164 • fine focus, vol. 3 (2) african swine fever virus • 167 supp. figure 5. diagram of mgf 505 organization. see supp. figure 11 for additional information describing this mgf. supp. figure 6. diagram of mgf compilation. see supp. figure 12 for additional information describing this mgf compilation. supp. figure 7. information sheet describing mgf 100. supp. figure 8. information sheet describing mgf 110 supp. figure 9. information sheet describing mgf 300. 166 • fine focus, vol. 3 (2) african swine fever virus • 169 supp. figure 10. information sheet describing mgf 360. supp. figure 11. information sheet describing mgf 505. supp. figure 12. information sheet describing mgf compilation. 168 • fine focus, vol. 3 (2) 1. achenbach je, gallardo c, nieto-pelegrín e, et al. 2016. identification of a new genotype of african swine fever virus in domestic pigs from ethiopia. 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african swine fever virus: current state and future perspectives in vaccine and antiviral research. vet. microbiol 185:15–19. we would like to thank chad smithson for his help and the many co-op students from the university of victoria who have built the viral bioinformatics resource center (virology.uvic.ca). acknowledgments references 170 african swine fever virus • 171 34 | fine focus genetic classification of two vibrio species isolated from florida gulf coast using multi-locus sequence analysis eric c. warricka, matthew j. thomasa, travis barlowb, alexis deanb1, ambar ramosb2, brian slivonikb, and brittany j. gasperb* *corresponding author department of natural sciences, state college of florida, bradenton, fl, usaa; department of biology, florida southern college, lakeland, fl, usab 1present address: alexis dean, georgetown university, washington, d.c., usa 2present address: ambar ramos, nova southeastern university, davie, fl, usa eric c. warrick – warrice@scf.edu matthew j. thomas – thomasm1@scf.edu travis barlow – tbarlow800@gmail.com alexis dean – alexisdean1995@yahoo.com ambar ramos – aramos18053@gmail.com brian slivonik – bwstennis@gmail.com brittany j. gasper – bgasper@flsouthern.edu manuscript received 16 september 2019; accepted 12 january 2020. volume six | 35 abstract prokaryotic species classification has lagged dramatically behind eukaryotic species due to the dependence on the difficult dna-dna hybridization technique required for species designation. this is disadvantageous for the general understanding of the diversity of microbial life and unique microorganisms, along with their metabolic abilities, like bioremediation of hazardous waste. this applies to the secondary metabolites microorganisms produce that can be beneficial for humans and other living organisms, such as antibiotics or other industrially important compounds. in an effort to isolate and identify new microbial species, environmental samples were collected from marine sediment environments as marine habitats are under-sampled compared to non-marine habitats. two marine isolates designated mi-1 and mi-2 were isolated from marine sediment environments off the gulf coast of florida and chosen for further study because of their antibacterial production and unique colony pigmentation. they were genetically analyzed through sequencing of the 16s rrna gene, shotgun cloning, and an eight-gene multi-locus sequence analysis comparison to 66 other species of the same genus. from these, mi-1 and mi-2 can be classified as members of the vibrio genus most closely related to vibrio ruber and a distinct strain from v. ruber type strain vr-1. 36 | fine focus introduction the goal of biologists and taxonomists for over a century has been to classify organisms into species and taxonomic groups, thus bringing order to the great biodiversity seen in the world. due to the lack of a broadly applicable species concept (56) and the historical reliance on the time-consuming and laborintensive dna-dna hybridization (ddh) test required for species delineation (6, 28, 41), prokaryotic species classification has lagged far behind that of eukaryotes in achieving this goal. compared to the number of known eukaryotic species (>1.2 million) (48), the ~12,000 recognized prokaryotic species (48, 67) are lacking, particularly when the number of prokaryotic species on the planet is estimated to be as high as 1011-1012 (39, 44). given the dominance of bacterial diversification as depicted in the recently expanded new tree of life (34), there is a need for increased attention to prokaryotic taxonomy to work towards classifying the remaining 99.999% of unclassified prokaryotic species (44). marine environments are a source of microorganisms with important roles including global nutrient cycling where they are responsible for half of the primary production on the planet (5, 14), production of industrially and medically important compounds (3, 10, 15, 19), and essential symbiotic relationships with larger organisms (14, 20, 32). as it is estimated that 91% of species in and around the ocean still await classification and the majority of these are microbial (48), marine environments represent a likely source for the isolation of novel microbes that may have medical or industrial importance. because the challenges of growth in a marine environment are different from that of terrestrial organisms, marine microorganisms have acquired unique metabolic capabilities that allow for production of compounds not present in terrestrial organisms (14, 24). antimicrobials are one type of compound that has generated considerable interest for production by marine microorganisms due to both the increased prevalence of pathogens resistant to currently available antibiotics (33, 43) derived from terrestrial organisms and the comparative lack of sampling for antibiotic-producing organisms from marine environments (35, 38). antibiotic compounds have been successfully purified from marine microorganisms (8, 21, 50), suggesting the marine environment offers an untapped source of antimicrobials that could help combat the impending antibiotic crisis. one of the most abundant groups of microorganisms in marine environments is the vibrio genus (64). organisms classified as vibrios are generally small gram-negative, facultatively anaerobic rods with a single polar flagella that are primarily aquatic and require na+ for growth (2, 17, 61, 64). more than 100 vibrio species have been validly described (22). of these, several have been found to have close relationships with higher organisms, with effects ranging from beneficial symbiosis (32, 47) to harmful pathogenesis (27, 37, 68). vibrios are believed to take part in nutrient cycling (62) and are particularly important for their production of polyunsaturated fatty acids that are vital for the aquatic food web but can only be produced by a handful of organisms (52) and their breakdown of chitin (60). some vibrio species have also been found to produce antimicrobial compounds (13, 45). in addition to the vibrio species already characterized, other new candidate vibrio species have been observed but not yet described (22). species determination in vibrios can be complicated as many different species have virtually identical biochemical phenotypes, and some species can have very similar genomes yet still represent evolutionary units in nature (2, 56). multi-locus sequence analysis (mlsa) can serve as a substitute for the cumbersome ddh test (23, 26). this technique compares multiple housekeeping gene volume six | 37 sequences, at least four (22), concantenated together end to end into one larger sequence for phylogenetic analysis. mlsa has been used extensively for species determination in vibrio (55, 61). the genetic classification of two antibiotic-producing vibrio species isolated from the gulf coast of florida through sequencing of the 16s rrna gene, shotgun cloning analysis, and mlsa is described here. materials and methods isolation of mi-1 and mi-2 mi-1 was isolated from a marine sediment sample aseptically collected from the intertidal zone near a nature park in pinellas county, fl. the gps coordinates of the mi-1 collection site were 27°53’19”n, 82°50’22”w. mi-2 was isolated from a salt flat sediment sample aseptically collected from leffis key in bradenton, fl, manatee county. the gps coordinates of the mi-2 collection site were 27°27’07”n, 82°41’20”w. one sediment sample was taken from each location. permission to collect samples was obtained from local authorities in both counties. both sediment samples were serially diluted in 0.5 m nacl to 10-5 grams soil/ml and grown on starch agar plated supplemented with 0.5 m nacl (mi-1) or modified potato dextrose agar (pda) with the following contents (mi-2): tryptone (2 g/l), potato starch (4 g/l), nacl (0.5 m), glucose (5 mm) filter sterilized and added to the media after autoclaving, and agar (20 g/l). the neidhardt mops salt components were prepared as described (51). to control for fungal growth from environmental samples, 100 µg/1 cycloheximide was added on the initial isolation plates. after four days of growth at 25°c, colonies were isolated and selected for their ability to inhibit the growth of staphylococcus epidermidis, pseudomonas putida, and chromohalobacter salexigens, a marine microorganism, by spread-plating s. epidermidis, p. putida, or c. salexigens onto the separate starch agar or modified pda plates supplemented with 0.5 m nacl and then patch-plating the environmental isolates on top. after initial isolation, mi-1 and mi-2 were maintained on 0.5 m nacl modified pda plates and 0.5 m nacl lb agar plates and stored at 25°c. chemical extraction of antimicrobial compound from mi-2 the antimicrobial compound from mi-2 was crudely extracted and tested for activity against five different bacterial species. isolates of mi-2 were continuously streaked onto 0.5 m nacl modified pda plates and incubated at room temperature for seven days. agar from the plates was cut into cubes and placed into 50 ml media bottles. the bottles were placed in -80°c freezer for 30 minutes. after freezing, 20 ml of ethyl acetate was added to the bottle and shaken on an orbital shaker for 24 hours. the organic ethyl acetate layer was decanted into 20 ml scintillation vials where the ethyl acetate was allowed to evaporate, leaving the extract behind. extracts were resuspended in 1 ml of ethyl acetate. four 20 µl portions of the resuspended extract were plated onto a single region on the side of an lb plate and allowed to dry. the same procedure was repeated on the other side of the plate with an ethyl acetate control. a tester strain culture was plated on top of the extract and ethyl acetate control be mixing 100 µl of an overnight culture of s. epidermidis, bacillus subtilis, citrobacter freundii, c. salexigens, or enterobacter aerogenes in with 10 ml of top agar and poured evenly over the plate. the plates were incubated for 24 hours at 30°c. the zones of inhibition surrounding the chemical extract were measured. genus identification of mi-1 and mi-2 identification of the genus of mi-1 and mi-2 was completed by pcr amplification of the 16s rrna gene sequence. the 16s primers 63f (5’cag gcc 38 | fine focus taa cac atg caa gtc – 3’) and 1387r (5’ ggg cgg wgt gta caa ggc – 3’) obtained from integrated dna technologies (idt) were used to amplify the gene along with the mytaqtm mix from bioline. the whole colony pcr reaction conditions were run as follows: 1 cycle of 95°c for 10 minutes; 30 cycles of 95°c for 30 seconds, 58°c for 30 seconds, and 72°c for 1 minute; and 1 cycle of 72°c for 5 minutes. successful pcr amplification was confirmed by agarose gel electrophoresis using a 1% agarose gel in 1x tae buffer and purified using the isolate ii pcr and gel kit from bioline. purified pcr products were sequenced at the dna analysis facility on science hill at yale university. sequencing results were analyzed by blast against the 16s rrna database of eztaxon (11). genotypic characterization of mi-1 and mi-2 for all genotypic characterization, genomic dna was obtained from overnight cultures of mi-1 and mi-2 grown in lb with 4% nacl using the phenolchloroform extraction method as described (70). shotgun cloning of mi-1 and mi-2 was completed. genomic dna from mi-1 and mi-2 was digested with eco-ri and hindiii (new england biolabs). the resulting fragments were ligated into pgem7 (promega) after digestion with the same enzymes. successful recombinant plasmids were transformed into escherichia coli dh5α and isolated after overnight growth using the genelute plasmid miniprep kit (sigma). dideoxy-sequencing of the isolated plasmids was performed by functional biosciences using the t7 promoter primer. the generated sequences were analyzed using the blast (1) feature in the patric, the bacterial bioinformatics resource center (69), comparing against the genomes of v. ruber vr1 (listed as dsm 16370 in patric), v. rhizosphaerae dsm 18581, v. gazogenes dsm 21264, and v. mangrove cect 7927. the shotgun clones from mi-2 were deposited in genbank (9) with the accession numbers shown in table s1 in the supplemental material. whole genome sequencing of mi-1 and mi-2 was completed. after ethanol precipitation of extracted genomic dna, the sample was checked for integrity by agarose gel electrophoresis and quantified using a nanodrop 2000 (thermo scientific). whole genome sequencing of the extracted dna was conducted using hiseq 2500 technology at purdue university. the resulting genomic sequence was analyzed using rast (7). mlsa of mi-1 and mi-2 along with 66 known vibrio species type strains was completed using eight proteincoding housekeeping genes: ftsz, gapa, mreb, reca, rpoa, rpod, topa, and toxr. the gene sequences for all eight genes from mi-1 and mi-2 were obtained from the whole genome sequencing results. gene sequences for the 66 known vibrio species type strains were obtained from patric (69). table s2 in the supplemental material contains the locus tag numbers or reference sequence numbers as available for the genes sequences found through patric. all sequences for each gene were aligned using clustal omega (29) and trimmed so each gene sequence covered the same aligned region. the aligned regions from each gene were concentrated to create an 8-gene chain. the concatenated sequences were aligned with clustal omega and analyzed phylogenetically with mega7 (42). nucleotide percent identity values were acquired using a percent identity matric in clustal omega. maximum likelihood trees with a phylogenetic test of 100 bootstrap replications was created using previously suggested parameters (22) in mega7. results isolation of mi-1 and mi-2 marine isolates 1 and 2 (mi-1 and mi-2) were isolated volume six | 39 from marine sediment samples collected in intertidal regions of the gulf coast of florida in pinellas county and manatee county, fl, respectively. these locations were selected because they are undersampled marine environments that do not receive a large amount of daily foot traffic. each isolate was selected for further study based on its ability to produce antimicrobial compounds that completely inhibit the growth of s. epidermidis, p. putida, and c. salexigens. these organisms were chosen as s. epidermidis and p. putida represent safe relatives of s. aureus and p. aeruginosa, two of the eskape pathogens whose increasing rates of antibiotic resistance represent a great threat to the healthcare field (54). c. salexigens is a halophilic marine microorganism with a broad range of salinity tolerance (4) that was chosen to see if mi-1 and mi-2 can inhibit halophilic organisms as well as halotolerant s. aureus and non-halophilic p. aeruginosa. mi-1 was initially isolated on naclsupplemented starch agar where is appeared orange while mi-2 appeared bright pink on the modifiedpda. different media were used for initial isolation to provide different environments and increase the chances of isolating unique microorganisms. colonies of mi-1 and mi-2 appear anywhere from pink, red, or orange in pigmentation depending on the type of media on which it is grown. fig. 1 shows the antibacterial inhibition and pigmentation of both mi-1 and mi-2. figure 2 shows the results of chemical extraction of the antimicrobial compound produced by mi-2 against five different bacterial species. the crude chemically extracted antimicrobial compound was capable of inhibiting all organisms it was tested against: two gram positive species (s. epidermidis and b. subtilis) and three gram negative species (c. freundii, c. salexigens, and e. aerogenes). its strongest inhibition was against gram positive species. similar zones of inhibition were seen for mi-1 (data not shown). a gram stain of both isolates showed small, slightly curved gram-negative rods. genus identification of mi-1 and mi-2 pcr amplification and sequencing of the 16s rrna gene of mi-1 and mi-2 showed both organisms are in the vibrio genus. this confirmed what was previously figure 1. the figure shows the antibiotic production of patch plated mi-1 against c. salexigens (panel a), and patch plated mi-2 against s. epidermidis (panel b) and c. salexigens (panel c). white arrows indicate the location of mi-1 and mi-2 on each plate. antibiotic production can be seen by the zones of inhibition surrounding the patch plated mi-1 and mi-2. the orange pigment production of mi-1 and pink pigment production of mi-2 can be seen. 40 | fine focus figure 2. the figure shows the inhibition of the crude chemically extracted antimicrobial compound from mi-2 against s. epidermidis (panel a), b. subtilis (panel b), c. freundii (panel c), c. salexigens (panel d), and e. aerogenes (panel e) which has been recently renamed as klebsiella aerogenes. zones of inhibition can be seen surrounding the pink-pigmented chemical extract of mi-2. the radius of the zones of inhibition is indicated in the table accompanying the figure. the zones were measured 24 hours after plating. volume six | 41 table 1 – 16s rrna analysis for mi-1 and mi-2. the table shows the three top matches for the 16s rrna gene sequence of mi-1 and mi-2 after blasting the sequence against the 16s rrna sequence database of eztaxon. the matches generated by eztaxon represent the type strains for each of the species. 42 | fine focus table 2 – shotgun cloning results for mi-1, mi-2, and v. ruber. the table shows the shotgun cloning results for mi-1 and mi-2 when sequences were on a blast in the patric database against the type strain genomes of v. ruber, v. rhizosphaerae, v. gazogenes, and v. mangrovi. the top organism match for each gene present on the shotgun clone is shown along with the information about the gene provided and the percentage identity of the match. * indicates the shotgun clone produced a sequence that contained two genes, each having a separate match. volume six | 43 table 3 – percent identity matrix from mlsa mi-1 and mi-2. the table shows the percent identity matrix for mi-1 and mi-2 and the six other vibrio species from the eight gene mlsa that showed greater than 80.0% identity to mi-1 and mi-2. percent identity was determined using clustal omega. 44 | fine focus figure 3 – molecular phylogenetic analysis from mlsa mi-1 and mi-2. the figure shows the phylogenetic tree resulting from the eight gene mlsa of mi-1 and mi-2 with 66 known vibrio species. symbols indicate the clade identity of each organism as assigned from the eight gene mlsa of sawabe, 2013; organisms without symbols were not included in this mlsa (61). evolutionary analyses were conducted in mega7 (42). the evolutionary history was inferred by using the maximum likelihood method based on the kimura 2-parameter model (40). the bootstrap consensus tree inferred from 100 replicates is taken to represent the evolutionary history of the taxa analyzed (18). all positions containing gaps and missing data were eliminated. there were a total of 9807 positions in the final dataset. volume six | 45 shown for mi-2 (49). the pcr sequencing was completed from three unique colonies with the same results obtained each time. the sequencing results were analyzed using the curated 16s rrna eztaxon database. sequencing results are shown in table 1. the match producing the highest similarity was vibrio ruber vr1 for mi-2 at 98.64% while the best match for mi-1 was vibrio gazogenes atcc 29988 at 97.58%. both of these values are below the suggested 98.65% similarity cutoff for species delineation with the 16s rrna gene sequences (39). as v. ruber vr1 was the best match for mi-2 and the second match for mi-1, it was obtained from the bcccm/lmg bacterial culture collection and used as a comparison for genetic characterization for mi-1 and mi-2. genotypic characterization of mi-1 and mi-2 genotypic characterization began with shotgun cloning to ascertain if mi-1 and mi-2 might be novel enough to warrant a larger investment through whole genome sequencing. results of the shotgun cloning are showing in table 2. there were four successful clones for mi-1 that matched up to six unique genes and eight successful clones for mi-2 that matched up to eleven unique genes. the top match for all clones when analyzed by blast through patric (69) was v. ruber. the percentage identity ranged from 91-99% with a median of 94% for mi-1 and 86-100% with a median of 95% for mi-2. these values are right around and below the suggested speciation cutoff of 95-96% for average nucleotide identity (ani) (28, 39, 59), suggesting mi-1 and mi-2 may be novel species and should undergo a more thorough genotypic characterization through mlsa. an eight gene mlsa of mi-1 and mi-2 was completed with 66 known vibrio type strains with genomes uploaded to the patric database. the eight genes used were ftsz, gapa, mreb, reca, rpoa, rpod, topa, and toxr. with the exception of rpod and toxr, these genes were previously used for an extensive eight-gene mlsa analysis of the vibrio clades (61). a percent identity matrix for all organisms showing greater than 80% identity to mi-1 and mi-2 from the mlsa is shown in table 3. mi-1 and mi-2 share 99.6% identity from the mlsa. the closest percent identity match from known vibrio species to mi-1 and mi-2 was v. ruber at 97.93 and 97.82% identical, respectively. the complete percent identity matrix for all 68 organisms is in table s3 in the supplemental information. the phylogenetic tree generated from the mlsa data is shown in figure 3. organisms with symbols on the figure were included in the eight gene mlsa of sawabe et al., 2013 (61), and the symbols indicate the vibrio clade to which they were assigned. mi-1 and mi-2 appear most closely related to v. ruber and belong in the gazogenes clade. v. mangrovi and v. quintilis were not included in the previous mlsa, but the results from this mlsa suggest they also belong in the gazogenes clade along with v. ruber, v. gazogenes, and v. rhizosphaerae. discussion marine bacterial isolates mi-1 and mi-2 cultured from two locations on the gulf coast of florida showed unique colony pigmentation and production of antibacterial compounds capable of inhibiting both gram positive and gram negative organisms. results of the 16s rrna gene sequencing indicated mi-1 and mi-2 are in the vibrio genus. v. ruber, v. rhizosphaerae, v. mangrovi, and v. gazogenes are the four most closely rated species by 16s rrna sequence homology, however none were above the 98.65% cut-off for species delineation (39). morphologically, mi-1 and mi-2 are small gramnegative curved rods. this along with their isolation from marine environments is consistent with all other vibrio species. the four most closely related vibrio species are known to produce red pigments similar to that seen from mi-1 and mi-2 (31, 57, 58, 63). v. ruber and v. gazogenes have been 46 | fine focus experimentally shown to produce the antibacterial compound prodigiosin (31, 46, 63), a chemical compound with red pigmentation responsible for the color produced by the colonies. it is best known for producing the characteristic red pigmentation of serratia marcescens (16). v. gazogenes is also capable of producing cycloprodigiosin, a cyclicized analogue of prodigiosin and fellow member of the prodiginine family of chemical compounds (25) capable of antimicrobial activities. prodiginine compounds have also been purified from cultures of mi-2 (49). it remains to be experimentally determined if mi-1, v. rhizosphaerae or v. mangrovi produce antimicrobial prodiginine compounds, but it is likely they do based on the color of the colony. v. gazogenes has also been shown to produce the antimicrobial compound magnesidin a (36). in an effort to contribute to the knowledge of marine bacterial species, these organisms were classified genetically through shotgun cloning and an extensive eight-gene mlsa. genotypic investigation of mi-1 and mi-2 was completed by performing an eight gene mlsa with 66 known vibrio species whose genomes have been sequenced. v. ruber showed the closest percent nucleotide sequence identity to mi-1 and mi-2, 97.93 and 97.82%, respectively. both of these values are below the 98% cutoff suggested by sawabe et al. as a species boundary (61), suggesting mi-1 and mi-2 may be novel species based on vibrio guidelines, though these values are higher than the accepted 9596% ani cutoff value (12, 28, 39, 59). the 99.61% identity between mi-1 and mi-2 indicate they are the same species. the phylogenetic tree generated from the mlsa (figure 3) showed mi-1 and mi-2 belong in the gazogenes clad along with v. ruber, v. gazogenes, v. rhizosphaerae, and v. aerogenes, v. magrovi, and v. quintilis were not included in the original mlsa of sawabe et al., 2013 (61), but based on their position in the phylogenetic tree, we suggest they be included in the gazogenes clade as well. there was general agreement between the clustering of organisms in this mlsa and the clade designations from sawabe et al., 2013 with the exception of the nereis and orientalis clades. the nereis clade was reported to contain v. nereis and v. xuii (61), however this mlsa shows v. xuii to be more closely aligned with v. orientalis of the orientalis clade and v. nereis to be more closely aligned with v. hepatarius, also of the orientalis clade. reasons for the differences could be explained by the use of rpod and toxr genes in this mlsa in place of gyrb and pyrh in sawabe et al., 2013. the substitution of gyrb and pyrh with rpod and toxr occurred based off data from pascual, 2010 that suggested rpod and toxr are better for reliable species identification of vibrio (53). this study also utilized sequences of the entire gene as opposed to smaller pcr fragments used in sawabe et al., 2013. v. neptunis of the coralliilyticus clade also aligned closely with v. nereis and v. heptarius; however the other member of its clade, v. coralliilyticus, was not included in this mlsa, so no conclusions regarding its appropriate clade can be made. other vibrio species not included in this mlsa of sawabe et al., 2013 that are closely aligned with members of the orientalis clade in this study include v. pacinii, v. galatheae, v. bivalvicida, and v. europaeus. more clade designation suggestions regarding the vibrio organisms not included in the mlsa of sawabe et al., 2013 can be made. it appears v. albensis belongs in the cholerae clade, not surprisingly considering it is a non-o1 serovar of v. cholerae (30). v. owensii, v. jasicida and closely related v. inhibens (66), v. diabolicus and the closely related v. antiquaries (65), and v. sagamiensis closely align with members of the harveyi clade; while v. algivorus and v. casei volume six | 47 appear most closely related to the rumoiensis clade. additionally, v. toranzoniae, v. celticus, v. atlanticus, and v. hemicentroti appear closely related to members of the splendidus clade. v. xiamenensis appears to be in an orphan clade much like v. proteolyticus (61). the genetic analysis has demonstrated that mi-1 and mi-2 are vibrio isolates that can be classified as a strain of v. ruber distinct from the type strain v. ruber vr1 used in this study. genotypically, they are on the threshold of being classified as distinct species. the refinement of the whole genome sequences of mi-1 and mi-2 are currently being completed to fill in the gaps and produce a complete genome sequence that can be confidently compared to v. ruber by whole genome ani or digital ddh, an electronic version of the classic ddh mechanism (12). this complete genotypic characterization will be included as a part of a polyphasic approach along with phenotypic and chemotaxonomic characterization (67). phenotypic characterization will consist of thorough biochemical and growth phenotype testing of mi-1 and mi-2 and comparison with v. ruber. chemotaxonomic characterization will be performed through analysis of the fatty acid methyl ester (fame) composition of membrane lipids in mi-1, mi-2, and v. ruber. additionally, future research will characterize the antibacterial secondary metabolites produced by mi-1 and mi-2. this will include confirming prodiginine compounds are responsible for pigment production in mi-1 as in mi-2 (49) and determining whether the antibacterial properties of these organisms is solely due to prodigiosin or if they produce additional antibacterial compounds, like v. gazogenes and its production of magnesidin a (36). acknowledgements the authors would like to acknowledge stephanie morgan for her work in the isolation of mi-2 and the state college of florida biotechnology classes of 2016 and 2017 for their work performing the shotgun cloning of mi-1 and mi-2. 48 | fine focus references 1. altschul sf, gish w, miller w, myers ew, lipman dj. 1990. basic local alignment search tool. j. mol. biol. 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department of chemistry, 2 department of biological sciences wellesley college, wellesley, ma 2 department of biology, suffolk university, boston, ma ‡co-first authors: lei wei, rebecca j. rubinstein manuscript received 30 june 2016; accepted 5 november 2016 copyright 2017, fine focus. all rights reserved. 10 • fine focus, vol. 3 (1) the microorganisms that inhabit cheese contribute greatly to the flavor and development of the final product. while the rind and curd microbiota have been characterized separately, there is limited information on how the structure and function of microbial communities in rinds and curds vary within and amongst cheeses. to better understand the differences in community structure and function between communities of cheese rinds and curds, we combined culture-based methods with culture-independent community profiling of curds and rinds. rinds contained greater taxonomic diversity than curds. lactobacillales dominated curd communities while members from the order actinomycetales were found in high abundance in rind communities. communities varied more between rinds and curds than among cheeses produced from different milk types. to better understand microbial community functions, we cultured and assayed isolates for antibiotic susceptibility and carbon source utilization. among european and u.s. cheeses, 70% of all susceptible isolates were cultured from u.s. cheeses. overall, our study explored the differences within and between rind and curd microbial communities of natural rind cheeses, provided insights into the environmental factors that shape microbial communities, and demonstrated that at the community and isolate level the cheese microbiome was diverse and metabolically complex. abstract corresponding authors vanja klepac-ceraj* vklepacc@wellesley.edu celeste peterson* cnpeterson@suffolk.edu keywords • cheese microbiome • rind • curd • diversity • antibiotic resistance • 16s rrna gene cheese production exemplifies a reproducible succession of microbial communities (10, 48, 49). microbes execute the biochemical transformation of milk from a liquid suspension of lactose, casein, whey, and fat into cheese which is a solid aggregate of amino acids, lactate and volatile flavor compounds and pigments (6). over centuries, humans optimized the cheese-making process to select for distinct and reproducible microbial communities that give cheeses their individual tastes, consistencies, colors and other desirable properties (6, 14, 43, 49). in natural-rind cheeses, endogenous microorganisms or the addition of bacterial starter culture to milk are needed to acidify and coagulate the milk into introduction bacterial community structure in cheese • 11 curds and enable future colonization by fungi and bacteria on the cheese surface (26, 49). the early process of curd formation is well characterized (11, 16) . ripening begins in milk, where lactic acid bacteria (lab) such as streptococcus thermophilus, lactobacillus helveticus, and lactobacillus casei ferment lactose into lactate, acidify the medium and digest proteins and milk (11, 25, 35, 36). lab can be introduced by a starter culture, or the milk can be permitted to acidify naturally from the microbial community in it (6). regardless of the addition of starter culture, most curds become inhabited by a simple community, dominated by lactose fermenters that may include organisms belonging to genera lactococcus, lactobacillus, leuconostoc, enterococcus, staphylococcus, enterobacter, and streptococcus (1, 6, 16). after the initial curd formation, the rind begins to form. throughout the ripening, excess lactate accumulates and dissolves into the medium, eventually migrating upward to the curd surface (22). once lactate becomes accessible at the outer surface of the cheese, aerobic fungal taxa including candida, penicillium, and scopulariopsis colonize the surface and metabolize lactate, leading to an increase in ph at the surface environment (22, 49). de-acidification facilitates the colonization and succession of microbes that prefer more alkaline and salty conditions including coryneforms such as corynebacterium, brevibacterium and brachybacterium (27, 37, 41). the composition of the external rind communities is governed by factors beyond ph, including microbe-microbe interactions. fungal species can contribute to cheeses’ unique characteristics, such as the blue-vein appearance of roquefort by the fungus penicillium roqueforti, and can be crucial to the survival of rind bacteria like corynebacterium, halomonas, pseudomonas, pseudoalteromonas, and vibrio spp. (4, 29, 49). fungi such as those belonging to the spore-forming penicillium species can also produce antibacterial compounds that can lead to selection for more antibiotic-resistant bacterial strains (28). likewise, bacteria on the rind such as those belonging to the antibioticproducing actinomycetes group can have a similar effect (32). the possibility of cheese bacteria developing resistance mechanisms has warranted the characterization of antibioticsusceptibility of bacteria on various types of cheeses (4, 29). while many studies have characterized the succession of early cheese community development from curd to rind in individual natural rind cheeses (1, 16, 20, 21), less is known about the comparison of microbial composition of a mature rind to a mature curd within and across natural rind cheese varieties. here we compare microbial community structure between the rinds and curds of seven naturalrind cheeses that differ by ph, moisture content, and milk source. to obtain a detailed understanding of the rind and curd microbial communities, we used culture-independent, high-throughput illumina sequencing of 16s rrna genes. we also sought to characterize specific bacteria cultured from the cheeses. these isolates were identified using sanger sequencing of the 16s rrna gene (40) and were assayed for antibiotic susceptibility and carbon utilization profiles using biolog’s ecoplates. through these analyses, we aimed to evaluate the following hypotheses: 1) cheese rind communities would exhibit higher taxonomical diversity than curd communities; 2) antibiotic susceptibility of cheese isolates would differ between cheeses and between the two regions where these cheeses originated, europe and the u.s.; and 3) cheeses with higher taxonomical diversity would be more metabolically active and can utilize more carbon sources. this study contributes to the characterization of the curdand rindassociated communities of natural rind cheeses and reveals patterns of microbial diversity according to cheese type as well as the overall metabolic and antibiotic resistance profile of isolates from the different cheeses. 12 • fine focus, vol. 3 (1) methods sample collection and environmental parameters seven natural rind cheeses—vermont shepherd, stichelton, sonnet, missouri truckle, maggie’s round, comte, and alpage gruyere— were obtained from wasik’s cheese shop in wellesley, massachusetts. a 100 +/10 mg sample from each of the rinds and curds of the examined cheeses was collected aseptically and processed immediately. rind samples were scraped using a sterile razor blade and curd samples were taken from the cheese center after scraping off the exposed curd layer. a solidstate ph meter (s175cd/bnc; sensorex, garden grove, ca) was used to determine the ph of each cheese rind and curd. samples were dried for 7 days and moisture was determined by subtracting the dry weight of the sample from the original wet weight of a 1 g sample. bacterial isolation and characterization the 100 +/10 mg of fresh rind and curd samples were crushed using a pellet pestle and diluted in sterile water to 10-3, 10-4, and 10-5 of their original concentrations. all diluted samples were grown aerobically at room temperature on three different media: plate count agar (pca) with milk (pcam: 5g peptone, 2.5 g yeast extract, 1 g dextrose, 1 g of whole milk powder, 15 g agar), nutrient agar (na: 0.5% peptone, 0.3% beef extract/yeast extract, 1.5% agar, 0.5% nacl) and pca with fermentation indicators for lactose (5 g peptone, 2.5 g yeast extract, 1 g dextrose, 10 g lactose, 0.03 g neutral red, 15 g agar). isolates were restreaked from single colonies three times in order to obtain pure cultures. dna analysis of isolates the genomic dna of selected bacterial isolates were extracted by suspending a colony in polymerase chain reaction (pcr)-grade water and freezing for 20 min at -80°c and then thawing. a 1465 base pair (bp) sequence of the 16s rrna gene was pcr-amplified from genomic dna (10-30 ng). mastermix reagents consisted of 1 μg/μl bovine serum albumin, 200 μm dntp mix, 1x buffer w/ mgcl2, 300 pm 27f primer (aga gtt tga tcc tgg ctc ag), 300 pm 1492r primer (acg gct acc ttg tta cga ctt) (idt integrated dna technologies, inc., coralville, ia) (30), and 0.2 u takara ex. taq polymerase (takara clontech, mountain view, ca). dna extracts from escherichia coli and sterile pcrgrade water were used as positive and negative controls, respectively. the thermal cycler program ran for 34 cycles in the following order: 1 cycle of initial denaturation (3 min at 95°c); 32 cycles of denaturation (30 sec at 95°c), annealing (25-30 sec at 50°c), and extension (1.5 min at 72°c); 1 cycle of final extension (10 min at 72°c); and hold (4°c). amplicons were detected from banding patterns on 1.5% agarose gel electrophoresis and quantified on the nanodrop 2000 uv-vis spectrophotometer (nanodrop products, thermo fisher scientific, inc., wilmington, de). excess taq polymerase, primer and nucleotides were precipitated from the amplicon solution by adding 1μl usb exosap-it reagent (affymetrix, inc., santa clara, ca) to 9μl of amplicon. purified products were identified via sanger sequencing of the 16s rrna gene using 27f primer (5’-agagtttgatcctggctcag-3’) (genewiz, madison, wi, usa). from each sequence we extracted >750 consecutive nucleotides with quality score q>20 and each chromatogram was visually inspected to insure there were no base caller errors (supplementary table 1). bacterial community structure in cheese • 13 is o la te n a m e se q u en c e id en ti fi c a ti o n m il k ty pe ch ee se n a m e c o u n tr y g en u s lw h w 227 f u nc ul tu re d br ev ib ac te riu m s p. c lo ne p ts -l -c l11 co w a lp ag e g ru ye re sw itz er la nd br ev ib ac te riu m lw h w 327 f u nc ul tu re d br ev ib ac te riu m s p. c lo ne p ts -l -c l11 co w a lp ag e g ru ye re sw itz er la nd br ev ib ac te riu m lw h w 427 f br ev ib ac te riu m a ur an tia cu m , i so la te 0 91 1t es 25 y 3 co w a lp ag e g ru ye re sw itz er la nd br ev ib ac te riu m lw h w 627 f br ev ib ac te riu m s p. e p1 1 s tr ai n ep 11 co w a lp ag e g ru ye re sw itz er la nd br ev ib ac te riu m lw h w 727 f c or yn eb ac te riu m g ly ci no ph ilu m co w a lp ag e g ru ye re sw itz er la nd c or yn eb ac te riu m z m bb 127 f st ap hy lo co cc us e qu or um co w st ic he lto n en gl an d st ap hy lo co cc us z m bb 227 f br ac hy ba ct er iu m a lim en ta riu m co w st ic he lto n en gl an d br ac hy ba ct er iu m z m bb 327 f br ac hy ba ct er iu m a lim en ta riu m co w st ic he lto n en gl an d br ac hy ba ct er iu m z m bb 427 f br ac hy ba ct er iu m a lim en ta riu m co w st ic he lto n en gl an d br ac hy ba ct er iu m z m bb 527 f la ct ob ac ill us p la nt ar um o r ca se i co w st ic he lto n en gl an d la ct ob ac ill us z m bb 627 f br ev ib ac te riu m li ne ns o r ep id er m id is co w st ic he lto n en gl an d br ev ib ac te riu m z m bb 727 f st ap hy lo co cc us s im ul an s co w st ic he lto n en gl an d st ap hy lo co cc us z m bb 827 f st ap hy lo co cc us p as te ur i co w st ic he lto n en gl an d st ap hy lo co cc us z m bb 927 f st ap hy lo co cc us e qu or um co w st ic he lto n en gl an d st ap hy lo co cc us z m bb 10 -2 7f st ap hy lo co cc us e qu or um co w st ic he lto n en gl an d st ap hy lo co cc us a x 127 f st ap hy lo co cc us e qu or um co w m is so ur i t ru ck le u sa st ap hy lo co cc us a x 227 f br ac hy ba ct er iu m a im en ta riu m co w m is so ur i t ru ck le u sa br ac hy ba ct er iu m a x 527 f br ac hy ba ct er iu m a lim en ta riu m co w m is so ur i t ru ck le u sa br ac hy ba ct er iu m a x 727 f st ap hy lo co cc us w ar ne ri co w m is so ur i t ru ck le u sa st ap hy lo co cc us r r sh 127 f st ap hy lo co cc us x yl os us co w m ag gi es r ou nd m as sa ch us et ts st ap hy lo co cc us r r sh 327 f br ac hy ba ct er iu m s p. co w m ag gi es r ou nd m as sa ch us et ts br ac hy ba ct er iu m r r sh 427 f ba ci llu s sp . co w m ag gi es r ou nd m as sa ch us et ts ba ci llu s r r sh 627 f st ap hy lo co cc us x yl os us co w m ag gi es r ou nd m as sa ch us et ts st ap hy lo co cc us r r sh 727 f st ap hy lo co cc us x yl os us co w m ag gi es r ou nd m as sa ch us et ts st ap hy lo co cc us r r sh 827 f ba ci llu s m oj av en sis co w m ag gi es r ou nd m as sa ch us et ts ba ci llu s c h 127 f st ap hy lo co cc us s p. u 13 71 -1 01 22 7x h 13 6 sh ee p v t. sh ep he rd u sa st ap hy lo co cc us c h 327 f br ev ib ac te riu m s p. e p1 1 sh ee p v t. sh ep he rd u sa br ev ib ac te riu m c h 727 f st ap hy lo co cc us e qu or um sh ee p v t. sh ep he rd u sa st ap hy lo co cc us supplementary table 1 14 • fine focus, vol. 3 (1) direct dna analysis of cheese microbial communities total dna from the cheese rind and curd samples was extracted using the mo bio power® soil dna isolation kit (mobio, carlsbad, ca). the 16s rrna gene from community genomic dna was amplified, detected, quantified, and purified as described for the isolates. samples were sequenced on a miseq instrument at the forsyth institute, cambridge, ma. the v3/v4 region of the 16s rrna gene was amplified from each sample using the forward primer 341f 5’-aatgatacggcgaccaccgagatctacac tatggtaatt gt cctacgggaggcagcag-3’; where italicized text indicates illumina adaptor, bold text indicates primer pad, italicized bold indicates primer linker and an underlined text a conserved bacterial primer 314f. the reverse primer was 806r 5’caagcagaagacggcatacgagat xxxxxxxxxxxx agtcagtcag cc ggactachvgggtwtctaat-3’; where italicized text indicates reverse complement of the illumina adaptor, 12 x-letters in bold are the golay barcode primer followed by barcode primer and primer linker (italicized bold letters). the conserved bacterial primer 806r is indicated by underlined letters. pcr products from respective samples were each tagged by a sample-specific 12-base barcode (9). all samples were amplified in triplicates with 5 prime hot master pcr mix (five prime) on an eppendorf master cycler pro pcr thermocycler using 0.2 μm of each primer and 10 ng template. reaction conditions were: 94°c for 3 min, followed by 35 cycles at 94°c for 45 secs, 50°c for 1 min and 72°c for 1.5 min. following the 35th cycle, samples were incubated at 72°c for 10 min. amplicons were purified using ampure magnetic beads according to the manufacturer’s instructions (agencourt from beckman coulter, danvers, ma), quantified by nanodrop, and further purified using the qiagen minielute gel extraction kit (qiagen, valencia, ca). libraries were quantified on a bioanalyzer instrument according to the bioanalyzer manual using a dna high sensitivity chip, pooled, and sequenced on a miseq illumina sequencer (illumina, san diego, ca). forward and reverse reads were joined using flash software (31). libraries were demultiplexed and filtered using a q-score cutoff of 20 using split_libraries_fasq.py in quantitative insights into microbial ecology (qiime) v1.8.0 (9). any reads that did not assemble or meet the q-score threshold were removed and were not used in subsequent analyses. sequences were clustered into operational taxonomic units (otus) using the uclust algorithm (15) at 97% sequence identity level with the generation of new clusters with sequences that match the reference, and classified using the greengenes 97% reference dataset released on may 2013 (12, 33). raw sequence data were submitted to sequence read archive in genbank under accession number prjna354727. antibiotic disc susceptibility assay we used a modified version of antibiotic disc diffusion susceptibility test to compare fungal and bacterial-derived antibiotic susceptibility of cheese bacteria isolates, focusing on antibiotic susceptibility trends among isolates cultured from cheeses of different regions (5, 7). ten morphologically diverse isolates were cultured in nutrient broth (nb; becton, dickinson and company, franklin lakes, nj, usa) for 40 48 hours. the liquid culture of bacteria was diluted using nb to match the turbidity of a 0.5 mcfarland standard to ensure roughly equivalent densities of each inoculum. bacterial culture was evenly streaked onto the dried surface of a nutrient agar plate using a sterile swab and allowed to be absorbed into the agar for at least 3 5 minutes. six antimicrobialbacterial community structure in cheese • 15 impregnated discs (am10: ampicillin 10 μg; p10: penicillin 10 iu/ie/ui; e15: erythromycin 15 μg; ra5: rifampin 5 μg; n30: neomycin 30 μg; nb30: novobiocin 30 μg) were evenly pressed onto the bacterial-containing agar surface using a disc dispenser. plates were incubated for one week at room temperature prior to examination for antibiotic susceptibility. diameter measurements in millimeters of the zone of clearance around the individual antibiotic discs for cheese isolates were used to categorize each cheese isolate into one of the three susceptibility levels based on the following zone clearance interpretation: resistant (13 mm or less); intermediate susceptible (14 – 16 mm); and susceptible (17 mm or more) (45). carbon source utilization profiling community-level physiological profiling (clpp), a metabolic profile, of both the cheese rind and selected curd communities and individual bacterial isolates were analyzed using biolog ecoplatetm assay (biolog, hayward, ca). the capacity of either a bacterial community or a single bacterial isolate to utilize 31 distinct carbon sources over a 7-day period was examined and compared between the community sample and isolates of the same cheese and among different cheese types. the 100 +/10 mg of fresh rind and curd samples were crushed with a pestle and diluted in sterile water to 10-3 in 10 mm phosphate buffer. subsequently, 100 μl of the solution was inoculated into separate biolog ecoplatetm wells. individual isolates were incubated in nb overnight at room temperature and diluted to 104 cells/ml and 100 μl was inoculated into each well of the new biolog ecoplatetm. growth was measured using the molecular devices spectramax 190 and the softmaxpro6.3™ program at a590 absorbance, for six consecutive days where day 1 was the day of inoculation. metabolic diversity (cmd) was defined as the number of carbon sources utilized by the sample. top carbon sources were defined as carbon sources that exhibited the maximum absorbance value for from fluorescence of tetrazolium salt reduction in the biolog ecoplatetm assay. cheese origin country milk curd ph rind ph curd % moisture rind % moisture alpage gruyere gruyere switzerland cow 7.45 6.42 9.71 28.43 comte french alps france cow 7.14 6.5 21 26 maggie’s round ma usa cow 5.34 5.04 4 26 missouri truckle mo usa cow 6.01 5.73 27 24 vermont shepherd vt usa sheep nd nd nd nd sonnet vt usa goat 6.44 5.55 nd nd stichelton nottingham england cow 8.17 blue 8.24 white 6.96 nd nd table 1. characteristics of the seven investigated natural rind cheeses. nd = not determined. 16 • fine focus, vol. 3 (1) environmental parameters of natural rind cheeses to explore the relationship between the rind and curd within and between natural rind cheeses, and to determine how the physical factors of cheese environments (ph and moisture) correlate with microbial community diversity, we analyzed the microbial communities from seven different cheeses. the seven natural rind cheeses varied in appearance, place of origin, and type of milk used in the cheese-making process. four cheeses came from the united states and three came from switzerland, france, and england. all of the european cheeses and two of the u.s. cheeses were made from cow milk. the remaining two u.s. cheeses were made from sheep (vermont shepherd) and goat (sonnet) milk. in each cheese the curd was slightly more acidic and contained more moisture than the corresponding rind (table 1). moisture in the curd varied slightly between cheeses, from 24% to 28%, while moisture in the rind had a larger range between cheeses, from 4% to 27% (table 1). to determine the types of bacteria that were present in the cheese communities, we analyzed the 16s rrna gene using illumina sequencing of the total extracted dna from cheese curd and rind, and sanger sequencing of cultured cheese isolates. there were more organisms identified to the genus level in the cultureindependent approach than culture-dependent approach (see “other” category, figure 1). unculturable organisms that were common to the cheese rinds included streptococcus spp. (comprising 24% of total bacterial cells in the comte rind and 55% of the maggie’s round rind), lactococcus spp. (39% of all cells in missouri truckle rind), yaniella spp. (20% of stichelton rind), prauseria spp. (18% of vermont shepherd rind), halomonas spp. (17% of stichelton rind and 7% of alpage gruyere rind) and lactobacillus spp. (16% of missouri truckle rind and 13% of comte rind). in the curds, prevalent uncultured taxa included lactococcus spp. (91% of missouri truckle rind and 89% of stichelton rind), streptococcus spp. (78% of maggie’s round rind, 73% of comte rind, and 26% of alpage gruyere rind), and lactobacillus spp. (71% of alpage gruyere rind, 35% of comte rind). in the curd, 43% (figure 1b), and in the rind, 65% (figure 1c) of bacteria were not culturable on pcam agar. thus, a significant fraction of bacterial species was not recovered on pcam plates. the rind harbors a more complex bacterial community than the curd in order to determine the bacterial diversity of the rind and curd, we carried out illumina sequencing of the 16s rrna gene. rarefaction curve analysis, which assesses species richness from samples, showed all samples approached the asymptote and revealed that the overall bacterial diversity was well represented (figure 2). the curds of alpage gruyere and comte had the fewest unique otus (figure 2a) while the largest number of unique otus was found in the rind of alpage gruyere cheese, followed by the rinds of stichelton and comte cheeses (figure 2b). the shannon diversity index, a measurement of overall diversity, of pooled data from the rinds and curds of the seven cheeses indicated more species richness and evenness in the rind communities than the curd (figure 2c). together, otu distribution and richness data demonstrate higher alpha diversity in rind communities. results through the identification of the organisms present in the cheese samples, we found that, with the exception of the sonnet cheese, the only phylum represented in the curd communities was firmicutes (figure 3). in contrast, firmicutes, actinobacteria, and proteobacteria were found in the rind communities. in a similar trend, no more than ten taxonomic units were found in each of the curd communities, while no less than ten genera were identified in each of the rind communities (figure 3). in general, dominant genera in the rinds and curds were widespread among sampled cheeses. cheese curds were dominated by lactic acid fermenters including lactobacillus, streptococcus, and lactococcus while the rind communities showed a greater relative abundance of brevibacterium and actinomycetaceae (figure 3; rind). however, variation was present within both the rind and curd communities. for example, while the missouri truckle, sonnet, and stichelton curds were almost completely dominated by lactococcus, this organism made up less than 2% of the maggie’s round, comte, and alpage gruyere cheeses (figure 3; curd, purple). we next sought to investigate whether the differences in community composition could be associated with other abiotic factors such as moisture content, ph, or milk type. principal coordinates of analysis (pcoa) demonstrated that communities were found to cluster by rind or curd (figure 4), but not by milk type or moisture content (supplementary figure 1). a trend towards clustering was seen with ph (supplementary figure 1). figure 1. prevalence (in % operational taxonomic units, otus) of cheese microbes successfully identified to the genus level through a) culture-dependent sanger sequencing of the 16s rrna gene of organisms isolated from both the rind and the curd, as compared to the prevalence of these organisms found through high-throughput community sequencing in either b) the curd or c) the rind. the green “other” category in figure 1 represents bacteria identified by illumina sequencing but not detected by culturing. brevibacterium corynebacterium staphylococcus brachybacterium bacillus other 39% 22% 17% 13% 9% a) 43% 33% 14% 9% 1%b) 65% 16% 10% 5% 4%c) bacterial community structure in cheese • 17 18 • fine focus, vol. 3 (1) figure 2. rarefaction curves of microbial populations from the a) curd and b) rind of natural rind cheeses show greater species richness in the rind than the curd. each line represents the standard error of the mean (±sem) of 10 samples from the rind or curd of a cheese sequenced using 16s rrna gene. c) rind communities are significantly more evenly and richly distributed than curd communities (t-test, tstat = 2.56, df = 14, p < 0.05). bar heights represent mean shannon-weaver diversity within communities sampled from the rind and the curd. rind: n = 9; curd: n = 10 for all means. error bars = mean ± sem. sequences per sample o bs er ve d o tu s 10000 20000 30000 40000 500000 200 400 600a) o bs er ve d o tu s sequences per sample 10000 20000 30000 40000 500000 200 400 600 800 1000 1200 1400 1600 1800 b) sh an no nw ea ve r di ve rs ity sampling location rind curd 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8 c) alpage gruyere alpage gruyere 2 comte stichelton maggies round sonnet missouri truckle vt. shepherd stichelton 2 comte 2 bacterial community structure in cheese • 19 figure 4. principal coordinate analysis of rind and curd microbial communities. clustering is seen with rind samples and curd samples, regardless of the cheese type. a 16s rrna gene dataset was analyzed with qiime and r was used to generate the principal coordinate analysis. each white or black mark represents averaged community composition data of the rind or curd for the cheese sampled. pc1 (76.4%) pc 2 (9 .3% ) rind curd figure 3. relative abundance of microbial phyla and genera from the rind and curd of six natural rind cheeses. horizontal bars represent microbiome samples from six cheeses in the rind and the curd and are colored according to the microbial phyla and genus found in these environments through illumina sequencing of the 16s rrna gene. darker shades represent actinobacteria while lighter shades represent either firmicutes or proteobacteria. represented organisms were found at ≥ 2% relative abundance. actinobacteria actinobacteria; arthrobacter actinobacteria; brevibacterium actinobacteria; corynebacterium actinobacteria; intrasporangiaceae actinobacteria; leucobacter actinobacteria; prauseria actinobacteria; sanguibacter actinobacteria; yaniella actinobacteria; branchybacterium firmicutes; aerococcaceae firmicutes; clostridiales firmicutes; clostridiisalibacter firmicutes; lactobacillaceae firmicutes; lactobacillus firmicutes; lactococcus firmicutes; planococcacus firmicutes; staphylococcus firmicutes; streptococcus protobacteria; halomonas proteobacteria; psychrobacter 1 10.8 0.80.6 0.60.4 0.40.2 0.20 vt. shepherd stichelton sonnet missouri truckle missouri truckle comte comte alpage gruyere alpage gruyere rind curd 20 • fine focus, vol. 3 (1) isolates from u.s. cheeses display higher overall susceptibility to antibiotics than european cheeses to explore the outcomes of potential interactions between bacteria and fungi in the cheese communities from regionally diverse cheeses, inhibition of bacterial growth by fungal and bacterially derived antimicrobials was examined for cheese isolates. isolate susceptibility levels were compared between cheeses and their two respective geographical regions of origin: the u.s. and europe (figure 5). thirty-five bacterial isolates sampled from six cheeses were tested for their susceptibility or resistance to six antibiotics on nutrient agar plates: ampicillin (am10), penicillin (p10), erythromycin (e15), rifampin (ra5), neomycin (n30), and novobiocin (nb30). among the six cheeses examined, french comte has the highest percentage of resistant isolates while u.s. maggie’s round and sonnet cheeses have the lowest percentage of resistant isolates (supplementary figure 2). between the two geographical regions, fifty-five percent of the total resistant isolates belong to the european cheeses while bacterial isolates from the u.s. cheeses constitute seventy percent of the total susceptible isolates (figure 5a and 5c). compared to european cheese isolates, american cheese isolates showed larger percentages of intermediate susceptibility when exposed to ampicillin (am10), penicillin (p10), and rifampin (ra5) (figure 5b). isolates from the three european cheeses exhibited higher percentages of resistant isolates, especially those isolated from french comte (supplementary figure 2a). in general, american cheese isolates revealed higher percentages of susceptible isolates, especially maggie’s round and sonnet (supplementary figure 2). however, the small and uneven numbers of cultured isolates and cheese types for each of the regions limited the ability to further analyze the correlation between the cheese origin and susceptibility to antibiotics. supplementary figure 1. principal coordinate analysis of weighted unifrac showing clustering based on other abiotic factors without consideration for rind or curd sampling location. a) milk type b) moisture and c) ph. values range from 5.04 to 8.24. pc1 (76.4%) pc 2 (9 .3% ) a) goat milkcow milk sheep milk pc1 (76.4%) pc 2 (9 .3% ) b) 10-25%5-10% 25-28% na pc1 (76.4%) pc 2 (9 .3% ) c) ph6-7ph 5-6 ph7-8 ph >8 bacterial community structure in cheese • 21 figure 5. antibiotics assay reveals u.s. cheese bacterial isolates constitute a larger percentage of the total susceptible cheese isolates than european cheese bacterial isolates. growth inhibition by six distinct antibiotics was tested among 35 bacterial isolates sampled from 6 cheeses, which represent two geographical regions, europe (blue) and usa (red). a) resistant isolates, b) intermediate susceptible isolates and c) susceptible isolates. diameter measurements of the zone of clearance (in mm) were grouped into the following susceptibility categories: a) resistant (13 mm or less); b) intermediate susceptible (14 – 16 mm); and c) susceptible (17 mm or more). cheeses: [france] comte, [england] stichelton, [switzerland] alpage gruyere, and [usa] sonnet, missouri truckle, and maggie’s round. am10 = ampicillin 10 ug; p10 = penicillin 10 iu/ie/ui; e15 = erythromycin 15 ug; ra5 = rifampin 5 ug; n30 = neomycin 30 ug; nb30 = novobiocin 30 ug. usa europe antibiotics % s us ce pt ib ili ty is ol at es % in te rm ed ia te su sc ep tib ili ty is ol at es % r es ist an t i so la te s am10 am10 p10 p10 e15 e15 ra5 ra5 n30 n30 nb30 nb30 overall overall 100 80 60 40 20 0 100 80 60 40 20 0 am10 p10 e15 ra5 n30 nb30 overall 100 80 60 40 20 0 a) b) c) 22 • fine focus, vol. 3 (1) supplementary figure 2. antibiotic assay shows a higher percentage of european cheese bacterial isolates that are resistant to the antibiotics tested than u.s. cheese bacterial isolates. susceptibility level to six distinct antibiotics was tested among 35 bacterial isolates sampled from 6 cheeses, which represent two geographical regions, europe: france (comte n=3 isolates), england (stichelton n=6 isolates), switzerland (alpage gruyere n=6 isolates), and usa: (sonnet n=6 isolates, missouri truckle n=6 isolates, and maggie’s round n=8 isolates). diameter measurements of the zone of clearance (in mm) were grouped into the following susceptibility categories: a) resistant (13 mm or less); b) intermediate susceptible (14 – 16 mm); and c) susceptible (17 mm or more). percentages of isolates belonging to one of the three susceptibility levels against each of the six antibiotics examined were plotted for all six cheese types. am10 (blue) = ampicillin 10 ug; p10 (red) = penicillin 10 iu/ie/ui; e15 (green) = erythromycin 15 ug; ra5 (purple) = rifampin 5 ug; n30 (cyan) = neomycin 30 ug; nb30 (orange) = novobiocin 30 ug. a) 0 20 40 60 80 100 comte (france) stichelton (england) alpage gruyere (switzerland) maggie’s round (us) missouri truckle (us) sonnet (us) b) 0 20 40 60 80 100 comte (france) stichelton (england) alpage gruyere (switzerland) maggie’s round (us) missouri truckle (us) sonnet (us) c) 0 20 40 60 80 100 comte (france) stichelton (england) alpage gruyere (switzerland) maggie’s round (us) missouri truckle (us) sonnet (us) am 10 p 10 e 15 ra 5 n 30 nb 30 bacterial community structure in cheese • 23 community level physiological profiling (clpp) on both cheese community samples and cultured isolates was performed to examine their metabolic potential and diversity through their utilization of 31 distinct carbon sources (46) (supplementary table 2). we expected that 1) cheeses with greater taxonomical diversity would also have community samples that are more metabolically active, with higher numbers of utilized carbon sources, than their counterparts; and 2) cheese community samples would be able to utilize higher numbers of carbon sources than their respective individual isolates within the same cheese. metabolic diversity (cmd), defined as the number of carbon sources utilized by the sample, increased for all isolates and for whole cheese communities over time (figure 6; supplementary figure 3). alpage gruyere community sample was found to be the most metabolically active, with the highest number of utilized carbon sources (23 carbon sources) (supplementary table 2). consistent with our first hypothesis, alpage gruyere rind was also the most taxonomically diverse out of all the examined rind communities, containing the most observed otus (figure 2b). however, the second and third most metabolically active cheese community samples, missouri truckle (21 utilized carbon sources) and maggie’s round (19 utilized carbon sources), respectively, contained the least numbers of otus in their curd samples (supplementary table 2 and figure 2a). this inconsistency may be attributed to the overall lower taxonomical diversity in the curd communities and that the community samples collected for metabolic analysis were mainly derived from cheese rinds, contributing higher diversity. in addition, with the exception of the stichelton cheese, cheese microbial community samples utilized more carbon sources than their respective individual isolates at the end of the 7-day sampling period, partially confirming our expectation that, in general, community samples are more metabolically active compared to their respective isolates (supplementary figure 3). a closer examination of the top carbon sources utilized by the cheese community samples revealed that there was large diversity of top carbon sources utilized. cyclodextrin, tween-40 and alpha-d-lactose were the top carbon sources most frequently found, though they were the top carbon sources in only two cheeses each. none of the cheeses metabolized the phosphate-activated substrates (glucose-1phosphate and alpha glycerol phosphate). discussion cheese is an excellent model system for studying the mechanisms and patterns of microbial diversity because the microbial communities form under controlled and easily manipulated conditions. while the succession of the microbial community during the early curd and rind formation has been well characterized, the differences between the microbial composition of the mature rind and curd are not well understood, especially when compared between different cheeses (1, 10, 16, 20, 21). we characterized the microbial communities present within the mature rinds and curds of seven different natural rind cheeses using culture-independent, highthroughput illumina sequencing and culture-dependent sanger sequencing of the 16s rrna gene. these seven cheeses vary in their geographic region of production and milk type used in the cheese-making process. additionally, we examined the antibiotic susceptibility and carbon source utilization of the microbial communities in each of the cheeses. 24 • fine focus, vol. 3 (1) we found that sampling site (curd or rind) was a strong predictor of community structure but milk type (cow, goat, or sheep), geographic origin, moisture content, and ph had little influence on the microbial community structure, in contrast to wolfe and colleagues, 2014. it was found that rinds of all seven of the cheeses had greater microbial richness, measured as the number of unique otus, compared to the respective curds of the seven characterized cheeses (figure 4b). the only phylum represented within the curds of all seven cheeses, except for that of sonnet, was firmicutes. streptococcus, lactobacillus and lactococcus, which are genera commonly used as starting cultures during cheese production, were the most abundant genera in the curd and have been previously shown to dominate the curd even in cheese made without starter cultures (1, 6, 16, 17). in contrast, the rinds of these cheeses hosted community members from the firmicutes, actinobacteria, and proteobacteria phyla and, compared to the curd, had a greater relative abundance of the known rind colonizers brevibacterium and actinomyceteacae (49). the differences in community composition and complexity between the rind and the curd is likely due to differential exposure to environmental conditions during ripening. while the curd is an anaerobic environment that is largely protected from environmental exposures, the rind is exposed to ambient air and is in direct contact with the surface on which the cheese is aged, providing opportunities for colonization and succession of the rind microbial community by secondary microorganisms from the environment. the colonizing microbes from the environment can influence characteristics of the rind such as ph that can influence further colonization and succession. for example, de-acidification of the rind by certain species of yeast facilitates the colonization and succession of microbes that prefer more alkaline environments such as corynebacterium, brevibacterium and brachybacterium, in keeping with our observation of an increase in relative abundance of brevibacterium in rind samples (27, 37). antibiotic assays another contributing factor to cheese community structure is the interaction between bacteria and fungi. in many cases, interactions among bacteria and yeast may prevent pathogens (27, 29), and opportunistic pathogens like staphylococcus aureus from dominating the rind community and spoiling food (3). a major means of interaction involves the release of antimicrobial chemical compounds from one microbe to the other (23). from the antibiotic susceptibility assays, we observed that isolates taken from microbial communities in the rind and curd of six of the seven cheeses were resistant to a variety of antibiotics. in order to survive on the rind, bacteria most likely develop resistance mechanisms to these antibiotics and mycotoxins produced by species known to inhabit cheese, such as that of penicillium nalgiovense (2, 8, 13, 18, 19, 24, 38). our limited analysis suggested that american cheeses overall have a higher susceptibility to antibiotics than do european cheeses. we speculate that the difference we observed could be ascribed to the different mechanisms by which european and american cheeses are aged. european cheeses are more commonly aged on older surfaces than american cheeses, such as wooden shelves or caves constructed centuries ago. thus, the microorganisms on such surfaces could have had an opportunity to develop resistance over a longer period of time. the documentation of antibiotic resistance within cheese microbiota is a public health concern due to the possibility of a transfer of resistance to pathogenic bacteria in the human colon upon consumption (42). consequently, an understanding of the stability, diversity, metabolism, and antimicrobial resistance of rind and curd microbiota would advance cheese production and safety. bacterial community structure in cheese • 25 supplementary table 2 m is so u r i t ru ck le br a ch y ba c te r iu m a im en ta r iu m st a ph y lo c o c c u s w a r n er i a lp a g e g ru y er e c o ry n eb a c te r iu m c a se i br ev ib a c te r iu m a u r a n ti a c u m c o m te u n c le a r u n c le a r m a g g ie ’s r o u n d s ba ci ll u s m o ja v en si s u n c le a r st ic h el to n g r ee n c u r d w h it e c u r d u n c le a r u n c le a r u n c le a r pyruvic acid methyl ester 1 1 1 1 1 1 1 0 1 1 1 0 1 1 1 1 1 0 tween-40 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 0 tween-80 1 1 1 1 1 1 1 1 1 1 1 1 0 0 0 1‡ 1‡ 0 cyclodextrin 1 1 1 1 1 1 1 1 1 1 0 1 1* 1 0 0 0 0 glycogen 1 1 1 1 1 1 1 1 0 1 1 0 0 0 0 0 0 0 d-cellobiose 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 0 alpha-d-lactose 1* 0 0 1* 0 0 1 1 0 1* 0 0 0 1 0 1 0 0 beta -methyl-d-dlucoside 1 1 1 0 1 1 0 1‡ 0 0 1 0 0 0 0 0 0 0 d-xylose 1 1 1 1 1 1 1* 0 0 1 1 1 0 0 0 0 0 0 i-erythroitol 1 1 1 1 1 0 1 1 0 1 1 1 1 0 0 1 1 1 d mannitol 1 1 1 1 1 1 1 1 0 1 1 0 1 1 1 1 1 1 n-acetyl-d-glucosamine 1 1 1 1 1 1 1* 0 0 1 1 1 0 0 0 0 0 0 d-glucosaminic acid 1* 0 0 0 0 0 0 1‡ 0 0 0 1‡ 1 1 1 1 1 1 glucose-1-phosphate 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 d,l-alpha-glycerol phosphate 0 0 0 0 0 0 0 1‡ 0 0 0 0 0 0 0 0 0 0 d-galactonic acid, gamma-lactone 0 0 0 0 0 0 0 0 0 0 0 0 1 1 1 1 1 1 d-galacturonic acid 1 1 1 1 1 1 0 0 0 1* 0 0 1 1 1 1 1 1 2-hydroxy benzoic acid 0 0 0 1* 0 0 0 0 0 0 0 0 0 0 0 0 0 0 4-hydroxy benzoic acid 0 0 0 1* 0 0 0 0 0 1* 0 0 1 1 1 1 1 1 hydroxybutyric acid 0 0 1‡ 1* 0 0 0 1‡ 0 0 0 0 0 0 0 0 0 0 itaconic acid 0 0 0 1* 0 0 0 0 0 1* 0 0 1 1 1 1 1 1 alpha-ketobutyric acid 0 0 0 1* 0 0 0 0 0 1* 0 0 0 0 0 1‡ 0 0 d-malic acid 0 0 0 1 0 1 1* 0 0 1* 0 0 1 1 1 1 1 1 l-arginine 1* 0 0 0 0 0 0 0 0 0 0 0 1 1 1 1 0 1 l-asparagine 1 1 1 1 1 1 0 0 0 1 1 0 1 1 1 1 1 1 l-phenylalanine 1* 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 l-serine 1 0 1 1 1 0 0 0 0 0 1 1 0 0 0 1‡ 1‡ 0 l-threonine 1 0 1 1 1 1 0 0 0 0 1‡ 0 0 0 0 1‡ 1‡ 0 glycyl-l-glutamic acid 1* 0 0 1 1 1 1* 0 0 1 0 1 1 0 0 1‡ 1‡ 0 phenylethyl-amine 1* 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1‡ 0 0 putrescine 0 1 1‡ 1 1 1 1* 0 0 1* 0 0 0 0 0 0 0 0 total 21 14 17 23 17 16 14 12 5 19 13 10 15 14 12 20 16 10 * carbon source utlized only in cheese community and/or curd community sample(s) ‡ carbon source utilized only in isolate sample(s) cheese community sample isolate sample curd community sample utilized carbon source 26 • fine focus, vol. 3 (1) supplementary figure 3. carbon source utilization profiling shows that, in general, cheese community utilizes higher number of carbon sources at the end of the seven day sample period than the individual isolates, with the exception of two isolated from stichelton. blue = cheese community sample; red = first isolate; green = second isolate; a) missouri trucke; b) vermont shepherd; c) alpage gruyere; d) comte; e) maggie’s round; f) stichelton: green curd (red), white curd (green), yellow isolate (purple), white isolate (cyan), orange isolate (orange). a) 25 20 15 10 5 0 0 1 2 3 4 5 6 7 8 av er ag e c m d time (days) c) 25 20 15 10 5 0 0 1 2 3 4 5 6 7 8 av er ag e c m d time (days) f) 25 20 15 10 5 0 0 1 2 3 4 5 6 7 8 av er ag e c m d time (days) b) 14 12 10 8 6 4 2 0 0 1 2 3 4 5 6 7 8 av er ag e c m d time (days) d) 14 16 12 10 8 6 4 2 0 0 1 2 3 4 5 6 7 8 av er ag e c m d time (days) e) 14 16 18 20 12 10 8 6 4 2 0 0 1 2 3 4 5 6 7 8 av er ag e c m d time (days) bacterial community structure in cheese • 27 carbon source utilization an alternative to measuring taxonomic diversity in a microbial community is measuring functional diversity, in this case the composite signature of various microbial metabolic pathways (34, 39). communitylevel physiological profiling (clpp) was conducted on microbiota and isolates from the rind and curd over a seven-day period using the biolog ecoplatetm assay (46). sources included detergents, amino acids, and simple and complex sugars, among other compounds. the number of unique carbon sources utilized, here described as metabolic diversity (cmd), increased for all isolates and communities over time, albeit at different rates (figure 6; supplementary figure 3). it appeared that digestion by some enzymes occurred more quickly than others. stichelton had the most unique metabolic signature, and was not able to metabolize any of the commonly used polysaccharides (cyclodextrin, xylose and n-acetyl-d-glucosamine). perhaps this metabolic signature is related to the fact it is a blue cheese and there is contact with the mold inside the cheese. further study is needed to elucidate a relationship between community composition and metabolic functions including mineralization. various rind communities have shown to be largely culturable and reproducible (49) suggesting that such study is possible. figure 6. diversity of carbon metabolism in cheeses over a seven-day period. utilization of a carbon source was determined by measuring the reduction of tetrazolium salts wts-1 and wts-2 to fluorescent purple formazans with the biolog communitylevel physiological profiling (clpp) kit and protocol. for a list of carbon sources see supplementary information. sonnet missoury truckle vermont shepherd alpage gruyere comte maggie’s round stichelton legend day c ar bo n m et ab ol ic d iv er sit y sc or e 0 1 5 10 15 20 25 2 3 4 5 6 7 8 28 • fine focus, vol. 3 (1) cheese type isolate total cmd cmd in isolate top carbon sources top carbon sources per isolate* missouri truckle brachybacterium 21 14 glycogen alpha-d-lactose d-mannitol d-cellobiose glycogen staphylococcus 21 17 glycyl-l glutamic d-cellobiose d-mannitol glycogen alpage gruyère corynebacterium 23 17 alpha-d-lactose tween-80 d-cellobiose d-mannitol n-acetyl glucosamine brevibacterium 23 16 d-cellobiose cyclodextrin tween-80 comté (unclear) #3 14 12 d-cellobiose cyclodextrin tween-40 d, l-alpha-glycerol, phosphate, beta-methyl-dglucoside, cyclodextrin (unclear) #9 14 5 tween-40, cyclodextrin d-cellobiose maggie’s round bacillus 19 13 d-mannitol cyclodextrin tween-80, beta-mthyl-d-glucoside, d-mannitol unclear#10 19 10 tween-40 cyclodextrin, tween-40, n-acetyl-glucosamine stichelton green curd 15 14 d-malic acid l-asparagine d-glucosaminic acid d-malic acid, l-asparagine, d-glucosaminic acid orange isolate unkown 20 d-glucosaminic acid, d-malic acid, l-asparagine white curd 12 d-glucosaminic acid, d-malic acid, l-asparagine white isolate unknown 10 l-asparagine, d-glucosaminic acid, d-malic acid yellow isolate unknown 16 d-glucosaminic acid, d-galactuonic acid, l-asparagine table 2. top carbon sources utilized by the cheese community samples and their respective isolates for five cheeses: missouri truckle, alpage gruyère, comte, maggie’s round, and stichelton. total cmd and top carbon sources refer to that of the whole cheese microbial community samples; cmd in isolates and top carbon sources per isolate refer to the individual isolate samples. bacterial community structure in cheese • 29 acknowledgements we would like to thank bisc 314 spring 2015 students: carley allen, becca berger, synthia v. hernandez, anita z. li, iris w. lin, zoe e. moyer, harini natarajan, and alice sun. special thanks to sherly veeragavan for helping with the course, and bruce paster (forstyth institute) and forsyth institute for sequencing our samples on a miseq illumina sequencer. we thank the two reviewers for their insightful comments. wellesley college’s bisc 314 environmental microbiology course funds were used to fund this project. cheese communities maintained higher cmd than isolates (supplementary figure 3). it is likely that the purified isolates did not represent the majority of the community. secondly, many isolates purified from cheese rinds were exposed to substrates typical of milk curds in the ecoplatetm assay (table 2). rind microbes are not selected for in an environment with a predominance of substrates found in raw milk. strikingly, in the missouri truckle cheese, isolates were able to digest two carbon sources that were inaccessible by the community 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(2014). cheese rind communities provide tractable systems for in situ and in vitro studies of microbial diversity. cell 158(2): 422-433. doi: 10.1016/j.cell.2014.05.041. characterization of antibioticproducing bacterium isolated from anthill sediment with activity against eskape pathogens cody a. postich, kevin b. kiser* university of north carolina wilmington, nc copyright 2018, fine focus all rights reserved manuscript received 07 june 2018; accepted 10 november 2018 189 healthcare institutions have seen an increase in infections caused by antibiotic-resistant eskape pathogens. current antibiotics have become less potent against pathogenic bacteria due to their overuse and misuse. in recent years, scientists have revisited local environments in search of novel antibioticproducing microbes to address the increasing threat of resistance. one species of bacteria was isolated from anthill sediment in coastal north carolina. this environment was selected for its abiotic properties, including organic substrates, moisture saturation and aeration. anthill isolate a2, inhibited various grampositive and negative eskape pathogens or their surrogates, including staphylococcus aureus, klebsiella pneumoniae, and acinetobacter calcoaceticus, in crossstreak tests. 16s rrna sequencing identified isolate a2 as pseudomonas koreensis. mass spectrometry and small-molecule analysis performed on ethyl-acetate extracts of culture supernatant were used to evaluate bioactivity and identify the probable structure of one potential antimicrobial compound, monolauryl maleate. discovery of novel antimicrobial compounds to replace overused antibiotics may help reduce the impact of antibiotic-resistant pathogens. abstract corresponding kevin b. kiser kiserk@uncw.edu keywords • antibiotic • eskape • monolauryl maleate • mass spectrometry • pseudomonas antibiotics are becoming increasingly ineffective in treating bacterial infections because of misuse and overuse, particularly in healthcare institutions. antibiotic-resistant bacteria, known as eskape pathogens, are a rising concern in healthcare settings (2). the eskape pathogens – enterococcus faecium, staphylococcus aureus, klebsiella pneumoniae, acinetobacter baumannii, pseudomonas aeruginosa, enterobacter sp. – have been designated as global threats because they have developed resistance mechanisms and “escape” death by antibiotics. discovering and developing novel antibiotics are one solution to combat antibioticresistant infections. unfortunately, there were introduction 190 • fine focus, vol. 4 (2) antimicrobial susceptibility of eskape pathogens • 191 only 16 broad-spectrum antimicrobial agents approved and utilized in the u.s. between 1983-1987, and only 5 novel antimicrobial compounds were approved for use from 2003-2007 (2). soil microbes are a potential source of new antibiotics. when microbes are in close quarters in terrestrial and aquatic environments, they compete for resources and utilize “chemical weapons” to inhibit growth of other microbes. the renewed search for antibiotic-producing microbes has focused on recruiting students at all levels to explore unique environments across the globe (4). furthermore, new approaches to identifying previously uncultured microbes have shown promise, as demonstrated by the discovery of teixobactin, an antibiotic with a novel mechanism of action against grampositive bacteria, with use of the ichip (11). in this study, bacteria were cultured from anthill soil in southeastern north carolina and evaluated for their potential to produce antibiotics. anthills were chosen for investigation because ant activity was shown to alter soil properties where they build their nests, and these soils were shown to contain an abundance of organic matter and cellulosic polysaccharides that could foster novel microbial communities (7). in fact, some ants that “farm” fungi for food have been shown to have mutualistic relationships with actinomycetes that produce antifungal agents to protect their “crops” against fungal parasites (1). however, little is known about antibacterial activities that may be associated with anthills. antibiotic activities of anthill isolates were characterized with use of inhibition assays against a variety of gram-positive and negative bacteria, including eskape pathogens. a cell-free extract from one anthill isolate was analyzed to identify the active compound and to determine whether this finding was novel. one previously unidentified compound, monolauryl maleate, appears to possess activity against the gram-positive pathogen, s. aureus. materials and methods isolation of antibioticproducing bacteria soil was collected from the surface of an anthill (red imported fire ant, solenopsis invicta) located in front of a residential area in shallotte, north carolina (33’58’36’n 78’31’30’w). 1:100 dilutions of soil in sterile water were plated on actinomycete isolation agar (aia; himedia laboratories, west chester, pa) and incubated at 35°c for 3 days. 100 µl of an overnight tryptic soy broth (tsb; hardy diagnostics, santa maria ca) culture of s. epidermidis was transferred to 8 ml of molten tsb soft agar (tsb with 0.7% agar), vortexed, and poured over the aia culture. after solidification, the plate was incubated at 35°c for 24 h, and then observed for zones of inhibition (zoi) in the overlay. isolates showing zoi were picked with a sterile loop and streaked for isolation on aia. s. epidermidis was used in initial screens because it has little natural resistance and should display zoi against a variety of antimicrobials. additionally, as a bsl-1 microbe, it is safer to work with as students are training in the lab and learning these techniques. cross-streak test each isolate was streaked down the center of mueller-hinton agar (mha; hardy diagnostics) and incubated at 35°c for 3 d. bacteria used in this study are listed in table 1. some were acquired from carolina 192 • fine focus, vol. 4 (2) table 1. test bacteria used in this study. gram-negative acinetobacter calcoaceticus** aeromonas hydrophila escherichia coli** klebsiella pneumoniae atcc 13883* proteus vulgaris pseudomonas aeruginosa atcc 27853* serratia marcescens * eskape pathogen ** eskape surrogate gram-positive bacillus cereus enterococcus faecalis atcc 29212** kocuria rhizophila staphylococcus aureus atcc 25923* staphylococcus capitis atcc 35661** staphylococcus epidermidis atcc 12228** biological supply company (burlington, nc) and strain identity was unknown. strains with known identity (indicated with atcc number) were purchased from fisher scientific (hampton, nh). six eskape pathogens or surrogates (table 1 and fig. 2) were streaked perpendicular to the isolate, and the plate was incubated at 35°c for an additional 24 h. isolate a2 was later retested by cross streak against a panel of 10 grampositive and negative bacteria, including some eskape pathogens and surrogates (table 1 and fig. 3). the inhibition distance was measured in mm. identification of anthill isolates each isolate was gram-stained and observed at 1000x magnification with a lecia dm750 compound light microscope. genomic dna was extracted from each isolate with the use of the ultra clean microbial dna isolation kit (mo bio laboratories, carlsbad, ca), according to manufacturer’s specifications. the cell disruption step was performed in a bead bug homogenizer (benchmark scientific, edison, nj) for 3 x 60 seconds at 320 rpm with cooling on ice between cycles. the 16s rrna sequence was amplified with use of primers eco8f (5’-agagtttgatcatggctcag-3’) and 1509r (5’-ggttaccttgttacgactt-3’) (5). each 50-µl pcr reaction (genemate reagents, bioexpress, kaysville, ut) contained: 1x pcr buffer, 0.025 u/µl taq dna polymerase, 1.5 mm mgcl2, 0.2 mm dntps, 0.5 µm of each primer, and 5.0 µl of genomic dna. pcr was carried out in an eppendorf (hauppauge, ny) master cycler under the following conditions: 95°c for 5 min; 30 cycles of 95°c for 1 min, 50°c for 30 s, 72°c for 1.5 min; and then 72°c for 5 min. the 1.5-kb pcr product was confirmed by gel electrophoresis, and then purified with use of the e.z.n.a cycle pure kit (omega bio-tek, norcross, ga), according to manufacturer’s specifications, and sequenced by eurofins genomics (louisville, ky). raw sequences were edited with finch tv software, v1.4 (geospiza). a contiguous sequence was created from the forward and reverse sequences from the same isolate with serial cloner software, v 2.6 (serial basics). sequences were analyzed with blast (national center for biotechnology information) with use of the 16s ribosomal rna sequences (bacteria and archaea) database to identify species with similar sequences antimicrobial susceptibility of eskape pathogens • 193 preparation of cell-free ethyl-acetate extract isolate a2 was cultured in tsb at 25°c for 7 d, and cells were pelleted by centrifugation for 10 min at 2000 rpm in an eppendorf 5804r centrifuge. the supernatant was passed through a 0.2 µm filter (corning, oneonta, ny). the filtrate was transferred to a separatory funnel and combined with 2 parts ethyl acetate (alfa aesar, haverhill, ma). the aqueous layer was removed, extracted again with 2 parts ethyl acetate, and the two organic extracts were combined. the ethyl acetate was evaporated in a rotary evaporator and the dried cell-free extract was dissolved in 100% methanol (alfa aesar). column chromatography a strata c18-e (55µm, 70 a) giga tube column (phenomenex, torrance, ca) was conditioned with three washes of 100% hplc-grade methanol to rinse the column, and then was equilibrated with three washes 20% methanol. 23.0 mg of isolate a2 extract was suspended in 5.0 ml of 20% methanol, then was pipetted onto the column. the crude extract was washed with increasing hplcgrade methanol gradient with pico-pure filtered h2o at 20%, 40%, 60%, 80%, and 100% meoh. a final rinse of 100% acetone removed everything else from the column. the column was placed in a supelco visiprep vacuum apparatus for eluting these compounds with vacuum pressure at 8 hg. the fractionated compounds were dried on a speed vacuum, then were resuspended in 1.0 ml of 100% methanol and tested for antibacterial activity by disk diffusion. disk diffusion assay s. aureus and e. coli were freshly cultured on tryptic soy agar (tsa; hardy diagnostics) at 35°c for 24 h. test bacteria were suspended in saline solution (0.9% nacl) to match a 0.5 mcfarland turbidity standard. a sterile swab was used to transfer test bacteria to mha. dried extracts of isolate a2 were resuspended in 1.0 ml of 100% meoh, then 30-40 µl of the mixture was pipetted onto sterile filter disks (whatman #3, ge healthcare life sciences, marlborough, ma) in 10 µl increments (10). dried disks were transferred to the mha plate, which were then incubated at 35°c for 24 h. diameters of zoi were measured in mm. small-molecule analysis small-molecule analysis of culture supernatants from isolate a2 involved preparation of cell-free ethyl-acetate extracts, as described above. ultrahighperformance liquid chromatography, low-resolution mass spectrometry, and small-molecule analysis yielded an s-plot, chromatogram of mass over ionization ratios, and uv absorption of a specific secondary metabolite. these results were analyzed with progenesis qi software (nonlinear dynamics, durham, nc) to compare known compounds to the secondary metabolites with potential antimicrobial capabilities (3). ultrahighperformance liquid chromatographylow resolution mass spectrometry (uplclrms) c-18 column chromatography elutions 3, 4, 5, and 6 were analyzed by mass spectrometry to search for similar patterns within the fractions that showed inhibition against s. aureus. the 60-100% methanol and 100% acetone washes were dried down on a speed vacuum, and then were suspended in 1 ml of 1:1 acetonitrile: pico-pure water. 30 µl of elutions 3, 4, 5, and 6 were run through 194 • fine focus, vol. 4 (2) results figure 1. soft agar overlay of s. epidermidis on aia culture of anthill soil. the reddashed circle shows the location of the a2 colony showing zoi. figure 2. cross-streak testing with cultured isolate a2 on mha plates against eskape pathogens and surrogates. isolate a2 was streaked vertically, and the test bacteria were streaked horizontally. a lack of culture growth from the horizontally streaked test bacteria revealed inhibition (represented by areas boxed by red-dashed lines). e1, e. faecalis; s, s. aureus; k, k. pneumoniae; a, a. calcoaceticus; p, p. aeruginosa; e2, e. coli. isolation of antibioticproducing bacteria from anthill sediment because anthills have unique characteristics compared to the surrounding soil and ants have been shown to have symbiotic relationships with antibiotic-producing microbes, bacteria were cultured from an anthill in southeastern north carolina colonized by red imported fire ants to search for antibiotic producers. the culture of anthill sediment on aia was overlayed with s. epidermidis and yielded a few small zoi (fig. 1). potential antibiotic-producers were picked and streaked on aia. in initial cross-streak tests against eskape species or surrogates, one of the anthill isolates, a2, demonstrated inhibition of five of six test bacteria, but not gram-positive e. faecalis, an eskape surrogate for e. faecium (fig. 2). overall, isolate a2 yielded the strongest inhibitory characteristics against three gram-negative eskape bacteria, particularly e. coli (surrogate for enterobacter sp.), k. pneumoniae and a. calcoaceticus (surrogate for a. baumannii). moderate inhibition was shown against gram-negative p. aeruginosa and gram-positive s. aureus. additional cross-streak tests with a2 showed inhibition of variety of gram-negative and gram-positive bacteria, including another species of staphylococcus (fig. 3). two gram-negatives, s. marcescens e1 s k a p e2 antimicrobial susceptibility of eskape pathogens • 195 and p. vulgaris, were not inhibited. identification of isolate a2 blast analysis of the 16s rrna sequence from the a2 isolate retrieved a sequence with only one mismatch in the 973-base alignment. this species, p. koreensis, was previously identified in soils from a korean farm (8) and a rice paddy (9). gram staining of a2 revealed a gram-negative bacillus, as expected for a pseudomonas. limited knowledge regarding antibacterial activity has been reported for this species, so it is likely the broad-spectrum inhibition seen in cross-streak tests represents a novel finding. to further characterize this activity, an attempt to isolate and identify the antibacterial compound(s) secreted by the a2 isolate was pursued. antibiotic activity of fractionated cell-free a2 extracts culture supernatants of isolate a2 (putative p. koreensis) were extracted with ethyl acetate. these cell-free extracts were tested for antibacterial activity in disk diffusion assays against two of the bacteria that were inhibited in cross-streak tests – gram-positive s. aureus and gram-negative e. coli. the a2 extract produced a 10-mm zone of inhibition against s. aureus (fig. 4, disk a); however, no inhibition was produced against e. coli (data not shown). the ability to detect inhibitory activity in cell-free extracts was useful for the preliminary isolation of antibacterial compound(s). c-18 column chromatography was used to fractionate compounds from 23.0 mg of a2 ethyl-acetate extract suspended in 20% methanol. the largest portion of the extract (9.1 mg) was eluted in the second fraction at 40% methanol, while the smallest portion (0.2 mg) was eluted at 80% methanol (table 2). elutions 2-6, were tested by disk diffusion against s. aureus to determine which fraction(s) contained the active compound(s). only 10% of each fractionated sample was loaded onto the sterilized disks (table 2). elution 2 produced a small and poorly defined zoi that could not be measured, while elution 3 and 6 yielded no zoi (fig. 4, disks 2, 3 and 6, respectively). elutions 4 and 5 both produced 10-mm zoi, similar to that displayed by the original cell-free extract figure 3. the inhibition distance (mm) displayed by isolate a2 in a cross-streak test against various gram-positive and negative test bacteria. bars represent data from a single cross-streak assay. there were no zoi detected when tested against s. marcescens and p. vulgaris. in hi bi tio n d ist an ce ( m m ) aero monas hydrophilia bacill us ce rus serr atia marce nses kleb siel la pneumoniae staphylococcu s aureu s esch eric ha coli proteu s vulga ris staphylococcu s ca pitis kocuria rhizo phila acin eto bacte r ca lco aceti cus 35 30 25 20 15 10 5 0 196 • fine focus, vol. 4 (2) figure 4. disk diffusion assay performed with cell-free ethyl-acetate extract of a2 (a) and its c-18 column fractions (2-6) against s. aureus. streptomycin was the positive control (+). methanol was the negative control (-). elutions [solvent] solvent dried sample weight (mg) amount loaded onto disk (mg) 1 20% meoh/h2o 2.0 0.02 2 40% meoh/h2o 9.1 0.91 3 60% meoh/h2o 2.1 0.21 4 80% meoh/h2o 0.2 0.02 5 100% meoh/h2o 0.9 0.09 6 100% acetone 0.4 0.09 table 2. c-18 column chromatography distribution of compounds from the cell-free, ethyl-acetate extract based on polarity with increasing methanol (meoh) concentrations. antimicrobial susceptibility of eskape pathogens • 197 (fig. 4, disks 4 and 5, respectively). mass spectrometry analysis of a2 column fractions c-18 column elutions 3-6 were selected for analysis by mass spectrometry. there were clear differences in number and location of peaks between the four fractions observed on the chromatogram, whereas elutions 4 and 5, which showed antibacterial activity by disk diffusion, had 13 peaks in common (fig. 5). there was an abundance of one compound at a 276.6 m/z ratio in both active fractions that was absent from the other two non-active fractions (fig. 5). there was another peak of interest at 294.5 m/z only present in elutions 4 and 5. although, this may represent an additional active compound, focus was directed at peak 276.6 m/z because of its greater abundance in the extract. future studies will isolate and assess the antibiotic potential of other peaks. small-molecule analysis of cell-free a2 extract small-molecule analysis was performed on cell-free, ethyl acetate extracts of a2 culture supernatant, which yielded over 1,000 different compounds, as displayed on the s-plot (fig. 6). the s-plot, where each dot represents a different compound, compared compounds from the a2 cell-free, ethyl-acetate extract and a matched tsb media control. focusing on the 13 outlier dots in the bottom left quadrant, each represented a compound unique to the a2 culture supernatant. one, if not more, of these 13 compounds may represent the antimicrobial agent produced by isolate a2. the potential active compound identified by mass spectrometry from elutions 4 and 5 was similar to one of the outliers (265.23 m/z) identifigure 5. uplc-lrms chromatogram results from 60%, 80%, 100% meoh fractions, and the 100% acetone extraction (elutions 3, 4, 5, and 6, from top to bottom). the boxed area is a selected peak of interest with a mass of 267.6 m/z in the 80% and 100% meoh fractions. elution 6 elution 5 active elution 4 active elution 3 198 • fine focus, vol. 4 (2) fied by small-molecule analysis. a cleavage product of this compound was also revealed with a 44 m/z ratio difference, which likely represents a potential carboxylic acid group in the complete compound (data not shown). this finding was consistent with qi progenfigure 6. s-plot from small-molecule analysis of triplicate cell-free extracts of a2 culture supernatants (bottom left quadrant) and the tsb media control for comparison (top right quadrant). arrow indicates compound at 265.23 m/z, which was identified as monolauryl maleate. esis prediction (97% probability) of the identity of the unknown 266 m/z compound as monolauryl maleate, which has a molecular formula c16h28o4 with a probable carboxylic acid group attached to a 12-carbon chain (fig. 7). figure 7. chemical structure of monolauryl maleate from progenesis qi software, a compound identified in bioactive c-18 column fractions from a2 cell-free, ethyl acetate extracts. tsb media a2 crude antimicrobial susceptibility of eskape pathogens • 199 discussion in response to the growing global problem of antibiotic-resistant eskape pathogens in healthcare institutions, soil samples were collected in coastal north carolina and investigated for the presence of novel antibiotic-producing bacteria. one promising isolate (a2) found in anthill soil inhibited a variety of gram-positive and gram-negative bacteria in cross-streak tests (fig. 3). broad-spectrum antimicrobial activity is likely beneficial to ants that become colonized by this microbe from sediment manipulation for protection against various pathogenic bacteria, as suggested previously (1). detection of this antibiotic-producing species on the heads or bodies of imported fire ants could reveal a new mutualistic relationship, similar to ones observed in other ant species. the fact that a2 also inhibited five of six eskape pathogens or their surrogates suggests that anthills may warrant further investigation for bioactive natural products. blast analysis of 16s rrna gene sequence of a2 showed a match with p. koreensis, a newly defined species first discovered in korean farming soil (8). genome sequence analysis of another p. koreensis isolate from a korean rice paddy revealed a putative gene cluster for the synthesis of cyclic lipopeptide (clp), which is a biosurfactant shown to have antagonistic properties (9). pseudomonas species are known to produce a variety of clps with antimicrobial activity that likely inhibit colonization of ants by pathogens in the environment (12). one of three p. koreensis isolates from a water basin in mexico demonstrated biosurfactant activity, but inhibition of bacterial growth was limited to two gram-positive species, bacillus subtilis and exiguobacterium aurantiacum (13). in fact, no broad-spectrum antibacterial activity has been described previously for this species. in this study, not only did the a2 isolate inhibit several different bacterial species, including eskape pathogens, crude and fractionated cell-free extracts from a2 inhibited s. aureus (fig. 3 & 4). genomic comparison of the two korean p. koreensis strains demonstrated genetic diversity among pseudomonas strains, with 631 putative genes unique to one isolate (9), so it is expected that the north carolina anthill isolate possesses genetic elements and metabolic capabilities not present in the korean or mexican isolates. one of the potentially active compounds, monolauryl maleate, produced by isolate a2 is structurally distinct from clps and represents a novel finding in p. koreensis. whether this new compound has similar biosurfactant activity to clps might be determined in the future with an in vitro biofilm inhibition assay, or potentially through the development of an ant colonization model. genomic sequencing of a2 would also reveal differences and help identify a gene cluster involved in antibiotic synthesis. it was curious that the a2 cell-free extract did not inhibit e. coli in a disk diffusion assay, since the a2 isolate did inhibit e. coli in a cell-based, cross-streak assay rather well. this finding indicates that the broad-spectrum activity demonstrated in cross-streaks may represent two or more distinct antibacterial compounds produced by a2 – one absent from cell-free extract that is active against e. coli and other gram-negatives, and another present in cell-free extract that is active against s. aureus and other gram-positives. the compound(s) that inhibit gram-negative bacteria may not have be soluble in ethyl acetate or may have remained with the cell pellet during collection of the culture supernatant. in hindsight, k. pneumoniae, which displayed the greatest inhibition in cross streaks, should have been first gram-negative tested. unfortunately, the extraction yielded only enough material to complete a preliminary round of disc-diffusion assays against one gram-positive and one gram-negative species. it will be important to revisit the extraction technique to 200 • fine focus, vol. 4 (2) acknowledgments this study was supported by funds from uncw center for the support of undergraduate research and fellowship (csurf), experiencing transformative education through applied learning (eteal), and department of biology and marine biology. c. postich was also supported, in part, by the paul e. hosier undergraduate fellowship. thomas coombs, jeffery wright, and jeremy morgan of uncw department of chemistry and biochemistry provided additional lab resources and encouragement. allison stewart and wendy strangman of marbionc assisted with small-molecule analysis and mass spectrometry analyses. this research was performed in compliance with federal regulations and institutional policies relating to the handling of infectious agents. explore other options for isolating bioactive metabolites and increasing yield, including modifying culture conditions and performing extractions on lysed cells in the pellet. furthermore, these extracts should also be tested for antibacterial activity against other gram-negative eskape pathogens, k. pneumoniae and a. calcoaceticus, which were also inhibited in the cross-streak assay. a potential novel antimicrobial compound, monolauryl maleate, was identified by small-molecule analysis and low-resolution mass spectrometry of a cell-free extract of isolate a2. mass spectrometry, uv light absorbance, and small-molecule profiling of isolate a2 provided evidence of a potential antimicrobial compound in the s-plot with a 266 m/z ratio that was characterized through qi progenesis software. there was a 97% match with the compound, monolauryl maleate (fig 7). this compound consists of a carbon chain connected to a carboxylic acid group, as confirmed by a 44 m/z fractionation with low-resolution mass spectrometry. no findings had been previously reported regarding the properties of monolauryl maleate as an antibiotic agent. monolauryl maleate is not available commercially for testing purposes at this time. synthesis of monolauryl maleate may be possible and could be valuable for future comparative studies. this is one of thirteen potential antimicrobial compounds residing within the active elutions 4 and 5 (fig. 5). hplc purification and nmr analysis (3) on the compounds of interest in these two fractions, including peak 294.5 m/z, will be required to ultimately confirm the identity of the active compound(s) and to characterize antimicrobial activity against the eskape pathogens that were inhibited in the cellbased assays. once identified, the potential of any active a2 compound as a therapeutic agent can be explored. in 2005, there were 8,987 cases of methicillin-resistant s. aureus, or mrsa, infections in hospital and communal locations across the united states, and there were a total of 988 deaths from complications of these infections (6). the constant battle against antibiotic-resistant infections in the healthcare community must be addressed, in part, through the discovery of novel antibiotics. one method includes studying environmental bacteria and their natural products from unique habitats around the world (4, 11). in this study, antimicrobial activity against s. aureus, and other eskape pathogens, was discovered in a pseudomonas species isolated from an anthill. the preliminary identification of monolauryl maleate may represent the initial step in the discovery of the next novel antimicrobial agent to address the increasing prevalence of antibiotic-resistant infections. antimicrobial susceptibility of eskape pathogens • 201 references 1. barke j, seipke rf, grüschow s, heavens d, drou n, bibb mj, goss rjm, yu dw, hutchings 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(2014) development of the eucast disk diffusion antimicrobial susceptibility testing method and its implementation in routine microbiology laboratories. european society of clinical microbiology and infectious disease. 20:255-266. 11. piddock lj. (2015) teixobactin, the first of a new class of antibiotics discovered by ichip technology? j antimicrob chemother. 70(10):2679-80. doi: 10.1093/jac/ dkv175. 12. raaijmakers jm, de bruijn i, de kock mjd. (2006) cyclic lipopeptide production by plant-associated pseudomonas spp.: diversity, activity, biosynthesis, and regulation. mol. plant. microbe interact. 19:699–710. doi: 10.1094/mpmi-19-0699 13. toribio j, escalante ae, caballero-mellado j, gonzález-gonzález a, zavala s, souza v, soberóncháveza g. (2011) characterization of a novel biosurfactant producing pseudomonas koreensis lineage that is endemic to cuatro ciénegas basin. systematic and applied microbiology 34:531– 535. doi:10.1016/j. syapm.2011.01.007 • fine focus, vol. 3 (2) isolation of halophilic bacteria from inland petroleum producing wells maedgen q. lindsey & jennifer r. huddleston* biology department, abilene christian university, abilene, tx 79699 copyright 2017, fine focus. all rights reserved. halophilic bacteria • 101 manuscript received 28 march 2017; accepted 20 may 2017 100 • fine focus, vol. 3 (2) the goals of this study were to isolate microorganisms from oil well-produced water, identify the microorganisms, and test the microorganisms’ salt tolerance. saltwater collected from two well locations producing from different zones in jones county, texas, was spread onto mannitol salt agar (msa). isolates showed a 16s rdna gene sequence identity of 99% with idiomarina baltica and marinobacter persicus. salt tolerance assays indicated an optimal growth concentration of 10-12.5% nacl for the idiomarina isolate and a decrease in growth beyond 5% nacl for the marinobacter isolate. in conclusion, organisms that are phylogenetically similar to marine microorganisms are present in oil well environments, and have variable salt tolerances, which may prove useful in microbialmediated hydrocarbon bioremediation of high salinity environments. abstract corresponding author jennifer r. huddleston biology department abilene christian university acu box 27868 abilene, tx 79699 jennifer.huddleston@acu.edu keywords • idiomarina • marinobacter • halophile • saltwater • petroleum as of 2015, there were an estimated 1.7 million oil and gas wells currently active in the united states (18). saltwater is produced as a byproduct of drilling as it is released from the geological formation along with oil and gas. the saltwater was originally sequestered as the respective formations were being laid down at the bottom of shallow oceans in past geological eras (11). in this study, saltwater samples were obtained from the flippen limestone and the king sand formations. the flippen limestone is located at a depth of 1850 feet at the drilling location, and is localized to fisher and jones county, texas. the limestone deposition process started in the late pennsylvanian and continued through the permian geological time periods (16), approximately 300 million years ago. sequestered saltwater was released from the limestone upon hydraulic fracturing, a process in which highly pressurized water is used to open fissures within the formation (12). the king sand was the second formation sampled in this study, and is introduction halophilic bacteria • 103 a pennsylvanian era channel-sand deposition located in central northern texas (5), and thus slightly older and deeper than the flippen limestone, measuring at a depth of 2000 feet at the oil well location. saltwater produced at these sites is pumped to the surface with oil and gas, where the components are separated and eventually utilized or discarded. a consequence of the prevalence of oil drilling is hydrocarbon contamination of soils and groundwater. microbial-mediated bioremediation of hydrocarbon contaminated environments is an effective treatment, however the efficacy is limited by the conditions necessary for the microorganisms to optimally biodegrade hydrocarbons (9). salinity of the environment, either indigenous or elevated by the contamination event, is an inhibitory condition for microorganisms responsible for the bioremediation (25). studies of optimum salinities for halophilic, hydrocarbon degrading microorganisms could lead to targeted bioremediation efforts. halophilic microorganisms are a type of extremophile which require a high salt content in order to survive. the extent of halotolerance is categorized by the percent salt concentration within the environment required for optimum growth. slight halophiles exhibit optimum growth at 1-5% nacl, moderate halophiles at 5-20% nacl, and extreme halophiles from 20-30% nacl (10). halotolerant organisms are resistant to the deleterious effects a high salt content environment poses, but do not require a high salt content for survival. an environment high in salt is inhibitory for most microorganisms, as water from inside a cell will diffuse through the semipermeable cell membrane into the environment via osmosis and ultimately result in plasmolysis (3). halotolerant microorganisms utilize haloadaptation to overcome this challenge, excluding salt from the cytoplasm where possible and biosynthesizing or accumulating organic osmotic solutes to remain isotonic relative to the environment (24). due to the range of salinities, temperatures, and possible carbon sources, oil fields and their related infrastructure pose a potential treasure trove of microbial diversity. previously identified microorganisms isolated from oil well associated environments include marinobacter aquaeolei, which was described after being isolated from the head of an offshore oil rig off the coast of vung tau, vietnam, but which also occurs in the water column of the same area (17). from the same oil field, desulfovibri vietnamensis was isolated from oil storage tanks, as well as oil well produced saltwater (8). members of the genus desulfovibri have been implicated in the corrosion of oil infrastructure via reduction of iron (13). the genus petrotoga, named for its outer toga-like sheath, is associated exclusively with oil production, having been found in offshore wells and inland oil reservoirs (23). the diversity of microorganisms in these salt rich, petroleum associated environments led us to look for variable halophilicity in microbe populations by sampling saltwater produced from different geological formations. in this work, we isolated two microorganisms from saltwater produced as a byproduct of oil drilling, belonging to the genera idiomarina and marinobacter, and characterized their respective salt tolerances. 102 • fine focus, vol. 3 (2) identification to identify the microorganisms, the 16s rdna genes were amplified and sequenced. dna was extracted using a zymo dna extraction kit (zymo research corporation, irvine, ca) according to the manufacturer’s instructions. recombinant vent dna polymerase (new england biolabs inc., ipswich, ma) was used in pcr to amplify a portion of the 16s rdna gene. pcr was executed utilizing the primers 5′-agagtttgatcctggctcag-3′ (f’-27m) and 5′-taccttgttacgactt-3′ (r’-1492) (positions 11–27 and 1489–1506, respectively, according to the escherichia coli 16s rrna numbering system of the international union of biochemistry) (14). primers were designed to our specifications and synthesized by invitrogen. the thermocycling conditions for amplification were as follows: initial denaturation: 95˚c for 5 minutes, 30 cycles of 95˚c for 30 seconds, 55˚c for 30 seconds, 72˚c for 1 minute 30 seconds followed by a final extension at 72˚c for 10 minutes. the products were then confirmed using gel electrophoresis of a 0.8% agarose (invitrogen; carlsbad, ca) gel containing ethidium bromide under a 70 to 90 voltage for 30 to 45 minutes. the resulting amplicons of 1.5 kb were further purified using dna clean & concentrator (zymo research corporation, irvine, ca) and 16s rdna sequencing was outsourced to dna analysis facility on science hill at yale university (new haven, ct). the identification of phylogenetic neighbors and the calculations of pairwise 16s rdna gene sequence similarities were achieved using ncbi blastn analysis (1). sequence alignment and creation of phylogeny were performed using molecular evolution genetics analysis 7. assay salt tolerance assays (figure 2.) were carried out in tryptic soy broth (tsb) (becton, dickinson and company, sparks, md); the idiomarina isolate was tested in 0.5-25% nacl while the marinobacter isolate was tested in 0.5-12.5% nacl. culture tubes containing 2 ml of tsb with concentrations of nacl from 0.5-25.0% were inoculated with 30 μl of an overnight culture and incubated at 37°c for 48 hours with aeration by a shaker at 200 rpm. the optical density at 600 nm (od600) of four replicates at each nacl concentration were measured using a hewlett packard 8453 uv-visible spectrophotometer. cultures were diluted 2-fold with sterile water to obtain accurate od600 readings as necessary. statistical analysis the error bars in figure 2 indicate standard deviation of measured od600 between four replicates. statistical analysis of the od600 for both data sets was calculated using microsoft excel’s data analysis add-in. an alpha of 0.05 was used as the cutoff for statistical significance. the idiomarina isolate data set has a p-value of 0.43238 and is therefore beyond the cutoff for statistical significance, while the marinobacter isolate data set has a p-value of 0.00017 and is therefore statistically significant. results two gram-negative, mesophilic, rodshaped isolates were obtained from saltwater samples originating in different geological formations utilized in oil and gas drilling. the isolate from the flippen limestone showed sequence similarity to the genus idiomarina, while the isolate from the king sand showed sequence similarity to the genus marinobacter. figures 3 and 4 illustrate phylogenetic relationships between the isolates and closely related species, chosen based on similar figures in the novel species reports of the isolates’ closest 16s rdna identities, idiomarina baltica and marinobacter persicus, respectively. the idiomarina isolate showed a 99.7% 16s rdna sequence identity with idiomarina baltica, originally isolated from the central methods sampling permission to sample, access to well sites, and instructions on equipment use were provided by the lease holder, jql energy, llc. saltwater, produced as a byproduct of oil drilling, was collected aseptically from two oil well locations in jones county, texas. locations, shown in figure 1 were chosen due to contrasting geological characteristics, as the first oil well pumps from a solid limestone formation, while the second pumps from a course-grain sand formation. the flippen limestone sample was obtained from a fiberglass saltwater storage tank, while the king sand sample was obtained from a water knockout, a horizontal tank which separates oil from saltwater based on varying density. four samples, two from each well site, were collected using 118 ml sterile glass jars and stored overnight at 4°c. the king sand samples were collected via release valve located on the bottom of the water knockout tank. the flippen limstone samples were collected via an access port on top of the saltwater tank, directly from the separated saltwater stored within. initial sampling took place in november, 2015. the idiomarina isolate has since been re-isolated, indicating that our method can be replicated. growth conditions samples of 1.0 ml were plated directly onto mannitol salt agar (msa) (hardy diagnostics, santa maria, ca), selected for its 7.5% salt content, and incubated overnight at 37°c. msa is typically utilized as a selective and differential medium for detection of pathogenic staphylococci, however it is also suitable for halophilic marine organisms (20). an incubation temperature of 37°c was selected by applying a formula designed to calculate the temperature of a geological formation: formation temperature = surface temperature + (temperature gradient * vertical well depth) (19). the average regional high temperature of jones county, texas in november, 2015 was 66 °f, roughly 19°c (6), which when plugged into the formula with the depth of the king sand well (2000 feet), and a temperature gradient of 0.015 °f/ft., yields a formation temperature of ~36°c. individual colonies were selected haphazardly and subsequently sub-cultured on msa. figure 1. map of sampled locations in jones county, texas. the muehlstein lease, located at 32°50’06.29”n, 99°43’51.26 w, elevation 1550 feet, is the location of the sampled saltwater storage tank from which the idiomarina isolate was isolated, and is used to store saltwater pumped from a flippen limestone oil field. the swenson lease, located at 32°50’48.75” w, elevation 1556 feet, is the location of a sampled water knockout tank from which the marinobacter isolate, and which separates saltwater from oil pumped from a nearby king sand well. halophilic bacteria • 105104 • fine focus, vol. 3 (2) baltic sea. morphologically, the isolate produced raised, circular, opaque, mucoid colonies, with white pigmentation that takes on a purple tint as it continues to grow on the msa. the rod shaped, gram negative cells were motile by a single polar flagellum. results from the salt tolerance assay indicate an optimum nacl concentration between 1012.5% (figure 2.), making the isolate a moderate halophile. the marinobacter isolate showed a 99.1% identity with marinobacter persicus, originally isolated from lake aran-bigdol, a hypersaline lake located in iran. the isolate grew on msa overnight incubated at 37°c and produced colonies morphologically characterized as small, convex, circular, mucoid, with a translucent leading edge, and white pigmentation that takes on a purple tint as it continues to grow on msa. the rod shaped, gram negative cells were motile by a single polar flagellum. results from the salt tolerance assay indicated a sharp decrease in growth beyond 5% nacl (figure 2.), and the ability to grow in tsb without supplemental salt, making the isolate moderately halotolerant. discussion the identification of the isolates as being moderately halophilic and moderately halotolerant was expected, given the salt-rich environment. however, the close phytological association of the isolates with classically marine bacteria genera was unexpected, as the figure 2. plot of idiomarina and marinobacter isolate salt tolerance. tubes containing 2ml tsb with varying concentrations of nacl were aseptically inoculated and incubated 48 hours at 37 °c with aeration. optical density at 600 nm was measured using a hewlett packard 8453 uv-visible spectrophotometer. the y-axis represents the optical density, while the x-axis represents the increasing salt concentration of each tube by weight/volume percent. error bars indicate standard deviation of the optical densities between four replicates. oil wells sampled are hundreds of miles inland and draw from reservoirs thousands of feet deep. given the origin of the saltwater present in these formations, perhaps it should not be unexpected, as a likely explanation is that the seawater was sequestered from ancient seas as the strata of the geological formations were being deposited approximately 300 million years ago. other less likely possibilities are that the oil wells could have become contaminated from fluid or equipment used to drill the well initially. drilling rigs and associated equipment move from site to site, increasing chance of cross-contamination. hydraulic fracturing, which used municipal water from albany, texas in both well sites, could have potentially introduced contaminants deep into the formations, and may change the indigenous chloride concentration, as the water is treated with 2% potassium chloride before use. a contemporary study of two appalachian basin shales also found idiomarina and marinobacter members in low abundance during their 328 day metagenomics and metabolic analysis study. findings from the study include niche differentiation among closely related strains, and interconnected metabolisms of microorganisms persisting in hydraulically fractured shales. a mechanism for aerobic oxidation of alkanes and respiration of sugars via nitrate and oxygen electron acceptors is proposed to be utilized by members of the genus marinobacter present in the studied environment (7). the salt tolerance assay shown in figure 2. utilized varying optical densities with nacl concentration as an indicator of microbial growth. as the bacteria have their growth inhibited with changing salt concentration, the optical density decreased as there were less cells present to scatter light from the spectrophotometer. this allowed us to chart the isolates’ growth from nacl concentrations of 0.5% w/v, which is tsb with no supplemental figure 3. unrooted phylogenetic tree generated from 16s rdna sequences of members of the genus idiomarina. phylogenetic tree illustrates an inferred evolutionary relationships with selected species. species were selected within the genus idiomarina based on relation to i. baltica, our idiomarina isolate’s closest 16s rdna identity match. a neighbor-joining algorithm with 500 bootstrap replications was used to produce the following topology. alignment and phylogeny preformed using molecular evolution genetics analysis 7. halophilic bacteria • 107106 • fine focus, vol. 3 (2) salt, to a supplemental nacl concentration of 25% w/v. the results of the experiment indicate decreased growth beyond 5% nacl for the marinobacter isolate, but the ability to grow with only 0.5% nacl, indicating that it was moderately halotolerant. the idiomarina isolate could not grow at 0.5% nacl, and could grow optimally at 10-12.5% nacl, indicating that it was moderately halophilic. an optimum salt concentration of 3-6% was initially reported for idiomarina baltica, compared to the idiomarina isolate’s 10-12.5% reported here, classifying this strain as moderately halophilic (figure 2). morphologically, the two are similar with idiomarina baltica producing colonies characterized as circular, smooth, opaque, and with a slight yellow pigmentation on marine agar. the most similar sequence reported for idiomarina baltica type strain was a 95-96% affiliation with idiomarina zobelli, while our idiomarina isolate showed a closer association with 98% identity to the same species (4). our idiomarina isolate also showed a 99.2% identity to idiomarina fontislapidosi, which is reported to grow optimally at 3-5% salt concentration, and is remarkable for being the first member of the genus idiomarina isolated from hypersaline soil rather than water (22). the isolate clusters closely with idiomarina fontislapidosi and strains os145 and os146 of idiomarina baltica (figure 3.), but has a much higher optimum salt concentration. an optimum salt concentration of 7.5-10% was previously reported for marinobacter persicus, making it moderately halophilic, while our marinobacter isolate has decreased growth beyond 5% supplemental salt. the two are morphologically different as well, with marinobacter persicus producing colonies characterized as raised, punctiform, contoured, and with a yellow-orange pigmentation when grown on hm agar. at the time of marinobacter persicus’s novel species report, m. hydrocarbonoclasticus was the closest relative with an identity of 97.7%, while our marinobacter isolate shows a 98.2% identity with the same species (2). the isolate also clusters closely with marinobacter sp. rs-29, and marinobacter rs-36 (figure 4.), isolated from yuncheng salt lake in china (21). given the sharp decrease in the marinobacter isolate’s growth beyond 5% nacl, it seems likely that the yuncheng salt lake isolates have a higher salt tolerance due to the lake’s inherent salt richness (27). hydrocarbon degradation abilities notwithstanding, results from the salt tolerance assays (figure 2.) suggest that the isolates would be suitable for a range of bioremediation efforts that might otherwise be inhibited by a high environmental salt content. the idiomarina isolate could prove useful in scenarios where separation of oil and saltwater does not occur before the contamination event, such as well site contamination due to leaking lines or storage tank failure, which ultimately results in the contaminated area being saltier than a typical oil spill. the marinobacter isolate grows well at a concentration of 3.5% salt, ocean level salinity, and would be well suited to cleaning up hydrocarbon contamination on beaches and marine environments. in conclusion, halophilic and halotolerant microorganisms phylogenetically associated with microorganisms present in saline and hypersaline bodies of water, such as the ocean and salt lakes, can be found in the saltwater produced as a byproduct of drilling for oil and gas. by sampling different geological formations, we were able to successfully isolate and characterize microorganisms with differing levels of halotolerance. results indicate the idiomarina isolate was moderately halophilic with an optimal nacl concentration of 10-12.5%, while the marinobacter isolate, was moderately halotolerant with decreased growth beyond 5% nacl. future studies will investigate hydrocarbon degradation by the new isolates, as the related idiomarina xiamenensis is known to act in a hydrocarbon degrading consortium with other marine bacteria (26), and the genus marinobacter has multiple members reported as capable of utilizing hydrocarbons (15). we would like to thank abilene christian university’s office of undergraduate research for funding this research, abilene christian university’s department of chemistry and biochemistry for use of its spectrophotometer, and jql energy, llc for permission to sample and instruction on equipment use. this research was done in compliance with federal regulations and institutional policies relating to recombinant dna and infectious agents. acknowledgments references 1. altschul sf, gish w, mille w, myers ew and lipman, d.j. 1990. basic local alignment search tool. j. mol. biol. 215:403–410. 2. bagheri, m., amoozegar, m.a., didari, m., makhdoumikakhki, a., schumann p, spröer c, sánchez-porro c, ventosa a. 2013. marinobacter persicus sp. nov., a moderately halophilic bacterium from a saline lake in iran. a. van leeuw. j. microb 104:47–54. 3. berestecky, j. hypertonic and hypotonic environments. university of hawaii: kapiolani community college. 4. brettar i, christen r, höfle mg. 2003. idiomarina baltica sp. nov., a marine bacterium with a high optimum growth temperature isolated from surface water of the central baltic sea. int. j. syst. evol. microbiol 53:407–413. 5. brown jr. lf, cleaves ii aw, erxleben aw. 1973. pennsylvanian depositional systems in north central texas: a guide for interpreting terrigenous clastic facies in a cratonic basin. guidebook number 14. bureau of economic geology at the university of texas at austin. 6. climate abilene. 2015. texas and weather averages figure 4. unrooted phylogenetic tree generated from 16s rdna sequences of members of the genus marinobacter. phylogenetic tree illustrates an inferred evolutionary relationships with selected species. species were selected within the genus marinobacter, based on relation to m. perscicus, our marinobacter isolate’s closest 16s rdna identity match. halophilic bacteria • 109108 • fine focus, vol. 3 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petrotoga halophila sp. nov., a thermophilic, moderately halophilic, fermentative bacterium isolated from an offshore oil well in congo. int. j. syst. and evol. microbiol 57:40–44. 24. oren a. 2008. microbial life at high salt concentrations: phylogenetic and metabolic diversity. saline sys 4:2. 25. qin x, tang j, li d., zhang q. 2012. effect of salinity on the bioremediation of petroleum hydrocarbons in a saline-alkaline soil. lett. appl. microbiol 55:210–17. 26. wang l, lai q, fu y, chen h, wang w, wang j, sun f, shao z. 2011. idiomarina xiamenensis sp. nov., isolated from surface seawater, and proposal to transfer pseudidiomarina aestuarii to the genus idiomarina as idiomarina aestuarii comb. nov. int. j. syst. evol. microbiol 61:969–973. 27. youli l, jingchun y, youli l, jingchun y. 1994. evolution of yuncheng saline lake (shanxi, china). geogr. res 13:70–75. halophilic bacteria • 111110 copyright 2018, fine focus all rights reserved “undergraduate perspective: the benefits of performing undergraduate research ellen wagner university of wisconsin madison undergraduate perspective • 225 226 • fine focus, vol. 4 (2) as is common for the majority of incoming biology majors, i began my undergraduate experience expecting to eventually find myself in medical school. now, almost five years after starting my undergraduate education, i am a first year graduate student pursuing a phd in cellular and molecular biology and could not be further from medical school. how did my career trajectory change? what were the key events during my four years as an undergraduate student that triggered the switch from medical doctor to basic biology researcher? when it came time to search for the university i would call home for the four years of undergrad, one of my priorities was the opportunity to work in a research lab. everyone i had spoken with at universities i was considering said research is a great resume builder for medical school. my only previous laboratory experience was in high school science labs. i had never seen a real research lab, nor did i understand what the research process truly was. the only perspective i had of research at this time was knowing pharmaceuticals are developed from research and go through clinical trials to be approved for use. there i was, 17 years old and putting top priority on something i did not understand. flashforward to my sophomore year of undergrad, when i took my first microbiology class. the major assignment for the lab section of the class was identifying an unknown bacterium using the skills we had learned throughout the semester. at first, this assignment seemed overwhelming. i kept thinking to myself, “how i am supposed to identify an unknown bacterium?” however, as i began to plan out the tests i wanted to perform on my bacterium, i discovered this would actually be fun. i began equating the research with solving puzzles (one of my favorite hobbies). in order to make discoveries in research (solve the puzzle), you need to be able to think critically about all of the data you have acquired (the puzzle pieces) and compile it into the bigger picture. this class taught me how to approach scientific questions, and i wanted to seek out a research lab to learn more about this side of science. this assignment was the turning point in my path of deciding between medicine and research. the process for joining a research lab as an undergraduate differs between universities and between labs at the same university. getting involved with research as an undergraduate was relatively easy for me. i simply looked up what type of research was occurring in the biology labs using the biology department website and emailed professors whose work sounded interesting to me. however, certain labs and universities have a more intensive application process. the professors at ball state university want “ “ i began equating the research with solving puzzles (one of my favorite hobbies). in order to make discoveries in research (solve the puzzle), you need to be able to think critically about all of the data you have acquired (the puzzle pieces) and compile it into the bigger picture. undergraduate perspective • 227 to be involved with the students and help them learn what they need to succeed, so they are very willing to talk to students about their research and joining their labs. that is one of the greatest benefits i enjoyed attending a smaller undergraduate university. moreover, the labs are not filled with post-doctoral fellows or phd students, so as an undergrad i was working directly with the professor. this helped me gain confidence in my abilities because i knew i was getting the best training possible. however, i do not believe this is solely a small university trait. since i began graduate school at the university of wisconsin-madison, i have also noticed that the professors at this top r01 research university care about giving both the undergraduate and graduate students the best education and training possible. i had an unusual undergraduate education in that i received training in two very different research labs. the first lab centered on studying the virulence and biofilm potential of bacillus amyloliquefaciens isolated from ultra-high temperature pasteurized milk. the second lab studied the mechanism and purpose of pseudouridine rna modifications in candida albicans. my time in the two labs partially overlapped, which is not something i would necessarily encourage a busy undergraduate student to do. i really enjoyed having the opportunity to work on two completely different projects, but it took time away from other parts of my life in order to equally commit time to both projects. while it is important for undergraduates to learn research techniques, the most important aspect of research an undergraduate student can learn is how to critically think about scientific questions. for any undergraduate student interested in pursuing a career in any type of research, my advice is to find a lab working on something interesting. i think it is very important for undergraduates to find a stimulating project to work on because this will ultimately determine if they want to continue pursuing research. if their first research experience is working on something they do not find intriguing, then they will not be motivated to commit time to working on the project. it’s important for undergraduates beginning in a lab to feel inspired by the science, so that they develop the desire to pursue answers for unknown questions. i encourage undergraduates to get involved in research as early as possible, so they have time to develop this curiosity, but i do not believe joining a research lab as soon as you start at your undergraduate institute is necessary. if you are truly interested in science and research, your curiosity to pursue questions will develop naturally and extra time in the lab will not provide benefit for that aspect of learning. the main benefit of joining early as an undergraduate is that it presents students with more opportunities to participate in summer research programs at other universities. i began reaching out to professors during my sophomore year of undergrad and did not begin working in the lab until the summer before my junior year. i believe my two years working in the lab was the perfect amount of time for me to discover that i enjoyed doing research and wanted to pursue graduate school. i was a competitive applicant when applying for graduate school, and i ultimately ended up at my firstchoice school. two years was enough time for me to develop a strong foundation of skills that i need to succeed as a graduate student. i encourage students to not be discouraged in their abilities when they are meeting 228 • fine focus, vol. 4 (2) new people at conferences, interviews, jobs, and graduate school who have experiences different from theirs. i have always struggled with impostor syndrome, the belief that your achievements occur by random chance and at any moment “truly” successful people will realize that you are not good enough to be where you are with your career. impostor syndrome is very prevalent in academia and extremely common in graduate students. during the first week of orientation for graduate school, i finally had the chance to meet the other students in my cohort. this was when my impostor syndrome really set in. to my surprise, i quickly learned that all but three of the students in my cohort had had at least one year off of school where they worked in a research lab. most of my peers had either attended r01 universities for undergraduate or worked at an r01 institute as a technician. they all talked about their incredibly interesting and diverse research, and it made me feel like i had not experienced or learned enough about research yet to be in the same place along our paths for our careers. however, something that i have had to continually remind myself is that everyone experiences a learning curve when beginning graduate school. we were all starting over in our research, so while it is possible that i may not know all of the techniques my classmates know, they still have to learn new techniques for their thesis projects, as do i. if i had not performed research as an undergraduate, i do not believe i would be prepared for graduate school. it provided me the exposure i needed to understand how research labs work, and it taught me the fundamental skill of thinking critically when approaching a research question. my ability to problem solve in research is not perfect, and i will continually work to develop it throughout graduate school, but i would be lacking in that skill if i had not had prior research experience. i am very grateful for the opportunity to perform research as an undergraduate student. it taught me many valuable skills and helped me determine what type of career i want. ultimately, i advise anyone entering a stem major to look into joining a research lab, even if they are not necessarily interested in a research career. conducting research supplements and solidifies undergraduate education because students learn to apply the theories they are taught in class. performing research as an undergraduate is beneficial because it teaches many critical thinking skills that can be used in any work environment. research also helps to diversify students’ experiences, making them more competitive when entering the workforce. when an undergraduate student conducts research for a significant period of time, it shows that they have experience with problem solving, big picture thinking, and collaborating with peers. i encourage students to branch out from their comfort zone, pursue something new (like research), and use that experience to help them develop new skills and discover what they enjoy related to their field. “ “ imy ability to problem solve in research is not perfect, and i will continually work to develop it throughout graduate school, but i would be lacking in that skill if i had not had prior research experience. 122 | fine focus undergraduate student perspective a pre-med graduate’s gap year of community immersion: finding a greater purpose naari jeong americorps city year teacher’s assistant, los angeles, ca current address: indiana university school of medicine, gary, in usa volume six | 123 i. introduction my eyes startle awake at the sound of my first alarm. 6:05 a.m. the sky is still dark, but i can already hear the lively bustle of my neighborhood on their way to work below my apartment in central los angeles. i begrudgingly get myself out of bed and prepare for a full day of school. this is a familiar routine. except this time, i’m not going as a student but as an educator. some people seem to have absolute clarity as to what careers they want to pursue while others may feel lost in the sea of possibilities. i belonged to the latter group. while i didn’t know the exact passions i wanted to pursue, growing up, i knew the things that i absolutely did not want to do – and that was anything involving math and/ or science. after barely scraping past basic high school chemistry and biology, i was relieved to know that once i entered college i would not have to meet those formidable foes again. as a lover of music, humanities, and the arts, i imagined that my life and career would be separated from science, which i thought was limited to the world of test tubes, pipettes, and moles. however, i was in for a surprise. having a close family member with a severe mental illness, i entered college with some interest in finding a field that would make mental health care more accessible given the extreme shortage of services in my area. in my freshman year, through a mandatory career planning class for psychology majors, i discovered the field of psychiatry. its ability to holistically treat patients with both psychotherapy and medication immediately piqued my interest, and it became a field i wanted to explore. after realizing that this career would require a medical degree, despite my earlier aversion to science, i decided to take an introductory chemistry course and see how i fared. i was surprised to find that i not only was capable of learning science, but that it also was a field that sparked curiosity and excited me. by my senior year, i had completed all the necessary coursework and testing to apply for medical school. however, having followed the tight trajectory of a pre-medical track, i felt the need for some space to seriously reflect on this career path, while also permitting myself to explore other interests. instead of jumping into a medical or graduate program immediately, i decided to take at least one gap year working. while the pressure to start my career was something that initially frightened me, this gap year has been the best gift of time for reflecting, exploring new hobbies, diversifying my professional skill set, and gaining clarity and motivation for a future career in healthcare. ii. a day in the life of my city year i currently work in the sunny city of los angeles with an educational non-profit called city year. in this national americorps-sponsored program, thousands of volunteers are placed in high-need public schools across the country to serve as nearpeer mentors for students in elementary, middle, and high schools. i chose to serve with city year because i was drawn to its vision that all children have equal potential for success and deserve a more equal opportunity towards the resources and support necessary for their growth and development. also, if i wanted to serve low-income populations in urban communities in the future, it was important that i spend time immersing myself in such a community. i am proudly serving at belmont high school in the westlake area, where i continuously gain inspiration from my students and am learning the importance and power of community building. 124 | fine focus my average day at school begins promptly at 7:15 a.m. after going through the team announcements for the day, we go outside to greet students to get them excited for the school day. this is one of my favorite parts of the day because i love watching students enter school with a smile! throughout the day we provide in-class support and plan one-on-one teaching activities to help strengthen our students’ foundational skills in reading and math. while the focus of our organization is academic, we also serve to support their socioemotional wellbeing and growth. thus, we not only assist students with classwork and after school enrichment programs, but oftentimes, we also get to have conversations about their jobs, families, and the stress and joy of being in love for the first time. after a long day, at 5:15pm, we gather as a team to share our triumphs and difficulties of the day before parting and getting ready for the next day. in the past eight months of service, i have had the honor of getting to know students like pedro [name has been replaced to protect identity], a vibrant tenth grader. when we first started the year, he refused to work on any assignments during class. after spending more time with him, i learned how he has been living and working in los angeles without his parents for the past year. while i can’t do much to change his personal situation, i try to make the classroom a welcoming environment so that he feels supported, encouraged, and accountable while at school. i am excited to say that through hours of practice spent in the classroom and after school, we both celebrated him passing all of his classes last semester. like pedro, most of our students at belmont move to los angeles from central america with the hopes of creating a better life for themselves and their families. many come here alone to this foreign country where they do not speak the language and are often not accepted by mainstream society. i often feel hesitant sharing stories of my students because i realize that these are not my stories to tell. however, pedro is more than a story. he is a valuable member of and asset to our community. acknowledging his history is not to gain sympathy, but rather done with the hope that there is a personal stake in very real, human lives. taking time to immerse myself in this particular community, i am learning that we are all interconnected. as my service year continues, my goal is to remind my students of their importance and encourage them to become the best advocate for themselves. i hope to be the best cheerleader i can for my students and let them know that they have someone supporting them in their corner. iii. seeing the bigger picture during my time taking biochemistry at university, i was intrigued by some life lessons it taught me. in the study of metabolism, i learned how the human body is one of the most efficient machines in the entire universe. the interconnectivity of metabolic pathways made me realize the intricate nature of how the body works. it depends on the function of each unique part; nothing can function on its own. my brief tour of the metabolism taught me more than just enzyme names and pathways. it radically changed the way i think about treatment and health delivery. just like our physical bodies, in order to thrive, human beings must rely on community. i now see that effective health treatment requires more than just looking at the symptoms and genetic history. it also requires an awareness of how volume six | 125 an individual’s relationships and living conditions are connected to the community to which they belong. this interconnectivity also made me realize that when one individual struggles, the entire community shares the weight. thus, it makes sense to acknowledge and advocate for those who may not be given equal resources to care for themselves. many times people who go into community service may come from places of privilege with a deficiency-centered outlook where we want to support a “struggling” neighbor. in my time of service, i have had first-hand encounters with injustices in educational practices that favor some neighborhoods while denying others with basic resources. while it is true that my school receives much less support, i am learning that our practice of labeling (e.g., use of deficit-based language like “achievement gaps”) significantly undermines student potential and ignores the larger structural issues. i am convinced that how we label others determines how we will treat them. i hope that we shift towards asset-based language (e.g. “opportunity gaps”), which acknowledges the inequities while respecting the dignity of our students and the community. with this shift, i see my community as an asset rather than a burden. if one takes the time to engage, my community offers strength and beauty. when walking around my school community in westlake, one will find that the streets are full of hardworking individuals who celebrate and share their vibrant culture and strong sense of family values. i have learned that through this year of service, i receive more than i give and am continuously growing through the support in my community. iv. gaining a greater purpose the gap year has radically changed my outlook on a future career in healthcare in a number of unexpected ways. one of these realizations is that i need to be intentional about engaging with diverse communities. living in los angeles, i have had the opportunity to be exposed and learn from multiple cultural groups every day. this has been a shift from my previous experiences living in the bubble of my college campus in indiana. it has taught me that diversity is much more than representation. diversity is the celebration and acknowledgement of those from all walks of life that may be completely different from my own. the beauty of seeing my students from different countries interact and connect has shown me that diversity teaches us to focus on unity and our need for each other rather than focusing on our differences. i have also discovered more about what motivates me to do the work i do. i have often heard the phrase, “knowledge is power.” in addition to power, knowledge is also responsibility. in my case, acquiring knowledge requires me to think about how and for whom i will use it. i want to use it for community building, as i learn more about what that means. these thoughts are new realizations that i have been spurred by my unique gap year experience. i encourage those reading to take time before you commit to a career and try a gap year. anyone in any field or level can engage in meaningful community work. by taking a non-traditional route and working in education, i have had the opportunity to broaden my perspective and learn that the impact of what we do as researchers, educators, and lifelong learners extends far past the domains of our individual sectors. v. acknowledgements i first thank professor john mckillip for extending me this opportunity to share my experiences. another big thank you to my program manager, 126 | fine focus lisa ines, my team leader, sarah perez, and my city year teammates who have taught me the positive power of collaboration and encourage me to grow into the best mentor i can be for my students. i would also like to extend special thanks to my mother, chin-sook pak, for being the best cheerleader and supporting me every step of the way of this journey. finally, i would like to thank my beautiful students who have embraced me. their determination to continuously challenge each day motivates me to push beyond myself and be the best advocate i can for my community. 6 | fine focus objective lens double-blind peer review john l. mckillip, ph.d. professor of biology, ball state university managing editor volume seven | 7 since our inception in 2013, fine focus has utilized a doubleblind peer review system for submitted manuscripts, ensuring that the most objective recommendations are made by members of our editorial board. your submitted manuscripts are redacted of all identifiers (including names, affiliations, and acknowledgements) before they are made available to 2-3 reviewers. this approach is unusual in scientific journals. etkin and colleagues (2017) have reported that for one wellknown academic journal publisher, 95 percent of physical science and health science journals operate singleblind peer review while 72 percent of life sciences journals are single blind. in fact, i am not aware of any journals in the fields of microbiology or science education other than fine focus which utilize a double-blind review process. data to substantiate the claim that double-blind minimizes bias in scientific review does vary according to what is specifically being measured, and by what methods. however, the general consensus viewpoint, reflected in several studies indicates that double-blind manuscript review effectively reduces or eliminates nepotism, gender bias, geographic bias, and personal bias against (or in favor of ) specific research groups, laboratories, and/or pis (thomkins et al., 2017; okike et al., 2017), and that most scholars prefer it over a single-blind review process (ware, 2008). the purpose of this writing is not to explore the topic comprehensively, but rather to inform and justify to our readers as to why the first international journal in undergraduate microbiology research, fine focus, has opted to use double-blind review over the traditional single – blind process. no system is perfect, but thus far, no more suitable manuscript review system has been adopted that has enjoyed any degree of practicality and persistence above that of singleor double-blind peer-review. the reason that the single-blind approach remains dominant in the sciences (but not in social sciences and humanities journals, where only 15% use single-blind review according to etkin et al., 2017) is unclear, but change comes very slowly in the global community of scientists and educators, where collaboration and information-driven action can generate a myriad of opinions and arguments with little hope of decisive action and fundamental change in this regard. arguments against double-blind peer review in the sciences include the notion that preparation of manuscripts with redacted identifiers (or blinding the papers by editorial staff after authors have submitted them) would be overly burdensome or too complicated. however, as any active author, or a managing or section editor for an academic journal could attest, this process is quite straightforward and certainly not as time-consuming or technically challenging as most of the other actions necessary during the manuscript submission/uploading process. critics of double-blind review also indicate that reviewers could probably guess the authors identity and/or affiliations based on the content of the research, key words, or the authors’ results. this is unlikely, and in any case a reviewer who would go to this length in a petty attempt to guess the authors is not someone who is likely to be an effective reviewer anyway, and probably should not be on the board. following my service as a reviewer, and/ or member of the editorial board for the cur quarterly, journal of food protection, journal of dairy science, biologia, and now managing editor of fine focus, i can submit that most reviewers are well-meaning, sincere, and ethical. they want to do a good job, and evaluate the science more than the authors or the place in which the work was done. doubleblind peer review is a good way to ensure that they stay honest and maintain confidence in our system by the submitting authors and editorial staff. 8 | fine focus references 1. etkin, a. , et al. 2017. peer review: reform and renewal in scientific publishing doi: http://dx.doi. org/10.3998/ mpub.9944026 2. tomkins, a., zhang, m., heavlin, w.d. 2017. reviewer bias in single-versus double-blind peer review. pnas 114(48):12708–12713. doi: 10.1073/pnas.1707323114. 3. okike, k., hug, k.t., kocher, m.s., leopold, s.s. 2016. single-blind vs double-blind peer review in the setting of author prestige. jama 316(12):1315–1316. doi: 10.1001/jama.2016.11014. 4. ware, m. 2008. “peer review: benefits, perceptions and alternatives.” publishing research consortium summary papers. web • fine focus, vol. 3 (2) undergraduate perspective : the grant application process amanda baskfield b.s. molecular/cell biology ball state university muncie, in usa current address: national center for advancing translational sciences, rockville, maryland usa copyright 2017, fine focus. all rights reserved. grant application • 173172 • fine focus, vol. 3 (2) the requirement for monetary aid has been needed in scientific research since galileo and prior. grant writing is an art form that scientists use in order to move their research along, regardless of the size of the award. one of the largest struggles is the ability to write the technical piece, describing the research being performed, the need for money, and what it will be used for. writing the piece in lay terminology can be one of the most challenging experiences new researchers face. i applied for a ball state university internal grant through sponsored projects administration office for funding to continue research in t-cell acute lymphoblastic leukemia. in this project, i analyze the process i went through, why i chose to write what i did, and what i would choose to do differently. my hope is that by explaining this process from an undergraduate perspective, others can learn from my challenges and experiences. in scientific research, as in many other areas of academia, it is imperative to receive grant funding in order to carry out the study to reach an end goal. as i am going into the research field, i thought it would be beneficial to study how to properly write a grant to aid in the research i am currently performing. the first step was finding the right grant program; for me, it was the ball state university’s sponsored projects administration internal grant. this gave me experience in two distinct areas: first, in general grant writing, and secondly, in tailoring the grant to an audience who had little to no science background. this caused me to analyze my work further and determine how to write what i was studying in a language the non-scientist could understand. this was a challenge, as i am accustomed to writing in a manner where scientists understand the complex terminology commonly used in research papers and presentations. because of this, i had to complete numerous rewrites of the final draft. after advice from my mentors, i was able to complete the grant proposal and received funding for my research project. i will now share with you, the reader, my overall experience. in order to identify which grant program is most relevant for your work, it is crucial to look into the types of grants available, the application requirements, and the audience who will be reviewing the proposed work. there are many types of external grants available from local, state, federal, private, and public agencies, as well as internal and organization-based grants. i chose an internal grant from the ball state university (bsu) sponsored projects administration, which gives students experience in grant writing without necessarily completing preliminary research. during the 2015-2016 fiscal year, the bsu sponsored projects administration received 27 undergraduate applications and 135 graduate applications, funding 19 and 101 grant requests, respectively. in total, $3,950 was awarded to fund 70% of the undergraduate applicants and $269,819 was awarded to fund 75% of the graduate applications (sponsored projects administration, 32). other grants available to student researchers include the ball state chapter of sigma xi (local), the indiana academy of science senior research grant summation process analysis statement the grant application process (state), or the sigma xi grant-in-aid grant (national), and the federal pell grant, which needs to be applied for in conjunction with the primary investigator. however, the federal pell grant is usually applied for as either a scientist who is a post-doc or lead researcher or as a secondary author on the grant. most of these will require accompanying recommendation letters from your research mentor or others familiar with your work and leadership skills (more information on this in the next article “how to write a good recommendation letter,” this issue of fine focus). the requirements for the bsu internal application included a cover sheet, a budget, a project design, references, letter of support, and a curriculum vita. the cover letter is designed to give a brief summary of the research and gives readers a quick glance as to what the rest of the documents are about (appendix i). attached to the cover letter was the budget, which explained what the money would be spent on (appendix i). it is common for granting organizations to have stipulations on how much can be funded. the bsu internal grant program limited undergraduate applicants to $300 or less and graduate applicants to $500 or less; it is important not to request more than the maximum allowable in the grant description. other grants, such as the indiana academy of science grant, allows up to $2500 to be requested by applicants. although more uncommon, another potential decision factor for the amount requested includes matching policies, where a granting agency will match that raised from additional resources, with these latter sources usually stipulating their contributions and support in an ancillary letter that will comprise part of the overall grant application package. there are five general categories for the bsu internal grant program: contractual, supplies, travel, participant costs, and other. contractual grants fund software licenses, such as needed statistical software to analyze data (or similar). supplies include any reagents, materials, or small benchtop equipment needed by the researcher. in this case, the bsu program would fund $300, while the remaining project costs would be funded by additional sources, such as the biology department and the honors college. this is typical for most primarily undergraduate institutions (puis). travel costs include travel to research sites or participation in research conferences. participant costs, such as conference registration or abstract submission costs, go hand-in-hand with travel costs. other costs include everything not described above, such as purchasing larger pieces of equipment. my advice would be to discuss the budget categories, needs, and spending timeline with your research mentor before drafting the budget, then carefully edit it together, including sources for each line item, quantity, and catalogue numbers. following the development of technical budget comes the budget narrative, where the applicant affirms why all of the costs described in the budget are necessary. in the budget narrative, it is important to describe why something has to be purchased, particularly items that seem conspicuously expensive, such as an antibody, enzyme, or unique reagent available from only a single source. these reasons should be clearly stipulated, and shipping costs (ex. dry ice shipping, hazardous material costs, and related) should be included and mentioned in the budget justification as well. it is important to note that some research grants have caveats, such as the funding cannot be spent on compensation. this means the funding received could not be paid to myself or any other lab assistant grant application • 175174 • fine focus, vol. 3 (2) to compensate for work completed. larger grants, such as those awarded by the national institute of health and national science foundation, have some additional funding categories, such as compensation and university costs. smaller grants usually will not pay for compensation because of the additional resources that are required to pay employees, and would require tax forms, benefits, or other logistics usually out of the realm of smaller internal grant opportunities. larger foundations have this resource and can go through the additional work to provide this benefit to researchers. another point worth mentioing would be indirect costs that go back to the applicant’s university. at bsu, this can go as high as 23% of the grant awarded, some of which will go back to the sponsored projects administration to fund student and faculty research, or to other administrative units. other universities have a much higher indirect cost, which may deter funding organizations from approving and awarding a grant. however, most or all internal funding opportunities for undergraduate projects will not involve any discussion of indirect costs being factored into the budget. when it comes to who applies for the grant, there is a distinct difference between the primary investigators and coinvestigators. the primary investigator is the one who comes up with the initial idea for the research and applies for the larger grants. typically, granting organizations want to hear from the person who had the idea for the overall project when granting large sums of money. however, for the bsu grant, the title of principal investigator was switched to the applicant and not the person who came up with the idea. in this instance, the student was the principal investigator instead of the professor. to decide what the idea for the project would be, my mentor, dr. james olesen (bsu biology) and i looked at previous work i had completed and decided to create a project that was a continuation of it. the third section of a grant is the project design, which has many distinct parts: an overview, background, goals and significance, and the research method timeline, each approximately a paragraph in length (appendix ii). the overview of the project is similar to the introduction of an essay, in that it explains what will be attempted in the proposal and why the research is significant. under background information, the information about the history of the project and the current status is explained. the writer lays out what is being studied in a way grant reviewers will understand. this is where it becomes imperative that one uses appropriate terminology. in terms of the bsu internal grant proposal, the audience included a panel of faculty from many different fields, such as history, math, english, and the sciences. not all grants have a wide range of reviewers, as most are discipline specific. when they are discipline specific, grants can be more detailed as the readers will be more knowledgeable as to what the project is designed to accomplish, what the grant will be spent on, and why the project is important to society at large. since the bsu grant proposal was open to a wide range of undergraduate majors, reviewers came from many different departments across campus to ensure a fair review of all proposals. thus, the writing had to be simplified to a level where most people reading could understand. if technical terminology had to be used, a glossary of terms was added to help without taking away the professionalism of the application (appendix iii). overall, this section should contain some level of detail, including any preliminary data, as it explains the concepts behind what is being studied or proposed. finally, appropriate references are included and formatted appropriately to the field in which the grant is being written (appendix iv). the third section of the project design includes explaining the overall goals or specific objectives of the research as well as the significance of the research. in research proposals, there may be two or three major goals that are presented. in this section, the applicant explains those goals to the reader and describes what they will do to attain them. in my lab, the ultimate end goal is to determine how the transcription factor tal-1 impacts leukemic t-cells and prevents cell death. the specific objectives are what the independent researcher is attempting to accomplish, such as what each lab tech is trying to accomplish to help reach the main goals. in dr. olesen’s lab at ball state, there are three other students all working on different projects, so my specific objectives are different from theirs. my goal is to understand if tal-1 influences caspase-10, thus preventing cell death. the significance of research is being able to take the knowledge outside of the laboratory and apply it to the real world. for this specific project, the significance is to be able to explain why the leukemic t-cells do not undergo death when treated with a chemotherapeutic drug and then use what we learn to help develop therapeutic treatments that may be able to treat t-cell acute lymphoblastic leukemia. the final section of the project design includes the research methods and timeline of the project. the methods section includes a description of what experiments and protocols will be utilized and are written in a way that others could follow. when detailing the protocols, not every step is included for sake of brevity, but other grants require that level of detail. typically, the larger the grant, the more detail the granting agency will require. this description of the methods shows reviewers the applicant has thought through the project, including what protocols will be best to run, and any additional experiments if the project does not go as planned (contingency plan). the timeline gives the granting agency an expected deadline as to when the project will be completed. this gives the reviewer a sense of how long the research will take and whether the timeline is feasible. after the project design is completed, at least one letter of recommendation is needed. in the case of the bsu internal grant, a letter of recommendation was required from the faculty mentor of the student who submitted the application (appendix v). the letter of support must include the viability of the project, a description of how the project will contribute to society, how the project fits with the aspirations of the student, and what role the student plays in the research of the faculty member. this letter of recommendation gives credibility to the student or applicant as to why the funding is needed and verifies the importance of the project. finally, a curriculum vita (cv) is required as documentation of the applicant’s abilities; the cv is generally required for all grant programs, regardless of the funding level or source (appendix vi). a cv is required to provide support as to the applicant’s background and to chronicle other work they have done in the chosen field of research. for the bsu grant, the cv included education, research experience, and awards and honors sections. under education, the university attended, majors, grade point average, and expected date of graduation is listed. additionally, examples of relevant coursework that have contributed to the skills or experience of the student may grant application • 177176 • fine focus, vol. 3 (2) be included in this section. the research experience section is where all of the applicant’s previous professional research is explained. the professional title, lab, location, and dates are listed for each professional lab in which the applicant has worked. this also includes a brief description of the research completed and the job responsibilities. responsibilities can range from skills, such as running specific experiments, to training new lab assistants. furthermore, attendance at conferences should be listed. by attending conferences, either local, regional, or national, this shows the applicant is capable of explaining his or her research to a group and is invested in the dissemination of the research. for some of the conferences, a peer-review of the applicant is done in order to determine if a presentation should be accepted. for example, our annual ball state university student symposium only accepted 100 presentations out of many more applications. by being accepted into a conference, the applicant has shown the significance of the work they are doing and the ability to communicate that significance to others. finally, many granting organizations want to see if the investigators have been publishing their work and how frequently prior to granting larger sums. finally, the last component of a grant is a final report that is submitted to the granting organization. after a designated amount of time, perhaps 12 months following the award, the recipient of the grant is required to submit a report describing how the project went, what was discovered, and where the project will go in the future. for the bsu internal grant, this gives the administration an opportunity to make sure the research was completed, while also validating the faculty mentor is worth funding in the future, should other students apply for a grant from his or her lab. if funding is denied, the hope is that the review panel would provide a ranking and/or a thorough overview of reasons on what aspects of the proposal were lacking or poorly written, in order to improve for a resubmission during the next cycle. occasionally, different reviewers may generate comments that contradict one another, or that seem out of place. however, a good review process will minimize the frequency of superficial or incorrect criticisms, which should be resolved during the panel’s deliberation and ranking discussion, in order to allow the submitter to learn how to make the proposal stronger and more clearly written after revisions. your research mentor will assist you with this phase as well, and offer encouragement along the way so that you are not hopelessly frustrated. overall, this exercise has helped me become a better researcher by providing me with experience in how to fund a research project. i now know how hard scientists have to work to come up with hypotheses and perform research. they have to continuously publish their work and make sure the significance of the work is known. however, most people do not realize the importance of grant funding and how it is the foundation of any research. by starting grant writing in my undergraduate years, i have been able to learn how to apply for grants, what is required, and why it is important. with this understanding of the process of applying for grants, i hope to be able to better fund the research of my future employers based on the knowledge i gained from writing the bsu grant. overall, grant writing is an art form that helps progress all areas of research, whether it is in the arts or in the sciences. this project has shown the importance of reworking different pieces of writing to cater to all those who may read the application and all the necessities that go into grant writing. it is not an easy feat for one to obtain funding for their research and should be a celebrated moment in the applicant’s life when he or she finally does receive funding. sponsored projects administration. (2016, november 17). ball state research 2016. https://issuu.com/ballstatespo/docs/research_2016_1116 works cited appendix i grant application • 179178 • fine focus, vol. 3 (2) appendix ii appendix iii executive summary: t-cell acute lymphoblastic leukemia (t-all) accounts for 15-25% of all acute lymphoblastic cases in children and adults. characterized by a resistance to chemotherapy, this cancer originates from white blood cells (t-lymphocytes) in the bone marrow. after becoming malignant, these cells continue to over-proliferate in the blood stream and upset the balance of the immune system. the development of new treatments for t-all has been stalled due to the complexity of the molecular signaling pathways involved (1). t-all is thought to occur from the ectopic expression of a transcription factor, known as tal-1, which has the ability to bind to dna and influence the expression of additional genes (2). it is thought that tal-1 may impact the expression of genes, especially those influencing the apoptotic or death cascade, thus allowing a cell to avoid death induced by chemotherapeutic treatments. thus, the influence of tal-1 needs to be further characterized, which is what this study is meant to accomplish. background information: further examination of important proteins that are potentially targeted by tal-1 is necessary. if the apoptotic-signaling pathway in a malignant t-cell is understood, this could serve as vital information for the development of a treatment therapy for t-all. in normal cells, the apoptotic pathway is key in removing unwanted or abnormal cells through a well-defined series of events. one protein that has shown to be critically important in the apoptotic cascade, which may be influenced by tal-1, is caspase-10. found in the cytoplasm of the cell (outside the nucleus), caspase-10 acts as an initiator of the apoptotic pathway. if activated, this protein can go on to activate other caspases responsible for the destruction of the cell (3). thus, caspase-10 has the ability to activate apoptosis in the cell. by determining expression changes in caspase-10, the characteristics of the apoptotic signaling pathway involved in t-all can be further determined. these insights are crucial to the development of better, more targeted drug treatments for this rapidly spreading cancer. goals, objectives, and significance: previous research from our lab suggests that tal-1 may negatively influence the induction of apoptosis, but further investigation is needed to understand the exact mechanism of this inhibition. the goal of this research proposal is to determine if tal-1 influences the expression of an important initiator caspase known as caspase-10, thus promoting survival after etoposide drug treatment. the jurkat t-cell line will be used to mimic t-all and inhibition of apoptosis will be assessed through examination of protein expression levels. in the end, a better understanding of the proteins and molecular interactions influenced by tal-1 may be realized. research methods and timeline: the jurkat t-cell line will be cultured in an rpmi/10% bovine growth serum media and maintained at 37o c and 5% co2 to promote cell growth and division. drug treatments with etoposide will be performed over a 24 hour period at concentrations of 0 µm (control), 1 µm, and 5 µm and whole cell lysate protein extracts will be created from all three cell populations. a bradford assay will determine the protein concentration of each extract. the extent of apoptotic induction will then be assessed using western blot analysis, immunofluorescence, and flow cytometry. for western blot analysis, polyacrylamide gels will be loaded with the extracts and proteins will be separated by electrophoresis. the separated proteins will be transferred to a nitrocellulose membrane using a semi-dry transfer apparatus. after transfer, the membrane will be exposed to a primary antibody against caspase-10. expression levels of caspase-10 will be analyzed and quantified using a li-cor imager system. furthermore, immunofluorescence will be performed where cells will be fixed with paraformaldehyde, air-dried onto slides, and incubated with the caspase-10 primary antibody. next, cells will be incubated with a secondary antibody containing a fluorescent tag, which will bind to the primary antibody. a zeiss fluorescence microscope, fitted with a uv light source, will be used to excite the fluorochrome, allowing for the visualization of caspase-10. finally, flow cytometry will be used where cells will be washed in pbs and then exposed to permeabilization buffer to disrupt the plasma membrane so the caspase-10 antibody will enter cells. a secondary antibody containing a fluorochrome tag will be added and will join to the primary antibody so caspase-10 can be visualized in cells. this research project will be completed during spring semester of 2017 and presented at the upcoming ball state student symposium and also at the 132nd indiana academy of science meeting in march 2017. glossary of terms: apoptosis: the highly ordered and timely process of programmed cell death, which can be beneficial to an organism in the removal of unwanted or damaged cells. caspase-10: a protein that activates/cleaves other proteins (protease) in the apoptotic cascade. concentration: the amount of a protein, in μg, that is present in 1 μl of whole cell lysate electrophoresis: a technique used to separate proteins through migration in an electronegative field ectopic: the expression of a biological molecule in an abnormal location in an organism or its cells. etoposide: a chemotherapeutic drug that stops cell growth and division, while also moving the cell into apoptosis. flow cytometry: a laser-based technology used in cell counting, sorting, and the detection grant application • 181180 • fine focus, vol. 3 (2) of proteins. immunofluorescence: a technique used to determine the presence of a protein or antigen biomarker inside of a cell through antibody binding. t-all: t-cell acute lymphoblastic leukemia is a rapidly spreading malignant cancer of the blood cells and bone marrow. tal-1: a transcription factor involved in blood cell development. abnormal expression may result in various cancers such as t-all. transcription factor: a protein that binds to dna sequences and controls the expression of other genes. western blot analysis (protein immunoblot): an analytical technique that can detect the presence of specific proteins isolated from cells. whole cell lysates: protein extracts created from cells, which can be used as samples in western blot analysis. appendix iv appendix v resources: 1) chiaretti s. foá r. t-cell acute lumphoblastic leukemia. haematologica. 2009;94(2):160162. doi:0.3324/heamatol.2008.004150. 2) soumyadeep, d., curtis, d., jane, s., & brandy, s. (2010, may). american society for microbiology. retrieved april 06, 2016. 3) overview of apoptosis (2016). cell signaling technology, inc. grant application • 183182 • fine focus, vol. 3 (2) appendix vi grant application • 185184 crispr • 23 how crisprmediated genome editing is affecting undergraduate biology education ethan s. pickerill1* caleb m. ebree2* ben a. evans1 elena r. north1 gennifer m. mager1 douglas a. bernstein1 1 department of biology, ball state university, muncie, in 47306 usa 2 department of molecular genetics, the ohio state university, columbus, oh 43210 usa copyright 2019, fine focus all rights reserved manuscript recieved 24 september 2019; accepted 12 decemeber 2018 abstract corresponding authors keywords crispr protects bacteria from viruses clustered regularly interspaced short palindromic repeats (crispr) are short dna repeats found in prokaryotic genomes (1). these repeats are interspersed with non-repetitive short sequences homologous to bacteriophage dna (figure 1) (2). the discovery that the interspersed elements possess homology to bacteriophage genomes lead investigators to propose that these short viral elements provide bacteria with immunity against invading viruses. indeed, crispr sequences were found to mediate bacteriophage resistance and investigators identified crispr-associated genes (cas genes) that were also important for this viral immunity (3, 4). bacteria can encode a wide variety of cas proteins which play diverse functions in bacteriophage dna cleavage reviewed in (5). briefly, 20 nucleotide crispr-rna (crrna) guides transcribed from the spacer regions inhibit bacteriophage infection by targeting a cas nuclease to the viral dna using complementary base pairing. some crisprmediated viral cleavage systems also require a transactivating crispr-rna (tracrrna) for maturation of crrna (6). in addition, cleavage of viral genome also requires a short, 2-6 nucleotide, protospacer adjacent motif (pam) in the viral sequence (79). the dual requirement of the 20 base guide and adjacent pam sequence provides a high degree of specificity limiting off-target or bacterial genome cleavage. douglas a. bernstein dabernstein@bsu.edu tel: (765)-285-8851 * authors contributed equally to this work • clustered regularly interspaced short palindromic repeats • crispr • genome editing • biology education • course-based undergraduate research experiences • cures in 2010, the crispr/cas system of streptococcus thermophilus was found necessary and sufficient to cleave bacteriophage dna. since this time, crispr went from a niche scientific field to the laboratories of major research institutions, undergraduate classrooms, and popular culture. in the future, crispr may stand along with pcr, dna sequencing, and transformation as paradigm shifting discoveries in molecular biology. crispr genome editing is technically uncomplicated and relatively inexpensive. thus, crispr-mediated genome editing has been adopted by and applied to undergraduate curricula in a wide variety of ways. in this review, we provide an overview of crisprmediated genome editing and examine some of the ways this technology is being leveraged to train students in the classroom and laboratory. 24 • fine focus, vol 5 2019 crispr-mediated genome editing the discovery that crrna guides, in coordination with cas nuclease mediate viral genome cleavage in bacteria provided an elegant method by which bacteria defend themselves from viral invasion. this discovery however begged the question, could this system cleave dna in vitro or in other organisms? investigators determined cas9, crrna, and tracrrna were sufficient to cleave plasmid dna in vitro in a sequence specific manner (8). building upon these results, multiple investigators tested and found cas9, crrna, and tracrrna could cleave dna in vivo in organisms other than bacteria including mammalian cells (10-12). this technique is now commonly referred to as crispr-mediated genome cleavage or merely crispr. for crispr-mediated genome cleavage to occur, a guide rna must target a cas9 nuclease to a dna sequence of interest. however, once cleavage occurs it is critical that the host cell’s dna repair machinery efficiently repairs the dna break or the cell will die. dna breaks are repaired by two distinct mechanisms. nonhomologous end joining (nhej), reviewed in (13), requires that the cleaved ends of the dna are trimmed (figure 2). the ends are then joined, resulting in deletion of the trimmed sequence. such deletions can shift the reading frame causing premature translation termination. homologous recombination (hr), reviewed in (14), is another mechanism used to repair cas9-mediated cleavage. during hr cells use a homologous dna sequence to repair the cut site. dna encoding a desired genome edit, the repair template, is cotransformed with the cas9 nuclease and guide rna. when the cell uses the repair template to fix the cleaved dna, changes encoded by repair template are introduced at the site of cleavage (figure 2). thus by designing the sequence of the repair template, the investigator can precisely edit the genome, introducing desired point mutations, insertions, or deletions. such precise changes are commonly referred to as crisprmediated genome editing. a number of nucleases can be used to perform genome cleavage, and these nucleases have distinct requirements. for instance, cas9 nuclease requires a tracrrna as well as guide rna to mediate cleavage (6, 9), while cpf1, another cas nuclease, does not require tracrrna (15). cas9 and cpf1 also use distinct pam sequences and cut at different lengths away from their pam sequence. in addition to genome editing, cas9 can be used to alter gene expression by creating a gene fusion a transcriptional activator or repressor to catalytically active cas9 and targeting these complexes to the gene of interest (16). furthermore, inactive cas9 nuclease has been used to fluorescently label the genomic loci enabling investigation of the 3d architecture of the nucleus (17). these discoveries have fundamentally changed the field of biology allowing researchers unprecedented flexibility to manipulate an organism’s genome and transcriptome. figure 1. crispr is used by bacteria to cleave viral dna. crrna is yellow and navy blue. tracrrna is in green. crispr • 25 figure 2. dna cleaved by cas9 is repaired by either nonhomologous end joining or homologous recombination. pam site is red. 26 • fine focus, vol 5 2019 has the potential to cause unforeseen consequences. beyond regulating the spread of disease, increased genetic engineering efficiency via crispr enables investigators to better model diseases in vivo (42-44). unlike transplant or in vitro experiments, crisprcas9 can be used to introduce a specific genotype. this is ideal for studying disease progression and variability, as such studies more closely maintain tissue microenvironments and can be performed in fully immunocompetent animals (45). genome editing systems in human pathogens such as trypanosoma brucei (46, 47) or c. albicans (21) may enable researchers to identify new therapeutic targets. collectively, these models have the power to open doors to new therapies and a better understanding of human health. finally, crispr-mediated genome editing could be used to alter the human genome to improve health and change phenotype (48). while crispr-mediated genome editing has the potential to improve human health in a variety of ways many such applications are provocative and controversial. the scientific community and public must wrestle with not only the technical but ethical challenges presented by such avenues of investigation (49). these potential applications can bring about significant technical challenges and require students balance complex ethical considerations as well. as such, it is imperative students are exposed to genome editing technology during their undergraduate education. this exposure will provide students the best opportunity to efficiently and responsibly apply crispr technology to important research problems. these experiences can come in a variety of forms (50). how crispr is being applied to undergraduate education crispr mediated genome editing becomes widespread the groundbreaking realization that crispr genome editing was broadly applicable has since lead to the development of crispr systems in numerous organisms. for instance, in the fungal kingdom, crispr-mediated genome editing systems have been established for traditional yeast model systems such as schizosaccharomyces pombe (18) and saccharomyces cerevisiae (19), as well as non-conventional yeast candida glabrata (20), candida albicans (21), yarrowia lipolytica (22), kluyveromyces lactis (23), pichia pastoris (24), naumovozyma castellii (25), ogataea thermomethanolica (26), candida lusitaniae (27), candida parapsilosis (28), cordyceps militaris (29), cryptococcus neoformans (30), shefferomyces stipites (31), and aspergillus fumigatus (32) as well as others. in addition, crisprmediated editing systems have been developed for many mammalian (33) and plant model systems (34), as well as other organisms whose genomes have historically been challenging to manipulate (21, 35). this expansion of the use of crispr is similarly demonstrated by online mentions. in 2004 a keyword search for crispr of the pubmed database would have retrieved under ten papers. in 2017 alone, over 3000 papers with the key word crispr were deposited and indexed on pubmed (figure 3). google searches for crispr have had a similar meteoric rise in popularity over the same time period (figure 3) and this rise in popularity has led to articles and features in popular media (36, 37). crispr’s appeal is in part due to the relative ease by which it can be applied to a wide variety of biological systems. such promiscuity gives crisprmediated genome editing the potential to effect human well-being in a multitude of ways (38). genome engineering in agriculture has the potential to increase yields and develop more nutritional food products (39). gene drive technologies that use crispr cas9 to spread genes in wild arthropod populations could fundamentally alter crop management strategies and reduce pesticide use (40). similar gene drive strategies could be employed to limit the spread of diseases such as malaria (41). however the release of genetically modified organisms to the wild is controversial and crispr • 27 figure 3. the increase in crispr related academic and social materials over time. a. crispr publications indexed on pubmed. number of publications submitted to pubmed indexed with the key word crispr by year. b. crispr google search trends. google search frequency from 2004 to 2017. each data point is divided by the total searches of the geography and time range it represents to compare relative popularity. time period with the highest search frequency will score 100, while the time period with the lowest frequency of searches will score 1. figure 4. crispr mediated genome editing of s. cerevisiae ade2. repair of the cleavage sites in the two strains of yeast were shown was done by homologous recombination. stop codons were inserted into the 5’ end of the ade2 gene generating a functional deletion. pink and red colonies indicate successful genome editing while white colonies are unedited s. cerevisiae. a. commercially available baking strain, red star quick rise instant yeast. b. commercially available brewing strain, lallemand nottingham ale brewing yeast. 28 • fine focus, vol 5 2019 one way students can be exposed to crispr-mediated genome editing is by applying it to an undergraduate research project. welldesigned undergraduate research projects both train students and move a larger research program forward. undergraduates typically have limited time they can devote to a research project and their tenure in a lab is likely to be shorter than that of a graduate student or more senior scientist. such limitations make it crucial that students generate strains and reagents required to test a hypothesis in a timely manner. in some fields, crispr-mediated genome editing has increased the speed by which investigators can generate such materials. for instance, the human fungal pathogen c. albicans is diploid (51), does not undergo meiosis, and cannot maintain plasmids. historically, genetic manipulation of c. albicans required multiple rounds of homologous recombination as each allele must be mutated independently and screened in succession (52). as ploidy increases genetic manipulation generally becomes more laborious. these characteristics made it considerably more challenging to work with than other fungal model systems such s. cerevisiae. the development of crispr-mediated genome editing in c. albicans has greatly increased the speed at which genetic engineering can be performed and allows undergraduate researchers to generate strains quickly (25, 53). such efficiency is important so students make progress on a project and are afforded the opportunity to witness the payoff of performing hypothesis-driven experiments. applying crispr to a mentored research project provides a tremendous opportunity for undergraduates to develop a deep understanding of how genome editing can be applied to a research problem. however, it is unrealistic to expect that all undergraduates could gain experience in this way. laboratory courses that employ crispr as a technique are an alternative method of teaching undergraduate research projects crispr in the classroom genome editing to larger groups of students. how crispr can be applied in an undergraduate curriculum depends upon the experience level of the students. introductory courses pose unique challenges due to students’ relative unfamiliarity with bench science and the financial and equipment constraints inherent to many such courses. thus, it is important that exercises yield easily interpretable results while staying within the course’s technical and financial boundaries. one example of a model system well suited for introductory biology courses is crisprmediated genome editing of yeast ade2. mutation of s. cerevisiae ade2 leads to a buildup of an adenine precursor pigmenting the yeast red (figure 4) (25). the intensity of the color and efficiency of gene editing will depend upon the length of the incubation as well as s. cerevisiae strain. this easily observable phenotype, and the practicality of yeast, make them an exceptional system to expose undergraduate students to crispr-mediated genome editing in introductory courses (54). as students gain more experience, the experiments they can perform and data they can interpret during laboratory classes broadens. this provides the instructor with significantly more latitude when designing exercises. there has been a shift recently in higher education away from laboratories that merely teach a technique towards course-based undergraduate research experiences (cures) (55). cures allow students to develop and address a scientific question using a variety of methodologies and techniques. the speed and relatively simple nature of crispr-mediated genome editing makes it particularly well suited for upper level biology cures where faculty address a research question of interest. crispr based cures have been developed using a variety of different model systems including zebrafish (56), drosophila melanogaster (57), and mammalian tissue culture (58). the framework for many of these courses is similar; students design and clone guide rnas into appropriate expression vectors and then use these vectors to edit the genome of their model system. these courses allow faculty to address an important research question and introduce cutting edge genome editing technology when appropriate to students concurrently. students then assess how edits they introduce affect phenotype. in the case that guide generation or editing takes longer than predicted, phenotypic assessment can be performed in other classes or in the research lab of the principal investigator leading the exercise. crispr • 29 in undergraduate curricula, the most immediate impact of cutting edge genome editing technologies like crispr is being felt in biology classrooms as the development of genome editing technologies directly affects course material being taught. however, the remarkable rise and accessible nature of crispr, combined with the powerful prospect that it will enable us to manipulate genomes at will, brings with it a variety of moral and ethical questions that society must examine. for all its promise, crispr raises apprehension regarding human germline manipulation, unforeseen risks, and the potential for these technologies to further social inequities (61). these are by no means novel concerns; since generation of the first transgenic organism, the scientific community has debated the lines that separate the unethical from ethical and the reckless from the enthusiastic. while these debates continue in laboratories and scientific conferences across the world, the discussion should extend beyond the broader science community to the general public and society at large. evidence for this shift can already be seen by the inclusion of crispr debates in academic courses on public policy and science law (62, 63). diverse enrollments in these types of classes will provide opportunities to engage in discussions and hear various viewpoints relating to genome editing technologies. it is imperative that the scientific community continue to inform the public of the remarkable promise of genome editing, as well as the limitations and hazards it presents. the inclusion of these discussions in classrooms outside of biology departments will provide the best chance for us to make informed decisions on how we choose to apply genome editing technology as a society. crispr and science policy another way crispr is being applied in the classroom is by examining how crispr helps bacteria defend against viruses. for example, labs have been developed where undergraduates infect streptococcus thermophilus with bacteriophage and sequence the bacterial genomic dna to see if viral dna has been incorporated to the crispr array (59). modified bacteria gain resistance to later phage infections and subsequent experiments can be performed that further examine the crisprmediated resistance. furthermore, cures have been developed to identify crispr repeats in uncharacterized strains of escherichia coli (60). the crispr loci found among different strains and species of bacteria should be distinct and would depend upon the viruses they have encountered. by examining the sequences of crispr arrays, students can identify phages the bacteria have encountered. while students that perform these exercises are not editing genomes, they learn about crispr biochemical mechanisms and gain an appreciation for how fundamental discoveries can have broad applications. the exercises described above provide examples of how crispr is being applied to undergraduate curriculum. however, crispr-based cures go beyond just genome editing, exposing students to a variety of other molecular biology techniques. for instance, to clone a guide rna into an expression vector students need to perform a variety of additional techniques including restriction digestion, plasmid purification, and dna ligation. crispr cures therefore represent comprehensive approaches that integrate and expose students to genome editing and foundational molecular biology techniques. crispr’s broad utility and practicality make crispr cures attractive pieces from which to shape undergraduate molecular biology curricula. as crispr is now part of the molecular biology tool box, instructors should consider adoption of it alongside classic molecular biology tools when developing classroom activities. summary when the students involved in writing and researching this article started high school, crisprmediated genome engineering had not been invented and now it is being taught in undergraduate curricula across the world. in just one decade, a little known bacterial immune response has profoundly changed 30 • fine focus, vol 5 2019 the pace and breadth of innovation in the genome editing field has made it challenging to include all relevant work. we apologize to those whose work was omitted. this work was supported by r15ai130950 to d.a.b. acknowledgments research and molecular biology undergraduate education in ways few envisioned. the potential for crisprmediated genome editing to profoundly affect research, medicine, and society over the next decades is significant. the initial version of any technology invariably requires refinement. advancements to crisprmediated genome editing will continue as more bacterial crispr systems are characterized and the molecular biology of systems in use is further refined. improvements to genome editing technologies along with the likely continued drop in the price of gene synthesis will open the door to further scientific advances. in addition, as crispr-mediated genome editing systems are developed for more organisms, the limitations traditional model systems impose on research may begin to dissipate. such progress is especially exciting in the context of undergraduate education. a recent paradigm shift has placed more focus on laboratory exercises that not only teach techniques, but also explore important biological problems through guided semi-independent research in the classroom. the confluence of this shift with advances in genome editing technology have the potential to allow students to make significant progress on research questions previously impractical for undergraduate curricula. crispr • 31 1. jansen r, embden jd, gaastra w, schouls lm. 2002. identification of genes that are associated with dna repeats in prokaryotes. mol microbiol 43:1565-75. 2. pourcel c, salvignol g, 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afflict impoverished populations and have therefore been previously understudied. one major focus of our research is to understand the influence that climate has on the distribution of disease causing microorganisms and their vectors, especially those in relation to american trypanosomiasis (chagas disease). chagas disease is caused by the hemoflagellate protozoan parasite, trypanosoma cruzi. for this study, we hypothesized that the increasing prevalence chagas in the state of texas is due to expanding distributions of vectors. to test this hypothesis, historical data on vector distribution and climate was used to determine the probable locations of prevalent vectors in texas. predictions for the future distributions were made using environmental niche models for bioclimatic variables with a maximum entropy algorithm. of the two triatominae species studied, the range and concentration of both decreased under a global warming scenario, a finding that is consistent with the current research of risk of chagas disease in venezuela. in future, this same procedure will be used on more chagas vectors to better understand if there is a northward shift for vectors, or if texas is becoming more inhospitable to all vectors of chagas. abstract corresponding author catherine a. wakeman department of biological sciences, texas tech university, 2901 main lubbock, tx 79409-3131. helen.scott@ttu.edu keywords • t. cruzi • chagas disease • triatomine • climate change • vector it has long been understood that the earth’s climate has wide ranging impacts on human health (9). one such impact that has been previously understudied is the effect climate has on microbial diseases. changes in temperature and rainfall, as we are currently seeing in anthropogenic climate change, may affect the distribution and abundance of disease vectors (9). introduction these vectors may carry microbial agents of disease, such as viruses (causative agents of diseases such as chikungunya, dengue, and west nile virus), bacteria (causative agents of diseases such as lyme disease), and protozoans (causative agents of diseases such as leishmaniasis) (7). the focus of this study is the vector-borne disease american trypanosomiasis, or chagas disease. chagas disease is a tropical infectious disease that is estimated to have affected as many as 18 million people worldwide, with more conservative estimates placing the number in the 8-10 million range (18, 12, 13, 7, 20). the majority of human infections occur in mexico and central and south america (1, 18). the variable estimates of disease prevalence are caused by underreporting of infection, often due to the fact that many people do not know they are infected and do not seek treatment (12, 5). this is, in part, due to the dual phased nature of the disease (1). the acute and the chronic phase are often separated by several decades (18). the first phase (the acute phase) may last anywhere from a few weeks to several months after initial infection (12, 18). the acute phase is asymptomatic or characterized by mild flu-like symptoms not unique to chagas, including fever, fatigue, body aches, headache, rash, loss of appetite, diarrhea, and vomiting (12, 18). the chronic phase, which develops in 30% of all people infected, may exhibit cardiac or gastrointestinal (gi) complications and can be life threatening (12, 18). common cardiac signs include cardiomyopathy, heart failure, altered heart rhythm, and cardiac arrest/sudden death (12, 18). less commonly, chronic gi symptoms include enlarged esophagus (megaesophagus) and enlarged colon (megacolon), both of which can lead to difficulties with eating or passing food (18). while there are cures for chagas, they are most effective in the acute phase (18). symptom management and treatments in the chronic phase may inhibit the progression of cardiomyopathy and lower the fatality rate, but the evidence for their efficacy is weak (18). chagas disease is caused by the hemoflagellate protozoan parasite trypanosoma cruzi (12, 20). infection may occur via organ transplants and blood transfusions from infected donors, and the consumption of uncooked food contaminated with feces from infected bugs and congenital transmission (12, 22). however, the most common route is vector transmission (12). t. cruzi lives in the gut of a variety of insects in the trypomastigote form, and is shed in the feces of the insect vector (4, 2, 4, 23). the parasite is then able to enter the bloodstream of a host via the wound left by the insect vector’s bite. once in the host cells, t. cruzi transforms from a trypomastigote, a mature flagellate form of the trypanosome, to an amastigote, when no external cilia or flagella are present, and multiplies within the tissue (4). progeny are released as trypomastigotes into the blood, to spread the infection to other tissues within the current host (4). while trypomastigotes are in the blood the host can serve as a reservoir, and any uninfected insect vectors that take a blood meal from this host may become infected, thus perpetuating infection to other organisms (4). the insect vectors, all from the triatominae family, are commonly called kissing bugs, due to their tendency to bite around the lips and eyes (1, 22, 7). in the endemic areas of the disease range, approximately 50% of permissive vectors (vectors capable of transmitting disease to humans) carry detectable burdens of t. cruzi (5, 20). the most common species that transmit chagas are triatoma sanguisuga, triatoma gerstaeckeri, triatoma protracta, and rhodinus prolixus (5, 7, 20). t. gerstaeckeri and t. sanguisuga were chosen to be the focus of the study as they are among the most common chagas vectors in texas, and are commonly linked with human american trypanosomiasis •131130 • fine focus, vol. 3 (2) and canine t. cruzi infection. t. gerstaeckeri has the highest t. cruzi infection rate in texas, with 58% of all t. gerstaeckei individuals testing positive, making it a clinically relevant vector (5, 7). historically, endemic chagas disease has been limited to rural areas of mexico, central america, and south america (1, 18). this range has been maintained due to the suitable environment for the reservoirs, triatominae vectors, and the t. cruzi parasite (12, 20). relevant reservoirs in texas include dogs, feral swine, woodrats, and armadillos (12). ideal climate parameters for the transmission of the parasite are tropical conditions, with mild winters, high humidity, and warm nights (1). with changing climates and global warming, there has been a recent shift in the distribution of vectors, including those transmitting diseases such as malaria, lyme disease, leishmaniasis, as well as chagas disease (1, 6, 20). the southern united states are already suitable habitats for the kissing bug vectors, as seen in recent autochthonous cases (cases acquired within the united states) of chagas disease (12). as the disease is underdiagnosed and unreported, estimates of the true prevalence of chagas in the southern united states vary wildly. in texas alone, estimates of infection range from 4 to 267,000 total locally acquired cases (1,15). the large range of suspected cases exists because chagas is often underdiagnosed and untreated. the two phase course of the disease, nonspecific symptoms, and possibility of totally asymptomatic infection makes diagnosis difficult (12). surveillance of donated blood supply in texas has highlighted that there are a number of chagas disease cases that were never treated. in texas, 0.01% of donated blood tested positive for t. cruzi. texas leads the united states in autochthonous cases, reported mainly in the southern regions of the state (12). the first autochthonous case of chagas disease in the united states was in southern texas in 1955, and since then the number of locally acquired cases have continued to grow. however, most studies use seroprevalence, which does not differentiate between locally acquired cases and those that were contracted in traditionally endemic countries. no recent publications specifically assessed locally acquired infection in a larger setting than individual case reports. while human data is far from complete, the use of sentinel species, including dogs, also shows an increasing number of cases over time since the mid 20th century. this figure 2. maxent model of distribution of t. gerstaeckeri in a) present day, b) 2050, c), 2070, white dots are training locations, warmer colors indicate higher ecological suitability for vectors. american trypanosomiasis • 133 figure 1. maxent model for distribution under current conditions (average of 1960 to present). white dots are training locations, warmer colors indicate higher risk of vectors being present for a) triatoma gerstaeckeri and b) triatoma sanguisuga a a b c b 132 • fine focus, vol. 3 (2) trend is expected to continue, with more and more cases occurring in the u.s. with the highest risk in texas (14). the main goal of this project is to assess the impact climate change is projected to have on bioclimatic variables in the years 2050 and 2070, and if this will change the ecological suitability of texas for t. gerstaeckeri and t. sanguisuga. to explain the increase in locally acquired chagas disease cases, we predicted that the ranges and concentrations of our species of interest would increase. however, our results do not agree with this prediction, indicating that the future chagas cases in texas are more likely to be caused by species that are coming to texas from latin and south america in response to a changing climate worldwide. methods data data of the occurrence and distribution of specific triatomine vectors was assembled from previous collections dating from present day to 1960. records of previously collected, identified, and t. cruzi tested specimens were obtained from museum collections (texas a&m university, college station; and university of texas brackenridge field laboratory, austin), and published peerreview journal articles (2, 5, 6, 7, 10, 11, 17, 20, 23, 14). only collections post-1960 were used because the worldclim (version 1.4, http://www.worldclim.org/) data used to in the environmental layers of analysis was an average of information since that year. model construction a maximum entropy algorithm was used to construct distribution models for each of the species of interest. the collected triatominae occurrence points and bioclimatic environmental layers were used with maxent software (version 3.3.3k) for a historical average of species distributions across texas. maxent was used as it is standard in the literature for use in constructing species distributions from a large number of collected specimens, a presence-only record (10). bioclimatic parameters were obtained for the state of texas from worldclim and are as follows: annual mean temperature, mean diurnal range, isothermality, maximum temperature of the warmest month, minimum temperature of the coldest month, temperature annual range, annual precipitation, precipitation of the wettest month, precipitation of the driest month, precipitation of the wettest quarter, precipitation of the driest quarter, precipitation of the warmest quarter, precipitation of the coldest quarter. the technical specifications of the maxent run include, a convergence threshold of 1.0 x 10-5, and use of the threshold and hinge features, without duplicates. future models bioclimatic predictions under the aim 6.0 pathway, rcp6.0, from the version 1.1 of the beijing climate center climate system model (bcc_csm1.1) gcm were retrieved in a 30 second spatial resolution from worldclim. results principle component analysis indicated that of 19 bioclimatic variables, the first two principal components (minimum temperature of the coldest month, and annual mean diurnal temperature for t. gerstaeckeri, and precipitation seasonality, followed by precipitation of the coldest quarter for t. sanguisuga) account for 44.9% and 47.2% of the explained variance for t. gerstaeckeri and t. sanguisuga, respectively. the most important bioclimatic factors for each species (after application of the jackknife procedure) differed between the two at all time points. for t. gerstaeckeri, the most important variable was the minimum temperature of the coldest month, followed by annual mean diurnal temperature, annual precipitation, and precipitation seasonality. for t. sanguisuga, the most important predictor variable was the precipitation seasonality, followed by precipitation of the coldest quarter, and minimum temperature of the coldest month. in 2050, the most important variable for t. gerstaeckeri changed to annual temperature range. annual temperature range remained as the most important variable for t. gerstaeckeri in 2070. for t. gerstaeckeri, estimates of suitability across the state ranged from 0.00 to 0.92 for the current conditions, between 0.00 and 1.00 for 2050, and between 0.00 and 0.77 for 2070. for t. sanguisuga, suitability ranged from 0.00 to 0.85 for the current conditions, between 0.00 and 0.75 for 2050 and between 0.00 and 0.68 for 2070. these both show an overall decrease in suitability. areas of current high suitability for t. gerstaeckeri, such as in south texas along the usa-mexico border, saw a decrease in suitability by 85% by 2070. whereas current areas at medium risk saw a dramatically localized increase of suitability, such as the 46% increase in the houston and galveston area, near trinity bay along the american trypanosomiasis •135 figure 3. maxent model of distribution of t. sanguisuga in a) present day, b) 2050, c), 2070, white dots are training locations, warmer colors indicate higher ecological suitability for vectors. a b c 134 • fine focus, vol. 3 (2) gulf coast. areas of current high suitability for t. sanguisuga decreased by 33% by 2070 in central texas where the highest suitability is currently. no areas saw an increase in suitability for t. sanguisuga. additionally, both species show unchanged low suitability in the texas panhandle in the northwest part of the state. discussion the current distribution of vectors of chagas disease is based on bioclimatic variables. of the bioclimatic variables studied to determine vector distribution, as listed above, different variables were more important to some species of vector than to others. triatoma gerstaeckeri’s niche was most heavily influenced by the minimum temperature of the coldest month, indicating a threshold temperature, below which the vectors die, most likely while in egg or nymphal stages. the niche of triatoma sanguisuga was determined by the precipitation variation throughout the year, favoring areas with constant and dependable rainfall. this requirement results in a distribution more toward the gulf coast region of texas and the east of the state, which tend to have more stable precipitation levels than the west and immediately along the coast. (figure 1a and 1b). presence of t. gerstaeckeri and t. sanguisuga will decrease and shift in response to climate change across the state of texas. under the rcp6.0 climate pathway, “aim 6.0”, the densities of both studied vectors will decrease and the distribution will shift towards the east. by 2050, t. gerstaeckeri will have a highly restricted range with an epicenter in central texas, as well as a large density on the gulf coast near houston. both trends are continued in 2070 (figure 2). in 2070, distribution of t. gerstaeckeri is restricted further, with the exception of a large increase of ecological suitability in the southern region, which may be caused by the presence of microclimates along rivers in the area or that the rural area is suited for the vectors than further north in more populous areas (figure 2). the trends observed for t. gerstaeckeri contrasts somewhat with t. sanguisuga which will have a much broader range in 2050 and 2070, comparable to its present day range, but with a much lower concentration (figure 3). the distribution for t. sanguisuga will be centered in central and east texas (figure 3). the presence of both vectors decreases along the texas-mexico border and southernmost regions of the state. this indicates that there is also a maximum suitable temperature being exceeded in these climatic scenarios. the consensus in the field is that chagas disease rates will increase in america, with texas being the first state affected. the finding of decreased vector range and density was thus very surprising and did not agree with our hypothesis that vector ranges will increase. however, the vectors studied do not represent all chagas competent vectors. and while the species studied have been major sources of concern in texas in the past, they may not be major vectors for disease in the future (14, 20). both of these species have been endemic to texas for many years, and are thus likely highly adapted to its current climate (10.1). future chagas infections may be caused more by invading species coming from latin america as the climate warms and becomes too hot to be suitable, and find refuge in texas with a warmer climate to match that of the previously inhabited endemic areas. therefore, for a better understanding of risk of infection, further research would open up this study to newer triatomine species to texas, including: t. indictiva, t. lecticularia, t. protracta, and t. rubida. this future direction would look to see if these species show the same northward trend over time. anthropogenic effects on the environment increase the risk of chagas transmission. the risk of chagas infection increases in cities, specifically in the largest cities of texas: houston, dallas, and austin due to the high human population in each of the cities. a high human population favors the establishment of a local infection cycle because the triatomine bugs are attracted to areas of human activity and the presence of infected reservoirs increases the risk of transmission to humans (20). while bugs are often found in rural areas, they are attracted to gaseous co2 which is emitted at higher levels in urban areas than rural ones (23). larger cities also tend to have high immigration rates; people raised where chagas is already endemic often are chronically infected without knowing it and can serve as a reservoir to infect naive vectors and perpetuate infection into their new area (4). larger cities also exhibit an urban heat island, where the inner city is warmer, specifically overnight than the outlying rural or suburban areas (9). this affects the mean diurnal temperature (the average temperature range for each day in any given month), which, as seen with t. gerstaeckeri, can have a strong effect on vector distribution (17). because of this a strong urban heat island, yields a stable overnight temperature, making cities more suitable than other areas for some vectors (20). american trypanosomiasis • 137 the authors would like to thank the center for the integration of stem education and research at texas tech university for the undergraduate financial support. we are grateful to nancy mcintyre (texas tech university) for maxent training and support, to jennifer vanos (university of californiasan diego) a geographical information systems consultant, and the texas tech writing center for language editing and proofreading. acknowledgements references 1. beard cb, pye g, steurer fj, rodriguez r, campman r, et al. 2003. chagas disease in a domestic transmission cycle in southern texas, usa. emerging infectious diseases 9: 103–105. 2. betz t. 1984. chagas disease investigation. texas preventable disease news 31: 1-4. 3. ceccarelli, s., & rabinovich, j.e. 2015. global climate change effects on venezuela’s vulnerability to chagas disease in linked to the geographic distribution of five triatomine species. journal of medical entomology 52: 1333-1343. 4. corral, ricardo s., jaime altcheh, sandra r. alexandre, saul grinstein, hector freilij, and alejandro m. katzin. 1996. detection and characterization of antigens in urine of patients with acute, congenital, and chronic chagas’ disease. journal of clinical 13microbiology 34: 1957-62. 5. curtis, r., & hamer, s.a. 2015. combining public health education and disease ecology research: using citizen science to assess chagas disease entomological risk in texas. plos negl trop dis 9: e0004235. doi:10.1371/journal. 136 • fine focus, vol. 3 (2) american trypanosomiasis •139 pntd.0004235 6. githeko, a.k., linsay, s.w., confalonieri, u.e., & patz, j.a. 2000. climate change and vector-borne diseases: a regional analysis. bull world health organ 78: 1136-1147. 7. gubler, d.j. 2009. vector-borne diseases. scientific and technical review of the office international des epizooties 28: 583-588. 8. hanford ej, zhan fb, lu y, giordano a (2007) chagas disease in texas: recognizing the significance and implications of evidence in the literature. social science and medicine 65: 60–79. 9. haines, a., kovats, r.s., cambell-lendrum, d., & corvalan, c. 2006. climate change and human health: impacts, vulnerability and public health. public health 120: 585-596. 10. hanford ej, zhan fb, lu y, giordano a. 2007. chagas disease in texas: recognizing the significance and implications of evidence in the literature. social science and medicine 65: 60 79. 11. hunter, p.r. 2003. climate change and waterborne and vector-borne disease. journal of applied microbiology 94: 37s-46s. 12. kjos, s.a. 2007. biogeography and genetic variation of triatomine chagas disease vecots and trypanosoma cruzi isolates from texas (doctoral dissertation). retrieved from tamu oaktrust library database. (avalable electronically from http : / /hdl .handle .net /1969 .1 /etd -tamu -1299.) 13. kjos, s.a., marcet, p.l., yabsley, m.j., kitron, u., snowden, k.f., logan, k.s., barnes, j.c., & dotson, e.m. 2013. identification of bloodmeal sources and trypansoma cruzi infection in triatomine bugs (hemiptera: reduviidae) from residential settings in texas, the united states. journal of medical entomology 50(5): 1126-1139. 14. kjos, s.a., snowden, k.f., & olson, j.k. 2009. biogeography and trypanosoma cruzi infection prevalence of chagas disease vectors in texas, usa. vector-borne and zoonotic diseases 9(1): 41-49. 15. medone, p., ceccarelli, s., parham, p.e., figuera, a., & rabinovich, j.e. 2015. the impact of climate change on the geographical distribution of the two vectors of chagas disease: implications for the force of infection. . philosophical transactions royal society b 370:20130560. 16. moffett a, strutz s, guda n, gonźalez c, ferro mc, et al. 2009. a global public database of disease vector and reservoir distributions. plos neglected tropical diseases 3: e378. 17. o’donnell, m.s., & ignizio, d.a. bioclimatic predictors for supporting ecological applications in the conterminous united states: u.s. geological survey data series 691. virginia: u.s. department of the interior. 18. pérez-molina, j. a., perez, a. m., norman, f. f., mongemaillo, b., & lópez-vélez, r. 2015. old and new challenges in chagas disease. lancet infectious diseases 15: 137-56. 19. pippin, w.f. 1970. the biology and vector capability of triatoma sanguisuga texana (usinger) and triatoma gerstaeckeri (stal) compared with rhodinus (stal) (hemiptera: triatominae). journal of medical entomology 7: 30-45. 20. sakar, s., strutz, s.e., frank, d.m., rivaldi, c.l., sissel, b, & sánchez-cordero, v. 2010. chagas disease risk in texas. plos neglected tropical diseases 4: e836. 21. tenney, t.d., curtis-robles, r., snowden, k.f., hamer, s.a. 2014. shelter dogs as sentinels for trypanosoma cruzi transmission across texas, usa. emerging infectious diseases 20: 1323-1326. 22. wozniak, e.j., lawrence, g., gorchakov, r., alamgir, h., dotson, e., sissel, b., sakar, s., & murray, k.o. 2015. the biology of the triatomine bugs native to south central texas and assessment of the risk they pose for autochthonous chagas disease exposure. american society of parisitologists 101: 520-528. 23. yaeger r. 1961. the present state of chagas’ disease in the united states. bulletin of the tulane university medical faculty 21: 9-13. 138 54 | fine focus the prevalence and identification of multidrug-resistant bacteria in adjacent ecological systems in the hocking hills region of appalachia orion d. brock and jennifer r. larson orion d. brock (obrock@capital.edu)1, jennifer r. larson (jlarson2@capital.edu)1* 1department of biological and environmental sciences, capital university, columbus, ohio, usa * corresponding author: capital university, 1 college and main, columbus, oh. 43209 manuscript received 12 september 2019; accepted 16 december 2019. volume six | 55 abstract multidrug resistance in clinical settings is a major threat to human health, but very little is known regarding the prevalence of multidrug-resistant organisms in the natural environment. studying antibiotic resistance in the environment is important for understanding the transfer of resistance between environmental microorganisms and those found in healthcare settings. in this study, soil samples from seven adjacent ecological zones were evaluated to determine if there were differences in the amount and types of antibiotic-resistant bacteria present. we hypothesized that we would find antibiotic-resistant bacteria in all ecological zones studied and that these bacteria would be unique to their specific niche. several resistant organisms from each site were also tested for multidrug resistance and subsequently identified through dna sequencing of the 16s gene. antibiotic resistance was discovered in all sites at varying percentages. some forms of bacteria were present at all sites, but there were differences in types of resistant bacteria found between sites. six different genera of bacteria were identified, and multidrug resistance was found in all the isolates studied. our findings indicate that multidrug resistance is prevalent in many different types of environments, including those that have never been directly used for agricultural or urban development. 56 | fine focus introduction antibiotic resistance (ar) has been declared a global health epidemic (53). resistant bacteria currently cause over 2 million infections and 23,000 deaths each year in the usa alone (8). multidrug resistance (mdr) has amplified this threat by making some infections difficult, if not impossible, to treat with antibiotics (33). the escalation of not only antibioticresistant organisms, but multidrug-resistant organisms has been linked to overuse and misuse of antibiotics in clinical and agricultural settings (48). this overuse has led to resistance to every class of antibiotics (8) and consequently, loss of antibiotic efficacy for treating common bacterial infections. in addition, lack of pharmaceutical investment in antibiotic discovery (48) has restricted the availability of novel treatment options. antibiotics have limited effectiveness as introducing them to bacterial populations creates a selective pressure, allowing resistant bacteria to survive and reproduce. ar can be acquired via a random mutation (52), preexisting efflux pumps, or by horizontal gene transfer (hgt). hgt has been linked to the spread of resistance since ar genes are often carried on mobile genetic elements such as plasmids (28), integrons (51), transposons (45), or bacteriophages (9). through hgt, these genes can disseminate and proliferate both within a population and between different populations of bacteria. most research has studied ar in clinical settings, yet there is evidence that some clinical ar genes have environmental origins (50). naturally occurring resistance is widely abundant but is still not entirely understood (10, 12, 41). specifically, it is not well understood if resistance is found only in specific environments or in certain groups of bacteria. previous studies have found antibiotic-resistant bacteria in pristine environments including remote alaskan soil (2) and multidrug-resistant bacteria in isolated cave systems (5). a more detailed surveillance of ar in the environment could allow us to determine the relationship between environmental reservoirs and clinical resistance. the centers for disease control and prevention (cdc) recommends combatting deadly infections and the spread of resistance by tracking ar patterns in all environments (53). previous research has also suggested there is a need for thorough environmental surveys, especially of those environments affected by human activities (17, 22, 32). these areas are of interest as urban environments with high population and building density often have higher levels of ar (19, 32), possibly due to antibiotic effluent from hospitals or from farms that pollute the surrounding environment and water bodies (26). people living or working in these areas also acquire resistant bacteria into their own microbiome (42), further spreading the prevalence of ar. to address the cdc’s recommendation, a project called the prevalence of antibiotic-resistant bacteria in the environment (pare) has created an infrastructure capable of organizing environmental data collection by large numbers of individual researchers (18). this project coordinates undergraduate and high school students across the usa to collect and compile data using identical methodologies. eventually, the pare project will make it possible to track resistance over time, identify areas with high ar, and find relationships between resistance and different environments. the merl and margaret primmer outdoor learning center (primmer), is an outdoor education and research property uniquely suited for a study on environmental drug resistance because of its location away from well-developed areas and its ecological diversity. more than 40 plants and 25 animals have been documented (c. anderson, capital university, personal communication), and much of this property has never been developed or used for agricultural purposes (mark laughlin, capital university, personal volume six | 57 communication). the primmer research property is located in appalachia, ohio, a primarily agricultural region. the hocking river, adjacent to the property, has been historically targeted by the environmental protection agency (epa) as it had been contaminated by industrial and sewer discharges, as well as mine drainage and agricultural runoff (39). with its seven different ecological zones located adjacent to one another and in proximity to a water source made this property an ideal and unique setting for quantitatively surveying both the prevalence of antibiotic-resistant bacteria in the different ecological zones and the diversity of these antibiotic-resistant organisms. select isolates were additionally tested for mdr. we hypothesized that antibiotic-resistant bacteria would be found in all ecological zones studied, and also that these bacteria would be unique to their specific niche. our findings not only supported our hypothesis by showing that antibiotic-resistant bacteria were found in all seven ecological zones studied, but that multidrug resistance can also be prevalent even in the absence of human development. materials and methods sample collection soil samples were collected from the primmer research property, located in logan, ohio. fig. 1 shows a map of primmer and the locations of the 14 collection sites from the seven ecological zones. the distinct ecological zones that have been identified include a grassland (g), a woodland (wl), a wetland (w), a prairie (f), a spring (s) and a riparian zone (r). since clinical ar genes have been detected at distances up to 15 km from the discharge point (13), samples were also collected from the hocking river (hr) to assess possible pollution from upstream locations. soil samples were collected between may and june 2017. gps coordinates in decimal degrees for each collection site were recorded (table 1). the sites were primed by loosening up the soil with a rock or piece of wood from the area. soil samples were isolated an estimated 2.5 cm below the surface from the upper soil horizon, or topsoil since previous studies have showed that bacterial populations are most abundant in this soil horizon (15). samples were then transported to the laboratory and stored at 4oc overnight. 58 | fine focus table 1. gps coordinates of sample collection sites. volume six | 59 fig 1. map of the primmer outdoor learning center and sample collection sites. primmer is a 0.3 km2 private research property with seven unique ecological zones. modified from the usgs national map viewer (2017). sample collection sites are noted by the pins and correspond to the ecosystems noted in the key. 60 | fine focus determining the prevalence of antibiotic resistance macconkey agar and nutrient agar plates without tetracycline (na), with 3 μg/ml (3tet), or 30 μg/ ml (30tet) tetracycline were prepared as per the manufacturer’s instructions (bd biosciences, san jose, ca). all plates also contained 10 μg/ml amphotericin b to prevent overgrowth on plates by fungi in the soil. one gram of soil was measured, serially diluted for five 1:10 dilutions in sterile distilled water, and 0.2 ml of each dilution were plated on all types of media using sterile glass beads. the plating of bacteria was done in duplicate. inoculated plates were wrapped in parafilm and incubated at 28oc for 72 hours. the total number of colony-forming units (cfus) was determined from countable plates without tetracycline (plates with 30-300 colonies and no overgrowth). the percent tetracycline-resistant colonies were determined by counting cfus from the plates containing tetracycline and comparing them with the plates lacking tetracycline. colony characterization after incubation, colonies were characterized according to morphological appearances as in breakwell, et al., 2007 (7). colonies of different morphotypes from each ecological zone were selected to test for additional ar. all colonies selected were resistant to either 3 or 30 µg/ml tetracycline. the kirby-bauer test for antibiotic susceptibility was performed as in hudzicki, et al., 2017 (24). tested antibiotic discs (thermofisher scientific, waltham, ma) contained one of the following: ciprofloxacin (5 μg), ampicillin (10 μg), penicillin (10u), and a tetracycline control (30 μg). these antibiotics were chosen based on their use in human and veterinary medicine (11, 54). the diameter of each zone of inhibition was compared to the standard set by the national committee of clinical laboratory studies (nccls) to determine if the isolate displayed resistance (r), susceptibility (s), or was intermediate (i). pcr and sequencing dna was extracted from isolates by a boiling lysis protocol as found in queipo-ortuño et al., 2008 (37). for each sample, pcr was performed using one of two different primer pairs for part of the 16s rrna gene. primer pairs were: (i): bakt_341f, (cctacgggnggcwgcag), and bakt_805r, (gactachvgggtatctaatcc) (21, 25) and (ii): pa/27f, (agagtttgatcctggctcag) (14, 27), and 1492r, (tacgggtaccttgttacgactt) (27, 44). pcr products were purified using the dna clean and concentrator kit (zymo research, irvine, ca) and then sequenced using the forward primer used for amplification by the ohio state university comprehensive cancer center genomics shared resource facility. resulting sequences were compared to previously published sequences using the basic local alignment search tool (blast) program (national center for biotechnology information). results percentage of antibiotic-resistant bacteria present in soil to assess the prevalence of ar in the seven different ecosystems, we collected samples from two different sites from each of the seven ecological zones present within the primmer research property (fig 1). soil samples were serially-diluted and plated onto media with or without tetracycline. two different types of media and two different concentrations (3 or 30 μg/ml) of tetracycline were used. macconkey agar was used so that a portion of our results could be contributed to the pare database, allowing future volume six | 61 comparisons between additional data collected from other sites. macconkey agar is the current standard media used by the pare project as it commonly results in more uniform colony morphology, which makes colony counting more accurate (18). nutrient agar was used to better assess the diversity of bacteria present. of the macconkey agar data, sites f2, hr1, and g1 exhibited the highest percentage of resistance to 3 µg/ml of tetracycline with the number of resistant colonies at greater than 10% (table 2). only five sites (s1, wl1, hr1, hr2, and r1) displayed resistance to the higher concentration of tetracycline (30 µg/ ml). with the exception of hr2, these sites showed a decrease in the percentage of resistant colonies to the higher concentration of tetracycline. all but one site (s2) showed resistant colonies at some level of tetracycline concentration. 62 | fine focus table 2. percentage of bacterial colomes plated on macconkey agar and nutrient agar media resistant to 3 µg/ml or 30 µg/ml tetracycline. and: not determined due to low coloy counts (<30) present on plates without tetracyline. btm: not determined due to high colony counts on plates with no antibiotic. volume six | 63 as expected, overall colony counts were higher on nutrient agar compared to macconkey, which facilitated the ability to accurately determine the percent resistance for more samples at more sites (table 2). every sample of soil showed some level of resistant bacteria. however, the percent resistance for one of the wetland samples (w2) could not be determined due to high colony counts on plates without antibiotic. both samples from the groundwater spring zone (s1 and s2) exhibited resistance to both concentrations of tetracycline on nutrient agar. s2 and hr1 had the highest percentage of resistant bacteria to 3 µg/ml tetracycline at greater than 10%. eleven of the fourteen samples showed less than 1% resistance to 30 µg/ml of tetracycline. when comparing results from both macconkey and nutrient media, samples s1, wl1, and hr1 had identical ranges for each concentration of tetracycline. all other samples, except f2, showed a slightly higher percent of resistant bacteria on nutrient agar compared with macconkey. these data suggest that naturally occurring ar is even more prevalent than originally expected. identification of isolated antibiotic-resistant bacteria we next tested our second hypothesis that tetresistant bacteria would be unique to their particular environment. tetracycline-resistant colonies on countable nutrient agar plates were analyzed and classified based on their phenotypic characteristics (table 3). thirty-six morphotypes were identified from the fourteen collection sites (morphotypes i-xxxvi). morphotype i, the most common, was found at all 14 collection sites and at percentages ranging from 54.05% (s1) to 98.9% (w2) of the total antibiotic-resistant population. morphotype i also represented 83.54% of all antibiotic-resistant bacteria. the woodland zone (wl1 and wl2) displayed the highest diversity of morphology with 12 unique morphotypes. these results suggest that, while at low frequencies, adjacent ecological zones contain distinct sub-populations of bacteria. 64 | fine focus table 3a. colony morphology for countable nutrient media plates. apercentage of antibiotic-resistant colonies with the population at a single soil sample collection site. *selected fror isolation volume six | 65 table 3b. colony morphology for countable nutrient media plates. apercentage of antibiotic-resistant colonies with the population at a single soil sample collection site. *selected fror isolation 66 | fine focus table 3c. colony morphology for countable nutrient media plates. apercentage of antibiotic-resistant colonies with the population at a single soil sample collection site. *selected fror isolation volume six | 67 to determine if the resistant bacteria were unique to their ecological zone, we selected a subset of colonies from different ecosystems and of different morphology. some colonies were selected from 3tet plates while others were selected from 30tet plates. we performed pcr on these isolates using primers designed to amplify the 16s ribosomal rna gene. sequence analysis of these genes allowed identification at the genus level. surprisingly, of the ten bacterial colonies successfully sequenced (table 4), nearly all of the bacteria identified were of different genera. as expected, streptomyces, which is prevalent in soil (23), was identified in four different ecological zones (g, wl, s, and hr). morphotype xxvi from the wetland zone (w2) and morphotype xxv from the riparian zone (r2) were identified as bacillus with 99% confidence. the duplicate genera suggest that antibiotic-resistant bacteria are not entirely specific to their ecological zone. these data showed that while some species of antibiotic-resistant bacteria were present in multiple zones, there were some that were specific to different zones, supporting our initial hypothesis. these data also showed that a variety of different bacteria are capable of possessing ar. antibiotic susceptibility test since all of our isolates were found to be resistant to tetracycline, we wanted to know if they were resistant to other antibiotics. to test this, we performed a kirby-bauer disk diffusion test allowing us to test other antibiotics used in human and veterinary medicine. the antibiotics chosen for testing were ciprofloxacin, which has a synthetic origin (49), and ampicillin and penicillin, which are both naturally occurring β-lactam antibiotics (1, 16). some of our isolates selected from 3tet plates were sensitive to the disk with 30 µg of tetracycline (isolates b, e, h, and i), showing that their resistance is concentrationdependent. since all isolates were resistant to some concentration of tetracycline and one or more antibiotics, 100% of the isolates were multidrugresistant (table 5). interestingly, every isolate displayed resistance to penicillin. isolates a, d, f, k, and n (36% of isolates) were resistant to all four antibiotics. discovery of mdr in each ecological zone indicates that there must be some selective advantage for this, even in naturally occurring environments. 68 | fine focus table 4. designation for identification and: not determined volume six | 69 table 5. zone of inhibition diameter measurements of selected bacterial colonies azone of inhibition diameter measured in mm bresistance determined by diameter standard (clsi, 2013) cresistant dsusceptible eintermediate 70 | fine focus discussion ar is naturally occurring, even in environments away from anthropogenic selection pressures. studying this naturally occurring ar is important for understanding and dealing with the growing resistance of pathogenic microorganisms in clinical settings. previous evidence of natural ar has been found in remote soil and isolated cave systems (2,5). while the primmer research property is not as isolated as a cave system, our samples were collected from a site that has never been developed. because resistant organisms were found in each of the ecological zones tested, our results support what previous research has found, that ar occurs naturally in bacterial populations. not much is known yet regarding how frequently ar gene transfer takes place in natural environments. mdr can occur through various mechanisms, one of which is efflux of the drugs by membrane transport proteins. efflux pumps are able to transport specific substrates or expel cytotoxic compounds (6, 29). while there is evidence that efflux pumps are physiologically important (35), they commonly mediate mdr. this has led to the occurrence of microorganisms intrinsically resistant to multiple antibiotics (33). our data support this claim, as resistance was found to ciprofloxacin, despite a seemingly absence of evolutionary pressures. interestingly, more of our isolates exhibited resistance to penicillin than ampicillin, yet both are β-lactam antibiotics. this could be attributed to the fact that penicillin has natural origins whereas ampicillin is semi-synthetic. overall, our results suggest that mdr in a bacterial population is a naturally occurring phenomenon. hgt of naturally occurring resistance genes to pathogens could also be a factor for the acceleration of resistance found in clinical and agricultural settings. to characterize the diversity of the bacterial populations present in the different ecological zones, we analyzed colony morphology present on our nonselective media plates and selected antibiotic-resistant isolates to be sequenced. previous research has shown a wide variety of taxonomic groups to have ar and mdr bacteria (31) and also that bacteria are capable of transferring ar genes either between bacteria of the same species or between different species (4). we found a high diversity of colony morphologies (table 3) and identified six unique bacteria genera (table 4) from a small subset of total colonies. since all selected isolates were found to be multidrugresistant, it is possible that hgt of ar genes occurs naturally at some frequency. these data support our hypothesis that unique bacteria would be found in the different ecological zones, and more importantly, that antibiotic-resistant bacteria can be from a highly diverse group. soil is rich in microbial abundance and species diversity. it has been estimated that a single gram of soil can have up to 1010 bacterial cells and more than 4×103 different bacteria species (20, 34, 36, 40, 46, 47). in addition, bacteria populations can differ between geography and altitude (30). using a site with seven adjacent ecological zones gave us the unique ability to directly compare both the prevalence and diversity of antibiotic-resistant organisms in these different ecosystems. we found a large range in the overall percentages of resistant organisms in each of the ecological zones. the highest percent resistant organisms were found in the spring (s), woodland (wl), and river (hr) zones. high percentages in the woodland areas could be due to availability of nutrients around the rhizosphere, an active region around a plant root that microorganisms inhabit (3). the river and spring zones could have high resistance from upstream pollution sources, especially since the primmer research property is located within an agriculturally focused region. this is not surprising as other bacteria have been previously found to survive volume six | 71 in water for a long time, such as those from the genera pseudomonas (38, 43). in conclusion, the differences we found between the adjacent sites show that while there is a relationship between ecology, prevalence of antibiotic-resistant organisms, and types of bacteria present, multidrugresistance was found in all sites and all types of bacteria tested and is likely more common in the environment than we thought. additional surveillance of the resistome present in different ecological locations will likely be essential for developing novel antibiotic treatments. acknowledgments the authors acknowledge dr. carol bascom-slack (tufts university) for collaborating on this project, the merl and margaret primmer outdoor learning center (capital university) for financial support, and dr. stephen osmani (the ohio state university) for use of his laboratory and reagents. we would also like to thank dr. kelly 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microbiol. infect. 13:5–18. 53. world health organization. 2014. antimicrobial resistance: global report on surveillance 2014. 54. world health organization. 2016. critically important antimicrobials for human medicine, 5th revision 2016. tatc2 is important for growth of acinetobacter baylyi under stress conditions justin m. langro1 megan m. chamberland2 celena m. gwin3 natalia prakash4 danielle t. velez5 nathan w. rigel6 1.department of biology, hofstra university, long island, new york city, usa 3. present address: department of microbial pathogenesis, yale university, new haven, ct, usa 5. present address: regeneraon pharmaceuticals, tarrytown, ny, usa copyright 2019, fine focus all rights reserved manuscript recieved 8 august 2018; accepted 8 november 2018 tatc in acinetobacter baylyi• 37 abstract corresponding authors keywords department of biology hofstra university 318a gittleson hall hempstead, ny 11549 nathan.w.rigel@hofstra.edu phone: 516-463-6542 fax: 516-463-5112 • acinetobacter baylyi • cell envelope • protein export • twin-arginine translocase • tatc2 protein export pathways are important for bacterial physiology among pathogens and nonpathogens alike. this includes the twin-arginine translocation (tat) pathway, which transports fully folded proteins across the bacterial cytoplasmic membrane. some tat substrates are virulence factors, while others are important for cellular processes like peptidoglycan remodeling. some bacteria encode more than one copy of each tat component, including the gram-negative soil isolate acinetobacter baylyi. one of these tat pathways is essential for growth, while the other is not. we constructed a loss-of-function mutation to disrupt the non-essential tatc2 gene and assessed its contribution to cell growth under different environmental conditions. while the tatc2 mutant grew well under standard laboratory conditions, it displayed a growth defect and an aberrant cellular morphology when subjected to high temperature stress including an aberrant cellular morphology. furthermore, increased sensitivities to detergent suggested a compromised cell envelope. lastly, using an in vitro co-culture system, we demonstrate that the non-essential tat pathway provides a growth advantage. the findings of this study establish the importance of the non-essential tat pathway for optimal growth of a. baylyi in stressful environmental conditions. 38 • fine focus, vol 5 2019 introduction transport of proteins between membrane-bound compartments is a fundamental biological problem faced by all cellular organisms, including bacteria. protein transport is carried out by multi-subunit, membrane-spanning molecular machines (12, 23). in gram-negative bacteria, most exported proteins cross the inner membrane via the general secretion (sec) pathway. proteins that take this path must remain unfolded in order to pass through the secyeg inner membrane translocase. however, some proteins are exported in a fully folded state by using a separate machine called the twin-arginine translocase (tat) (15). proteins destined for export via sec or tat contain a recognizable tripartite signal peptide composed of a positively charged region at the n-terminus, followed by a hydrophobic region in the middle and a polar region at the end (30). however, there are several key differences between sec and tat signal peptides; among them the charged region of canonical tat signal peptides contains an arginine dipeptide within a srrxflk sequence motif (6, 8). signal peptides are recognized by the sec and tat machinery during the initial stages of protein translocation, and are cleaved by periplasmic signal peptidases upon export (29). the tat machinery is composed of tata, tatb, and tatc proteins; some bacteria lack tatb, but in these cases the role of this protein is performed by tata (17, 22, 45). other organisms may also encode tate, which acts as a functional homolog of tata (39). to achieve substrate transport, the tatb and tatc proteins form a subcomplex in the inner membrane that recognizes the tat-specific signal peptide (1). recent evidence suggests that tata protomers are part of the tatbc receptor complex and that after binding signal peptide, additional tata subunits are recruited to form the translocase (2, 18). the active translocase requires the proton motive force for translocation of the substrate across the inner membrane. upon export of the substrate, the tata protomers dissociate from the recognition complex and the cycle can begin again. to date, most studies of this protein transporter have been performed using escherichia coli as the model. recent analyses of bacterial and archaeal genomes reveals that in some organisms, multiple tat homologs are present (29). in fact, there are reports of gram-negative bacteria with two distinct tat translocases (24, 33). similarly, there are two distinct tat translocases in the gram-positive bacterium bacillus subtilis, though in this case only tata and tatc components are found. in some cases, these extra tat components are functionally redundant (4), while in other cases they are used to export specific substrates (13). there are few studies of protein transport in organisms with multiple tat components, and thus the significance of possessing two distinct tat pathways is not fully appreciated. in order to better characterize the importance of dual tat pathways, we used the gram-negative soil bacterium acinetobacter baylyi as a model. scanning the genome of a. baylyi reveals the presence of two distinct tat pathways. remarkably, comprehensive mutagenesis of a. baylyi suggests that the components of one tat machine are essential for growth, while the components of the other machine are dispensable (11). for clarity, henceforth we refer to the essential tat genes as tata1, tatb1, and tatc1 and the non-essential tat genes as tata2, tatb2, and tatc2. in this report, we describe experiments characterizing the non-essential tat pathway of a. baylyi. specifically, we sought to understand how tat-dependent protein export enables the growth of a. baylyi under different environmental conditions. we uncovered a role for the non-essential tat pathway in maintaining normal cell envelope integrity and cellular morphology under stress conditions. using co-culture experiments, we found that the non-essential tat pathway provides a growth advantage to a. baylyi in competition with pseudomonads that also possess two tat machines. taken together, our findings provide insight into the function of the non-essential tat pathway of a model soil microbe. tatc in acinetobacter baylyi • 39 materials & methods strains and growth conditions. unless otherwise noted, growth of a. baylyi wild-type strain adp1 (strain 33305 from the american type culture collection; manassas, va) was performed at 30 degrees celsius. for routine culturing, all bacterial strains used in this study were grown in lb lennox (10 g bacto-tryptone, 5 g yeast extract, and 5 g nacl per liter; fisher scientific). for growth curve experiments, each strain was grown overnight in lb and then diluted 1:100 in fresh media. these cultures were incubated with intermittent shaking at 200 rpm and optical density (od) measurements (at 600 nm) were taken every 30 minutes for 8-10 hours. all strains were grown in triplicate. construction of an a. baylyi tatc2::kan insertion-deletion mutant. the a. baylyi tatc2 mutant was constructed using an overlap-extension pcr strategy described previously (3). briefly, upstream and downstream sequences flanking tatc2 (aciad0521) were amplified by colony pcr from the a. baylyi wild-type strain adp1 (fig. 1a). the upstream flanking sequence was amplified using primers a (5’-tggagtatataaaaatggc-3’) and b (5’-attgttttagtaccgagctccttgggcaggcatgatgtc-3’). the downstream flanking sequence was amplified using primers c (5’-gccatttattatttccttcgatcctcgaaaaacgtag-3’) and d (5’-gattacctttggcatcaac-3’). a 795 bp kanamycin-resistance cassette was amplified from plasmid pim1445 (gift from ichiro matsumura) (28) using primers e (5’-gagctcggtactaaaacaat-3’) and f (5’-gaaggaaataataaatggc-3’). these three products were mixed together in equal molar ratios in a new tube and joined together in a final pcr using the outer-most upstream and downstream flanking primers (a and d), resulting in the ::kan insertion-deletion allele. this pcr product was purified and then used to transform naturally competent wild-type a. baylyi (14). incorporation of the insertion-deletion allele was confirmed by pcr analysis and dna sequencing. construction of ptatc2 expression plasmids. to perform complementation experiments on the tatc2::kan mutant, the wild-type tatc gene was cloned into the bamhi site of pwh1266 by custom gene synthesis (genscript, piscataway, nj) using sequences from the a. baylyi genome (www.biocyc. org). plasmids were transformed into a. baylyi as follows. from an overnight culture of wild-type strain adp1, 0.1 ml of cells was gently mixed with 100 ng of plasmid dna. the mixture was allowed to incubate at room temperature for 1 hour. to allow outgrowth of any transformed cells, 0.9 ml of lb was added to the mixture followed by overnight incubation at 30°c while shaking. the next day, 0.1 ml of the transformation mixture was plated onto lb-ampicillin agar and grown overnight at 30°c to select transformants. detergent sensitivity assay by efficiency of plating method. cultures of wild-type, tatc2 mutant, and complemented tatc2 mutant bacteria were grown overnight in lb broth, then diluted in series in fresh lb in a 96 well plate. using a multi-prong metal replicator, approximately 2 °l of each dilution was transferred to the surface lb agar or lb agar supplemented with 2% sodium dodecyl sulfate (sds) and 1 mm ethylenediaminetetraacetic acid (edta). plates were incubated overnight at 30°c and growth on the plate was assessed the next day. microscopic examination of cellular morphology. wild-type and tatc2 mutant starter cultures were grown at 30°c overnight. the next day, each strain was diluted 1:100 in fresh lb broth in duplicate. one set of strains was grown at 30°c while the duplicates were grown at 42°c until late exponential phase. ten microliters of each culture was spotted onto a glass slide and the smears were allowed to air dry. following heat fixation, the smears were stained with crystal violet and visualized using an olympus bx41 microscope under the 100x objective with immersion oil. images shown are a representative field from at least three independent experiments (figure 2). competitive co-culture experiments. overnight cultures were used to inoculate flasks containing 50 ml of fresh lb broth for competition assays. the optical density (od600 nm) of each culture was normalized to obtain a final od of 0.1 in the flask. flasks were incubated at 200 rpm on a platform shaker at 30°c for 24 hours. one hundred microliter aliquots were taken from the initial inoculum and at 40 • fine focus, vol 5 2019 the 24-hour time point, diluted in 10-fold series to a final dilution of 10-7. to enumerate colony forming units (cfus), 0.1 ml aliquots were plated from the dilutions to obtain well-isolated colonies. individual competition assays were performed in triplicate. input and output cfu counts were used to determine the competitive index (ci) for each a. baylyi strain using the following formula: ci = (af/ai)/(bf/bi), where a represents the a. baylyi strain and b represents the competitor strain, with f and i denoting the final and initial cfu counts, respectively. ci values were subsequently analyzed using one-way anova. figure 2. the tatc2 mutant displays abnormal cellular morphology under high temperature stress conditions. overnight cultures of wild-type (left column) and tatc2 mutant (right column) bacteria were diluted 1::100 into fresh lb broth and grown to late exponential phase at 30°c (top row) or 42°c (bottom row). aliquots from each culture were heatfixed to a glass slide and stained with crystal violet. images were captured using the 100x objective. each tick in the scale bar equals 1 um. figure 1. strategy to construct a tatc2::kan insertion-deletion mutant. a) primers used to generate the upstream (a and b) and downstream (c and d) regions flanking tatc2 are indicated by solid arrows. primers used to for pcr analysis of kanr recombinants are indicated by dashed arrows. b) colony pcr analysis of wild-type and tatc2::kan strains. pcr products were resolved on a 0.7% agarose gel and visualized by staining with ethidium bromide. molecular weight (mw) size standards are shown (in kb) for comparison. tatc in acinetobacter baylyi • 41 figure 3. colony morphology of wild-type and a. baylyi tatc2 mutant strains. wild-type (left) and tatc2 mutant (right) a. baylyi strains were grown overnight in lb broth cultures at 30°c. then serial dilutions of each strain were plated onto lb agar. the plates were incubated overnight at 30°c prior to imaging. figure 3. genome architecture of the tat genes of acinetobacter spp. homologs of tata, tatb, and tatc have light shading. putative alkaline phosphatase phod has dark shading. flanking genes of unknown function are labeled with aciad genome reference numbers. 42 • fine focus, vol 5 2019 the genome of acinetobacter baylyi encodes homologs of the twin-arginine translocation (tat) pathway. the genus acinetobacter has recently drawn increased attention due to the rise of drug resistant isolates of acinetobacter baumannii (31). in addition, acinetobacter baylyi is of growing interest as a model gram-negative organism due to its ease of genetic manipulation and diverse metabolic capabilities (14). while exploring the genome of a. baylyi for evidence of known protein export machines, we located homologs of the tat pathway. interestingly, when we examined the a. baylyi genome, we noted two homologs each of tata, tatb, and tatc (fig. 3). we found that the tata homologs have the highest sequence identity to each other at 86%, while the sequence identity of the tatb and tatc homologs is lower scoring 62.3% and 70.8%, respectively (table 1). in addition to the differences in amino acid sequence, there was also a reported difference in function between these two putative tat pathways. de berardinis and colleagues reported that the genes encoding one tat pathway were essential for growth (tata1b1c1), while genes encoding the other (tata2b2c2) were dispensable for growth (11). this finding suggests the possibility that each of these tat pathways has a different function in secreting proteins out of the cytoplasm. in particular, it could be the case that some tat substrates are specifically routed through one tat pathway rather than the other. as precedent, the dual tat pathways of bacillus subtilis are known to display substrate specificity (32). to clarify the role played by each tat pathway in a. baylyi physiology, we focused on the non-essential tat machine. given the established role of tatc homologs in substrate recognition (19, 20, 35), we started our analysis by creating a loss-of-function tatc2 mutant strain. construction and phenotypic analysis of a tatc2 mutant. we constructed an insertion-deletion tatc2 mutant using a previously described method (3). the tatc2::kan mutant allele was transformed into the wild-type strain of a. baylyi adp1, and kanamycin-resistant colonies were selected on lb agar supplemented with kanamycin. recombination of each mutant allele into the bacterial chromosome was confirmed by pcr and dna sequence analysis (fig. 1). we obtained numerous transformants for the tatc2::kan allele, suggesting that this gene is dispensable for growth under standard laboratory conditions, in agreement with previous findings (11). to begin characterizing these mutants, the mutant cultures were assessed for their growth properties. colonies of the tatc2 mutant are indistinguishable from wildtype a. baylyi when grown on lb plates at 30°c (fig. 4). when grown in lb broth at 30°c, the tatc2 mutant exhibited similar growth kinetics as wildtype a. baylyi (fig. 5a). a similar growth trend was observed when we plated for viable counts (data not shown). in some organisms, loss of a functional tat pathway has been associated with defects in virulence, while in other organisms tat mutants exhibit aberrant morphological and physiological phenotypes under in vitro stress conditions, including when subject to high salt or detergent concentrations (10, 42, 44). we next tested the ability of the tatc2 mutant to grow when challenged with various environmental stresses. the tatc2 mutant is sensitive to detergents and growth at high temperature. defects in bacterial protein export machines, including the tat pathway, are often accompanied by increased sensitivity to toxic small molecules (21, 25, 26, 36). in e. coli, the tat pathway exports proteins that help maintain the structural integrity of the cell envelope. failure to export these proteins weakens the cell envelope, thereby increasing its permeability. to determine if the non-essential tat pathway is important for cell envelope integrity in a. baylyi, we tested the ability of the tatc2 mutant to grow in the presence of the detergent sds. using an efficiency of plating assay, we found that growth of the tatc2 mutant is impaired by treatment with detergent (fig. 6). importantly, we observed that complementation of the detergent sensitivity phenotype of the tatc2 mutant could be achieved by constitutive expression of tatc2 on a plasmid. given the increased cell envelope permeability associated with the tatc2 mutant, we wondered if the non-essential tat machine was important to resist temperature stress. although the tatc2 mutant grew as well as wild-type at 30°c, its growth was impaired at 42°c (fig. 5b). at later time points, growth of the tatc2 mutant slowed as compared to wild-type cultures grown at 42°c. this finding is consistent with the the function of tat in other organisms and suggests that the non-essential tat pathway of a. baylyi plays a role in cell envelope biogenesis (10). given the established role of tat in maintaining normal cellular morphology (7), we next examined the cellular morphology of the tatc2 mutant by microscopy (fig. 2). we found that the tatc2 mutant cells grown at results tatc in acinetobacter baylyi • 43 30°c were indistinguishable from the wild-type cells. both strains appeared as small coccobacilli, which is characteristic of the acinetobacter genus (41). at 42°c, cells of wild-type a. baylyi were slightly larger than those grown at 30°c. cells of the tatc2 mutant were longer and thicker when grown at 42°c. thus tatc2 is important for maintaining normal cellular morphology of a. baylyi under high temperature stress conditions. the non-essential tat pathway provides a competitive advantage to a. baylyi grown in coculture. we showed that tatc2 is important for maintaining normal cellular morphology under stressful growth conditions in the laboratory. this led us to wonder how important the non-essential tat machine is for a. baylyi to grow in the environment. the soil is home to complex microbial communities; these microbes are often in competition with each other for scarce nutrients. in such an environment, some protein secretion systems are important for nutrient acquisition (5, 27, 37), while others are used to directly inhibit growth of microbial competitors (16, 38, 43). with this in mind, we tested whether the non-essential tat pathway, and tatc2 in particular, was important for the growth of a. baylyi in a competitive co-culture system. pseudomonas species are closely related to acinetobacter species, and often share similar environmental niches. therefore, we selected a subset of pseudomonas species for the coculture experiments. as shown by the competitive index (ci) values in table 2, all three pseudomonas strains tested grew significantly better than both wildtype and tatc2 mutant strains of a. baylyi. however, when comparing the relative ci values between wildtype and the tatc2 mutant, we noticed that the tatc2 mutant was significantly impaired when grown in competition with p. putida. in competitions with both p. aeruginosa and p. fluorescens, there was no significant difference in ci between wild-type and the tatc2 mutant. the difference in ci does not appear to be due to a secreted toxin as p. putida grown in spent media from either wild-type a. baylyi or the tatc2 mutant showed no difference in growth compared to control (data not shown). 44 • fine focus, vol 5 2019 tatc in acinetobacter baylyi • 45 figure 5. growth of wild-type and a. baylyi tatc2 mutant strains in liquid culture. wild-type (●) and tatc2 mutant (▲) a. baylyi strains were grown at 30°c (a) or 42°c (b) in lb broth cultures. overnight cultures of each strain were diluted 1:100 in fresh lb at the start of the experiment. optical density (od600 nm) was measured periodically. each data point represents the mean of triplicate experiments. error bars indicate standard deviation from the mean. *, p < 0.05 by one-way anova. 46 • fine focus, vol 5 2019 figure 6. the a. baylyi tatc2 mutant is sensitive to detergents. the indicated strains were tested for detergent sensitivity using an efficiency of plating assay. overnight cultures of each strain serially diluted and plated onto lb agar or lb agar supplemented with 2% sds + 1 mm edta. each plate was incubated overnight at 30°c prior to imaging. shown is a representative experiment from triplicate trials. tatc in acinetobacter baylyi • 47 despite the fundamental nature of protein export systems, the important contributions of these pathways to bacterial physiology and growth is often underappreciated. since its discovery, much has been learned about the structural features and mechanistic details of how the tat pathway transports proteins. yet there are still key questions to be addressed. specifically, how do organisms with two tat pathways sort their cargo to the correct translocase? what is the benefit to having two tat pathways? using a. baylyi as a model system, we have started exploring these questions in detail. by disrupting the non-essential tat pathway by mutation of tatc2, we uncovered a role for this machine during stress conditions. when exposed to detergents, viability of the tatc2 mutant was decreased as compared to wild-type. this finding supports a role for the non-essential tat pathway in maintaining integrity of the bacterial cell envelope. we also found that the a. baylyi tatc2 mutant displays aberrant cellular morphology under high temperature stress. similar phenotypes have been observed in tat mutants of other bacterial species; notably, cells of e. coli tat mutants form long chains because the cell wall amidases important for proper cell division are tat substrates (7). our search of the a. baylyi genome did not reveal homologs to these particular amidases. given that e. coli only possesses a single tat machine, it is possible that there is a more diverse collection of tat substrates in organisms with two tat machines like a. baylyi. we believe that biochemical methods will be needed to identify the full suite of tat proteins in a. baylyi. while the role of protein export systems in bacterial pathogenesis has been well-studied, the importance of these machines with respect to growth in polymicrobial communities is poorly understood. by using a simple co-culture assay, we uncovered a role for the non-essential tat pathway when grown in competition with other bacterial species. interestingly, this phenotype was only evident when a. baylyi was grown in competition with p. putida, an organism that also encodes two distinct tat translocases. to explain this difference in ci among the pseudomonas strains, we considered the basic physiology of all three organisms. all three grow at a similar rate and under similar conditions. p. aeruginosa is a human pathogen but can also be isolated in environmental samples (40). neither p. fluorescens nor p. putida are human pathogens, however, both are associated with the plant root microbiome (9, 34). interestingly, when we examined the genomes of each species for tat gene homologs, we found that p. aeruginosa and p. fluorescens possess genes encoding a single tat pathway, while p. putida encodes genes for two separate and distinct tat pathways. perhaps the non-essential tat pathway of a. baylyi is important when facing competition from other dual-tat pathway bacteria, particularly when growing in complex microbial communities in soil. more experiments are needed to rigorously test this possibility. discussion acknowledgments the authors would like to thank elana galassi for assistance with the competition experiments. this work was supported by the national science foundation under grant no. 1615822 to nwr. mmc was supported by a summer fellowship from 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virulence: identification and characterization of tatdependent translocation proteins. phytopathology. 107(9):phyto-02-17-005 45. wu l, ize b, chanal a, quentin y, fichant g. 2000. bacterial twin-arginine signal peptidedependent protein translocation pathway: evolution and mechanism. j mol microbiol biotechnol. 2(2):179–89 50 • fine focus, vol 5 2019 fine focus 74 i fine focus antibacterial effects of bitter melon extract in combination with commonly prescribed antibiotics olivia mae ambrose, tiffany thanh mai nguyen, and emily m. nowicki department of natural sciences and mathematics, curry college, milton, ma 02186 keywords: antibiotics, antibacterial, bitter melon, synergy, antagonism manuscript received 31 january 2022; accepted 1 june 2022 vol 8 i 75 abstract: antibiotics are commonly overprescribed or taken incorrectly, which has resulted in an alarming increase of antibiotic-resistant bacteria. one potential solution to combat this problem is administering multiple antibiotics together to achieve antibiotic synergy; when two or more antibiotics work together to increase antibacterial efficacy. when considering potential synergistic combinations of antibiotics, one possibility is to utilize antibacterial plant extracts in addition to common antibiotics. the goal of our research was to compare the antibacterial properties of the chinese medicinal plant bitter melon (momordica charantia) and four common antibiotics alone or in combination with bitter melon against micrococcus luteus, pseudomonas putida, and escherichia coli. we hypothesized that combining the antibiotics with bitter melon extract would result in increased antibacterial effects against one or more bacterial strains. oil from dried bitter melon was prepared using the soxhlet extraction method. antibacterial properties of bitter melon extract and carbenicillin, streptomycin, colistin, and tetracycline alone or in combination with the extract were determined by performing disk diffusion assays. diameters of the resulting zones of inhibition for the two treatments were measured and analyzed for statistical significance by performing a two-tailed, paired sample t-test using rguroo. we found that bitter melon extract individually had little to no antibacterial effect against any of the organisms tested. interestingly however, combining bitter melon extract with common antibiotics resulted in synergistic effects in some cases, as well as one example of antibiotic antagonism. these results demonstrate that plant-derived extracts can enhance the antibacterial effects of commonly prescribed antibiotics if paired correctly. 76 i fine focus introduction the injudicious prescription and use of antibiotics creates selective pressure for bacteria to evolve resistance. according to the centers for disease control and prevention, each year over 2.8 million people are infected with antibiotic-resistant bacteria (4). in the united states alone, a person acquires an antibioticresistant infection every 11 seconds, while every 15 minutes someone dies from such an infection (4). this startling statistic can be attributed in part to the fact that nearly one-third of the antibiotics prescribed are not appropriate for the conditions being treated. the continuous misuse of readily available conventional antibiotics is proving to be a catalyst for the persistence, evolution and spread of antibiotic resistance. this severely limits the efficacy of antibiotics that were once used to treat life threatening bacterial infections and is now resulting in increased hospitalization rates and even deaths due to the rise in resistance. in turn, this leads to the requirement for heavier dosages and new antibiotic drugs to treat infections that were once readily treatable (4). health care professionals, scientists and the public must do their part in combating this growing problem as antibiotic resistance is evolving into a serious threat to the human healthcare field globally. one potential solution to combat the rising number of antibiotic-resistant strains is administering multiple antibiotics together to achieve antibacterial synergy. if an antibiotic demonstrates stronger antibacterial effects in combination with another antibiotic rather than when used alone, the combination treatment can be deemed synergistic. alternatively, antibiotic antagonism occurs when the overall antibiotic efficacy of two or more antibiotics administered together is decreased relative to the effects of each when used alone (1). combining extracts from plants with commonly prescribed antibiotics is one possible way to achieve antibacterial synergy. plant-derived compounds can exhibit a direct antibacterial activity as well as indirect activity as antibiotic resistance modifying compounds that, when combined with antibiotics, can potentially increase their efficacy (13). the organic compounds in plants are referred to as biologically active substances and include phenolic compounds, terpenes, and alkaloids. these substances can be isolated into crude extracts, some of which have prominent antibacterial activity (13). in this study, we tested the antibacterial properties of an organic extract prepared from the chinese medicinal plant bitter melon (momordica charantia) alone and in combination with commonly prescribed antibiotics. while this is not the first investigation of the antibacterial properties of bitter melon, to our knowledge this is the first study in which antibiotic synergy was tested between bitter melon and antibiotics. we hypothesized that bitter melon would itself have antibacterial properties against one or more organisms, as well as a synergistic effect with at least some of the common antibiotics tested. materials and methods bacterial strains and growth conditions escherichia coli, micrococcus luteus, and pseudomonas putida were tested in order to compare antibiotic efficacy against a variety of organisms. all of these organisms meet the current biosafety guidelines for curry college in that all are designated bsl-1, in addition to being clinically relevant organisms. bacterial cells were suspended in luria-bertani (lb, miller) or mueller-hinton broth and incubated with continuous shaking at 225 rpm, or grown on solid agar plates of the same media. e. coli and m. luteus were incubated at 37 ºc, while p. putida was grown at a temperature of 30 ºc. media preparation lb (miller) broth and agar (fisher) used for routine growth of bacterial strains was prepared according to the manufacturer’s instructions. mueller-hinton broth and agar (fisher) used for the antibacterial susceptibility assays was prepared according to the manufacturer’s instructions. broth was stored at room temperature (68-72 ºf), while plates were stored at 4 ºc. preparation of antibiotic solutions the following common antibiotics were tested against each organism: carbenicillin, streptomycin, colistin, and tetracycline. antibiotics were obtained from midwest vol 8 i 77 scientific (midsci) and prepared by dissolving the antibiotic powder in molecular grade water (invitrogen) or 70% ethanol (fisher) for tetracycline, and then aseptically passing the sample through a 0.22 μm filter (whatman) using a 10 ml syringe (fisher). antibiotic solutions were prepared in the following stock concentrations: carbenicillin (100 μg/ μl), streptomycin (10 μg/μl), colistin (30 μg/μl), and tetracycline (30 μg/μl). preparation of bitter melon extract oil from bitter melon (momordica charantia) was extracted using the soxhlet method, following a previously developed protocol (19). the bitter melon was purchased at kam man supermarket (quincy, ma), cut up, then dried in the oven at 200 ºf overnight. 12.45 g of bitter melon was extracted in 250 ml of 80% ethanol and subsequently concentrated by boiling off the ethanol to a final volume of 9 ml. disk diffusion assays disk diffusion assays were performed according to a previously published protocol (7). briefly, bacteria were grown in an overnight culture in the appropriate conditions for the bacteria as described. the next morning, cultures were diluted to an od600 of 0.05 and swabbed as a lawn on a mueller-hinton agar plate, swabbing the entire surface area of each plate three times to ensure complete coverage. then each antibiotic, bitter melon extract, or combination was tested by gently placing 6mm paper disks (thermoscientific oxoid blank disks) containing the appropriate antibiotic solution in the center of each plate using sterilized forceps. to prepare the disks, 20 μl of antibiotic stock solution, extract, or control (molecular grade water, 70% ethanol, or 80% ethanol) was aseptically pipetted onto each disk tested, and then allowed to dry in sterile conditions for approximately one hour. for disks with both common antibiotic and bitter melon extract, 20 μl of the common antibiotic solution (or water/70% ethanol as control) was first pipetted onto the disk, and then allowed to dry for ~1 hour. then, 20 μl of bitter melon extract (or 80% ethanol as control) was pipetted onto the disk, and the disk was dried for an additional ~1 hour. plates were wrapped individually in parafilm, and incubated overnight (~16-24 hours) at the preferred growth temperatures for each organism as described. four to six replicates were performed for each control, antibiotic, bitter melon extract, or combination tested against all three organisms. data analysis after overnight growth, the diameter of the zone of inhibition (zoi), or area resulting in no observable bacterial growth, was measured in millimeters using a ruler and recorded. average (mean) zoi diameters were calculated for all replicates along with standard error of the mean to analyze variance in the data. data were then categorized by each different organism and further by each common antibiotic tested. the zoi measurements for each replicate of the common antibiotic alone was included, along with the measurements for the same common antibiotic tested in combination with bitter melon extract. each spreadsheet was uploaded into the statistical analysis program rguroo (22). a mean inference analysis between two populations (with population 1 as the common antibiotic zoi diameters and population 2 as the same common antibiotic tested with bitter melon extract) was selected in order to perform a two-tailed, paired sample t-test evaluating the difference between each population mean and generate p-values. unequal variance was assumed when performing all t-tests. tests for normality of each dataset were also performed. results: bitter melon extract alone has little to no effect at preventing bacterial growth since our research aimed to test whether or not bitter melon extract enhances the antibacterial properties of commonly prescribed antibiotics, we first tested the efficacy of four different common antibiotics (carbenicillin, streptomycin, colistin, and tetracycline) at preventing growth of e. coli, p. putida, and m. luteus by setting up disk diffusion assays. for each organism tested, four to six replicates were performed. controls were also tested for all three organisms by soaking disks in either molecular grade water or 70% ethanol. none of the control disks produced a zone of inhibition. table 1 shows the average zoi diameter 78 i fine focus measurements for all antibiotics tested for e. coli, p. putida, and m. luteus, respectively. unsurprisingly, while different antibiotics demonstrated varying degrees of efficacy against each organism, all were at least somewhat effective at preventing growth and resulted in a zoi surrounding the antibiotic-soaked paper disk. next, we performed disk diffusion assays on the same three organisms using disks soaked with bitter melon extract or control disks soaked in 80% ethanol, the solvent used to prepare the bitter melon extract. bitter melon extract had no effect at preventing growth of either e. coli or p. putida, since no zoi was found around the paper disks (reported as “0 mm” in table 1). bitter melon had only a small effect at preventing the growth of m. luteus, resulting in an average zoi diameter of 5 mm (table 1). all commonly prescribed antibiotics tested against m. luteus were found to produce a substantially greater average zoi diameter than bitter melon, ranging from 22.1 mm for colistin to 47.25 mm for tetracycline (table 1). as before, none of the control disks soaked in 80% ethanol as the control for bitter melon produced a zoi. e. coli p. putida m. luteus antibiotics: average zol diameter (mm): standard error of the mean: average zol diameter (mm): standard error of the mean: average zol diameter (mm): standard error of the mean: carbenicillin streptomycin colistin tetracycline bitter melon 31.25 18.77 21.4 40 0 1.4930 1.2925 1.7709 2.1909 0 25.2 18.15 19.6 23.35 0 1.5979 0.2872 0.9055 1.0874 0 41.45 33.45 22.1 47.25 5 1.3525 1.8715 0.1291 2.2867 2.8868 bitter melon extract used in combination with commonly prescribed antibiotics influences their antibacterial efficacy although bitter melon extract itself had minimal to no effect against the three bacterial strains tested, we decided to test whether bitter melon extract could enhance the antibacterial properties of any of the four commonly prescribed antibiotics when used in combination. disks for the combination disk diffusion assays were prepared by first soaking with 20 μl of the common antibiotic solution (carbenicillin, streptomycin, colistin, and tetracycline). once dry, the same disk was then soaked in an additional 20 μl of bitter melon extract and set out to dry completely in a sterile environment. control disks were also prepared and consistently resulted in no zoi. table 1. zone of inhibition measurements for antibiotics and bitter melon extract tested alone against e. coli, p. putida, and m. luteus. the zone of inhibition (zoi) diameter measurements (in mm) for each common antibiotic tested (carbenicillin, streptomycin, colistin, and tetracycline, respectively) or for bitter melon extract are reported here. for each antimicrobial substance tested, a total of four to six replicates were performed. the average zone of inhibition diameter was calculated from all replicates performed, and the standard error of the mean was calculated as a relative measure of how consistent the measurements were. vol 8 i 79 for e. coli, all four commonly prescribed antibiotics tested in combination with bitter melon extract showed enhanced ability to prevent bacterial growth. the most striking difference was for streptomycin, as the zoi diameter more than doubled with the combination resulting in an average zoi of 40 mm relative to the 18.76 mm observed for streptomycin alone (figure 1). interestingly, bitter melon extract used in combination with commonly prescribed antibiotics did not always result in enhanced antibacterial properties. for p. putida, bitter melon extract actually decreased the average zoi diameter from 25.2 mm to 19.55 mm when tested in combination with carbenicillin (figure 2). the antibiotic effects of the other three antibiotics against p. putida, however, were enhanced when combined with bitter melon extract. the most striking enhancement was for colistin, which increased from an average zoi diameter of 19.6 mm for colistin alone compared to an average of 33.65 mm for the combination (figure 2). when tested for efficacy at inhibiting m. luteus growth, bitter melon extract combined with carbenicillin showed only a modest increase in the average zoi diameter from 41.45 mm to 49 mm (figure 3). all other combinations resulted in either the same effect against m. luteus growth or a decreased effect against m. luteus growth (figure 3). antibacterial synergy between bitter melon extract and some commonly prescribed antibiotics is statistically significant since our data suggested that bitter melon extract can influence the antibacterial properties of the commonly prescribed antibiotics tested, our final goal was to determine whether the differences observed between the average zoi for the common antibiotics alone compared to those for the combinations with bitter melon extract were statistically significant. to determine this, the raw data derived from the disk diffusion assay zoi measurements was compiled in an excel spreadsheet and categorized by each bacterial species and common antibiotic. the data for each replicate was included, and each spreadsheet was uploaded into the figure 1. e. coli average zone of inhibition statistical analysis between common antibiotics alone and in combination with bitter melon extract. the average diameter (in mm) of the zones of inhibition produced in the disk diffusion assays by each common antibiotic or common antibiotic combined with bitter melon extract are represented by the black and grey columns, respectively. average diameters were calculated from the four to six replicates performed for each assay. error bars represent the + and values for standard error of the mean. a t-test was performed (with an assumption of unequal variance) to calculate the difference between the mean values for common antibiotic tested alone or in combination with bitter melon extract; those resulting in statistically significant p-values are indicated with asterisks (* indicates a p-value of <0.05, and *** indicates a p-value of <0.001). the t-test for carbenicillin resulted in a p-value of 0.0295; for streptomycin, 0.0004; for colistin, 0.0002; and for tetracycline, 0.3106. 80 i fine focus figure 2. p. putida average zone of inhibition statistical analysis between common antibiotics alone and in combination with bitter melon extract. | the average diameter (in mm) of the zones of inhibition produced in the disk diffusion assays by each common antibiotic or common antibiotic combined with bitter melon extract are represented by the black and grey columns, respectively. average diameters were calculated from the four to six replicates performed for each assay. error bars represent the + and values for standard error of the mean. a t-test was performed (with an assumption of unequal variance) to calculate the difference between the mean values for common antibiotic tested alone or in combination with bitter melon extract; those resulting in statistically significant p-values are indicated with asterisks (* indicates a p-value of <0.05, and *** indicates a p-value of <0.001). the t-test for carbenicillin resulted in a p-value of 0.0296; for streptomycin, 0.1386; for colistin, 0.0001; and for tetracycline, 0.0212. figure 3. m. luteus average zone of inhibition statistical analysis between common antibiotics alone and in combination with bitter melon extract. the average diameter (in mm) of the zones of inhibition produced in the disk diffusion assays by each common antibiotic or common antibiotic combined with bitter melon extract are represented by the black and grey columns, respectively. average diameters were calculated from the four to six replicates performed for each assay. error bars represent the + and values for standard error of the mean. a t-test was performed (with an assumption of unequal variance) to calculate the difference between the mean values for common antibiotic tested alone or in combination with bitter melon extract; those resulting in statistically significant p-values are indicated with asterisks (** indicates a p-value of <0.01). the t-test for carbenicillin resulted in a p-value of 0.0068; for streptomycin, 0.1002; for colistin, 0.1284; and for tetracycline, 0.9333. vol 8 i 81 statistical analysis program rguroo (22). a mean inference analysis between two populations (average common antibiotic zoi diameter relative to the average diameter for each tested with bitter melon extract) was used to perform a t-test evaluating the difference between each population mean and generate p-values (figures 1, 2, and 3). for all organisms and antibiotics tested, data was highly repeatable with relatively low variance as measured by standard error of the mean (table 1, figures 1, 2, and 3). despite the low observed variance, t-tests were performed with an assumption of unequal variance, which allowed for a more rigorous comparison. a test for normality of each dataset was also performed in rguroo, with each set of replicates found to be normally distributed (22). the data for e. coli show that there is a statistically significant difference between the mean zoi diameter for the common antibiotics carbenicillin, streptomycin, and colistin alone relative to the mean zoi diameter for each of these combined with bitter melon extract, with p-values of 0.0295, 0.0004, and 0.0001, respectively (figure 1). for p. putida, bitter melon extract was found to significantly increase the mean zoi diameter of colistin and tetracycline relative to the common antibiotics alone, with p-values of 0.0001 and 0.0212, respectively (figure 2). on the other hand, bitter melon extract combined with carbenicillin when tested against p. putida resulted in a significantly smaller average zoi diameter compared to carbenicillin alone, with a p-value of 0.0296 (figure 2). for m. luteus, bitter melon extract only significantly increased the mean zoi diameter when combined carbenicillin, with a p-value of 0.0068 (figure 3). discussion: the extensive use of antibiotics in healthcare has led to a dramatic rise in antibiotic resistance and requires immediate action. the development and discovery of new drugs to treat such resistant infections has risen to be the top priority of the world health organization (who) (23, 25). it is critical to test new antibiotic combinations in order to treat a wider range of pathogens. the goal of our research was to test the antibacterial properties of bitter melon extract alone and in combination with four commonly prescribed antibiotics (carbenicillin, streptomycin, colistin, and tetracycline) against three different bacterial species: e. coli, p. putida, and m. luteus. the disk diffusion data show that bitter melon alone had little to no antibacterial properties when tested against the bacteria individually (table 1). however, when tested in combination with the common antibiotics, there were in some cases significant effects on the efficacy of the antibacterial properties (figures 1, 2, and 3). the data primarily showed a statistically significant enhancement in the antibacterial activity of the common antibiotics (i.e. antibacterial synergy), but also one example of antibacterial antagonism. in this study, we aimed to test a broad range of organisms to explore differences in the potential synergistic or antagonistic effects of bitter melon extract. although the three organisms selected are all biosafety level 1 organisms, all have important clinical relevance. e. coli is a gram-negative bacterium that is resistant to many prescribed antibiotics (18). one study found that the rate of adaptive mutations in e. coli is about 10-5 per genome per generation, which is 1000 times higher than previous estimates (18). these antibiotic-resistant e. coli strains are passed on, often carrying multiple drug resistant plasmids that can easily transfer to other organisms (20). m. luteus is a gram-positive organism that is detected as a commensal organism in the mucous membrane as well as in soil and water (27). although m. luteus is described as low virulence, the bacterium can become pathogenic under certain conditions (5). p. putida is found in soil and water and has been reported as an opportunistic pathogen that can occasionally cause hospital-acquired infections (11, 12). strains of this species are known to exhibit resistance to many antibiotics through the presence of plasmids that encode antibiotic resistance factors (12). carbenicillin, streptomycin, colistin, and tetracycline were the common antibiotics chosen to test in our study. carbenicillin is an antibiotic among the semisynthetic penicillin group and is used to treat illnesses such as bladder infections. it has gram-negative coverage but limited gram-positive coverage (17). streptomycin is an antibiotic that is commonly used to treat aerobic gram-negative bacterial infections such as tuberculosis. the original wide spectrum of activity exhibited by streptomycin against both gram-negative and gram-positive bacteria has severely decreased due to the rise of antibiotic resistance; commonly 82 i fine focus to enterobacteriaceae such as e. coli (24). colistin is a polymyxin “last line” antibiotic used to treat infections caused by multi-drug resistant gram-negative bacteria such as p. aeruginosa. being a “last line” antibiotic, this means there are no other novel antibiotics to treat such infections and research for an alternative treatment is crucial (16). tetracycline is a protein synthesis inhibitor antibiotic that is used to manage and treat bacterial infections. originally tetracycline showed antibacterial activity against most medically relevant aerobic and anaerobic bacteria, both gram-positive and gram-negative (21). many plants used in chinese traditional medicine have been shown to have antibacterial properties. in one study, essential oils prepared from 21 different plants were all found to have at least some degree of antibacterial activity against 10 pseudomonas species tested, with oil from cinnamomum zeylanicum displaying the highest inhibitory effect (9). in another study, berberine, an alkaloid derived from goldenseal (hydrastis canadensis) was itself found to have antibacterial activity against staphylococcus aureus. flavonoids isolated from a different part of the plant were found to have a synergistic effect on berberine efficacy despite having no inherent antibacterial effect when used individually (8). previous studies have explored the various biological activities of bitter melon such as antiviral, antioxidant, and antibacterial components (3, 6, 15, 26). for example, one study determined that essential oils of bitter melon have significant inhibitory effects on s. aureus growth, as well as significant antibacterial activity against several other bacteria including pseudomonas multocida, salmonella typhi, and staphylococcus epidermidis(6). bitter melon pulp extract also was proven to have a broader spectrum of antibacterial activity against e. coli, staphylococcus, and pseudomonas (6). in addition to plant-derived compounds themselves exhibiting antibacterial properties, many plant extracts have been shown to have synergistic effects with antibiotics, thus warranting our research (8, 13). as we expected, all commonly prescribed antibiotics tested had at least some effect at inhibiting the growth of each bacterial species tested (table 1). it was also unsurprising that the efficacy of each antibiotic tested varied depending on the organism. for example, zois for p. putida were typically smaller for all antibiotics tested. pseudomonas species are known for their high degree of intrinsic antibacterial resistance, which can account for this observation (14). bitter melon extract tested alone had no effect against either e. coli or p. putida, and only a very minimal effect against m. luteus (table 1). we found this result surprising given that previous research has shown antibacterial activity of bitter melon against some microorganisms, including e. coli and pseudomonas species (6). differences in bitter melon’s antibacterial activity however, have been observed depending on which part of the plant was tested (i.e. seeds, leaves, fruit), and whether mature or immature plants were tested (15, 26). it is thus possible that by using the entire plant, antibacterial properties were diluted out and not detected in our assays. it is also possible that differences in the methodology and solvent used for extraction of oils as well as the amount of plant used and extent to which the oils were concentrated account for the lack of antibacterial activity we observed. although bitter melon extract had little to no antibacterial activity against the strains tested when applied individually to paper disks, we nonetheless decided to test whether bitter melon could enhance the antibacterial activity of any of the four commonly prescribed antibiotics tested (figures 1, 2, and 3). while most studies that assay for synergistic combinations of antibacterials test two or more compounds that have antibacterial activity individually, a study by junio et al. (2011) found that some compounds can have synergistic effects on other antibacterials without themselves having any direct antibacterial properties (8). interestingly, our results do suggest that bitter melon extract can have indirect effects on some of the antibiotics tested, when used against specific organisms. for e. coli, bitter melon extract significantly increased the zoi size when used in combination with carbenicillin, streptomycin, and colistin, compared to each of these antibiotics administered alone (figure 1). for p. putida, statistically significant enhancement in zoi size for antibiotics used in combination with bitter melon extract was seen for colistin and tetracycline (figure 2). for m. luteus, significant enhancement of growth inhibition was seen when bitter melon extract was used in combination with carbenicillin, compared to carbenicillin alone (figure 3). this suggests that bitter melon extract has bioactive components that work synergistically in combination with vol 8 i 83 commonly prescribed antibiotics, when used against certain microorganisms. of note, we did find one potentially antagonistic combination, as carbenicillin combined with bitter melon extract resulted in less inhibition of p. putida growth than carbenicillin alone (figure 2). our finding of significant antibiotic synergy between colistin and bitter melon extract when used against p. putida is of particular interest. colistin is commonly used as a “last line” antibiotic to treat multidrugresistant pseudomonas aeruginosa infections, but resistance to colistin has been steadily increasing over the years (10). if our results are found to be repeatable when tested against p. aeruginosa, this synergistic combination could provide another potential avenue for treatment of these deadly infections. testing for synergistic antibiotic combinations to treat p. aeruginosa is already underway. for example, the combination of streptomycin and cefadroxil were reported to be synergistic against pseudomonas aeruginosa isolates in vitro when compared to the effects of each of these antibiotics alone (2). further testing will be the key to determining which combinations work best in vivo, when treating patients. on the other hand, our result of an antagonistic effect of bitter melon extract when used with carbenicillin relative to carbenicillin used alone against p. putida is also noteworthy. since carbenicillin is a commonly prescribed antibiotic for gram-negative pathogens such as pseudomonas species, it is important to be aware of the limitations of combination treatments and have a thorough understanding of both synergistic and antagonistic combinations in order to optimize treatment outcomes (1, 17). antibiotic antagonism is not unexpected when combining antibiotics. for example, murray and colleagues found that treating p. aeruginosa first with low concentrations of polymyxin b resulted in its decreased antibiotic susceptibility when later exposed to gentamicin, neomycin, tobramycin, or ciprofloxacin (14). it is therefore critical that combinations of antibiotics are tested prior to use either combinatorially or subsequently to avoid the possibility of antagonistic effects such as this. our research presents a possible solution to combat the growing problem of antibiotic resistance. although the results of our study are preliminary, they contribute to the foundational understanding necessary for future advancements in furthering the discovery of synergistic antibiotic combinations. future directions of this work include repeating assays using isolated and purified components of bitter melon. bitter melon has many phytochemicals with antioxidant, anti-inflammatory, and antibacterial properties (3, 15). further research on the efficacy of bitter melon’s antibacterial properties when used either alone or combinatorially should thus be done through fractionation and chromatography assays, as well as minimum inhibitory concentration analysis (mic). ultimately, our findings will need to be tested using in vivo models and with physiologically relevant concentrations of antibiotics and purified bitter melon components. in conclusion, the findings of this research give one potential solution to help combat the drastic increase in antibioticresistant pathogens that are threatening our global healthcare. acknowledgements we would like to thank dr. jennifer mcnally for her guidance on the statistical analysis and for sharing institutional access to rguroo with us. we would like to thank dr. jennifer mcnally and dr. adam hilterbrand for critical review of this manuscript. this research was performed in compliance with institutional policies relating to biosafety level. 84 i fine focus references 1. acar, j. f. 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science, technology, engineering, and mathematics (stem) faculty. peer mentoring is not widely adopted in graduate professional development, yet incorporating this approach can better facilitate the transition from graduate student to faculty member. using evidence from experience as peer mentors (2011-2017), we examine established characteristics of peer mentoring and evaluate their strengths in the context of a future faculty professional development program. peer mentors coached mentees by sharing common experiences related to teaching and learning, provided a safe space for mentees to discuss their experiences, and acted as a liaison between mentees and faculty advisors. these benefits translate into increased competency for future faculty to engage in research, teaching, and mentoring. a call for improved graduate professional development recognition that future faculty preparation is insufficient to meet the changing expectations of education has led to a re-envisioning of professional development programs for graduate students aiming at careers in academia (austin 2002; cooper et al. 2015). the focus of many existing professional development programs is preparing future faculty to implement and advance effective teaching practices (pfund et al. 2009). however, few of these programs emphasize mentoring in their goals (wulff and austin 2004; ebert-may et al. 2011). given that new faculty are expected to fulfill diverse roles that include mentoring as well as teaching and research (austin 2002), it is essential that professional development programs provide opportunities for future faculty to develop mentoring skills (schussler et al. 2015). many professional development programs employ leadership structures led by faculty advisors, which are an integral part of graduate education (grant-vallone and ensher 2000). traditional mentoring is generally characterized by a one-way flow of expertise and skills from mentor to mentee (mcmanus and russell 2008). alternatively, peer mentoring is by nature a reciprocal relationship and thus offers the potential for additional mentorship owing to a similar hierarchical level, complementary knowledge and skills, and reciprocity between peer mentors and mentees (mcdougall and beattie 1997; holland et al. 2012). for peer mentors, immersion in the academic process allows further development as independent scholars (campa et al. 2000). 16 • fine focus, vol 5 2019 including peer mentoring relationships in professional development infrastructure can provide unique benefits to mentees and peer mentors. to demonstrate these benefits, we examine the established characteristics of peer mentoring and evaluate their strengths in the context of future faculty professional development. evidence for the efficacy of peer mentoring in future faculty preparation is provided via reflections collected from mentees and peer mentors (i.e., the authors), who are now early-career faculty and instructional consultants contributing to teaching and learning at academic institutions. fast: a model professional development program the future academic scholars in teaching (fast) program is a year-long high engagement professional development program that complements the efforts of the center for the integration of research, teaching and learning (cirtl) network (www.cirtl.net). the fast program has been in existence for 13 years at michigan state university. throughout the fast program, a selected cohort of 10-14 stem graduate students participated in workshops on pedagogy and instructional design, sessions on professional development for academic careers, and completed a teaching-as-research project in a course within their discipline (vergara et al. 2014). participants are mentored by faculty advisors and a graduate student that has previously completed the program (the peer mentor). the peer mentor acts as a liaison between the faculty advisors and mentees and leads a reading group attended only by peer mentors and mentees, that serves as a time to discuss literature on teaching and learning, and to address successes and challenges in teaching-as-research project development. throughout the program, peer mentors gain practice in building a supportive learning community, providing constructive criticism, and tailoring mentoring to the specific instrumental (i.e., logistical and academic) and psychosocial (i.e., emotional and interpersonal) support needed by individual mentees. as a result, peer mentoring benefits the mentees as well as the peer mentors themselves (figure 1). these gains contribute to peer mentors’ development as future stem professionals, particularly as the skills gained through peer mentoring are not often included in formal graduate education (schussler et al. 2015). benefits of peer mentoring for the mentee peer mentoring relationships provide psychosocial support that traditional mentoring relationships are unlikely to provide (table 1; grant-vallone and ensher 2000). research on peer mentoring has shown that mentees are more likely to display vulnerability when interacting with peer mentors than with traditional mentors (mcmanus and russell 2008). the fast reading group, attended only by mentees and the peer mentor, provided a space in which mentees were comfortable displaying vulnerability and taking risks (table 1). interviews of past program participants have indicated a larger degree of comfort sharing insecurities compared to meetings when faculty advisors were present. for instance, one participant reflected: it’s kind of less pressure to just say how you’re feeling about things to group of your peers... one of the more impactful parts of the [reading group] was just having a forum, a venue to talk about your experiences with people who are experiencing similar things, or have experienced similar things. figure 1. diagram illustrating the roles and relationships between peer mentors, mentees, and faculty mentors in future faculty professional development. perspective • 17 as both mentees and peer mentor are new to the scholarship of teaching and learning, there is reduced fear of judgment when communicating challenges of their experience (colvin and ashman 2010). the peer mentor has often encountered and resolved similar challenges either personally or in their interactions with other participants. the empathy inherent in this relationship encourages mentees to display vulnerability and in turn allows the peer mentor to provide encouragement and shared experience (table 1). evidence that peer mentoring can increase the confidence of mentees is demonstrated by a participant that noted: [the peer mentor] definitely provided that… i personally need that kind of encouragement to know, ‘okay, you’re seeing what i’m doing. what i’m doing is okay.’ otherwise i start to second-guess myself. the vulnerability and empathy displayed between peer mentors and mentees gives rise to several instrumental support roles (table 1; colvin and ashman 2010). by working closely with program participants in a comfortable setting, the peer mentor can monitor mentees’ progress closely and relay challenges to faculty mentors. in this way, the peer mentor acts as a liaison between the two groups (figure 1). the faculty mentoring team can then resolve issues on an individual basis or shift the focus of future group meetings to address common challenges. small hierarchical differences between the peer mentor and mentee allows the peer mentor to fill a unique role as a learning coach (colvin and ashman 2010). while traditional mentoring by faculty advisors provides a learning coach role through the sharing of expertise, the peer mentor’s role as a learning coach is more commonly embodied by sharing recent experience (figure 1). for instance, the peer mentor in the fast program has often observed a specific strategy for success through interactions with their own cohort of participants and can help mentees overcome a problem by using this strategy in a similar context. however, it is common for mentees to encounter issues that are outside the expertise of the peer mentor. in these cases, the peer mentor can act as a connecting link with faculty advisors (sanft et al. 2008). this role often involves consulting with a faculty advisor with relevant expertise who then works with the mentee to address topics such as assessment of student learning, innovative instructional techniques, experimental design, or data analysis. benefits of peer mentoring for the mentor given the increasingly interdisciplinary nature of education and research in academia (adams 2007), developing skills to engage diverse audiences in a common dialogue is essential. serving as peer mentors in the fast program contributed to our ability and confidence to work with a diverse academic community by providing a psychologically safe environment where the fear of misguidance would not have career-ending consequences (mcmanus and russell 2008). opportunities for us to explore our own developmental needs and having nonjudgmental and supportive feedback was instrumental in facilitating our growth as mentors. the expectations for us as peer mentors were clearly outlined at the onset of the mentoring relationship, which was a crucial component in our development of self-efficacy (hall et al. 2008). further, positive reinforcement provided by both mentees and faculty advisors for our willingness to pass along information that we had learned to others was personally rewarding (sensu mcmanus and russell 2008, colvin and ashman 2010). having a safe space for peer mentoring to take place helped to foster both professional and personal empathy for our mentees. the ability to engage in dialogue with a diverse group of peers and express empathy was significant given that survey responses from mentees suggest that they found our peer-mentoring relationship to be more valuable for psychosocial support than technical or logistical support related to teaching and learning. these findings are consistent with empirical research on peer mentoring which have shown that psychosocial support in peer-mentoring relationships is a unique characteristic that distinguishes them from traditional mentoring (kram and isabella 1985, grant-valione and ensher 2000). collectively, these experiences have contributed to our ability to bridge differing perspectives (e.g., vocabulary, theoretical framework) and work toward a common goal, a skill that has been critical in the transition from graduate student to professionals. as peer mentors, we gained a depth of knowledge related to teaching and learning, leadership skills to facilitate instructional improvements, and experience working with university administrators on programmatic priorities. through this process, we not only became better equipped to contribute to the curricular goals of our future institutions but also to work across multiple administrative levels to act as agents of change (sensu healey 2012). as a 18 • fine focus, vol 5 2019 result, we have been able to transfer many of these skills to a variety of research and education settings, such as advising teaching assistants and mentoring undergraduate and graduate researchers. table 1. peer mentoring offers unique benefits to professional development programs that supplement traditional mentoring by faculty. experiences from the future academic scholars in teaching (fast) program provide evidence to support for the inclusion of peer mentoring as part of future faculty professional development. perspective • 19 mentoring is a critical skill in academia, and peer mentoring in the fast program provided a formal opportunity to train as a mentor that would not be available in most graduate programs (schussler et al. 2015). the unique role and support structure demonstrated by peer mentors in the fast program represents a model that is transferable to a wide range of graduate professional development programs across disciplinary and institutional boundaries. given increasing recognition for the need to prepare future faculty as independent teacher-scholars (cooper et al. 2015) and the potential for peer mentoring opportunities to facilitate the transition from mentee to mentor, we advocate that formal peer mentoring opportunities should be built into existing professional development frameworks. we thank the fast steering committee members and numerous fast fellows for their guidance and inspiration. the fast program was supported by the graduate school at michigan state university and the national science foundation (nsf-0717768). institutional review board (irb) authorization was obtained for all data collection procedures (irb x06-481/app# i025197). we also thank rique campa and diane ebert-may for helpful comments throughout the writing process. acknowledgmentsconclusion 20 • fine focus, vol 5 2019 1. adams, j. u. (2007). interdisciplinary research: building bridges, finding solutions. science, 318, 13151318. doi: 10.1126/science.opms.r0700032 austin, a. e. (2002). preparing the next generation of faculty: graduate school as socialization to the academic career. journal of higher education, 73(1), 94-122. 2. campa, h. iii, raymer, d. f., & millenbah, k. f. (2000). models for preparing graduate students in natural resources management to be educators in agencies and academia. in m. r. ryan & w. b. kurtz, (eds.), proceedings of the third biennial conference on university education in natural resources (pp. 28-32). columbia, mo: university of missouri-columbia. 3. colvin, j. w., & ashman, m. (2010). roles, risks, and benefits of peer mentoring relationships in higher education. mentoring & tutoring: partnership in learning, 18(2), 121-134. doi: 10.1080/13611261003678879 4. cooper, m. m., caballero, m. d., ebert-may, d., fata-hartley, c. l., jardeleza, s. e., krajcik, j. s., laverty, j. t., matz, r. l., posey, l. a., & underwood, s. m. (2015). challenge faculty to transform stem learning. science, 350(6258), 281-282. doi: 10.1126/science.aab0933 5. ebert-may, d., derting, t. l., hodder, j., momsen, j. l., long, t. m., & jardeleza, s. e. (2011). what we say is not what we do: effective evaluation of faculty development programs. bioscience, 61(7), 550558. doi: 10.1525/bio.2011.61.7.9 6. grant-vallone, e.j., & ensher, e.a. (2000). effects of peer mentoring on types of mentor support, program satisfaction and graduate student stress: a dyadic perspective. journal of college student development, 41(6), 637-642. 7. hall, k.m., draper, r.j., smith, l.k., & bullough, r.v. (2008). more than a place to teach: exploring the perceptions of the roles and responsibilities of mentor teachers. mentoring & tutoring: partnership in learning, 16(3), 328–345. 8. healey, m., cook-sather, a., gerum, n. & yanagida, k. (2012). students as change agents. in international society for the scholarship of teaching and learning conference (pp. 24-27). 9. holland, j. m., major, d. a., & orvis, k. a. (2012). understanding how peer mentoring and capitalization link stem students to their majors. career development quarterly, 60, 343-354. doi: 10.1002/j.2161-0045.2012.00026.x 10. mcdougall, m., & beattie, r. s. (1997). peer mentoring at work: the nature and outcomes of nonhierarchical developmental relationships. management learning, 28(4), 423-437. 11. mcmanus, s. e., & russell, j. (2008). peer mentoring relationships. in b. r. ragins & k. e. kram (eds.), the handbook of mentoring at work: theory, research, and practice (pp. 273-298). thousand oaks, ca: sage publications, inc. 12. pfund, c., miller, b. k., bruns, p., chang, a., ebert-may, d., fagen, a., gentile, j., gossens, s., khan, i., labov, j., pribbenow, c., susman, m., tong, l., wright, r., wood, w. b., yuan, r., & handelsman, j. (2009). summer institute to improve university science teaching. science, 324(5926), 47071. doi: 10.1126/science.1170015 13. sanft, m., jensen, m., & mcmurray, e. (2008). peer mentor companion. boston, ma: houghton mifflin. 14. schussler, e. e., read, q., marbach-ad, g., miller, k., & ferzli, m. (2015). preparing biology graduate teaching assistants for their roles as instructors: an assessment of institutional approaches. cbe life sciences education, 14, 1-11. doi: 10.1187/cbe.1411-0196 15. vergara, c. e., urban-lurain, m., campa, h. iii, cheruvelil, k. s., ebert-may, d., fata-hartley, c., & johnston, k. (2014). fast-future academic scholars in teaching: a high-engagement development program for future stem faculty. innovative higher education, 39, 93-107. doi: 10.1007/s10755013-9265-0 16. wulff, d.h., & austin, a.e. (2004). paths to the professoriate: strategies for enriching the preparation of future faculty. hoboken, nj: jossey-bass. references perspective • 21 volume seven | 25 nylon oxygen barrier tubing reduces biofouling in beer draught lines parker heger and andrew r. russell* department of biology northern state university aberdeen, sd 57401 manuscript received 1 february, 2021; accepted 4 august 2021 *corresponding author: andrew.russell@northern.edu keywords: biofilm; biofouling; beer spoilage; nylon oxygen barrier; beer draught line; beverage tubing 26 | fine focus abstract beer draught lines are frequently contaminated with biofilm-forming microorganisms, which forces retailers to spend considerable time and money cleaning and replacing lines. in light of this financial burden, draught tubing composition was examined for its role in the prevention of biofouling in beer lines. three types of draught tubing vinyl, polyethylene, and nylon barrier were inoculated with a combination of biofilm-forming microorganisms (hafnia paralvei, raoultella planticola, pediococcus damnosus and saccharomyces cerevisiae) and used to simulate a bar environment for sixteen weeks. following simulation, the degree of biofouling in each draught line was determined by spectrophotometry and microscopy. absorption values and fluorescence images showed that nylon barrier tubing was superior to the other lines at resisting biofilm maturation.these results suggest that tubing composition plays a significant role in the prevention of biofilm formation in beer draught lines and supports the adoption of nylon barrier tubing as an effective strategy against biofouling in a variety of applications. volume seven | 27 introduction biofouling creates a considerable financial burden at various levels of the beer industry. arguably, the most challenging environment to keep clean is found in beer draught lines where yeast and spoilage organisms readily adhere, altering the taste, aroma, and quality of the beer. although the deleterious effects of microbial biofilms have been recognized for several decades, relatively little scientific research has been applied to combat these issues, and most methods focus on routine maintenance of beer draught lines. for example, the brewers association recommends cleaning draught lines, at a minimum, every two weeks. the cleaning solution should be recirculated through the line for at least 15 minutes at a velocity of two gallons per minute. additionally, all draught lines should be replaced annually as, despite regular and consistent cleaning, biofilms can still form (1). beer spoilage can be caused by a variety of microbial species. perhaps the most notorious beer contaminants are lactic acid bacteria such as lactobacillus brevis, and pediococcus damnosus. these species are able to survive the harsh conditions of the brewing environment due to the presence of genes for hop resistance and polysaccharide production (2; 25). while the biofilm formation of most lactic acid bacteria is relatively weak, their persistence and prevalence is enhanced by the presence of ubiquitous environmental species that have also been isolated from brewing equipment (14). many isolates, such as stenotrophomonas maltophilia, pseudomonas putida, and citrobacteri freundii, are known to be prolific biofilm producers and may aid lactic acid bacteria as secondary colonizers (11; 24; 28). p. damnosus was recently isolated in our lab as a component of a multispecies biofilm in beer draught tubing from a local brewery (russell et al, unpublished). notably, a human commensal organism, halfnia paralvei, a common soil inhabitant, raoultella planticola, and brewer’s yeast, saccharomyces cerevisiae were the other predominant species in this biofilm. as many retailers and distributers know, standard draught line cleaning procedures are very time-consuming, expensive, and only moderately effective (20). although proper maintenance can delay biofilm growth and formation, cleaning of beer draught lines needs to be done consistently, as biofilms can form quickly and are more difficult to remove when mature due to the vast and multi-faceted defense mechanisms they exhibit (5). consequently, alternative treatments have been developed for beer draught lines and other industrial applications that utilize enzymatic digestion (10; 16; 29; 31). these approaches are moderately effective; however, they are often marketed as an additional step to a routine maintenance schedule, adding unnecessary time and expense. recently, the medical and industrial fields have shifted their focus to proactive methods of biofilm prevention rather than reactive treatment options. for example, a number of natural and artificial chemicals have been shown to possess antibiofilm properties that block quorum sensing (9), disperse extracellular polysaccharide (19), inhibit curli biosynthesis (6), or alter membrane permeability (13). a variety of studies also have been published in recent years describing options for making surfaces more resistant to biofouling, including development of novel materials (17; 18), improvement of manufacturing methods (15; 32), and creation of postproduction coatings (7; 8; 22). likewise, beer-draught line manufacturers have begun to experiment with various materials and manufacturing procedures to create lines that are more resistant to biofilm formation and, consequently, require less routine maintenance. one such product, known as gen-x (valpar), utilizes both a novel manufacturing method and addition of a nylon barrier layer to stop oxygen permeation and preserve beverage characteristics. consequently, gen-x tubing promises to ensure draught quality and hinder microbial growth 2-3 times more effectively than other alternatives (30). while this and other new proprietary compositions show promise at combatting biofouling, no peer-reviewed research has been done to characterize the effectiveness of these lines. our project aims to test the qualitative and quantitative effect that various tubing compositions have on biofilm formation in beer draught lines. we hypothesized that nylon barrier tubing would exhibit reduced biofouling compared to other tubing materials due to its potential to reduce oxygen permeation. to test this, three beer draught lines—vinyl, polyethylene, and nylon barrier (gen-x)—were inoculated with a microbial cocktail consisting of hafnia paralvei, 28 | fine focus raoultella planticola, pediococcus damnosus, and saccharomyces cerevisiae liquid cultures. following inoculation, the lines were connected to a keg-draught system from which beer was regularly dispensed for sixteen weeks to simulate a bar environment. next, spectrophotometry and microscopy were used to determine the ability of the different draught tubing compositions to resist biofouling. results from our experiments consistently showed that nylon barrier tubing was significantly better at resisting biofilm growth than traditional vinyl or polyethylene lines. the nylon barrier draught line showed impressive resistance to biofouling after the sixteen-week simulation, supporting the use of nylon oxygen barriers as a preventative measure against beerspoilage biofilms. methods microbial cultures four microbial species were obtained and cultured for this study as follows. hafnia paralvei (atcc 29927) and raoultella planticola (atcc 33431) were streaked onto nutrient agar plates and incubated at 37°c. pediococcus damnosus (atcc 29358) was cultured on lactobacilli mrs plates and incubated at 25°c. saccharomyces cerevisiae (wyeast 1728) was streaked onto a malt agar plate and incubated at 25°c. individual 500ml cultures of each species were inoculated with a single colony from each streak plate and incubated at the appropriate temperature until cultures reached late exponential phase (~24 hours). following incubation, all four cultures were mixed in a 1:1:1:1 ratio at od600 ~4.8 to create a microbial cocktail for inoculation of our beer draught lines. simulated dispensary system to simulate a standard dispensary system, three types of beer-draught lines, vinyl (micromatic 550c), polyethylene (micromatic 550ne), and nylon barrier (micromatic 550bf), were attached to a single keg—containing an amber ale from dakota territory brewing company, aberdeen, sd—and kept at 4ºc. before being connected to the keg, all lines were cleaned with alkaline liquid beer line cleaner (micromatic mm-b68), inoculated with a biofilm-forming microbial cocktail (described above), and left to incubate at room temperature for one hour. after inoculation, the culture was drained, and the lines were connected to the same keg using line splitters; the lines were then filled with beer. once connected, 500 ml of beer was drawn from each line everyother day for sixteen weeks to simulate a bar environment, and kegs were replaced as needed. after the sixteen-week simulation, the lines were disconnected from the keg, wrapped in parafilm, and stored at 4ºc for further processing; some beer was left in the line to prevent the line from drying while in storage. quantifying biofilm formation to quantify biofilm formation in each tube, 25 mm long segments were cut at various regions from each line using a completely randomized design. two segments were cut from each end and two segments were cut from the middle for a total of six samples in each treatment group.the samples were, then, rinsed lightly with deionized (di) water, and one end was sealed with parafilm. next, the samples were filled with 0.1% crystal violet and allowed to incubate for 10 mins. after incubation, the crystal violet was removed, and the samples were rinsed with di water a second time to remove any unbound dye. next, the tubes were filled with 1 ml of dimethylsulfoxide (dmso) and incubated for 10 minutes. a stainless-steel spatula was used to gently agitate and break uplarger chunks of biofilm. the sample was diluted 1:1 with more dmso, loaded into a 2 ml cuvette, and the absorbance was measured at λ600 on a spectrophotometer. results were analysed using a one factor anova to assign statistical significance. fluorescence microscopy one foot of tubing was taken from each type of beer line and three 12.5mm samples were cut from each foot-long segment. the samples were then cut longitudinally into semicircles and stained using the live/dead baclight bacterial viability kit (invitrogen), according to the manufacturer’s recommendations. the sections were then attached to a slide using tape and observed with fluorescence microscopy. images volume seven | 29 were acquired using a semi-randomized, single blind method on a leica dm8 confocal microscope with an excitation wavelength of 480 nm and a 63x oil immersion objective. results quantifying biofilm formation after disassembling our simulated draught system, each draught line was stained with crystal violet to compare total biofilm formation between the three tube types.visual inspection of each line showed a dramatic difference between the nylon barrier tubing and the other two types. some biofouling was evident in the nylon barrier tubing, but it was visibly cleaner than both the polyethylene and the vinyl lines (fig. 1). to quantify this difference, each tube was stained with crystal violet and the absorbance was measured using a spectrophotometer. matching its visible appearance, the vinyl beer draught line had the highest average absorbance value at 2.771 with a standard deviation of ±0.464 (2.771 ± 0.464, n=3). biofilm from the polyethylene line measured an absorbance value of 1.601 with a standard deviation of ±0.447 (1.601 ± 0.447, n=3). notably, the nylon barrier line exhibited significantly lower absorbance than both the vinyl (p= 0.00001) and the polyethylene (p= 0.0004) lines, measuring an average value of 0.253 with a standard deviation of 0.139 (0.253 ± 0.139, n=3) (fig. 2). it should be noted that crystal violet stain does not differentiate between viable and dead cells in this assay. therefore, the mean absorbance values reported herein are a measurement of cells that are alive and those that are not. fluorescence microscopy while the crystal violet staining of the nylon barrier tubing was significantly different from the other two materials, these results gave little insight into the biofilm characteristics and microarchitecture on each tube type. in particular, they did not indicate if bacteria and yeast were prevented from adhering to the nylon barrier tubing altogether, or if microbial cells were just concentrated in small, localized biofilms dispersed along its surface. therefore, confocal fluorescence microscopy was utilized to obtain three-dimensional images of the biofilms on each tube type. after removing three 25 mm segments from each line, the cells were stained with a fluorescent dye and observed by confocal microscopy to view the characteristics of biofilms in each microenvironment. image analysis revealed that most of the inner surface on the vinyl line was covered with a thin, but mature biofilm (fig. 3, left column). by comparison, the polyethylene line exhibited substantially less biofouling, but still had a moderate level vinyl polyethylene nylon barrier figure 1. crystal violet staining of draught lines. following a sixteen-week simulation, each draught line was drained, rinsed with di water, and stained with crystal violet. qualitative images were taken to compare relative biofouling. 30 | fine focus of bacteria adhered to its surface (fig. 3, middle column). similar to the polyethylene line, the nylon barrier tubing exhibited considerable resistance to biofouling. only single cells were adhered to its surface, with no evidence of a cohesive biofilm (fig. 3, right column). discussion the maintenance of beer draught lines is an expensive and time-consuming process that affects thousands of businesses around the globe. standard line maintenance is essential to ensure taste, aroma, and quality of the product, but it involves cleansing of the line using acidic or alkaline chemicals at a minimum every 2-3 weeks and complete line replacement yearly. improper cleaning of beer draught lines allows for microorganisms to adhere in the lines while feeding off the nutrients provided by the beer. the adhered microorganisms multiply in the line, and once there are sufficient organisms in the surrounding environment, the organisms begin to produce a polysaccharide matrix around themselves for protection. this biofilm makes the beer-spoiling organisms difficult to eradicate,costing retailers and distributors money and time. for these reasons, the prevention of biofilm formation is of great importance. in recent years, tubing manufacturers have been developing new chemical compositions that promise increased resistance to microbial biofilms and, therefore, longer intervals between line cleanings.to our knowledge, our results represent the first peer-reviewed study to support the claims that nylon barrier tubing with reduced oxygen permeability does, indeed, resist biofouling. our findings indicate that the nylon barrier line figure 2. quantifying biofouling of draught lines. six 25 mm segments were taken from each draught line and stained with crystal violet. the stained biofilm was solubilized with dmso, transferred to acuvette, and absorbance was measured at λ600. errorbarsrepresentstandarddeviation in each group. significant differences wereobtained between groups indicated with different letters; *p<0.01. volume seven | 31 is roughly six times more resistant to biofilm formation than polyethylene tubing and ten times better than standard vinyl tubing (fig. 2). fluorescence microscopy revealed that this is due to fewer cells adhering to the surface of the nylon barrier line compared to the others (fig. 3). while moderate levels of microbial cells still adhere to the surface of both polyethylene and nylon barrier tubing, the microenvironments in these tubes do not promote biofilm maturation. it is likely that the microbial population density required to establish a biofilm has not been reached. although the results from this experiment show that the structural and chemical composition of beer line affects biofouling, there are several caveats that deserve consideration. first, it could be assumed that the nylon barrier line helps preserve the quality of the beer longer because it inhibits biofouling. however, the taste and quality of the beer was not evaluated as a part of this study. more research might be needed to substantiate the benefit to consumers. second, only one style of beer – an amber ale – was used in this study; results may vary with beer styles containing higher alcohol content, greater alpha-acid levels, differing ph, etc. research suggests that each beer style may provide a unique growth environment, supplying specific micronutrients and antimicrobial compounds (21). for example, hops content is known to have a significant effect on the types of microorganisms that survive in different beer styles (23; 26; 27). knowing these differences could be useful to optimize cleaning protocols for beer draught lines dedicated to certain beer styles. third, each line was directly inoculated with a very high concentration (od600~4.8) of microorganisms and those cells were allowed to establish themselves in the absence of a regular cleaning regimen for sixteen weeks. these conditions were chosen to ensure adequate adherence of primary biofilm colonizers and to accentuate the potential impact of each tubing material. these conditions may not fig. 3. confocal imaging of adhered biofilm. six 25 mm segments were taken from each draughtline, stained with a fluorescent dye, and observed on a confocal microscope with an excitation wavelength of 480 nm and a 63x oil immersion objective. 32 | fine focus reflect the typical biological conditions that most beer retailers and distributors encounter. finally, there are many anaerobic organisms such as megasphaera and pectinatus species that are known to adhere to brewery surfaces and cause beer spoilage (3; 4). since all four of the test organisms in our study were aerobic or facultative anaerobes, our results cannot predict the potential impact that nylon barrier tubing might have on biofilms formed by strict anaerobes. given that the benefit of nylon barrier tubing is due to a presumed reduction in oxygen permeability (12), it is likely that lines containing this barrier would show little to no impact on the growth of anaerobic microorganisms. despite the caveats described above, our study provides convincing evidence that nylon barrier tubing substantially reduces biofouling when used for beer dispensing. the adoption of such tubing is a low-cost option for retailers to improve the quality of their product and reduce the frequency of line cleaning, resulting in less long-term expense. moreover, the use of such nylon barriers could have wideranging benefits in other applications such as water and soft drink lines, diagnostic equipment, and medical devices. acknowledgments: we would like to thank brodie mueller and dakotaterritory brewing company for providing used draught lines, equipment for the simulated draught system, and the ale for experimentation. we would also like to acknowledge dr. darla goeres at montana state university for her advice on experimental design. disclosure statments: the authors declare no conflict of interest. funding: this work was supported by the national science foundation epscor research infrastructure improvement program under the rii track-1 grant # 1849206. volume seven | 33 references 1. a1. association, b. 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(2015). bacterial biofilm asapossiblesourceofcontaminationinthemicrobreweryenvironment.foodcontrol,50,809-814. doi:10.1016/j. foodcont.2014.10.032 16. narayana, p. s. v. v. s., & srihari, p. s. v. v. (2019). biofilm resistant surfaces and coatings on implants: a review. materials today-proceedings, 18, 4847-4853. 17. nijland, r., hall, m. j., & burgess, j. g. (2010). dispersal of biofilms by secreted, matrix degrading, bacterial dnase. plos one, 5(12). doi:artn e1566810.1371/journal.pone.0015668 18. park, h. h., sun, k., lee, d., seong, m., cha, c., & jeong, h. e. (2019). cellulose acetate nanoneedle arraycoveredwithphosphorylcholinemoietyasabiocompatibleandsustainableantifoulingmaterial. cellulose, 26(16), 8775-8788. doi:10.1007/s10570-019-02681-w 19. park, h. h., sun, k., seong, m., kang, m., park, s., hong, s., . . . jeong, h. e. (2019). lipid-hydrogelnanostructureh ybridsasrobustbiofilm-resistantpolymericmaterials.acsmacroletters,8(1),64-69. doi:10.1021/acsmacrolett.8b00888 20. park, s. c., park, y., & hahm, k. s. (2011). the role of antimicrobial peptides in preventing multidrug-resistan tbacterialinfectionsandbiofilmformation.internationaljournalofmolecular sciences, 12(9), 5971-5992. doi:10.3390/ ijms12095971 21. quain,d.e.(2016).draughtbeerhygiene:cleaningofdispensetapnozzles.journaloftheinstituteof brewing, 122(3), 388396. doi:10.1002/jib.335 22. riedl, r., goderbauer, p., brandl, a., jacob, f., & hutzler, m. (2017). bavarian wheat beer, an example ofaspecialmicrobehabitat-cultivation,detection,biofilmformation,characterizationofselected lactic acid bacteria hygiene indicators and spoilers. brewing science, 70(1-2), 39-50. 23. sae-ung, p., wijitamornloet, a., iwasaki, y., thanyasrisung, p., & hoven, v. p. 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(2005). isolation of a hop-sensitive variant of lactobacillus lindneri and identification of genetic markers for beer spoilage ability of lactic acid bacteria. appl environ microbiol, 71(9), 5089-5097. doi:10.1128/aem.71.9.5089-5097.2005 28. suzuki, k., iijima, k., sakamoto, k., sami, m., & yamashita, h. (2006). a review of hop resistance in beer spoilage lactic acid bacteria. journal of the institute of brewing, 112(2), 173-191. doi:doi 10.1002/j.2050-0416.2006.tb00247.x 29. thompson, l. j., gray, v., lindsay, d., & von holy, a. (2006). carbon : nitrogen : phosphorus ratios influence biofilm formation by enterobacter cloacae and citrobacter freundii. journal of applied microbiology, 101(5), 1105-1113. doi:10.1111/j.1365-2672.2006.03003.x 30. tsiaprazi-stamou, a., monfort, i. y., romani, a. m., bakalis, s., & gkatzionis, k. (2019). the synergisticeffectofenzymaticdetergentsonbiofilmcleaningfromdifferentsurfaces.biofouling,35(8), 883-899. doi:10.1080 /08927014.2019.1666108 31. valpar beverage tubing. (2000-2019, 2019). retrieved from https://www.pasp.com.br/valparbeverage-tubing.html 32. walker, s. l., fourgialakis, m., cerezo, b., & livens, s. (2007). removal of microbial biofilms from dispenseequipme nt:theeffectofenzymaticpre-digestionanddetergenttreatment.journalofthe institute of brewing, 113(1), 61-66. doi:doi 10.1002/j.2050-0416.2007.tb00257.x 33. zhou, c., song, h. q., loh, j. l. c., she, j. q., deng, l. h., & liu, b. (2018). grafting antibiofilm polymer hydrogel film onto catheter by sara si-atrp. journal of biomaterials science-polymer edition, 29(17), 2106-2123. doi:10.10 80/09205063.2018.1507268 fine focus 100 i fine focus bacteriophage: an underutilized bacterial combatant rachel pittsley m.s. (and fine focus alum) research associate i pharmaceutical cro vol 8 i 101 introduction viral infections are commonly known to occur in humans, but what most people do not know is that viral infections are just as common in bacteria [1]. bacterial viruses are termed “bacteriophage” or “phage” for short and can be found in any environment: soil, water, snow, food products, and even sediments. viruses are generally 1000x smaller than their bacterial hosts and can possess a wide variety of different genotypic and phenotypic characteristics. such diversity includes morphology (size, shape, and structure), the number and type of open reading frames (translational portions of the genome), host range, and mode of infection. phage differ from animal viruses in a few distinct aspects. phage attach directly to the bacterial cell wall, inject their viral nucleic acids into the cytoplasm, and become synthesized by bacterial components. in contrast, most animal viruses have to bind to a cell membrane protein, become engulfed by the mammalian cell, outer protective layer digested by enzymes, and genetic material finally synthesized in order to cause infection [2]. bacteriophage were first discovered in 1915 by f. w. twort and have yet to be utilized to their full potential over 100 years later [3]. bacteriophage characteristics and their detection bacteriophage harbor a few unique physical and genetic characteristics. their physical characteristics (phage morphology) can be separated into 5 categories: icosahedral, hexagonal, spherical, tailed, and filamentous [1]. the length of the phage tail varies among phage types as some have no tail, some possess a short tail, and others have a tail twice the length of the capsid head. phage morphology and tail formation dictate their functional capabilities. genetically, genome types are variable and may consist of either doublestranded dna (dsdna), single-stranded dna (ssdna), double-stranded rna (dsrna), or single-stranded rna (ssrna) which ultimately determine how the phage will function in the host, and what particular mode of infection they harbor. in contrast, viruses that infect humans are typically single-stranded rna (ssrna) that function as messenger rna (mrna) and can be directly translated into a polyprotein [4]. another unique characteristic is that some phage can infect multiple species of bacteria while others are more speciesspecific [1]. this is termed ‘host range’ and can be of great importance when trying to find a way to combat multidrug resistant infections. due to the ease with which phage can acquire genes from bacterial hosts, phage can evolve to infect a wide variety of bacteria; advantageous to the phage, disadvantageous to the bacterial hosts. the mode of infection differs among phage types as they can be either lytic, lysogenic, or latent [1]. before entering either the lytic cycle or lysogenic cycle, all phage must first exhibit the latent stage in which the phage attaches to the cell surface, penetrates the membrane, and incorporates its dna into the host genome. lytic phage replicate inside of the bacterial host before lysing (breaking open) the cell, releasing more phage into the environment. lysogenic phage incorporate their dna into the bacterial genome in order to have the bacteria, unknowingly, replicate the phage genetical material numerous times. this allows the phage to have its genome carried within the bacterial progeny, an advantage to the phage itself. however, lysogenic phages can, at times, become virulent due to the protection they obtain from “hiding” inside the bacterial cells and going undetected. this could deem problematic as this is a similar mechanism to how mrsa (multi-resistant staphylococcus aureus) infects: s. aureus “hides out” inside immune cells, going undetected by the immune system, and ultimately rendering antibiotics useless. this gives mrsa time to replicate before the immune cell undergoes autophagy, releasing the mrsa into the bloodstream. one method used by scientists for detecting phage is by observing plaque formations within agar [5]. plaques are formed when phage lyse bacterial cells, spread to neighboring bacterial cells, and lyse again [6]. the lysing of the neighboring cells forms a circular, clear “lawn” that is visible to the human eye and is known as a plaque. plaques can be counted to determine how infectious a phage is to the host and to help determine host range. plaque formation is dictated and influenced by the phage genome, rate of 102 i fine focus adsorption (attachment), time spent inside of the host cells, and phage morphology [5]. depending on the phage genome, different genes could be present that ultimately affect the size of the plaque. adsorption rate impacts plaque size as well because as the phage is slowly adsorbed into the bacteria, the phage have more time to reproduce before lysing the cell. this phenomenon leads to a higher concentration of phage within the plaque formations. the density of phage inside the host cell dictates the formation of the plaque. in a study done by gallet, kannoly, and wang, it was found that “the higher the adsorption rate then the lower the phage concentration within the plaques." the timing of the lysis stage determines how long the phage has to reproduce inside the cell beforehand [5]. gallet et. al. found that there was a linear relationship between lysis time and burst size (plaque formation). phage with a low adsorption rate spend more time externally to the bacterial cells, therefore, by the time the phage dna is inserted into the cell, there is less time for the virus to cause infection. the physiology of the bacterial cells themselves could have changed by the time the phage infects, resulting in a smaller plaque size. the less time the phage have to diffuse out of the lysed cell, the smaller the plaque. phage morphology and architecture also determine the plaque size and formation after lysis. the longer the sheath and legs of the phage, the smaller the plaque size. this is due to the ability of the virus to diffuse through the agar layer when forming a plaque. a smaller virion would lead to a larger plaque due to the ease at which it can diffuse through the matrix of the agar. phage classification as of 2011, approximately 750 different phage types have been successfully isolated and fully sequenced, with about 5,000 phage types yet to be elucidated or identified [7]. there are two types of comparative analyses that allow for a better understanding of a specific phage: viral metagenomics and prophage mining. viral metagenomics is the process by which phage are harvested in large numbers and sequenced at random. this allows for large numbers of phage samples to be examined together from a specific environment and compared. utilizing the mining method entails comparing phage genomic sequences against portions of a complete bacterial genome that harbor areas of prophage sequences. to attempt to isolate an individual phage genome, it is best to extract dna from individual plaques following host-range experiments. most interestingly, bacteriophage can possess either dna or rna genomes, however, the vast majority possess linear double-stranded dna genomes (dsdna) and a tail (sheath): order caudovirales [7, figure 1]. the dsdna of this phage order is generally comprised of 55%-70% g+c. most of the order caudovirales fit into the siphoviridae family (55%) possessing dsdna and flexible non-contractile sheaths, while others make up either the myoviridae (25%) family who possess contractile sheaths, or the podoviridae (20%) family that exhibit short, stubby sheaths. the structure of the phage sheath has been found to reflect the phage genome, mode of infection, assembly, and maturation [8]. it has been determined that the long non-contractile tails of the siphoviridae were assembled first then added to the head, whereas the short non-contractile tails were assembled after the phage heads. the similarity of the phage types in this order is currently being described as a result of horizontal gene transfer among phage. other, less common phage families include tectiviridae (lipid-containing), corticoviridae (lipid-containing, circular genome), plasmaviridae (enveloped), lipothrixviridae (rod-shaped), rudiviridae (non-enveloped), and fuselloviridae (non-enveloped, lemonshaped) [9]. these are the current, known and classified phage families but more remain undiscovered. the myoviridae family is comprised of many species of phage: t4, t2, p1, p2, mu, bacillus phage sp01, and halobacterium phage ɸh [10]. phage in this family are characterized as having an elongated head (90-110nm in diameter) and a long tail to match (100-120nm in length). the species share about 50-70% of their genomes to one another, making characterization of unknown phages difficult. meanwhile, the siphoviridae family is comprised of phage t1, t5, mycobacterium l5, lactococcus c2, λ, and ψm1 [9]. these phages are characterized by having a smaller head (60nm in diameter) and longer sheath (70-90nm in length). what is most interesting is that the myoviridae and siphoviridae families are the only phage known to infect both bacteria and archaea. it is hypothesized that this is due to their contractile tails and the evolution of phages from vol 8 i 103 prokaryotes [11,12]. the lesser known family, podoviridae, is comprised of phage t7, enterobacteria phage p22, and bacillus phage ɸ29 [9]. podoviridae are characterized as having a head of about 60nm in diameter and a short tail with maximum length of about 20nm. conclusions bacteriophage have been studied for years but have yet to be utilized to their full potential. phage can be beneficial to both the food industry and pharmaceutical industry. phage with a wide host range can be used to combat multiple bacterial infections, while phage who are selective to a certain bacterial host can be used against one particular infection. additionally, when antibiotics are rendered useless, phage could be the answer. phage can more easily bind to and attack bacterial cells. phage could also be used in combination with antibiotics to fully rid a patient of an infection. phage do not target mammalian cells, therefore, they may also be a safer therapeutic. in the food industry, bacteriophage could be used when pasteurization and antibiotics fail to rid the product of bacteria. bacteria can form a protective outer layer (biofilm) that antibiotics and high temperatures fail to penetrate. when the environment is right for bacteria to disperse from the biofilm, phage can be used as a final protective measure for food products. with science and technology rapidly advancing, now is the time to begin adding phage to the list of more commonly used biological control agents. figure 1. bacteriophage familial identification [7] phage are classified into 13 distinct families, 3 of which have been grouped together into the order caudovirales [7]. caudovirales make up the vast majority of phages (roughly 96%) that have been reported [13]. they are comprised of the families myoviridae, siphoviridae, and podoviridae. the other 10 phage families make up only 4% of reported and classified phage. 104 i fine focus references 1. hyman, p. and s.t. abedon, smaller fleas: viruses of microorganisms. scientifica (cairo), 2012. 2012: p. 734023. (4) 2. cohen, f. h., how viruses invade cells. biophysical journal: ncbi, 2016. 110: p. 1028-1032. 3. duckworth, d.h., who discovered bacteriophage? bacteriol, 1976. rev. 40, p. 793-802. 4. pellett p.e., mitra s., holland t.c. basics of virology. handb. clin. neurol. 2014;123:45–66. 5. ács, n., gambino, m., and brønsted, l., bacteriophage enumeration and detection methods. frontiers in microbiology, 2020. 11. 6. gallet, r., s. kannoly, and i.n. wang, effects of bacteriophage traits on plaque formation. bmc microbiol, 2011. 11: p. 181. 7. hatfull, g.f. and r.w. hendrix, bacteriophages and their genomes. curr opin virol, 2011. 1(4): p. 298-303. 8. shao y, w., bacteriophage adsorption rate and optimal lysis time. genetics, 2008. 180: p. 471-482. 9. maniloff, j.a.a., h.w., taxonomy of bacterial viruses: establishment of tailed virus genera and the order caudovirales. arch virol, 1998. 143(10): p. 2051-2063. 10. ackermann, h.w., frequency of morphological phage description in 1995. arch virol, 1996. 141: p. 209-218. 11. ackermann, h.w.a.d., ms, viruses of prokaryotes. crc press, 1987. 12. zilling, w., prangishvilli, d., schleper, c., elferink, m., holz, i., albers, s., janekovic, d., and gotz, d., viruses, plasmids and other genetic elements of thermophilic and hyperthermophilic archaea. fems microbiol rev, 1996. 18: p. 225-236. 13. matsuzaki, s., et al., bacteriophage therapy: a revitalized therapy against bacterial infectious diseases. journal of infection and chemotherapy : official journal of the japan society of chemotherapy, 2005. 11: p. 211-9. © 2025 mckillip. fine focus, 11(1), 2-4. doi: 10.33043/r2y588ab shared with cc-by-nc-nd 4.0 license. objective lens: insect antimicrobial peptides as a promising source for antibiotic “substitutes” currently, at least 2.8 million infections and over 700,000 deaths are reported from amr bacterial infections globally (1,2). if no new antibiotics are isolated and made available by 2050, the cdc estimates that 10 million annual deaths will occur globally as a result of this inaction (5,8). yet, surprisingly few have even heard of antibiotic-resistant bacteria, or understand the implications for global health. in fact, no new classes of antibiotics have been developed to treat microbial infections in well over 30 years, as pharmaceutical companies have instead pursued research and development of more lucrative drugs for non-infectious diseases. since this trend is likely to continue into the foreseeable future, this crisis must be addressed using alternative creative approaches. unfortunately, this problem is exacerbated poor antibiotic stewardship practices by healthcare providers and consumers for decades. the world health organization (who) has declared multidrug-resistant (mdr) bacterial and fungal pathogens to be one of the principal threats to global public health (5,7-9). alexander fleming’s discovery of penicillin in 1928 was met with his own prediction that bacterial resistance to this “miracle drug” would soon be documented (3,4). in the 70+ years since penicillin was introduced, overuse and misuse of antibiotics have contributed to the problem of mdr bacterial infections, as has the widespread use of antibiotics in agriculture for prophylaxis and growth promotion (8). in fact, the cdc has reported that over 70% of antibiotics used in the u.s. are in production animal environments (8-10). clinically, antibiotic stewardship and surveillance programs have shown limited success in addressing the mdr crisis (9). in recent years, both the cdc and the white house have outlined clear goals and objectives for directly addressing antibiotic resistance in order to slow the spread of mdr bacteria, while offering a timeline on collaborative international efforts required to make this happen by 2020 (5,6). unfortunately, this executive order signed by president barack obama (#13676) has not been addressed, during which time mdr bacterial infections have worsened and become more frequently diagnosed (6). the global crisis of antimicrobial resistance (amr) is fueled by antibiotic overuse in agriculture and human clinical applications. eskape pathogens, identified by the world health organization as a priority for new antimicrobial drugs (11), are central to this pending catastrophe. however, it may be prudent to open a new line john l. mckillip, fine focus managing editor https://creativecommons.org/licenses/by-nc-nd/4.0/ mckillip | objective lens 3 of investigation in ernest evaluating insects as a largely unexplored source for novel antimicrobial peptides (amps), which show tremendous promise as an alternative for the use of antibiotics. with over one million described species, insects are the largest class of organisms on the planet, and thus represent tremendous untapped potential for describing novel antimicrobial compounds (12). insects adapt in remarkable ways to constant changes in their environments, including to a wide range of pathogens. this adaptability is due in large part to their immune systems, which respond quickly to invading microorganisms that gain entry into the hemocoel. a variety of amps comprise the key elements of an insect’s humoral immune response; some of these molecules are involved in binding to bacterial cells, and facilitating their inactivation or elimination in unique ways. for example, many insect amps are positively charged and are thus attracted to the negative charge of bacterial surfaces, where they may serve as membrane antagonists (disrupting bacterial cell structure), or are involved in a hemolymph coagulation response that prevents the bacterial pathogen from disseminating or growing within the insect host. insect amps are not only antibacterial, but have also been shown to have potential against fungal pathogens, some viruses, and cancer cell lines as well (12), although few if any of these molecules have been investigated more deeply with regard to their biotherapeutic practicality. because these amps are unique in their mechanisms of action against target pathogens, little if any resistance issues have been observed, in stark contrast to traditional antibiotics (13), and this trait is arguably one of the most compelling reasons to investigate amps further as a promising means of treating the growing roster of drug-resistant bacterial pathogens. this issue’s cover features an artist’s conceptualization of various insect amps. references 1. antibiotics resistance threats in united states, 2019. https://stacks.cdc.gov/view/cdc/82532 2. church, n.a., mckillip, j.l. 2021. antibiotic resistance crisis: challenges and imperatives. biologia 76, 1535–1550. 3. khardori, n., stevaux, c., & ripley, k. 2020. antibiotics: from the beginning to the future: part i. ind. j. ped. 87:39-42. 4. tan, s. y., & tatsumura, y. 2015. alexander fleming (1881-1955): discoverer of penicillin. singapore med. j. 56:366-367. 5. https://www.cdc.gov/drugresistance/pdf/threats-report/2019-ar-threats-report-508.pdf accessed 8.26.2024 6. https://obamawhitehouse.archives.gov/sites/default/files/docs/national_action_plan_for_ combating_antibotic-resistant_bacteria.pdf accessed 8.26.2024 7. floris, l., cluck, d., & singleton, a. 2020. understanding antimicrobial resistance. u.s. pharmacist 45: hs10-hs16. 8. abadi, a.t.b., rizvanov, a.a., haertlé, t., & blatt, n.l. 2019. world health organization report: current crisis of antibiotic resistance. bionanoscience 9:778-788. 9. michael, c.a., dominey-howes, d., & labbate, m. 2014. the antimicrobial resistance crisis: causes, consequences, and management. front. public health 2:1-8. https://stacks.cdc.gov/view/cdc/82532 https://www.cdc.gov/drugresistance/pdf/threats-report/2019-ar-threats-report-508.pdf https://obamawhitehouse.archives.gov/sites/default/files/docs/national_action_plan_for_combating_antibotic-resistant_bacteria.pdf https://obamawhitehouse.archives.gov/sites/default/files/docs/national_action_plan_for_combating_antibotic-resistant_bacteria.pdf fine focus | volume 114 10. venter, h., henningsen, m.s., & begg, s.l. 2017. antimicrobial resistance in healthcare, agriculture, and the environment: the biochemistry behind the headlines. essays biochem. 61:1-10. 11. world health organization. 2017. global priority list of antibiotic-resistant bacteria to guide research, discovery, and development of new antibiotics. 12. manniello, m.d., moretta, a., salvia, r., scieuzo, c., luchetti, d., vogel, h., sgambato, a., & falabella, p. 2021. insect antimicrobial peptides: potential weapons to counteract the antibiotic resistance. cell. mol. life sci. 78:4259-4282. 13. patyra, e, & kwiatek, k. 2023. insect meals and insect antimicrobial peptides as an alternative for antibiotics and growth promoters in livestock production. pathogens 12:854. objective lens: insect antimicrobial peptides as a promising source for antibiotic “substitutes” references molecular pathogenesis of bacillus spp., with emphasis on the dairy industry alyssa a. grutsch, pierre s. nimmer, rachel h. pittsley, katherine g. kornilow, & john l. mckillip department of biology & biotechnology certificate program, ball state university, muncie, in copyright 2018, fine focus all rights reserved manuscript received 07 june 2018; accepted 10 november 2018 203 the bacterial species bacillus cereus accounts for 1.4-12% of foodborne illness outbreaks worldwide, a statistic that is certainly an underestimate. this bacterial genus is capable of contaminating a wide range of food products, including rice, chicken, vegetables, spices, and dairy products. b. cereus endospores are partially resistant to pasteurization, dehydration, gamma radiation, and other physical stresses used in food processing, and their adhesive characteristics promote biofilm-forming capability on a variety of substrates in dairy operations. b. cereus and other closely-related species produce several types of exotoxins, including at least four hemolysins, three phospholipases, a heat/acid stable emetic toxin called cereulide, and three well-studied heat-labile enterotoxins that all cause gastroenteritis following ingestion. while a great deal of information on virulence gene presence and expression is known in b. cereus, very little has been done to explore the virulence potential of thermoduric spore-formers that may be found in ultrahigh temperature (uht) pasteurized milk, and their ability to produce biofilms. biofilm production is understood to be under similar regulation as toxins and other extracellular virulence determinants. this chapter describes the current status of knowledge with bacillus spp. relevant to the dairy industry, virulence potential, and biofilm production from the perspective of food safety. abstract corresponding katherine g. kornilow kgkornilow@bsu.edu keywords • bacillus spp. • enterotoxins • food pathogen bacillus spp. bacteria show a wide range of characteristics that allow them to live in most natural environments (griffiths, 2010). bacillus comprise a large group of ubiquitous gram-positive, rodshaped, aerobic-to-facultatively anaerobic endospore-forming saprophytes (weber & rutala, 1988). although the majority of bacillus spp. are nonpathogenic, a few (bacillus cereus, bacillus anthracis, and bacillus thuringiensis) opportunistically infect animal hosts (mammals and insects) (vilian et al., 2006). bacillus microscopic morphology may be individual or as long chains in primary isolates from soil or water samples (weber & rutala, 1988). the size bacillus – general information 204 • fine focus, vol. 4 (2) bacillus spp. virulence • 205 of an individual rod can range from 0.5 x 1.2 um to 2.5 x 10 um. spores produced by bacillus spp. are resistant to heat (including to some extent, pasteurization conditions), cold, ionizing radiation, dehydration, and many disinfectants (griffiths, 2010). the endospores are oval or cylinder shaped and are found centrally, sub-terminally or terminally. over 30 species of bacillus spp. are recognized, and are divided into two groups based mostly on the 16s rrna/dna sequences: the bacillus subtilis group and the bacillus cereus group. bacillus subtilis, bacillus amyloliquefaciens, bacillus licheniformis and bacillus pumilus are mesophilic, have ellipse shaped spores, and are the most common members of the b. subtilis group. b cereus, b. anthracis, b. thuringiensis, bacillus weihenstephanensis, and bacillus mycoides do not ferment mannitol, produce lecithinase, and comprise the b. cereus group. the colony morphology of bacillus spp. is diverse across species. bacillus spp. grow on nutrient agar or peptone media and exhibit ideal growth at a ph 7; however some bacillus spp. grow at a ph of 9, while other species can endure ph 2. bacillus spp. grow best within a temperature range of 30°-45°c, but thermophilic variants grow optimally at 65°c. all bacillus spp. metabolize organic substrates such as amino acids, organic acids and sugars by aerobic respiration, anaerobic respiration, or fermentation, depending on species and environment. the enzymatic processes and metabolic characteristics are typically the criteria for bacillus species differentiation. bacillaceae family members demonstrate a wide range of characteristics, including the ability to produce a battery of enzymes, antibiotics, and other secondary metabolites (schallmey et al., 2004). for example, bacillus spp. have unique abilities to synthesize and/or secrete many substances which are beneficial and show great success in agriculture and industry. many bacillus species exhibit antibacterial and antifungal activity against phytopathogens through secretory products (yu et al., 2002), a logical evolutionary strategy since bacillus spp. are soilborne or are found in epiphytes (plant that grows non-parasitically on another plant) and/or endophytes (living within a plant host)(fravel, 2005). many antimicrobial compounds are well recognized in the biotechnology and biopharmaceutical industries for their surfactant properties are derived from b. subtilis (jacques, 2007). a surfactant lowers surface tension between two liquids or a solid and a liquid (singhal, 2007). surfactants are used for foam creation and stabilization in food processing, household products (paint, detergent, fabric softener), solubilization of agrochemicals, oil recovery, crude oil drilling lubricants, and bioremediation of water insoluble pollutants. phenotypically, the genus is difficult to delineate into species, but using genotypic methods, determination of relatedness has been revisited in recent years (sneath, 1986). the mole % g + c content of the dna is a well-regarded metric by which organisms may be compared genetically. the bacillus genus is diverse and has a g + c content from 33-69% (winn et al., 2006). sequencing of16s rrna genes and dnadna hybridization methods have been used to assign species names (goto et al., 2000). 16s rdna has a hypervariant region (hv region) on the 5’ end. this hv region is highly specific to each bacillus spp. and is a good genotyping target. overall, much emphasis has been placed in recent years on defining criteria for species determination within the genus bacillus, although no single accepted system or approach has been established yet. bacillus in clinical settings through biofilm production, b. cereus has been implicated in contaminating intravenous catheters (hernaiz et al., 2003) resulting in b. cereus-mediated sepsis (kuroki et al., 2009; ozkocaman et al., 2006). the formation of biofilms also allows the release of planktonic bacteria that produce additional biofilms increasing the severity of the infection (costerton et al., 1999). in addition to catheter contamination, b. cereus and its endospores have been shown to contaminate air filtration and ventilation equipment (bryce et al., 1993), fiber optic bronchoscopy equipment (goldstein & abrutyn, 1985; richardson et al., 1986), linens (barrie et al., 1994), gloves (york, 1990), specimen collection tubes and balloons used in manual ventilation (vanderzwet et al., 2000), alcohol-based hand wash solutions (hsueh et al., 1999), plasterimpregnated gauze (rutala et al., 1986), and many antiseptics such as chlorhexidine and povodone iodine (dubuoix et al., 2005). the most common types of infections b. cereus causes, other than foodborne illness, include fulminant bacteremia, central nervous system (cns) involvement (meningitis and brain abscesses), pneumonia, gas gangrene-like cutaneous infections and endophthalmitis. other molecular techniques are also used to identify bacterial species including bacillus spp. the following molecular techniques are used to confirm identify of bacillus and other bacterial species: (1) polymerase chain reaction (pcr)(adzitey et al., 2013); (2) pulsed field gel electrophoresis (pfge); (3) random amplified polymorphism deoxyribonucleic acid (rapd); and (4) matrix assisted laser desorption/ionization time of flight (maldi-tof)(murray, 2012). pcr is a dna replication process that amplifies small portions of dna (amplicons) exponentially with the help of oligonucleotide primers and dna polymerase. pcr has many different variations, including real time pcr (qpcr). qpcr is a powerful approach wherein template bacterial dna is amplified and quantified at the same time using a standard curve-based comparison of type strain standards. pfge is an agarose gel electrophoresis method that separates large pieces of genomic dna. this separation of dna is done by applying an electrical current that periodically changes between three different directions, providing a means to accurately resolve small differences in genomic sequences for bacterial community analyses. rapd is a pcr based method that uses arbitrary primers to randomly amplify segments of target dna, essentially acting as a dna fingerprinting system for bacterial species. maldi-tof is a simple and rapid technique. bacterial colonies are removed from the plate, mixed with a uv absorbing matrix (saturated solution of α-cyano-4hydroxy-cinnamic acid in 50% acetonitrile and 2.5% trifluoroacetic acid) and dried on a target plate. the target plates are exposed to laser pulses that develop an energy transfer from the matrix to the nonvolatile analyte molecules. the analyte is removed in the form of gas. the molecules are enhanced in a flight tube to the mass spectrometer. maldi-tof is accurate, rapid, and after initial purchase, inexpensive. these characteristics perhaps explain why maldi-tof is being used in hospitals for quick identification of bacterial infections. 206 • fine focus, vol. 4 (2) bacillus spp. virulence • 207 b. cereus-mediated endophthalmitis b. cereus is not only capable of causing foodassociated toxicoinfections, but can cause endophthalmitis as well (davey & tauber, 1987; hermandy et al., 1990; ullman et al., 1987). b. cereus is not the only pathogen capable of causing endophthalmitis, but is considered one of the most aggressive pathogens causing this condition. because there is a limited immune response when a pathogen enters the eye, a wide spectrum of pathogens can enter and elicit a wide array of effects. symptoms can range from a relatively painless anterior chamber inflammation (aaberg et al., 1998), to an explosive ocular and periorbital infection caused by b. cereus (schemmer & drebe, 1987). specific toxin production by a particular microorganism is theorized to account for the difference in symptoms. b. cereus induced endophthalmitis is characterized by a corneal ring abscess followed by increased pain, chemosis, proptosis, retinal hemorrhage, and perivasculitis (callegan et al., 1999). fever, leukocytosis, and general malaise often appear as the systemic manifestations of this condition (martinez et al., 2007). b. cereus induced endophthalmitis can be divided into two categories: exogenous and endogenous. an exogenous source is due to blunt trauma that penetrates the eye, which may occur due to occupation (for example, metal workers), in an agricultural setting (david et al., 1994) or infection resulting from unsterile instruments during cataract surgery. in one example in rome, an ophthalmologist had four of his cataract patients lose vision in their treated eye one day after their cataract surgery (simini, 1998). b. cereus is ranked second behind staphylococcus aureus which is responsible for about 70% of post-cataract surgery endophthalmitis (han et al., 1996). the three main risk factors surgeons need to be aware of to reduce posttraumatic endophthalmitis are the presence of an intraocular foreign body, delay in closure of the globe, and the location/extent of the laceration of the globe. endogenous sources represent about 2-8% of all endophthalmitis cases (romero et al., 1999) and are due to bacteria entering the posterior segment of the eye. the most common pathogen to enter the posterior segment of the eye is candida albicans but other common pathogens include s. aureus, b. cereus, escherichia coli, neisseria meningitides and klebsiella spp. b. cereus can accomplish this route of entry through blood transfusion, contaminated needles/illicit drug injection paraphernalia (grossniklaus et al., 1985), or by iatrogenic administration of medications such as b vitamins or insulin (motoi et al., 1997). moyer et al (2009) demonstrated that b. cereus is capable of disrupting tight junctions between endothelial cells and the basement membrane of retinal capillaries and retinal pericytes as early as 4h post-infection. such changes are hypothesized to be responsible for causing the loss of retinal structure and function (kopel et al., 2008; moyer et al., 2009). the exact toxins from b. cereus responsible for causing this breakdown of the blood retinal barrier are unknown but are theorized to consist of the following molecules that may be working individually or in concert to achieve this effect: the hbl enterotoxin, the nhe enterotoxin, a crude exotoxin (cet) derived from cell-free b. cereus culture filtrates, phosphatidylcholinepreferring phospholipase c (pc-plc), collagenase, cereolysin o (shany et al., 1974), or cereolysin ab (scott et al., 1996). however, only the hbl enterotoxin protein has been identified for its role in endophthalmitis (callegan et al., 1999a). 208 • fine focus, vol. 4 (2) hbl enterotoxin has been shown to cause irreversible tissue damage to the photoreceptors of the retina in less than 1224h causing blindness in the infected eye (beecher et al., 1995; davey & tauber, 1987). b. cereus is capable of disrupting the blood retinal barrier as early as 4h in retinal tissues, 6h post-infection in aqueous humor, and in all other ocular tissues 12h postinfection (callegan et al., 1999b). b. cereus has been shown to be a more rapid and virulent endophthalmitis pathogen compared to s. aureus and enterococcus faecalis. additionally, b. cereus seems to exhibit an almost immediate inflammatory response despite low numbers of the organism present at the early stages of infection. limited research exists addressing the exact role the immune system plays in endophthalmitis, but the eye is known to be an immunoprivileged site as was first described by medawar in 1948 (cunha-vaz, 1997). the eye restricts both the adaptive and innate immune systems in such a way to balance the challenge of pathogen infection against inflammation-induced vision loss (streilien, 2003). in most instances of b. cereus induced endophthalmitis, vision loss occurs regardless of the type of therapeutic or surgical intervention utilized because the severity of the disease has progressed to such a condition, that too many toxins have been released by b. cereus and many bacteria will have migrated in the eye out of the reach of antibiotics (callegan et al., 2006). thus within a 12-18h time frame, massive tissue destruction occurs to the retina and surrounding ocular tissues resulting in antibiotics no longer being maximally effective (callegan et al., 2002). in addition, the inflammatory response inside the eye is so aggressive that even if the antibiotics control b. cereus, the inflammation produced causes damage to surrounding ocular structures thus making it difficult to manage ocular infections. bacillus in food foodborne illness from a variety of microorganisms affects on average 76 million individuals in the u.s. each year resulting in some 5,000 deaths (mead et al., 1999). worldwide statistics on bacillus cereus foodborne illness are underestimated due to a variety of factors, including emetic symptoms similar to staphylococcus aureus intoxication and diarrheal symptoms similar to those elicited by clostridium perfringens type a. most affected individuals do not seek medical attention due to the short duration of signs and symptoms. b. cereus seems to account for between 1.4-12% of foodborne illness outbreaks worldwide (stenfors et al., 2008). bacillus spp. are capable of contaminating a wide range of food products, including rice, chicken, vegetables, spices, and dairy products. contamination in the dairy industry may occur when b. cereus spores come in contact with the udders of cows (andersson et al. 1995), if the spores colonize feed or bedding, or if the spores survive pasteurization (claus & berkley, 1986; sneath, 1986). this is a serious problem in the food industry because b. cereus endospores are in many instances partially resistant to the bacillus spp. virulence • 209 heat of pasteurization, dehydration, gamma radiation, and other physical stresses. this resistance is due to the ultrastructure of the endospore of course, but also in part to the hydrophobic nature of the spores that allows them to adhere strongly to surfaces and develop biofilm-like properties (mattson et al., 2000; ronner et al., 1990). for example, an irradiation dose of 1.25-4 kgy needs to be administered to reduce spores by 90% (de lara et al., 2002). also, pasteurization may result in the activation and germination of spores (hanson et al., 2005). in addition, b. cereus endospores germinate in response to particular nutrients such as glycine or in response to physical stress such as temperature (spore germination can occur over 5-50°c in cooked rice) (granum, 1994) and high pressures (i.e. 500 mpa). thus foods need to be cooked at least at a temperature of 100°c (212°f) or above to kill most of the endospores (griffiths & shraft, 2002). thermoduric sporeformers have many important reasons to be the subject of great interest within the dairy industry (burgess et al., 2010). thermophilic bacilli produce heat-resistant (80-100°c) and highly heatresistant (>106°c) endospores in uht treated products, which can lay dormant for years. heat, chemicals, and ph levels can activate a spore for germination and outgrowth. this is particularly important in the dairy industry because heat is used as a preservation mechanism. b. subtilis has a low activation temperature of 65-70°c. once the spores are activated, germination is elicited by nutrients that bind to germination receptors. a nutrient mixture of asparagine, glucose, fructose and k+ (agfk) triggers b. subtilis spore germination (setlow, 2003). many people consider b. anthracis, b. thuringensis, and b. cereus to be the same species (helgason et al., 2000). b. anthracis is found in the soil and infects primarily herbivorous animals, causing human disease (winn et al., 2006; kolsto et al., 2009). this disease may be contracted by local infections of skin lesions, through the gastrointestinal (gi) route, or by inhalation. respiratory and gi-acquired routes are highly lethal forms of anthrax. b. anthracis virulence mechanisms easily allows for the spread of the bacteria to the lymph nodes. once in the lymph nodes, the bacteria disseminate via the bloodstream and internal organs. b. anthracis spores are highly resistant to adverse environmental conditions and it is difficult to be certain that the organism has been fully eradicated from endemic areas (winn et al., 2006). the endospores are maintained in soil and stay dormant indefinitely. the virulence determinants produced by b. anthracis are composed of three proteins: a protective antigen (pa), an edema factor (ef), and the lethal factor (lf). virulent strains are also typically capsule-producers. toxin expression and production is enhanced by elevated co2 and growth temperatures of 35-37˚c. b. anthracis strains harbor two large plasmids, pxo1 and pxo2 (kolsto et al., 2009). these plasmids are needed for full virulence. pxo1 contains the coding for the pa (pag), ef (cya), and lf (lef). pxo2 contains a five-gene operon for the biosynthesis of a polyglutamate capsule. this capsule is important for the ability to escape the host immune system, by protecting the vegetative cells from phagocytosis. b. thuringensis classification has been accomplished by h serotyping, which utilizes bacterial flagellar antigens (sneath, 1986). this species has unique insecticidal properties demonstrating activity against several insect orders, as well as nematodes, mites and protozoa. b. thuringensis produces protoxins during sporulation (aronson et al., 1986). these toxins are either parasporal inclusions or found on the spore surface. b. 210 • fine focus, vol. 4 (2) thuringensis produces parasporal crystals during sporulation, which are inclusions of insecticidal toxins. the midgut of the larvae have proteases that convert protoxins to toxins, activating the toxin to bind to receptors on columnar midgut cells. this binding event results in pore formation of the midgut epithelium, and susceptible insects die from this extensive damage and ph changes as midgut contents mix with the hemocoel cavity. three common subspecies variants have been recognized and well characterized over the last 40 years: (1) b. thuringiensis subsp. kurstaki; (2) b. thuringiensis subsp. israelensis; and (3) b. thuringiensis subsp. japanensis. each produces crystalline endotoxin specific for a unique order of insect for selectively toxic biological control. interestingly, each is also extremely genetically similar to the type strain pathogen in this family, b. cereus. b. cereus and other bacillus spp. are a major cause of foodborne illness globally and a major cause of endophthalmitis (weber & rutala, 1988; stenfors et al., 2008; moyer et al., 2008). b. cereus has an optimum growth temperature of 30°-40°c, although psychotrophic members can grow in temperatures as low as 4°c. b. cereus can grow in a ph of 5.0-8.8 with optimal ph of 6.0-7.0. food poisoning due to b. cereus is underreported because it is short-term and self-limiting. in 2005, bacillus spp. were responsible for 1.4% of foodborne illness in europe. in the netherlands 12% of foodborne illness was caused by b. cereus between 1993-1998. in 2006, an average of 63,400 (0.4%) people were domestically affected with b. cereus food poisoning (scallan et al., 2011). reports of b. cereus induced food poisoning has increased in industrialized countries, however reporting and testing is variable. in the us passive surveillance is usually performed due to low hospitalization of b. cereus food poisoning. foods frequently contaminated by b. cereus include milk, dairy products, dry foods, rice, egg products and legumes. two types of food-related illnesses are caused by b. cereus: (1) type 1: short-incubation “emetic” and (2) type 2: long incubation “diarrheal.” type 1 has an incubation time of 2 hours and lasts approximately 9 hours. type 2 has an incubation time of 9 hours and lasts 24 hours. type 1 is mostly associated with contaminated rice and type 2 is associated to contaminated meat or vegetables. the main virulence factor for type 1 food poisoning caused by b. cereus is cereulide (cueppens et al., 2011). cereulide is a small molecular weight heat stable exotoxin that can withstand treatment at 121°c for 2 hours at a ph of 7.0. this stability means the toxin can withstand frying, roasting, and microwave exposure, eliciting a foodborne emetic intoxication in susceptible individuals. the main causes of type 2 foodborne illness are hemolysin b (hbl) and non-hemolytic enterotoxin (nhe), both comprised of three components encoded by separate operons (fig. 1) – typical ab toxin architecture. hbl is made of the cytolytic subunits hblc and hbld, and the protein b binding domains. the hbl operon also has a fourth member, the hblb gene. however, hblb is not transcribed and is likely a pseudogene. nhe is made of the cytolytic protein nhea, and the protein b binding sections nheb and nhec. in recent research, 7.5% of reported emetic symptoms have been linked to hbl and nhe. these toxins are a product of aerobic, spore forming b. cereus. aerobic spore formers in food are ubiquitous. this ubiquity makes it impossible to prevent aerobic spore formers from being present in many fresh foods. spore counts in raw milk vary throughout the year, but are highest in winter when dairy cows are primarily indoors. pasteurization is effective figure 1. bacillus spp. hbl and nhe operons. the plcr region is the regulator gene as described in the text [24].” plcr plcr hblc hbld hbla nheb nhecnhea hblb in inactivating vegetative cells in raw milk, but fails to kill many spores. the spores have no competition from vegetative cells, so they proliferate rapidly if the product is mishandled or improperly stored. the sporulated bacillus, upon germination, can adhere to pipelines and equipment, causing biofilm formation. these spores and vegetative cells in equipment and raw milk may be tolerant to sterilization. biofilm extrapolymeric substances (eps) offer a significant survival strategy to established populations of bacteria. these counteractive techniques include ultra-high temperature (uht) processing, previously known to inactivate all living material, however spores are now known to survive uht-processing. uht-processing is achieved by treating fluid milk at 135-150°c for 1-8 seconds. the milk flows continuously during this process and is packaged into pre-sterilized containers (aseptic packaging). the uht process is designed to kill almost all organisms including spores. the concern is that some spores still survive and there is no competition for these spores, giving them an ideal environment to proliferate. the growing concern for psychrotolerant spore formers is that they show potential to induce foodborne illness and produce spoilage defects caused by enzymatic activity. these concerns are due to a combination of the following reasons: (1) longer refrigeration storage pre-pasteurization; (2) higher temperatures used for pasteurization; (3) prolonged shelf life; and (4) pasteurization activates the germination of spores. a combination of these “advantages” are beneficial for b. cereus endospores to form from vegetative cells or vegetative cells to form endospores. production length of milk treatment has been reduced to 6-8 hours to help reduce thermophile growth (burgess et al., 2010). once a production cycle is complete, a cleaning-in-place (cip) method is performed on the equipment. cip consists of the following steps: (1) a warm water rinse; (2) a 1.5% caustic wash at 75°c for 30 minutes; (3) a water rinse; (4) a 0.5% nitric acid wash at 70°c for 20 minutes; and (5) a second water rinse. these steps have helped with growth within the equipment, but not within the milk itself. table 1 indicates the time and temperature requirements laid out by the food and drug administration for pasteurization regimes, including uht pasteurization. bacillus spp. virulence • 211 bacillus spp. biofilms adherence of microbial biofilms to dairy production surfaces makes sanitization more difficult, and increases cost via labor and chemical usage along with lost production time. fda involvement and subsequent product recalls can also occur causing further financial problems for dairies. araújo et al. (2009) have proposed a basic mechanism for biofilm adhesion based on six general stages. first, the biofilm surface must be primed for adhesion with the existence of food deposits. the biofilm-producing microorganism must then come into contact with the primed surface. positive and negative biochemical forces including van der waals forces and other electrostatic forces then allow the biofilm to make a non-permanent attachment to the surface when microorganism are between 20 and 50 nm away. irreversible adhesion results within 1.5 nm when extracellular polysaccharide production, ionic bonds, and hydrophobic forces occur. the fourth stage is described by the multiplication of bacterial cells and an increase in secreted polysaccharides and the fifth stage involves strong metabolism in the biofilm. lastly, microorganisms begin to be released from the biofilm during the sixth stage, shedding bacteria to generate new biofilms elsewhere. several authors have identified a variety of mesophilic bacillus subspecies capable of surviving ultra-high temperature pasteurization via endospore formation (araújo et al. 2009; lindsay et al. 2002; scheldeman et al. 2006; sutyak et al. 2008). using bacterial cultures sampled from dairies, 16s rrna, and pcr amplification some of the most prevalent and potentially problematic species, in regards to biofilm production, have been characterized. these species include b. cereus, b. amyloliquefaciens, and several others. the level of virulence activity in b. cereus cells is due to a number of different environmental factors, including temperature, ph, oxygen tension, glucose concentrations, and specific antimicrobial chemical compounds (glatz and goepfeort, 1976; sutherland and limond, 1993). biofilm production is understood to be under similar regulation as toxins and other extracellular virulence determinants, which suggests that subinhibitory stress may have great influence on overall potential for bacillus spp. to become problematic in dairy microbiology settings. 212 • fine focus, vol. 4 (2) quorum sensing quorum sensing is a regulatory system where the bacterium (b. cereus) recognizes an extracellular signal caused by an autoinducer (ai) to sense the density of b. cereus in the immediate environment. quorum sensing is used to govern cell density, and the corresponding regulation of relevant gene expression that would enhance survival during the log-to-stationary phase transition in dense cultures, or in a natural environment such as soil, food, or within a host [28]. quorum sensing mechanisms control many processes in the bacterial cell, including sporulation, biofilm production, and virulence factor secretion [27]. quorum sensing involves direct or indirect activation of a related receptor protein by the ai (graumann, 2012). this activation results in upor down-regulation of specific genes. all quorum sensing routines are dependent on three principles: (1) the bacterial species produces ais; (2) ais are detected by membrane or cytoplasm receptors; and (3) ais produce a positive feedback loop (rutherford et al., 2012). gram-positive bacteria use small, post-translationally modified peptides as same species ais, called autoinducing peptides (aips) (graumann, 2012). aips are expressed as large, precursor peptides and processed into smaller, cyclic, thiolactone-containing peptides that are transported across the membrane. this transportation can happen in two ways: (1) two-component signaling (fig. 2) or (2) aip-binding transcription factor signaling (fig. 3). in the two-component signaling method, once the aips are transported outside of the cell they are too hydrophilic to cross the membrane without help. the aips remain in the extracellular matrix. the bacteria sense the aip as it binds to the receptor protein (histidine kinase) located in the neighboring bacterial cell surface. this binding induces phosphorylation of the kinase. the phosphoryl group is then transferred to an aspartate residue of the response regulator. then this binds to the promoter region of target genes, which activates or represses transcription. figure 2. two-component quorum sensing of gram-positive bacteria. ai synthase is used to process and transport the pro-aip out of the cell. once the concentration of aip outside the cell is high, aip binds to histidine kinase receptors. this binding activates the kinase activity of the receptor, inducing autophosphorylation. the phosphoryl group binds to the response regulator and activates transcription of the quorum sensing system genes [27]. aip aip aip aip aipaip aip ai synthase b pro-aip b cytoplasmic response regulator -histidine-kinase receptor phosphoryl quorum sensing regulation ins ideoutsid e bacillus spp. virulence • 213 quorum sensing & bacillus spp. pathogenesis quorum sensing in b. cereus is dependent on a protein plcr. plcr is a pleiotropic regulator of most virulence factors specific to the b. cereus group (nhe and hbl) (rutherford et al., 2012). the activity of plcr depends on binding to the aip that is produced from the papr protein. papr is a small signaling peptide that acts as a quorum sensing effector (slamti & lereclus, 2005) (fig. 3). papr is 48 amino acids long and is encoded by an open reading frame located downstream from plcr. papr is secreted from the cell forming a papr pro-aip. papr pro-aip is processed by neutral protease b (nprb) to form the active aip. the aip is transported back into the cell by an oligopeptide permease system (opp). aip then binds to the transcription factor plcr, activating the protein. this plcr-aip complex regulates the production of virulence factors and a positive feedback loop for papr. it has been shown that plcr expression is positively regulated by cody expression (frenzel et al., 2012). cody is a global transcriptional regulator that facilitates advantageous changes in response to variations of available nutrients in gram-positive bacteria (sonenshein, 2005). cody is a gtp and isoleucine binding protein that also initiates endosporulation. the binding of gtp and isoleucine act as co-repressors of the transcription of many genes. figure 3. aip binding plcr (transcription factor) quorum sensing signaling in b. cereus. a high population density outside the cell activates papr (pro-aip) and then the papr (proaip) is secreted outside of the cell. papr (pro-aip) is processed by the protease nprb to become a heptapeptide aip. aip is transported back into the cell using an opp. once aip is inside the cell, it binds and activates plcr. this plcr-aip complex regulates virulence factors and also produces a positive feedback loop for papr secretion [27]. 214 • fine focus, vol. 4 (2) aip ins ideoutsid e b pclr-aip opp processed by nprb papr (pro-aip) high-population density papr (pro-aip) a ctivates virulence factors nhe, hbl figure 4. cody regulates papr and plcr expression in b. cereus. nutrient availability for the cell regulates the expression of cody [27, 30]. endosporulation happens when there is a response to bacterial starvation by limited levels of carbon, nitrogen or phosphorous. endosporulation is a defense mechanism of gram-positive bacteria, like a turtle hiding in its shell, to protect its genome. nucleotide synthesis is dependent on carbon, nitrogen, and phosphorus. this spore keeps the genome dormant until the environment is favorable enough to replicate. cody is a transcriptional activator of the plcr gene [30] (fig. 4). in a δcody b. cereus strain, plcr expression was strong in exponential, late exponential, and stationary phases of growth. in contrast, a wildtype b. cereus strain showed expression of plcr in only the stationary phase of growth. cody was first found in b. subtilis to control expression of more than 100 stationary phase genes. thus, it is generally accepted that (like plcr), cody is widely conserved among bacillaceae family members. b. amyloliquefaciens belongs to the b. subtilis group (priest et al., 1987). members of this group exhibit similar behaviors physiologically, although b. amyloliquefaciens is not a subspecies of b. subtilis due to the difference in β-amylase production. b. amyloliquefaciens has been found to share less than 5% homology at the dna level with b. subtilis. b. subtilis has been shown to express cody (serror & sonenshein, 2002; ratnayake-lecamwasam et al., 2001). phelps and mckillip (2002), using dna pcr, found that hblc, hbld, hbla, nhea, and nheb genes or gene homologues were present in a different strain of b. amyloliquefaciens obtained from a louisiana creamery, although expression of these genes was not measured. thus, the potential for this species to harbor and express these or other virulence factors (via global effectors cody and/or plcr) is a realistic possibility, despite this species being placed (at least currently) in the b. subtilis group rather than the b. cereus group. nutrient availability cody papr plcr virulence factors high population density bacillus spp. virulence • 215 the debate over proper identification and understanding of bacillus virulence has been ongoing for over 50 years (rasko et al., 2005). recent public awareness of potential bioterrorism using the anthrax toxin produced by b. anthracis has lead government agencies to fund multiple studies aimed at rapidly differentiating b. anthracis from other closely related bacillus species such as b. cereus and b. thuringiensis, since b. anthracis produces the anthrax toxin encoded by two plasmid-based operons, pxo1 and pxo2. the anthrax toxin primarily kills herbivore mammals, but can also kill humans. not to be underestimated, b. cereus can cause severe food poisoning through its production of emetic and diarrheal toxins. while heavily used as an insecticidal agent in crops with its cry crystalline toxins, b. thuringiensis has also recently been demonstrated to cause food poisoning symptoms in humans similar to b. cereus. ironically, species like bacillus coagulans, which has been found to harbor the nhea gene, are readily used as probiotics in human health. bacillus spp. were originally differentiated into species at a time when biologists did not possess the molecular tools to delve deeper than biochemical tests and phenotypical observations. while this strategy worked well for other genera, 16s rrna analysis of differences among b. cereus, b. thuringiensis, and b. anthracis have shown these species to have a nucleotide sequence difference of less than 1%. recent advances in molecular biology have allowed scientists to scrutinize the genetic properties of these three “species”. after exhaustive studies using dna-dna hybridization, 16s and 23s rrna comparative analyses, multilocus sequence typing (mlst), fluorescent summation & future work amplified fragment length polymorphism analysis, rep-pcr, and small nucleotide polymorphism (snp) analyses, scientists have been unable to reliably differentiate these three bacillus species. while many methods have been pursued, most results have suggested that b. cereus, b. thuringiensis, and b. anthracis should be considered the same species due to highly conserved nucleoidal genetic sequences. due to the easily identifiable symptoms of b. anthracis and b. cereus, there is recent concern among biologists that the “b. anthracis” species may in fact be an oversampled subset of b. cereus. other scientists speculate that b. anthracis may have only recently evolved to the point to be considered distinct from b. cereus. unfortunately, recent literature is contradictory when discussing how similar two separate bacillus genomes need to be in order to be considered the same species. there are claims that b. thuringiensis, b. cereus, and b. anthracis should be considered one species on the basis of genetic evidence. alternatively, other scientists claim that current taxonomy has not divided bacillus strains enough, suggesting that more species or subspecies than currently listed in literature exist. no commonly accepted definition that separates these species on genetic evidence has been found. there are three nhe genes that are encoded on the nheabc operon, and have been shown to remain conserved as a cluster during genetic recombination. it can reasonably be assumed that the presence of the most proximal subunit of nhe indicates the presence of the other two genes. in the literature, all genes encoding the nhe and hbl enterotoxins have been readily located downstream in both b. cereus and b. 216 • fine focus, vol. 4 (2) thuringiensis (phelps & mckillip, 2002). the presence of the nheabc operon does not necessarily indicate a virulent strain, but has a very high likelihood of expressing these genes in a host environment or in food under permissive conditions. thus, future work to determine the pathogenicity of nhea positive samples could include the use of a tecra via to detect enterotoxin proteins. without this step, the virulence of nhea positive samples cannot be definitively determined. a large degree of genetic variation exists in nhe sequences among bacillus spp., giving rise to false negative results in pcr-based detection assays. strains negative for nhea in real-time pcr have been found to produce the enterotoxin nhe as determined using a tecra via kit. the nheabc operon is mobile among bacillus spp. through horizontal gene transfer (hgt). indeed, hgt has been observed among bacillus spp. and can serve as a mechanism explaining the incidence of non-b. cereus samples positive for nhea. while no data has been found to suggest that this gene transfer mechanism uses an integron, the anthrax-like operon pxo16 found in b. thuringiensis is part of a conjugative plasmid. it is reasonable conjecture that other bacillus species may also harbor conjugative plasmids that aid in hgt. within bacillus, most virulence factors are encoded on plasmids (55), which have been demonstrated to readily transfer between differing species. indeed, a recent study indicated that the virulence genes associated with b. cereus infection undergo frequent rearrangement both within the bacterial nucleoid and between species. thus, a better method than traditional biochemical tests to detect pathogenic bacillus strains is to screen for virulence operons present in plasmids or in nucleoidal dna. bacillus genomes that have been sequenced display a high level of genetic synteny in their gene order. two genes that encode for bacterial ribosomes, 16s and 23s rdna, contain genetic sequences that are less than 1% different when compared between b. cereus, b. thuringiensis, and b. anthracis (12). a dissimilarity of 3% between 16s or 23s rdna sequences is the minimal “cut off” between two strains to be considered as distinct species. additionally, the gyrb gene sequence shared among these species is very homologous. because these genes are shared among different species within the bacillus genus, they cannot be used to differentiate species. however, 16s and 23s rrna can be used to differentiate between different strains of b. anthracis. interestingly, there are a number of mechanisms that facilitate the movement of genes between different members of the bacillus genus. one such mechanism is through the natural action of bacteriophage. after lysing its host cell, the bacteriophage will insert its genes into bacillus genomes. while normally either lytic or lysogenic, it is possible for prophage to undergo random mutation, which renders it unable to enter the lysogenic cycle. in this way, genes from one species of bacteria can be transferred to bacillus spp. as previously mentioned, bacillus operons may be on conjugative plasmids. additionally, bacillus spp. are naturally competent, allowing these microbes to naturally take up random dna in their vicinity. the virulence genes for nhe are present in more strains of bacillus than is currently accepted within the scientific community. this research identified several “species” of bacillus that were not previously known to harbor the nhe enterotoxin operon. given bacillus spp. virulence • 217 218 • fine focus, vol. 4 (2) that a debate is currently underway about the very identity of b. cereus and other strains, it is improper for food safety experts to screen food products only for b. cereus. phenotypicbased classification techniques have failed to accurately differentiate bacillus species. additionally, no molecular-based approach can accurately differentiate bacillus. the bottom line is the determination of species within bacillus does not even matter when concerned with food safety. molecular techniques should instead screen for virulence determinants in microbes instead of identifying said microbes. since endospore formation enables bacillus spp. to be ubiquitous in the environment and on food, all foods should be examined in this way. this is the only true way to determine whether food products are safe for human consumption. 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acquired immunodeficiency syndrome. journal of clinical microbiology, 28(9), 21142116. 222 • fine focus, vol. 4 (2) 18 | fine focus embracing ignorance and failure promotes research excellence hanna dery* hanna_dery@taylor.edu chase holland* chase_holland@taylor.edu, jessica baker** jessica_baker@taylor.edu taylor university 1846 s main st, upland, in 46989 key words: failure, humility, ignorance, integrity, research * co-first authors **corresponding author vol 9 | 19 abstract pursuing research as an undergraduate student deepens and enhances both your college experience and your post college plans. whether someone hopes to go to graduate school, medical school, or into the workforce, the lessons you learn during a research project will further your abilities and potential. in this essay, we reflect on four lessons that we learned during our first fulltime nine-week research project: (1) do it, (2) learn to thrive at the interface of knowledge and ignorance, (3) embrace failure, and (4) build confidence for the future. we use our experiences to identify key areas of growth, both personal and professional, that we gained as we moved from class-based students to researchers exploring at the edge of current knowledge. introduction in college, most course-based labs are fun, hands-on, achievable, and success-oriented. as students, we were, more often than not, set up for success. because of this, we naively assumed that this was how all research worked. so, as we entered into our first non-course based research opportunity, our expectations were similar to that of our other college labs. we were expecting basic benchwork, small write ups, and strong step-by-step directions from our principal investigator (pi) that would culminate in novel insights and clear conclusions. our first foray into research was a nineweek, full-time summer research project. we both had our own individual projects to work on, but overall we focused on drosophila melanogaster (fruit fly) wings as a model of the cell biology process planar cell polarity. with such a short timeframe, we had to jump into quickly learning new and technically challenging skills, including how to perform microdissections and how to use a confocal fluorescence microscope. learning these skills was stressful; there was always a looming feeling that our ability was inadequate to the project at hand. our fears proved true; for the first few days and even weeks, we were not able to collect useful or quality data. we wrestled not only with failure due to experimental and human error, but also with failures that we had no control over regardless of skill level. alongside learning lab skills, we were working through previous literature that provided a basis for our projects. we had to grasp complex concepts and find the connections between previous projects and our own. taking together our technically difficult experiments and the complexity of the literature, we were in a position where we faced small failures throughout the entirety of our summer. despite the uncomfortable nature of failure, being pushed outside of our comfort zones taught us that failure is a common, perhaps even a necessary, component of research. as we reflected on our experience and discussed research with other students and professors, we found that ignorance and failures were not unique to our situation. rather, we realized that they are intrinsic to research, and when embraced more fully equip us as researchers. stuart firestein wrote a book, “ignorance: how it drives science” (1) and a sequel, “failure: why science is successful” (2). in the introduction to his first book, he says of the scientific process: 20 | fine focus “it’s not facts and rules. it’s black cats [being searched for] in dark rooms. as the princeton mathematician andrew wiles describes it: it’s groping and probing and poking, and some bumbling and bungling, and then a switch is discovered, often by accident, and the light is lit, and everyone says, “oh, wow, so that’s how it looks,” and then it’s off into the next dark room, looking for the next mysterious black feline.” (1) and yet, as we learned, exploring questions shrouded in uncertainty is not a depressing process, but rather “somehow exhilarating” (1). to be honest, we were initially and at times, still are frustrated by the difficulties we encountered because we had a naïve expectation of easy success and a misplaced fear of failure. however, we now realize that groping, bumbling, and bungling are what make research... research! we were and are! ignorant because research asks novel questions in dark rooms that have not yet been explored! that is liberating! we did and will! have failure because we study an incredibly complex and creatively beautiful realm of the natural world. that is exhilarating! of course, embracing ignorance and failure is not to say that researchers are to be intentionally uninformed, indifferent, or incompetent. rather, as said by louis pasteur in 1854 when he became dean of the faculty of sciences at the university of lille, “dans les champs de l’observation, le hasard ne favorise que les esprits préparés” (3), which roughly translates to “in the fields of observation, chance favors only the prepared mind.” for us, some of the mistakes in our summer projects were due to lack of preparation or individual carelessness. while we would have preferred to avoid preventable mistakes, the lessons we learned through these mistakes shaped our growth as experimentalists. further, our research experience taught us the importance of embracing broader, communal ignorance as a key step of the research experience. as student researchers, the valuable lessons we learned during our project allowed us to expand our mindsets to better understand the complexities of research. in the paragraphs to come, our goal is to share several of the lessons we learned in order to encourage fellow undergrads to explore and excel in the field of research. suggestion 1. do it. undergraduate research is a rare opportunity to do research without the expectations and burdens of graduate school. if you get the chance to join a lab, our best advice would be to do it. research as an undergraduate student will be hard and frustrating at times, but it is also an exciting and fulfilling experience that can have a major impact on your future. by his sophomore year, chase was already leaning towards a career in research. so, a fulltime research opportunity was a great way to solidify his desire and bolster his grad school application. when hanna decided to find a project, she was on a pre-med track and hadn’t even considered a career in research. her advisor mentioned that she might want to find a research opportunity so she decided it would be worth a try. after doing a nine-week project over the summer vol 9 | 21 she found a new career interest in research. for many college students, doing research might not have such a dramatic effect on career interests or future prospects, but it is still a valuable experience. as we will expand on below, research can broaden your understanding of how science works. in a relatively low-stakes environment, you will learn how you respond to failure and be mentored through these failures by professionals. although getting involved in research can be intimidating, the experience will build resiliency, knowledge, and skills that will benefit your life and career. suggestion 2. learn to thrive at the interface of knowledge and ignorance at the start of our summer research, we felt like we were jumping into the deep end of our projects. being naive college students, we thought we would have an extensive amount of time learning the introductory material. instead, we jumped immediately into the lab before we could fully grasp the relevance of our projects. that led us to be dependent on our pi’s lead. despite our inexperience, right from the beginning, our pi nudged us to take ownership and responsibility for our projects. of course, because of how little we knew, we often sought out guidance and direction which our pi was happy to provide. over the course of the summer, we realized that our pi was very intentional in encouraging us to jump right into the research. it wasn’t that she didn’t want us to eventually dig deep into the framework of our project. indeed, she had high expectations that we would. but, initially, she wanted us to experience learning at the edge of our comfort zone. while this was new and daunting to us, it is or ought to be common in research. in college courses, you are expected to study in order to know all the material and comprehend every concept; if you don’t know something, it’s usually because you did not study enough or did not learn the material that you are expected to know. in contrast, good research expands and works at the boundary of the known and unknown. the point of research is to discover and learn the unknown, about things we are currently ignorant of. excellent researchers understand that they do not know everything, yet they still strive to learn more. even though we came into our summer not knowing much, we had that drive to learn and discover the importance and biology behind what we were doing. we learned how to balance the tension of not understanding the bigger picture while also striving to learn as much as we could to do the best research possible. that skillset prepares us well for future research contexts. suggestion 3. embrace failure. in most areas of life, failure is shown in a very negative light. the same is often true for research; it is emotionally difficult to throw out data, have an incorrect hypothesis, or realize halfway through a project that you missed a confounding factor. however, failure plays an important role in research. failure often feels like a step back, but with persistence it can lead to larger steps in the right direction. one of our peers used a 22 | fine focus wagon wheel analogy to better understand failure in the research process. we expect a wheel to always be moving forward to reach our desired destination. but if you focus on any particular spoke on a wheel, about half the time the spoke will be moving backwards. when the spoke is moving backwards, it is easy to believe that we are being delayed from reaching our destination. however, without that backwards motion of the individual spoke, the wheel would not move forward. as with the wagon wheel, when we are not getting the data we expect, we often see that as a setback. but in reality, it is part of the process of moving the wheel of understanding forward. in the beginning of our project, we failed many times learning new lab techniques. we naively believed that most of our failures would only be early in the research process, but we slowly realized that failures occur through all stages of research. about halfway through her project, hanna had to throw out a good amount of her data when we realized the genotypes we needed were very difficult to identify. a few days before our research wrapped up for the summer, hanna once again had to throw out data, again due to unexpected genetics. while this may seem like avoidable failure, the lesson we learned about the genetic nuances was a necessary and informative step in furthering our conclusions. as we talked to our pi about struggles in research, she recounted one of her own projects. after a year and half of work on her project, she discovered that the data she collected did not correctly address her research question. although the data set was robust and thorough, it was deemed useless and was set aside. however, a couple of years later, she and her collaborator realized the data could be used to explore and test a new research question. within a few months, the “deemed useless” data became the central framework for a peer-reviewed journal article. experiencing our own struggles and hearing our pi’s story allowed us to understand that because you are exploring the unknown, there will always be failures in research. failure is important and sometimes inevitable in all steps of the research process. how you view and respond to failure is key. with a negative perspective, you can see failure as a hindrance and allow it to hold you and your research back. in contrast, realizing that it is an expected part of novel, boundary-pushing research, helps minimize our instinctive fear of failure. suggestion 4. build confidence for the future. as we encountered our lack of knowledge and failure time and time again throughout the summer, it felt like we would never grasp the main goal or collect the data we needed to test our hypotheses. yet, with lots of patience and persistence, we gained the lab and critical thinking skills needed to do good research. in the beginning we struggled with perfecting lab techniques and we doubted whether or not we would get quality data. however, when we started to obtain quality data and were able to confidently draw conclusions, we felt as if we had succeeded regardless of our earlier failures and doubts. indeed, by the end of vol 9 | 23 our project, we had discovered significant insights and contributed to our research field. our pi is currently working on a manuscript for publication that includes research from each of our projects. improving our research abilities not only sets us up for success in these projects, but also for success in our post undergraduate endeavors. looking back at our projects and experiences, we each saw personal growth in how we handle failure and success. for example, initially, we both saw failure as a hindrance, not as a stepping stone to progress. as we shared above, mistakes and failure are often shown in a very negative light, especially in our college course system. because of this, hanna often worried about making mistakes or failing in her work. when she got an unexpected result, she spent considerable time trying to pinpoint her assumed mistake and worrying about what our pi might think. however, our pi encouraged her through the mistakes and shared a personal mantra she tries to live by, “be cautiously optimistic, but emotionally detached.” by this, she did not mean that we should be apathetic, but rather that we should begin each experiment with optimism and finish each day in the lab with joy, regardless of the failures we may have experienced in our experiments. over time, hanna incorporated this mindset and learned to stay optimistic under stressful conditions and be confident in her abilities even when things go awry. in contrast to hanna, chase found it somewhat easier to not take failures personally. however, that ability to dismiss failures prevented him from taking larger steps in the pursuit of excellent research. through his research, he learned to apply ideas and solutions he discovered in past failures, to current problems. although chase and hanna naturally deal with failure very differently from one another, both learned that failure is an opportunity for growth and research progress. chase grew in terms of using failure as a building block for problem solving; hanna learned to see failure as part of the discovery process, rather than as an indicator of ability or as a source of self-doubt. with this experience, we also now feel confident in our lab skills and in our ability to be successful in another lab. during our nine weeks in the lab, we developed our skills from novices who did not know what we were doing to capable researchers. we now understand that becoming a good researcher depends less on academic prowess and more on persistence, grit, critical thinking, and new perspectives on ignorance and failure. with this new knowledge of how biology research works, we now have newfound confidence in our ability to succeed as we pursue further research. acknowledgements we thank the taylor university fmus program for funding the research project we discuss throughout this article. in addition, we thank the following professors and peers for reviewing this manuscript: dr. sarah justice, dr. lauren woodward hartzler, afia asamoah, natalya tropea, and natalie schmitt. 24 | fine focus references 1. firestein, s. (2012). ignorance: how it drives science. usa: oxford university press. 2. firestein, s. (2015). failure: why science is so successful. usa: oxford university press. 3. pasteur vallery-radot, ed. (1939). oeuvres de pasteur. paris, france: masson and co. vol. 7, p. 131. © 2025 smith, wheeler. fine focus, 11(1), 41 -47. doi: 10.33043/42dz3qzx shared with cc-by-nc-nd 4.0 license. stepping into the unknown: a graduate’s guide to the real world abigail smith b2s life sciences, franklin, in usa madison wheeler b2s life sciences, franklin, in usa https://creativecommons.org/licenses/by-nc-nd/4.0/ fine focus | volume 1142 1. introductions author smith: when you are young and the question of what you want to do when you grow up is posed, for some people, one answer comes to mind and it is as sure as the seasons change. as a former student convinced of my future, the path to realizing my actual interests has been winding. the year i turned 13, an under-calculated jump off a trampoline landed me a hospital bed with a broken ankle, a visit during which i questioned each nurse and doctor that was assigned to me about their job. it was then, sitting in the hospital, that i realized that helping to ease people’s pain was exactly what i wanted to do. as my high school years passed, i began to focus more on the challenging science classes and academic experiences that would provide access to any program or degree i pursued in university. i can vividly remember conversations with my mom about different types of doctors and the commitments i would have to make to become one, and we both discovered the magical field of neuroscience. within a week of researching and visiting hospitals to bug the doctors about their professions, i was completely convinced neurosurgery was going to be my career. when it was time to apply to college, i chose a small school that had a reputable neuroscience program and began eagerly moving through my classes, taking on extracurriculars and classes that medical schools value. after three semesters of slowly coming to the conclusion that a small school was not the best fit for me, i transferred to a larger school in hopes of finding research experience that would propel me further into my medical school dreams. it was not until i stumbled upon an opening in a graduate student’s lab conducting research in the adaptability of demographics to robotic surgery tools that i allowed myself to consider anything other than my “dream” career. being a part of a whole different branch of research opened my eyes to the possibility that i didn’t have to go to medical school to have an interesting and fulfilling career. desperate for this new discovery to not sway my goals, i fought the excitement and growing interest in the idea of pursuing research. it was not until senior year of college that i confronted the reality of my interest in research and made the decision that i would take some time off after graduation to work and gain research experience before applying to a phd in neuroscience research. this decision led me to become a scientist at b2s life sciences, learning the tactical skills that accompany the theoretical ideas i had been taught in school. the different knowledge and hands-on training i have learned in my industry job has given me confidence in my own ability to learn and perform at high levels, giving me the last push i needed to submit applications for graduate schools. as those application dates are steadily moving closer and i have given myself permission to explore careers in the research world, i have become increasingly ecstatic about the idea of being on the cutting edge of discovery of the brain and its incomprehensible abilities. i am grateful that i trusted myself to follow that small grain of curiosity, as it ultimately led me to confidently pursue my interests and find a career that engages and excites me. author wheeler: my journey started in franklin, indiana in a high school chemistry class. at first, it seemed like my teacher was much too excited about chemistry than anyone in their right mind should be. however, as the school year wore on, i found his enthusiastic energy rubbing off on me and awakening my interest in the logical ways that science and math weaved together to explain how even the tiniest bits of the universe works. it just made sense to me. fast forward 2 years: i stepped onto memorial mall at purdue university, marching excitedly to the first day of biology i. over the course of my four years at purdue, i pursued courses that interested me, even if they were not part of the typical biology degree path. one of these was a graduate-level ecology lab typically reserved for environmental engineers and ecology majors, and it ended up being my favorite class by far. we went on field trips to indiana dunes to collect data on succession and walked from campus to the nearby wildlife preserve to investigate migration of milkweed seeds. although i participated in various activities that were related to my major such as medical scribing, biology club, and career smith & wheeler | stepping into the unknown: a graduate’s guide to the real world 43 seminars, my favorite parts of my education were the most unexpected: the out-of-the-box courses, mentorship programs, purdue old masters, and exchange student club. the commonality among all of these favorite memories is that they allowed me to step outside of my comfort zone and learn about myself through the different lenses these unique experiences offered. as i continue to pursue a path of lifelong learning, i keep challenging myself to actively contribute to both my career and community. i am a scientist at b2s life sciences, a biotherapeutic company that develops quantitative assays and creates custom reagents to aid research teams in the drug development process. outside of work, i am a court-appointed special advocate (casa) volunteer advocating for children that have experienced neglect or abuse. i identified a need for improving my programming and statistics skillset, so i am also enrolled in a data science master’s program. in my free time, i thrift, mend, and resell vintage clothes. in essence, even if i’m not sure what my next career step may be at a given moment, i’m learning more about myself and my interests each day with the confidence that this knowledge will guide me in the right direction. 2. post-grad transition transitioning from student life to the workforce is one of the most complicated and tumultuous life adjustments that society leaves us exceptionally unequipped for. over and over during this period, i expressed my worries to other adults, with their only attempt at reassurement being a simple and unencouraging “welcome to the real world.” college sends students off with a resume stuffed full of technical skills and awards; however, there is no blueprint for what comes next. until this point, the paths of academics had been carefully laid out, starting on the first day of elementary school and ending with university graduation. in order to start navigating the shift from school to work, it’s important to first highlight the differences between the two. as every job is different, it can be hard to get a clear picture of the day-to-day until you actually begin working; nevertheless, there are a few common challenges detailed in the following sections about transitioning into the workforce that apply to most professions. in addition to outlining these obstacles, we have compiled our recommendations for overcoming them based on our personal experiences. a. becoming adjusted to working life: the time you spend for yourself is so crucial to a healthy work-life balance, and it tends to be one of the largest differences between school and a job. most college experiences have structured class schedules, but you are completely free to fill the time between and after classes however you choose. the abundance of student activities on campuses make occupying free time an easy choice between interests and friends who share them. however, in the setting of a typical adult working life, clubs and organizations are less accessible. while finding groups that share your interests is not quite as easy as it is in undergraduate programs, the exciting thing about being a true adult is having complete control over your free time and deciding how to best use it without limitations of campus resources and with more of an access to personal funding. similarly, there are a few more barriers to making and spending time with friends after college. during undergraduate school, if you take courses and join clubs that interest you, then by default you are surrounded by others with similar interests. this dynamic makes for easy connection and camaraderie; you share the same struggles, interests, and schedules. after college, a general sense of loneliness emerged as my friends scattered across the country and everyone was suddenly living according to starkly different timelines. some of my friends are mid-way through professional school, some of them are traveling the world, some of them are married with kids, and some of them are completely changing their career path. although this may sound scary, in reality it is such an exciting time in which you are free to write your own story, learn who you are, and cultivate intentional and meaningful friendships. fine focus | volume 1144 our solution: finding friendships as an adult can feel daunting, but the reality is that it just takes a new approach. you would be surprised how almost all post-graduate adults feel the exact same way! all it takes is a bit of intentionality and bravery to take the first step, like reaching out to an acquaintance on social media or talking to the other new person in your yoga class. essentially, i found it the most helpful to put myself in environments where i was guaranteed to meet people with shared interests. for example, if you like to read, go to a library event, join a book club, or visit your local independent bookstore. if you like to workout, join a workout class, go every week, and get to know the other people in the class. just like a muscle, it’s hard to push yourself outside of your comfort zone at first, but after each repetition, it gets easier and easier until it’s second nature. b. individualism versus being a team player: while directly opposite to the individualistic mindset in school, a productive and hireable employee needs to be a team player and have a sense of community effort in the work that they do. when working in a collaborative environment, it is important to understand that your work reflects your peers and supervisors and that doing your best boosts yourself and everyone around you. in school, oftentimes, the work you submit is solely yours and it is a direct correlation with the information you have learned, meaning your grades only reflect on you. there is instant and constant gratification of your work in academia that acts as a direct gauge for your success. in a work environment, there is much less chance for recognition or gratification in your work. in most work environments and depending very much on the career, group recognition is much more common than individual appreciation, as the company is wholly dependent on the productivity of the team. for a recent graduate entering the workplace, it can be disheartening when your contributions are not acknowledged and instead lumped into the whole. learning to change expectations of acknowledgment can be very difficult for some. immediate, measurable feedback for every single assignment and project gives a sense of linearity and achievement to students; recognition in the workplace is more subtle and less personal, often only occurring for major milestones. however, many people find this type of recognition, like being selected to lead a major project or receiving encouragement from a boss, to be more meaningful and fulfilling as it is tied to real world impact and is a direct result of your deliberate efforts. our solution: when beginning to work for a common goal among peers, as is the case for most careers, your small successes are often lost to the bigger picture. because of this transition, it is very common to feel overlooked and undervalued unless your individual role is singled out and praised. i have found that celebrating your own victories and work can change your outlook on how valuable you are to your company. it is important to begin with clearly outlined personal goals and make a point to celebrate them once you achieve them. many direct supervisors will congratulate and encourage you, but being your own cheerleader helps you ultimately happier with the effort you put forth. c. input versus output: yet another key difference between the learning stage and career stage of life is the type of work expected. during school, a student is asked to assimilate loads of information and absorb the bigger pictures and connections between the concepts they are learning. in a working environment, one is expected to produce by means of information, data generation, reports, presentations, etc. for young adults coming from a phase of life that expects constant intake, it can be overwhelming to consistently yield productivity. our solution: continue to prioritize learning after graduation. if there are opportunities at work to learn something new, seize it and ask as many questions as you can. if there aren’t, identify a need or gaps in knowledge and consider asking your boss for an opportunity to learn about it through a class or conference. it’s also been smith & wheeler | stepping into the unknown: a graduate’s guide to the real world 45 incredibly invigorating to explore my passions and hobbies after work hours. in the year since we graduated college, we attended events with our local bookstore where we learned to bedazzle books; we visited a yarn store downtown for a beginners knitting class; we started learning the native languages of countries that we are soon traveling to. in the transition from the mass intake of information to constant output and productivity, it’s been vital to continue to foster opportunities for learning, both professionally and in our personal lives. d. communication: in college, professional communication is somewhat limited and rarely formal; however, at work, efficient and effective communication is single-handedly the most valuable skill. emails, meetings, newsletters, and direct messages are all frequently used throughout a workday to ensure work is being done correctly. however, during school, the most formal means of communication are occasional emails to professors or fellow students. at our job, if there is a simple miscommunication between the principal investigator (pi) and scientist regarding experiment setup resulting in assay failure, it can cost thousands of dollars to re-run it, and our company is forced to absorb that cost. additionally, being able to summarize data analysis from the day’s experiment and relay it effectively to the pi is imperative to guarantee that everyone involved is on the same page. similarly, if a scientist does not accurately record and convey reagent requirements for a project, it can cause a weeks-long setback and completely disrupt timelines. it becomes absolutely vital to communicate every single detail, no matter how miniscule. our solution: this shift in expectations presents quite the learning curve. thus, it is important to be proactive and hone these skills throughout school as you prepare for the transition from academics to work. to improve your ability to present complex information in a straightforward manner, take that presentation course you’ve been avoiding because you hate talking in front of people. to learn to break down information quickly and effectively, try teaching or tutoring your favorite subject for practice. while you have access to your university’s career center, work on a resume and cover letter template and ask for feedback or schedule a mock interview. you’ll have a head start after graduation if you commit time and energy to refine your communication skills as a student. when you arrive at your first job, ask as many questions as you can come up with (especially the “dumb” ones), don’t be afraid to repeatedly clarify expectations or plans, and be meticulous in both your written and verbal communication. fine focus | volume 1146 3. navigating the job market after graduation, embarking on the job search journey can be intimidating. imposter syndrome, determined confidence, and enthusiasm of a first job can coalesce to create an uncomfortable mix of worry and excitement. before starting a job search, it is important to clarify which features of a job are the most important to you. after giving an overview of our journeys through this process, we hope to provide some actionable tips to help prepare for navigating the job market. author smith: as graduation grew nearer and the daunting task of applying to jobs and entering the workforce became more than a distant thought, i sought advice from those around me who had been through the process. a few of my older friends who had recently graduated from my purdue school of science program were more than happy to share their experiences while job hunting. their advice was to be patient and to be diligent about following up. while applying is the first step of finding your spot in your chosen industry, remembering to check back in is often overlooked. it’s important to show employers that you are eager and intentional throughout the entire process. while this seems like a small detail, companies look for committed workers who are willing to go out of their way to achieve their goals. when asking my father what he would look for in a hirable candidate for his company, he described that good communicators and team players are always moved to the front of the line as potential employees. keeping these tips in mind as i began to put out applications and sit for interviews, i found that oftentimes i was complimented on these very aspects after meeting with several companies. i am a strong believer that an attractive personality, an eagerness to be involved and learn any way you can, and the ability to take direction and communicate well will be key to any person participating in the difficult endeavor that is job hunting. author wheeler: upon graduation, i began the tedious process of finding my first “big girl” job. initially, i was overwhelmed by the sheer volume of job listings requiring a phd or 10+ years of experience. it felt as though everything i had worked so hard for over the past 4 years had amounted to nothing. the strong sense of pride i felt during graduation was in sharp contrast to the lack of confidence i felt immediately upon embarking on my job search. i sought advice from anyone that i thought could relate– peers grappling with similar emotions, family that constantly encouraged me, and older friends that had experienced the exact same challenges. in retrospect, i recognize that i was not seeking advice, but reassurance. i desperately wanted reassurance that i was on the right path, that i could truly offer value to an employer, and that the right job opportunity would eventually come. i realized that i needed a major shift in my mindset. rather than simply viewing it as asking a company to give me a job, i began to see all of the ways my skillset would benefit an organization. in place of worrying whether my qualifications and experience was enough to impress an employer, i shifted my focus to how the role could help me grow and develop. in this time of stress and worry, it took a great deal of intentionality to elucidate exactly what i wanted from a job and transform both my mindset and my confidence. with this newfound perspective, i was able to approach my job search in a new light, with a sense of purpose and self-assurance. ultimately, this shift in mindset not only helped me find a job that aligned with my needs and goals but also set me on a path of continual professional and personal growth, with the confidence that the right opportunities would present themselves when the time was right. the most important tip, albeit cliche, is to be patient. from submitting an application and awaiting a response to completing various rounds of interviews and discussing pay and benefits, the process of securing a job is often lengthy and sluggish. as many companies receive hundreds of applications each day, it may take a while to hear back at all. this is the case regardless of your smith & wheeler | stepping into the unknown: a graduate’s guide to the real world 47 experience or qualifications; it is simply a requisite element of the process. nonetheless, if there is a position you are especially excited about, it can be helpful to reach out to the hiring team again to check in and restate your interest in the position. before ever browsing indeed or linkedin, it is imperative that you have a concrete list of your non-negotiables and needs that you are looking for in a job. you are interviewing the company just as they are interviewing you. ensure that you know exactly what you need from an employer by investigating the aspects of a job that are the most important to you. for example, if you know you enjoy travel, make sure that the company’s pto policy will accommodate your needs. in addition to technical aspects such as salary, pto, and benefits, it is important to seek work environments and teams that best suit your personality. some companies greatly value teamwork, while others reward individual accomplishment. some people thrive in competitive environments while others flourish in more collaborative workspaces. while interviewing, inquire about things like the work-life balance, the flexibility of work hours, the possibility of hybrid/remote work, relocation needs or opportunities, and any other aspect that would help you reach your individual life goals. while it is common to discuss salary and pay before committing to a job, many recent graduates are not taught that they have the ability to negotiate. it is perfectly acceptable to make a counter offer that would better meet your needs with regard to salary or pto, for example. there will be times when an employer is unable to meet a request for negotiation outside the offer they have already made, at which time, you will need to examine if you are happy accepting the job as is, or if you feel that your experience and time would be better suited in another place. a crucial part of finding the best fitting job for you is to advocate for yourself and your needs. if you are fulfilled and find your company and work community rewarding, it will be much easier for you to be productive and in the mindset to learn and participate. 4. final reflections ultimately, the transition from college to the workforce is challenging, but it is also rewarding and fulfilling to see the culmination of years of work. although we dove into various tips and solutions that may help in the job search process, there is no need to overcomplicate it. you’ve done the work and are deserving of the job you are looking for! in times of doubt and worry, it is vital to lean on your friends or peers who are experiencing the same lengthy process as you, find comfort and encouragement that most job-hunting journeys take time. we sincerely hope that our experience provides the helpful groundwork needed to succeed in transitioning from university to the workforce, as the advice included here are the exact tips we desperately wished we had during that period of change. after securing your first job in the industry, prioritize a clear boundary between work and the rest of your life. the habits developed at the start of a new job lay the foundation for your entire tenure at that company. fulfilment at work is equally influenced by your life outside of work as by your work itself. the freedom and opportunities that working life provides are unparalleled– take advantage of it! embrace a well-rounded life, explore new hobbies, and be purposeful with the time you spend with those you care about. stepping into the unknown: a graduate’s guide to the real world 1. introductions 2. post-grad transition 3. navigating the job market 4. final reflections © 2024 ebrahimbabie. fine focus, 10(1), 6-8. doi: ff.10.1.6-8 shared with cc-by-nc-nd 4.0 license. parisa ebrahimbabaie’s academic tenure at bethune-cookman university (b-cu) unfolds as a captivating narrative intricately woven with challenges stemming from her international background as a woman from iran. this exploration of her experiences at the historical black university (hbcu) delves into the complexities of identity, cultural adjustments, and the unique dynamics of an academic environment rich in historical significance. adapting to b-cu requires more than adjusting to the physical surroundings; it involves a deep understanding of various facets of the local culture, including communication dynamics, social nuances, and aligning academic expectations with prevailing norms. this cultural transition demands a significant reassessment of approaches to teaching, research collaboration, and interpersonal relationships for a seamless integration into the academic and cultural environment at b-cu. parisa ebrahimbabie parisa ebrahimbabaie’s academic sojourn at bethune-cookman university: unraveling the complexities https://creativecommons.org/licenses/by-nc-nd/4.0/ parisa ebrahimbabaie’s academic sojourn at bethune-cookman university 7 navigating the pervasive stereotypes linked to her nationality and gender poses a persistent challenge for parisa. overcoming preconceptions requires a delicate blend of assertiveness and cultural education to foster a more accurate understanding of her identity. the intersection of parisa’s iranian identity introduces challenges within the academic sphere, requiring a continuous negotiation of her identity within a landscape not always attuned to the complexity of her experiences. this intersectionality weaves into her daily interactions, shaping her role and experiences within the academic community. building authentic connections with colleagues and students at b-cu becomes a multifaceted challenge for parisa. overcoming cultural barriers and establishing professional rapport within the context of an hbcu necessitate an awareness of cultural diversity and an appreciation for the historical foundations shaping relationships in this distinctive academic community. the unique setting of an hbcu exposes parisa to an environment steeped in historical significance and cultural traditions. navigating this context requires more than academic understanding; it demands a deep immersion into the institution’s ethos. grasping and appreciating the historical commitment to the african american community becomes fundamental to her role as an academic contributor. engaging with the predominantly african american local community at b-cu presents challenges connected to cultural sensitivity. navigating community engagement demands dedication to academic outreach and a keen awareness of diverse perspectives within the community. despite challenges, parisa is very happy working at bethune-cookman university as an assistant professor, appreciating the positive and enriching academic environment. achieving a harmonious equilibrium between her diverse cultural identity and institutional expectations remains a continuous negotiation. striking this delicate balance involves leveraging the strengths of her iranian heritage while aligning with the cultural fabric of the hbcu. this intricate dance of identity within the academic realm is central to her ongoing experience. gender dynamics, evident within the institution and broader cultural context, significantly influence parisa’s professional journey. navigating gender-related challenges, advocating for equity, and fostering an inclusive environment are integral components of her academic role. establishing a professional network within the academic sphere poses distinctive challenges. cultural differences and potential biases add complexity to networking dynamics, necessitating active participation in opportunities to overcome barriers and forge meaningful connections within the university community. access to leadership positions for women, particularly from underrepresented backgrounds, poses nuanced challenges. navigating the path to leadership roles demands professional acumen and a proactive approach to contribute meaningfully to fine focus | volume 108 decision-making processes within the academic landscape. accessing research funding, laboratories, and resources presents parisa with a multifaceted challenge. adapting to constraints and identifying alternative avenues to pursue research goals becomes a crucial aspect of her academic journey. this adaptive resilience characterizes her approach to academic research within the distinctive institutional context. diverse levels of institutional support for diversity and inclusion at b-cu add complexity to parisa’s academic experience. advocating for initiatives that advance diversity and actively participating in creating an inclusive environment are essential components of her role, requiring a nuanced understanding of institutional dynamics. in conclusion, parisa’s academic journey at bethune-cookman university has been marked by diverse challenges extending beyond traditional academic boundaries. this narrative unravels the complexities inherent in her experience, shedding light on the multifaceted nature of her identity intersectionality within the unique landscape of an hbcu. as she continues navigating this intricate journey, her story stands as a testament to the resilience and adaptability required to thrive in the dynamic and diverse academic environments shaping the modern educational landscape. importantly, she emphasizes her happiness and appreciation for the positive and enriching academic environment at bethune-cookman university. 12 | fine focus utilizing your academic advisor to maximize your opportunities and success as an undergraduate stem student. by sarah r. williams msc. biology, academic advisor for biology, ball state university vol 9 | 13 twenty years ago, i was an undergraduate student studying biology, working through my program mostly timely, if somewhat aimlessly (throwing in a random class or two along the way), checking off requirements in a ragged paper copy of the 20002001 undergraduate course catalog at northern kentucky university. back then, at most institutions, there were no dedicated, full-time academic advisors. we had faculty advisors that we were expected to seek out (if you were able to figure out who your faculty advisor was). ultimately, most undergraduates were left on their own to navigate the requirements of their major, minor, and bachelor’s degree. because of this, my experience as a firstgeneration college student could be, at turns, overwhelming and confusing. how much more could i have gotten out of my undergraduate years if i’d had someone helping me navigate each semester while also working with me on the big picture of life after graduation? since becoming an academic advisor six years ago, i’ve thought about this question a lot, and now it defines how i approach my job working with undergraduate biology students each day. according to a 2018 article in edsurge, a recent student engagement survey found that 78% of students reported having met with an academic advisor, and 65% reported that their advisor helped them create an academic plan (johnson, 2018). this is a huge step forward for student success in college. in this perspective, i’ll explain why the increase in academic advising and advisors is so beneficial to you. i’ll discuss what a typical advising meeting looks like, and how you can gain the most benefit from meeting with an advisor. i’ll also provide some advice on other ways you can get engaged in your major and be successful in your college courses. a typical advising meeting lasts about thirty minutes and can be in-person, virtual, or a phone appointment based on your preference. each meeting is usually a combination of problemsolving (“my classes are conflicting in my schedule” or “the class is full, what can i take instead?”) as well as bigger picture topics (“i love my microbiology class! what future classes can i take that are related, and what types of jobs can i get?”). when preparing for an appointment, i utilize the university’s advising tools and suggest students do the same. this way, the conversation feels more like a twoway street, since we’ll have the same points of reference when discussing requirements. at most universities, this will include access to a degree-tracking software program like degree works, starfish, or achieve. these programs list what is required of your major, minor, and bachelor’s degree, as well as keeping track of what you’ve already completed, your grades, and gpa. another tool 14 | fine focus we use allows students to book advising appointments, as well as to review notes that were entered by the advisor or other support staff in previous meetings. ball state currently uses navigate but has used aviso in the past. prior to an advising appointment, and when looking at a student’s degree and note tracking records, i consider the following: • current course schedule (is the student taking the right courses, and not too heavy of a load?) • current gpa (is the student on academic probation and needs support, or is their gpa competitive for medical school or other graduate or professional programs?) • past course loads (did they do well at 18 credit hours or do they need to lighten up in future semesters?) • academic plan (is the student’s semester-by-semester plan reasonable and up to date?) • updates (are there any changes to the degree program or course offerings i need to make the student aware of ?) when meeting with the student, i consider these additional topics for discussion, but base it off the situation and the student’s main goals for the meeting: • do you enjoy the major and feel connected to classmates, clubs, faculty, or other opportunities related to the major? • when do you want to graduate? • are you a commuter or living on campus? • are you employed full-time? • do you have scholarships or financial aid requiring certain credit hours each semester? • is your physical or mental health causing academic struggles? • do you have family support and/ or pressures affecting academic decisions? • what do you want to do after graduation? go directly into the workforce, or apply to graduate or professional programs? many times, after having a conversation with a student, it becomes clear to them they aren’t sure of a direction moving forward. they may enjoy biology, but may not be certain of what a future career looks like for them. if a student wants to continue moving forward in the major, but is unsure of their interests, i usually suggest they consider participating in undergraduate research. undergraduate research is a great place to start, since it can be completed on or near campus, and the faculty mentor will usually work around your existing school or work schedule. also, most faculty don’t expect you to come in with vol 9 | 15 skills or experience. they will train you on specific lab skills, while reinforcing topics you’re already learning in your science courses, such as the scientific method and how to complete a literature review. to get involved in undergraduate research, i suggest students take 3040 minutes to check out the faculty biographies and research interests that are listed on a university’s departmental webpage. take notes on what research topics piqued your interest, and then construct an individualized (but brief) email to two or three faculty. in the email, you’ll want to introduce yourself and mention you read about their research and would like to participate. it is also helpful to mention any relevant coursework you’ve taken that could help in the laboratory (e.g. “i have completed principles of biology 1, so i am familiar with pipetting technique”, or “i have a foundational knowledge of cell division”). finally, you can decide to attach a résumé, but it’s not necessary. another approach is to have a conversation with one of your faculty after class. if there is one class or instructor that stands out to you, ask what type of research they do and if they are taking on any new students. being flexible on the research topic can be worthwhile if you’re still exploring, but want to gain new insights from a mentor you feel you already have a good rapport with. even students who don’t see themselves in a research-related career after college report positive outcomes after completing at least one semester of undergraduate research. in a 2007 web-based survey of 367 undergraduate stem students and faculty mentors, six categories of benefits emerged: • personal/professional gains • gains in thinking and working like a scientist • gains in skills • demonstrating norms of professional practice and understanding how scientists practice their profession • gains in career clarification • enhanced career preparation. further analysis found, “strong congruence across the student, faculty, and alumni data sets” (hunter et al, 2009). my own advisees have reported similar benefits. they report more confidence in their problem-solving abilities and in working independently. they also report that undergraduate research made them consider other factors that relate to their ultimate career goal. for example, premedical preparation students who’ve completed microbiology research have a deeper understanding of aseptic technique and bacterial resistance, and will undoubtedly carry this knowledge into their medical training and future patient care. additionally, skills and knowledge gained in undergraduate research make a student a more 16 | fine focus competitive candidate in many fields. i’ve had several students report back to me after graduation that their undergraduate research experience gave them something extra to talk about in an interview, making them stand out to an employer. in conclusion, your advisor can be a valuable part of your undergraduate experience. they will help you problem solve, while also thinking of ways to dovetail your interests and strengths to make the most of your undergraduate experience. ultimately, you can expect your advisor to: • know your degree requirements and university policy and procedure, and keep you updated on changes • keep your academic plan updated • check in with you each semester • have knowledge of, and refer you out to other services, such as financial aid, tutoring, counseling, registrar’s office, etc. in return, an advisor expects you to: • utilize the tools the university has provided you, such as degree tracking software and appointment apps, and ask if anything seems confusing • respond to check-ins and let your advisor know if you are struggling or need help with something • read emails from your advisor or the university for important updates, such as registration and withdrawal deadlines examining my own experience as an undergraduate, feeling supported and advocated for by an advisor at the university would have given me hope when i felt discouraged, struggling with course material or a bad grade. having now met with hundreds of undergraduate biology majors, i know a good advisor can help students find several successful paths forward, regardless of perceived setbacks. upper-level students reflecting on their own struggles also support this point of view. a 2018 study of successful pre-health students’ views of academic success showed that participants “almost all agreed that incoming students should not allow a single challenge or failure to deter them from pursuing their goals” and “did not emphasize natural gifts, talents, or intelligence as the keys to academic success.” rather, successful students demonstrated traits such as adjusting study strategies, seeking help through professors or supplemental instruction, and time management (dumke et al, 2018). one student in the study summed it up best, saying, “you can’t always do it on your own…sometimes, it’s not enough to just read a textbook and try and comprehend the information on your own; you need an entirely different perspective” (dumke et al, 2018). vol 9 | 17 references 1. dumke, e. k., tyndall, c., naff, d., crowder, a., & cauley, k. m. (2018). a qualitative exploration of pre-health students’ perceptions of academic success and persistence. nacada journal, 38(2), 5–19. 2. hunter, a.-b., laursen, t. j., & thiry, h. (2009). urssa: evaluating student gains from undergraduate research in the sciences. cur quarterly, 29(3), 15–19. 3. johnson, s. (2018, december 27). report: advising attendance is up, but more ‘in-depth’ student support is still needed edsurge news. edsurge. retrieved september 30, 2022, from https://www.edsurge.com/news/2018-02-13report-advising-attendance-is-up-but-more-in-depth-student-support-isstill-needed 102 | fine focus opinions and attitudes of serbian high school students regarding usage of antibiotics nemanja r. kutlesic and dimitrije milosavljevic nemanja kutlesic nemanja.kutlesic@studenti.unicam.it school of biosciences and veterinary medicine, university of camerino, italy manuscript received 18 march 2020; accepted 13 may 2020. volume six | 103 abstract proper usage of antibiotics is a rather important subject that is often overlooked in secondary education, which can provoke misconceptions among youth towards this important topic. the aim of the study was to examine the knowledge and habits regarding the usage of antibiotics in high school population. the research hypothesized that the students would have an acceptable level of basic knowledge on antibiotics and their usage, as students from all study programs are taught equally about antibiotics. the research also examined whether parents’ affiliation in healthcare influences their knowledge. the study has discovered that most of the students have an acceptable knowledge on the matter, with 59% being acquainted with the antibiotics’ mechanism of functioning. the largest part (78%) was able to distinguish paracetamol from antibiotics. however, as much as 41% identified that they have taken antibiotics from different time than prescribed and 31% admitted to having used antibiotics to treat viruses. a majority have identified their doctor as their source for advice about treatment (67%). the research shows that students’ knowledge is generally constant among the groups, regardless of the study course or parents’ affiliation in healthcare. the only exception is that more students with parents’ working in healthcare have taken antibiotics for the time prescribed (p<0.05). overall, students have demonstrated good theoretical knowledge with habits that should be improved. students need to be better acquainted with guidelines of proper usage through informative campaigns and school projects. 104 | fine focus introduction clinical effectiveness of antibiotics is directly dependent on the manner of their usage [2, 5, 42]. any improper, irrational use of antibiotics could lead to the treatment failure, adverse side effects, or development of antimicrobial resistance [5, 13, 19, 20, 40, 42]. according to the world health organization (who), resistance to antibacterial/viral/parasitic/ fungal drugs occurs when microorganisms change in ways that render the medication used to cure the infections they cause ineffective [39]. during the first 20 years after fleming’s discovery of penicillin (1928), followed by the discovery of several other classes of antibiotics, it seemed that bacteria-induced diseases were defeated. unfortunately, since then, almost every type of bacterium had developed resistance to one or more antibiotics in clinical use [3, 7, 30], escherichia coli, klebsiella pneumoniae, staphylococcus aureus, streptococcus pneumoniae being the most reported [14]. it has been estimated that antimicrobial resistance causes 700 000 deaths per year; by 2050 this number may rise to 10 000 000 deaths per year [29]. bacterial resistance to antibiotics is a global health emergency [33], since there are more and more infections that become harder or even impossible to treat. as in the pre-antibiotic era, the most common infections and minor injuries could become lethal again [3, 11]. antibiotic misuse can be the consequence of physicians’ error, like over-prescription [3]. it can also be associated with consumers’ drug use habits and their insufficient/incorrect knowledge of antibiotics indications/contraindications, storage, dosage, possible side effects and duration of therapy [6, 18, 42]. taking antibiotics without a prescription (selfmedication), together with over-prescription, leads to increased antibiotic consumption in the community, which has been positively associated with bacterial resistance [7]. the association is particularly strong in south european countries, including serbia, where antibiotic consumption is higher than in northern europe [7, 30]. strict guidelines for prescription and antibiotic use resulted in lowest level of resistance in scandinavian countries, uk, and the netherlands [4, 18, 19, 21, 36]. belief that antibiotics can prevent and cure any disease, that all types of infection (including viral or fungal) should be treated with antibiotics, that they can be effective as antipyretics, antitussics, painkillers, are common misconceptions that lead to the development of antibacterial resistance and, consequently, increased morbidity, duration of treatment, side effects, even premature mortality [5, 6, 9, 10, 15, 22, 23, 26, 27, 31, 40]. these worrying data related to antibiotics usage have been the motivation for the conduction of this research, which has encompassed a sample of 130 students of different age groups within a serbian high school located in the country’s third-largest city that features a specialized clinical center [38]. the study’s main objective was to examine the overall knowledge, beliefs and habits of serbian high school students from the sample regarding antibiotics usage. the secondary objective of the study was to determine the influence of the study course as well as the students’ family background (i.e. if a person engaged in a medical profession is present in the family) on the students’ overall knowledge of antibiotics. currently, in the high school education program in the country of study, antibiotics are briefly mentioned in the chemistry course in the fourth year of the course of study as drugs used to treat common infections of bacterial origin (population of 17 to 18 years of age) [34]. at the time of the study, no participants had completed this chapter. similar cases of only a small part of the curricula devoted to study of antibiotics have been reported in other european countries, such as portugal [5]. however, the base of biology courses in serbian high schools include a volume six | 105 chapter on bacteria and viruses, which also includes parts devoted to usage and functioning mechanism of antibiotics. information reported in this chapter include the division of bacteria into gram-positive and gram-negative species. some examples of bacterial diseases are cited along with the bacterium that causes it, and a short passage is devoted to the advice about treatment of bacterial diseases, emphasizing the importance of medical guidance. in some textbooks [8], a particularly strong emphasis is put on the fact that antibiotics cannot be used to treat viral diseases. during the course of this study, all the participants have already completed the study of this chapter, as it is studied in the first semester of the first year of high school. therefore, we hypothesize that serbian high school population will demonstrate knowledge of clear distinction between the two types of diseases, unlike some comparable groups in the region. in addition, we expect that students will demonstrate an acceptable level of basic knowledge on the matter, which would include basic understanding of mechanism of functioning, dosing regimens and importance of medical advice when antibiotics are used. the importance of the study lies in the fact that the studied age group represents a sample of a future main antibiotic-consumer population. assessing their current knowledge helps determine their capacity to use the medication in a way that is acceptable for both them and their future generation. furthermore, the analyzed data on antibiotics usage undoubtedly examines educational system’s attitude towards the very important topic of antibiotics usage. the revealing of possible misconceptions and bad habits regarding the use of antibiotics would enable to update the school curriculum, devise appropriate awareness campaigns and therefore ameliorate the students’ knowledge on such an important topic. materials and methods prior to any data gathering, the study was approved by the school board of the public high school in serbia where the study was performed. the main method for gathering data was conducting a questionnaire among the surveyed parties. students taking part in the survey were between 15 and 18 years of age. in order to comply with legislative norms of the country where the study was performed, all the students under the legal age of 18 were given consent forms for parents to fill in. by signing these forms, parents gave informed consent for their children to participate in the survey by answering the enclosed questionnaire form, and for this information to be processed and studied for research purposes. students of legal age have signed the consent themselves. participation in the study was completely voluntary and included no compensation or reward for participating parties. participating students were chosen randomly based on the envelope method for each grade of high school. total number of participants was 130. class names were inserted in envelopes that were divided into groups in reference to the study course. from each grade and study course, one envelope was chosen, to give a total number of eight surveyed classes. one class was not surveyed due to a low number of students in the class. prior to taking the survey, students were asked whether they have ever taken antibiotics, to ensure that students would be able to give answers to the survey. surveys were answered in a classroom setting. language of the survey was serbian. all the questions referred to the habits of students regarding recent antibiotic usage throughout their 106 | fine focus no question possible answer 1. have you ever taken an antibiotic for a longer or shorter time period than prescribed? yes/no 2. do you consume alcohol when using antibiotics? * yes/no 3. does any member of your family nucleus work in a healthcare institution? yes/no 4. have you ever used antibiotics when a virus causes your disease, such as in case of common flu and cold? yes/no 5. have you ever acquired antibiotics without prescription? yes/no 6. after you consume an antibiotic, do you feel calmer? yes/no 7. is the paracetamol** antibiotic? yes/no no question possible answer 1. i use antibiotics often on a monthly level for repeated treatments. 1-2-3-4-5 2. i am well acquainted with antibiotics’ method of function. 1-2-3-4-5 3. i often miss my doses when taking an antibiotic. 1-2-3-4-5 4. antibiotics can help us with most diseases (headache, fever, hypertension, flu, etc.) 1-2-3-4-5 5. name all the applicable advisors for your treatment when ill: parents in healthcare parents not in healthcare friends media/internet official doctor * legal age in serbia for acquiring both cigarettes and alcohol is 18 years of age. however, it is not uncommon that younger individuals have access to these substances. ** paracetamol is a commonly used analgesic or antipyretic drug in serbia, whose usan is acetaminophen. this drug is commonly marketed in the usa as tylenol. table 1. yes-no questions used in questionnaire table 2. assesment of agreement questions volume six | 107 no question possible answer 1. have you ever taken an antibiotic for a longer or shorter time period than prescribed? yes/no 2. do you consume alcohol when using antibiotics? * yes/no 3. does any member of your family nucleus work in a healthcare institution? yes/no 4. have you ever used antibiotics when a virus causes your disease, such as in case of common flu and cold? yes/no 5. have you ever acquired antibiotics without prescription? yes/no 6. after you consume an antibiotic, do you feel calmer? yes/no 7. is the paracetamol** antibiotic? yes/no no question possible answer 1. i use antibiotics often on a monthly level for repeated treatments. 1-2-3-4-5 2. i am well acquainted with antibiotics’ method of function. 1-2-3-4-5 3. i often miss my doses when taking an antibiotic. 1-2-3-4-5 4. antibiotics can help us with most diseases (headache, fever, hypertension, flu, etc.) 1-2-3-4-5 5. name all the applicable advisors for your treatment when ill: parents in healthcare parents not in healthcare friends media/internet official doctor high school studies. the questionnaire distributed among the groups included two main question groups: simple questions requesting yes/no answers, and questions in which the surveyed individual was to express the level of consent with the phrase given by answering from 1 to 5 on the likert rating scale, with 5 expressing highest level of agreement. furthermore, the questionnaire, although fully anonymous, included personal questions that would help process the data among groups for more effective analysis. design of the questionnaire was based on many previous studies that have encompassed interviewees from different backgrounds [1, 5, 9, 15, 21, 29, 36, and 40]. first question group: yes-no answer the first question group was designed to be simple in appearance so that quantitative data can be easily obtained. the questions are listed in the table 1. questions were designed in a manner so that they can mostly assess multiple points of interest at the same time. furthermore, the form of the question was laid out in a manner so that it does not in any way resemble a knowledge test, but rather a survey. this was performed so that students do not put much thinking when giving their answer, thus altering the truth in order for their answers to seem more plausible. second question group: assessment of agreement – likert rating scale in the second group of questions, students were asked to assess how much they agree with a given sentence linked to antibiotics usage. these questions were designed with the same idea as the questions in the group one, with an aim to provide slightly more in-depth information about students’ habits about antibiotics’ usage. the possible answers were numbered from 1 to 5, with number representing levels of agreement, such as: 1 – i do not agree at all, 2 – i mostly do not agree, 3 – unsure/don’t know, 4 – i partially agree, 5 – i agree completely, as per the five-degree likert rating scale. the questions are enumerated in the table 2. statistical methods in order to better assess the results of the questionnaire, all results have been quantified, first represented by the number of students who gave a particular answer to the question, then by percentile, showing the percentage of a particular response in the overall population of students. for the questions where a scale of answers was expected, differently to the simple yes/no questions, a mode was determined in order to show the most frequently chosen option, similarly to a study conducted on awareness of antibiotics in norway [36]. in order to determine the statistical importance of data obtained and its connection to the studied factor, a common χ2-test for independence (chi-squared test) was used where value p<0.05 was considered significant. results the study was conducted encompassing 130 participants, who have been offered a questionnaire to fill in regarding their habits and knowledge of antibiotics usage. of 130 participants asked, 128 participants have completed the questionnaire form as they have stated they had previously used antibiotics. as to comply with the legislative norms, all participants aged under 18, 72 of them (56%), provided a parental consent, or a self-signed form if over 18 years of age. the information about the participants of the survey is provided within table 3. participants in survey are students of the science and mathematics study course (62%), languages and 108 | fine focus number of students percentage in total population all surveyed 128 100% students with a medical profession present in the family nucleus 69 54% students with no medical profession present in the family nucleus 59 46% students of the sciences and mathematics study course 80 62% students of the language and humanities study course 43 34% not affiliated in a distinct study course 5 4% table 3. the number of surveyed individuals participants in survey are students` of the science and mathematics study course (62%), languages and humanities study course (34%) or not affiliated in any of the two main courses of study (4%). overall, the participants of the study are equally distributed in respect to the presence of member of the family affiliated in a medical profession (54% who have compared to 46% who do not). volume six | 109 figure 1. answers to yes-no questions 110 | fine focus humanities study course (34%) or not affiliated in any of the two main courses of study (4%). overall, the participants of the study are equally distributed in respect to the presence of member of the family affiliated in a medical profession (54% who have compared to 46% who do not). as seen in the figure 1, most of the participants of the study are aware of the risks of alcohol usage under a therapy including antibiotics (91%). it is worth noting that of all the participants, 63 (48%) were of the legal drinking age, which is over 18. only 26% have stated a calming effect of antibiotics. furthermore, only 22% have incorrectly classified paracetamol as antibiotic. however, as much as 31% have tried to treat viral diseases using antibiotics and as much as 32% have admitted to acquiring antibiotics without an official prescription. moreover, 41% identified to have taken antibiotics for different time than prescribed. as seen in the figure 2, only 18 participants (14%) have admitted to a frequent monthly usage of antibiotics (mode: 1 – completely disagree). no other question has, however, given such a polarizing answer, where as much as 46% (n=59) of population expressed doubt towards the regularity of their doses (mode 2 – partially disagree). most of the population claims they are aware of the functioning mechanisms of antibiotics (59%, n=76, express a high level of certainty (answers 4, 5; mode 4 – partially agree), which is slightly opposed to the given opinions whether the antibiotics can be used against most common everyday diseases, such as headaches, high body temperature, hypertension, flu etc. (mode 3 – unsure/don’t know, 38%, n=48). figure 3 represents the answers to the question asking who the participants’ choice is when it comes to health and disease treatment advice. for these questions, participants were asked to select all applicable answers, giving a total of 163 answers. the majority of the surveyed population answered official doctor (86/128 participants or 67%). the next most frequent answer was family (69/128 participants or 54%). of this number, only slightly more numerous are participants advised by parents not working in healthcare (38/128 participants, 30% of total population or 55% within the answer family). a small number of participants also identified friends as a source of information and advice (2% of the total population) and internet or media (3% of the total population). this study also aimed to compare the answers of participants who have and who do not have a family member affiliated to medical profession, as well as answers of participants following different study courses. for a difference in answers to be considered statistically significant the p value must be less than 0.05. no statistically significant difference has been detected between the groups following different study courses (p>0.05 constantly) and almost no statistically significant difference was discovered between the groups with a different family composition regarding medical professionals. a statistically significant difference was discovered, however (p=.045, p<0.05) in answering question about taking the antibiotic for shorter or longer periods than the prescribed time. in the group of students with medical professionals in the family, 23 out of 69 respondents answered that they have done so (35%), while in the group of students without medical professionals in the family, 30 out of 59 students answered affirmatively (52%) with a larger obedience to the therapy period detected in the group whose family members are affiliated to the medical industry. discussion this study aimed to examine the habits of antibiotic volume six | 111 figure 2. answers to questions with assessment of agreement figure 3. sources of advice for disease treatment 112 | fine focus use among students and the knowledge they possess. furthermore, we wanted to explain the obtained results as well as compare them to similar studies done around the world. nevertheless, to our knowledge, our study is one of the rare few that bases itself on responses of high-school students [12]. by studying these results, we would be able to observe if there is a widespread problem of misuse and misunderstanding of antibiotics among students that would bring into question the way we currently approach the subject. the results show whether a campaign is necessary to improve students’ knowledge on the subject. the questions were meant to assess: (a) students’ knowledge of antibiotics functioning mechanism, (b) students’ habits when antibiotics usage is concerned, (c) students’ understanding of effects of improper antibiotics usage and (d) students’ capacity to distinguish antibiotics from some commonly used medicines. the questions have been designed in compliance with some previous studies in indonesia [40], portugal [5], italy [21] and oman [15]. the following table 4 enumerates all the questions in the study with the points that they were meant to assess. the questionnaire included the usual questions regarding the acquisition of antibiotics, the implementation of a dosing regimen and the usage of alcohol. the question about members of the family nucleus working in health institutions would help assess whether a presence of a medical professional would in any case contribute to a more controlled usage of antibiotics. other questions were added in reference to the particular mood regarding usage of medication in serbia. being aware of the existence of a belief that antibiotics are extraordinary medicine, able to prevent and cure any disease [40] (representing a “magic bullet”, as put by the famous german nobel prize winner p. ehrlich), we wanted to examine whether the students would feel calmer believing the antibiotics are bound to help them cure the disease. in reference to this belief, a question about using antibiotics to treat viral diseases was also added. it has been reported that a common practice exists to use additional selfmedication even after receiving an official prescription from a medical doctor [9, 15, and 40]. a large study encompassing several developing countries by pechere et al [27] has also reported that antibiotics have been used to treat viral diseases, which generally include common flu and cold. this is why the students were asked if they had used antibiotics to treat a diagnosed viral disease. a similar tendency was observed in a study in oman [15] and indonesia [40], where the latter reported a very high rate of 74% of surveyed parties believing antibiotics are useful in combat against any disease. furthermore, the question about paracetamol was included as this is one of the most commonly used analgoantipyretic drug in serbia, especially for treating young children. this is one of the medicines that students are most acquainted with from early childhood, and this is why it was important to assess their knowledge on its nature, since some studies reported that antibiotics are being used as antipyretic, antitussives or analgesic drugs [9, 15]. students from serbia have demonstrated an acceptable knowledge of antibiotics, as seen in figure 1. this is similar to the studies on the general public in indonesia [40] and norway [36], and unlike studies in general public in kosovo [41] and a study encompassing chinese university students [37], which has reported a massive cross-country misuse. a majority of students from our sample (73%) do not report feeling calmer after taking them and do not consume antibiotics on their own (67%). of the volume six | 113 statement / question points statement / question points statement / question points does any member of your family nucleus work in a healthcare institution? a, c have you ever taken an antibiotic for a longer or shorter time period than prescribed? a, b, c do you consume alcohol when using antibiotics? b, c after you consume an antibiotic, do you feel calmer? b, d have you ever used antibiotics when a virus causes your disease, such as in case of common flu and cold? a, b have you ever acquired antibiotics without prescription? b i am well acquainted with antibiotics’ method of function. a, c is the paracetamol an antibiotic? d i use antibiotics often on a monthly level. b, c name all the applicable advisors for your treatment when ill. b i often miss my doses when taking an antibiotic. b antibiotics can help us with most diseases. a, c, d table 4. study questions with their points key. a – students’ knowledge on antibiotics’ mechanism, b – students’ habits on usage, c – students’ understanding of effects of improper usage, d – students’ capacity to distinguish antibiotics from other drugs. 114 | fine focus surveyed students, 78% were able to differentiate analgoantipyretic paracetamol – acetaminophen from antibiotics, displaying their knowledge of the most commonly used pharmaceutical drug in serbia that can be acquired without an official prescription. among surveyed students, 91% do not consume alcohol during treatment. a study conducted in a serbian hospital [16] reported that 90% of underaged high school students have tried/occasionally used alcohol, with 52.5% stating that they have consumed alcohol in the 30 days preceding the study. taking these numbers into account, our results could demonstrate awareness of the effects of alcohol usage during treatment of illnesses. in case of certain antibiotics, these include inhibition of the drug’s effect or even violent physical reactions with alcohol [32]. additionally, alcohol usage in general can cause fatigue, lack of energy, and therefore prolong recovery from the illness, as described by the national health service of uk (nhs) [24] and by the mayo clinic of usa [35]. in general, these numbers reinforce our hypothesis that students have a good basic knowledge of antibiotic use, as they are aware that antibiotics have no calming or analgoantipyretic effect and that alcohol should not be consumed during treatment. however, they seem to be less aware of the fact that antibiotics are inefficient in treatment of viral infections since 31% of them reported that they had used antibiotics to treat viral diseases. this would indicate that, contrary to our hypothesis, a large number of students are still unaware that antibiotics are not helpful against viruses. in addition, a worrying 41% has reported tampering with the dosing regimen. so, despite their acceptable level of theoretical knowledge, it seems that the students do not apply it to their habits when it comes to consumption of the drugs. as seen in the figure 2, 59% express a high level of certainty when it comes to understanding the effects of antibiotic usage, similarly to a study conducted in indonesia [40]. it seems that the most students consider themselves to be well informed about antibiotics and their function. this is in agreement with the result that only 5% agree completely with the statement that antibiotics cure most diseases. figure 2 indicates that with only 14% of students reporting frequent usage of antibiotics on a monthly level, it is safe to assume that the overuse of antibiotics is not a major concern. what should be concerning, however, is the fact that almost a half of surveyed students were not aware of the importance of the adherence to dosing regimens. this coincides with the results of a study performed in china in a similar age group where half of the questioned university students admitted ceasing treatment 1-2 days after clinical improvement, without finishing the full course of treatment [17]. it is worth noting that antibiotics often display the desired effect before the entire treatment is complete, as discussed by bin zaman et al [42] in reference to the antibiotic resistance, and it is also highlighted in the guidelines by the nhs [25]. figure 3 shows that 67% of students address their family doctor for medication, but 54% also report that they consult their parents, with no significant difference between those who are and are not affiliated in the healthcare industry. these figures might be explained by students’ belief that some everyday ailments are not worth going to the doctor for, and that their family members have enough knowledge and experience to help them. similar findings were reported in studies done in turkey [2], italy [21] and cameroon [9] while in kosovo only 0.6% consulted family members [41]. furthermore, with friends and media/the internet being reported as 2% and 3% respectively, it is clear that students do not consider these sources as relevant when looking for advice concerning treatment. this has reinforced our belief volume six | 115 that the education and the family nucleus remain as the most prominent sources of knowledge on antibiotics usage of the surveyed students. the disparity among the answers of students with and without a family nucleus member affiliated in the healthcare industry was insignificant for most questions. the only significant difference found refers to the adherence to dosing regimen. students who have a family nucleus member in healthcare have reported fewer disruptions of the prescribed regimen, probably because they were warned (by a family member) that in order to obtain the complete effect of the prescribed medication, the full dose must be taken [1]. it is worth noting that no dedicated health class exists in serbia. thus, the only knowledge of the subject that the surveyed students could have acquired through their school studies would come from biology classes, where antibiotics are relatively scarcely covered, [8, 34]. even though the study course of science-mathematics is oriented towards life sciences and chemistry more than the language-humanities course in terms of time devoted to these subjects, the differences between responses across all questions were minimal. this is likely due to the fact that antibiotics, unlike most topics related to science, are covered equally by the two courses. the subject of bacteria and viruses is studied during first year of biology [8] where the program is identical for both language-humanities and science-mathematics courses. in the time of conducting the study, all the participants had already completed the study of this chapter. even though the science-mathematics course is geared more towards medicine or pharmacology, these fields are never directly studied based on the official textbook [34]. a group of similar studies on high school students’ knowledge on the subject of antibiotics has been conducted in portugal [4, 5], where the topic is covered once during the biology course. the surveyed students from serbia have demonstrated an overall better level understanding of the subject of antibiotics and their use than their peers despite the overall similar distribution of the topic in the two curricula. compared to a large study done on university students in china, the surveyed students in serbia were significantly less likely to buy antibiotics without a prescription – 3/10 compared to 7/10, as well as tending to consult their doctor more often compared to the university students in china [28]. conclusion the results of the study lead to the conclusion that a majority of interviewed high school students understand the basic properties and use of antibiotics. they are able to tell apart the other commonly used analgopyretic paracetamol from antibiotics and do not consume alcohol during treatment. a majority go to their doctor for advice and prescription. however, many also choose to seek advice from their family, regardless of whether the family member is affiliated with the healthcare industry. the research has shown that a premature interruption of prescribed treatment is common, however it is less frequent among those students whose family is in healthcare as compared to others. therefore, students need to be better acquainted with the subject through informative campaigns and school projects. conflict of interest the authors have no conflict of interest to declare. 116 | fine focus acknowledgments the authors thank the following teachers of svetozar markovic high school in nis, serbia for their work in the distribution of questionnaires and data gathering: slobodan petrovic (chemistry) snezana mitic (biology) and dragana jelenkovic (physics). also, the authors thank the following students for their efforts in the distribution of questionnaires: stasa petrovic, luka ilincic (svetozar marković high school in niš, serbia) and mila zivkovic (faculty of medicine, university of niš, serbia). volume six | 117 references 1. adorka, m., kirk, a., lubbe, m., serfontein, j. 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(2018). antibiotic misuse among university students in developed and less developed regions of china: a cross-sectional survey. global health action, 11(1), 1496973. doi: 10.1080/16549716.2018.1496973 29. pereira, j. q., silva, m. t., & galvão, t. f. (2018). use of antibiotics by adults: a populationbased cross-sectional study. sao paulo medical journal, 136(5), 407–413. doi: 10.1590/15163180.2018.0168060818 30. sabuncu, e., david, j., bernède-bauduin, c., pépin, s., leroy, m., boëlle, p.-y., … guillemot, d. (2009). significant reduction of antibiotic use in the community after a nationwide campaign in france, 2002–2007. plos medicine, 6(6). doi: 10.1371/journal.pmed.1000084 31. spellberg, b., & gilbert, d. n. (2014). the future of antibiotics and resistance: a tribute to a career of leadership by john bartlett. clinical infectious diseases, 59(suppl 2). doi: 10.1093/cid/ciu392 32. steckelberg, j. m. (2018, february 6). can i drink alcohol while taking antibiotics? retrieved from https:// www.mayoclinic.org/healthy-lifestyle/consumer-health/expert-answers/antibiotics-and-alcohol/faq20057946. 33. the world is running out of antibiotics, who report confirms. (n.d.). retrieved from https://www.who. int/news-room/detail/20-09-2017-the-world-is-running-out-of-antibiotics-who-report-confirms. 34. velimirovic, s., petrovic, j. (1994). hemija za cetvrti razred gimnazije (chemistry for the fourth grade of grammar school). belgrade, serbia: zuns. isbn: 978-86-17-20032-7 35. ventola, c. l. (2015). the antibiotic resistance crisis part 1: causes and threats. pharmacy and therapeutic, 40 (4), 277-283, pubmed: 25859123 36. waaseth, m., adan, a., røen, i. l., eriksen, k., stanojevic, t., halvorsen, k. h., … nordeng, h. m. e. (2019). knowledge of antibiotics and antibiotic resistance among norwegian pharmacy customers – a cross-sectional study. bmc public health, 19(1). doi: 10.1186/s12889-019-6409-x 120 | fine focus 37. wang, x., peng, d., wang, w., xu, y., xudong, z., hesketh, t. (2017). massive misuse of antibiotics by university students in all regions of china: implications for a national policy, international journal of antimicrobial agents, 50 (3), 441-446. doi: 10.1016/j.ijantimicag.2017.04.009. 38. web presentation of clinical centre of nis, serbia (2019). retrieved from http://kcnis.rs/en/. 39. what is antimicrobial resistance? (2017, december 14). retrieved from https://www.who.int/features/ qa/75/en/. 40. widayati, a., suryawati, s., crespigny, c. d., & hiller, j. e. (2012). knowledge and beliefs about antibiotics among people in yogyakarta city indonesia: a cross sectional population-based survey. antimicrobial resistance and infection control, 1(1), 38. doi: 10.1186/2047-2994-1-38 41. zajmi, d., berisha, m., begolli, i., hoxha, r., mehmeti, r., mulliqi-osmani, g., … raka, l. (2017). public knowledge, attitudes and practices regarding antibiotic use in kosovo. pharmacy practice, 15(1), 827–827. doi: 10.18549/pharmpract.2017.01.827 42. zaman, s. b., hussain, m. a., nye, r., mehta, v., mamun, k. t., & hossain, n. (2017). a review on antibiotic resistance: alarm bells are ringing. cureus. doi: 10.7759/cureus.1403 volume six | 121 fine focus 6 i fine focus objective lens john l. mckillip, managing editor, fine focus professor, microbiology, ball state university vol 8 i 7 fine focus was not only the first international journal for undergraduate microbiology research, but also the ball state university chapter of the american society for microbiology (bsuasm) – that is, a campus recognized student organization, and an honor’s course at bsu. students in both the club and the class manage the journal in all ways – from stewardship of our double-blind peer review system for manuscript management (via our ojs – open journal system) – to marketing, promotion, and design changes inside and out on the physical print copy of each issue. this new issue represents a completely new design for fine focus. for example, the artwork on the cover and on each title page is an original watercolor by one of our student marketing team conveners, sharon cung. this design represents the industrially important yeast saccharomyces cerevisiae, as explained further inside the front cover. since our inception in 2013, fine focus is managed as an “immersive learning” course at bsu – that is, a productbased course with the following unique attributes: • a project offered to undergraduate students for academic credit • is conducted under the guidance of a faculty mentor • fills a need or solves a problem for a community partner • results in a specific outcome or deliverable • is conducted by a team or teams of undergraduate students (in classes of 10 or more) working collaboratively these faculty-led, student-driven team model puts the students at the helm of day-to-day decision making (1). what better way to further introduce the effectiveness of this model than to introduce you to the team of students who put together the issue you are reading now. their profiles are on the following pages. fine focus 86 i fine focus the impact of petase’s active site disulfide bond on pet biodegradation kreesha saha and clark gedney 915 w. state street, lilly hall of life sciences, purdue university, west lafayette, in 47907 manuscript received 4 july 2021; accepted 24 august 2021 vol 8 i 87 abstract plastic pollution is one of the largest problems globally, with polyethylene terephthalate (pet) plastic as one of the main sources. effective depolymerization of pet to its monomers for upcycling is a challenge. petase is reported to be an effective enzyme for biodegradation of pet via c-o bond cleavage of ester linkage. the role of the disulfide bond, present in petase’s active site sequence, is unknown in the cleavage of pet’s ester linkage. to understand the role of this bond, two separate versions of petase – one containing the disulfide bond, and the other without the disulfide bond were modeled using pymol™, synthesized, and tested for degradation of pet surrogate compound, bis (2-hydroxyethyl) terephthalate (bhet). several experiments were performed in the presence and absence of phenylmethylsulfonyl fluoride (pmsf), a serine protease. the results reveal that the role of the disulfide bond in the degradation of bhet’s ester linkage is insignificant and the variation in the results (ethylene glycol yields, bhet degradation per microgram of enzyme) are within the experimental uncertainty. this finding is a stepping-stone to further modifying petase and improving its activity towards commercial adaption of this technology for pet upcycling and creating a circular carbon economy, improving the world’s carbon footprint, and mitigating ocean and environmental plastic pollution. 88 i fine focus introduction polyethylene terephthalate (pet) is one of the most common consumer plastics, which has a variety of uses ranging from plastic water bottles to polyester clothing items.(paydar & olfati, 2018) in 2013, the worldwide production of plastic was 299 million metric tons.(chen et al., 2018) by 2015, the worldwide production had reached 322 million metric tons per year, and this amount will continue to increase. despite plastic’s versatile uses, it is also an environmentally hazardous substance because it does not biodegrade in natural ecosystems.(chen et al., 2018) a small fraction of post-consumed plastics is currently recycled, mostly by physical recycling. physical recycling refers to the process of melting and then re-shaping the plastic into new consumer products. for example, waste pet plastic bottles can be melted to make polyester carpeting.(tullo, 2019) this method of recycling introduces impurities, ultimately reducing its quality and value. as a result of these challenges, only 18.4% of pet is recycled annually. (tullo, 2019) all large-scale pet recycling today is physical recycling, and due to the lower quality end product, most manufacturers produce new pet from monomers, derived from crude oil, to meet the market demand. chemical recycling of pet plastic is now being explored as an alternative. it involves degradation of pet into its constituent monomers, ethylene glycol (eg) and terephthalic acid (tpa) (scheme 1). the produced monomers can be chemically re-bonded to form pet plastic of the same caliber as new pet. this advantage makes chemical recycling preferable to physical recycling. chemical recycling is not used in large-scale facilities due to the lack of an effective technology as well as use of corrosive chemicals such as sodium hydroxide, and harsh reaction conditions (e.g., high pressure requiring expensive reactors).(khoonkari et al., 2015) glycolysis is the most efficient method of depolymerizing pet,(khoonkari et al., 2015) which is done at high temperature (180°c to 240 °c).(khoonkari et al., 2015) ionic liquids such as 1-butyl-3-methylimidazolium bromide ([bmim] br) have been used as catalysts for pet glycolysis. wang et al. used [bmim] br to degrade 100% of pet in 8 hours at 180 °c.(khoonkari et al., 2015) this reaction depolymerized long ester chain of pet into short chain ester intermediates and did not fully degrade to produce desired eg and tpa. despite glycolysis showing high depolymerization yields, because it does not totally degrade pet, and necessitates high energy (up to 240 °c), more energy efficient and effective alternative methods have been explored for chemical recycling of pet. one such method is enzymatic degradation of pet.(kawai et al., 2020; taniguchi et al., 2019) this method reduces the activation energy of the reaction, requires no pressure, and involves equipment which is inexpensive and commonly available. enzymatic degradation of pet film was first accomplished by muller in 2005.(kawai et al., 2019; müller et al., 2005) their method depolymerized two kinds of pet films by approximately 40–50% at 55 °c in 3 weeks using cutinase obtained from the bacterium thermobifida fusca. (kawai et al., 2019; müller et al., 2005) since then, several thermostable cutinases have been discovered with an ability scheme 1. pet breakdown to terephthalic acid (tpa) and ethylene glycol (eg). vol 8 i 89 to break down pet film,(furukawa et al., 2019; ribitsch et al., 2012) but their degradation rates for pet are lower than that with cutin. recently, a gram-negative bacterium, ideonella sakaiensis 201-f6 was isolated that can consume pet as an energy and carbon source to survive.(yoshida et al., 2016) research revealed that this bacterium contains the enzyme, petase, which can biodegrade pet into mono(ethylene terephthalate (mhet) and (bis(2-hydroxyethyl) terephthalate) (bhet). mhet and bhet are then hydrolyzed into eg and tpa. (yoshida et al., 2016) 200 nm of wild type petase enzyme was found to degrade 3.7 mg/l of pet at 30 °c over the course of 72 hours.(seo et al., 2019) when compared with pet degradation activity of all reported enzymes, petase demonstrated the best performance (table 1). petase’s active site is composed of a serine-histidineasparagine (ser-his-asp) triad, which is one of the most commonly studied catalytic triads, and is often found in ɑ/β hydrolases.(jones & solomon, 2015; rauwerdink & kazlauskas, 2015) it catalyzes a redox reaction to break the ester bond in pet.(yoshida et al., 2016) the redox process is initiated when asparagine removes a hydrogen atom from histidine; thus, effectively removing a proton and an electron. the histidine then replaces its hydrogen by taking a proton and an electron from serine, creating a nucleophile.(yoshida et al., 2016) the ser-his-asp triad removes an electron from the oxygen atom of pet’s ester linkage and passes the electron to serine.(yoshida et al., 2016) then this oxygen atom takes an electron from its neighboring atom, making a free radical on the carbon atom, and weakens the c-o bond of the ester linkage. this results in cleavage of the ester linkage and complete the redox cycle. the extra electron gained by asparagine at the beginning of the reaction is given to o2, an electron acceptor, resulting in formation of two molecules of h2o. petase does not degrade pet film at a rate high enough to be utilized in recycling facilities. commercial adaptation of this process would need much higher degradation rate. (o’brien, 2019) in 2018, austin et al. modified wild type petase and improved the activity of the modified petase to 2.5 times the wild type.(ma et al., 2018) in the modification process, width of the active site cleft of the modified petase was lowered through mutagenesis of two amino acids (phenylalanine and serine). the results indicated that specificity of the active site caused by a narrower cleft allows the substrate (pet) to better interact with the enzyme. (austin et al., 2018) while other enzymes (e.g., cutinases) have already been perfected by nature because of their long existence, petase, a recently developed enzyme, can be further modified through appropriate protein design. ma et al. also modified wild type petase. in their modification, hydrophobicity was increased near the active site of the enzyme through mutagenesis under the hypothesis that hydrophobicity would increase pet degradation. this modification led to a 15-fold increase in degradation of pet as compared to the wild-type petase.(ma et al., 2018) although significant achievements have been made by researchers in improving wild type petase’s activity, more research is necessary in developing enzymes with higher thermostability, and better degradation capability, enabling their utilization for pet recycling by waste management facilities and chemical industries. to develop petase for effective degradation of ester linkages, the following section summarizes desirable features, e.g. isoelectric point, amino acid sequence, thermostability etc. of modified petase. petase shares 52% sequence identity with t. fusca cutinase, its closest homolog.(austin et al., 2018; yoshida et al., 2016) this sequence can be used to identify features of petase which make it an effective degrader of pet. these features include 3-fold wider active site cleft and higher isoelectric point (caused by dipole; isoelectric point of petase is 9.6 while this value of t. fusca cutinase is 6.3) of petase as compared to t. fusca cutinase.(austin et al., 2018; yoshida et al., 2016) in addition, petase contains many basic amino acids such as lysine and arginine, which are charged and form salt bridges to make petase stable.(yoshida et al., 2016) (austin et al., 2018) another important feature is thermostability. for example, leaf branch compost cutinase (lcc), another cutinase with similar sequence to petase, has high thermostability at 90 i fine focus 70 °c.(shirke et al., 2018) because glass transition temperature of pet plastic is around 70 °c, and pet becomes more pliable and its bonds weaken, lcc can degrade pet effectively at this temperature. in contrast, petase is stable and active at much lower temperature (~37 °c) at which pet is not pliable and its bonds are rigid. modified petase with high thermostability would be beneficial. table 1 entry enzymes reaction conditions degradation amount (methods used) ref 1 2 3 4 5 6 petase contains a disulfide bond in its active site. lcc and t. fusca cutinases do not have this disulfide bond.(fecker et al., 2018) simulations predicted that this disulfide bond increases flexibility around the active site.(fecker et al., 2018) austin et al. (austin et al., 2018) have shown that a narrower binding cleft of the active site, arising from flexibility of enzyme, can improve enzyme’s affinity to substrates, e.g., pet or bhet. modified petase (mutant i179f) pet-g/tfh* pet-g/rtfh ab300432, ab298783, ab300774 est119 est1 modified petase expressed in e. coli, 5 μg petase reacted with pet (1.5x1 cm2 size pieces) in bicine buffer; ph 8.5, 48 hours, 30°c 20-25 mg pet (12 mm diameter disks) reacted with enzyme of concentration 0.1 mg/ml in buffer; 5 ml buffer; ph 7.0; 21 days; 55 °c 20-25 mg pet (12 mm diameter disks) reacted with enzyme of concentration 0.1 mg/ml in buffer; 5 ml buffer; ph 7.0; 21 days; 55 °c 7 × 7 cm2 biomax® films in compost; 55-60 °c; 70-100cm below surface; 3 weeks and 4 weeks 1 × 1 cm2 pet film reacted with sufficient amount enzyme in the presence of 300 mm ca2+;50°c; ph 7; 3 h 1 × 1 cm2 pet film reacted with sufficient amount enzyme in the presence of 300 mm ca2+;50°c; ph 7; 3 h mutant i179f has 2.5 times more degradation than wild-type pet [22.5 mg μmol-1·l−1 petase per day biodegraded by mutant] (sem) 49.7 ± 1.0 % mass loss (mass weighed) 54.2%% mass loss (mass weighed) all films fragmented in 4 weeks no weight loss or visible surface change (mass weighed and sem) no weight loss or visible surface change (mass weighed and sem) (austin et al., 2018) (müller et al., 2005) (müller et al., 2005) (hu et al., 2008) (thumarat et al., 2015) (thumarat et al., 2015) vol 8 i 91 entry enzymes reaction conditions degradation amount (methods used) ref 7 8 9 10 thc_cut1 thc_cut2* thf42_cut1 tha_cut1 10 × 100 mm2 pet film reacted with 6.75 μm enzyme in 13 ml buffer; 50 °c; ph 7.0; 2 hours 10 × 100 mm2 pet film reacted with 6.75 μm enzyme in 13 ml buffer; 50 °c; ph 7.0; 2 hours 10 × 100 mm2 pet film reacted with 6.75 μm enzyme in 13 ml buffer; 50 °c; ph 7.0; 120 hours 10 × 100 mm2 pet film reacted with 6.75 μm enzyme in 13 ml buffer; 50°c; ph 7.0; 2 hours wca decreased from 74.2° ± 1.6° to 66.3° ± 2.7° (wca analyzer) wca decreased from 74.2° ± 1.6° to 71.2° ± 0.9° (wca analyzer) crystallinity loss (ftir-atr) wca decreased from 87.7° ± 4.8° to 45.0° ± 6.0° (wca analyzer) (herrero acero et al., 2011) (herrero acero et al., 2011) (herrero acero et al., 2011) (ribitsch et al., 2012) *tfu = t. fusca; **tfcu = t. fusca cutinase; sem = scanning electron microscopy; ftri = fourier-transform infrared spectroscopy; wca = water contact angle it is hypothesized that the flexibility provided by the disulfide bond attributed to petase’s degradation performance. the role of the disulfide bond, which is unique to petase, has not been experimentally tested in the degradation of pet, despite it predicted positive traits from simulation. the present work experimentally demonstrates the effect of disulfide bond of petase to the degradation of ester linkage of pet. first, amino acid sequence of a petase protein without disulfide bond was modeled. then petases with and without disulfide bond were synthesized following their sequences and their activities in the degradation of ester linkage of pet have been experimentally tested using bhet as a model compound. bhet, a building block oligomeric unit of pet which contains eg and tpa monomeric units in 2:1 molar ratio, is used as a surrogate substate in this study because (1) it is expected to have faster degradation rate than pet and (2) it can completely degrade to eg and tpa, which can be quantitatively measured to accurately determine the role of the disulfide bond. controlled experiments were performed without petase and in the presence of phenylmethylsulfonyl fluoride (pmsf) to evaluate if pmsf disrupts petase’s activity by interacting with their catalytic triad serine. the results indicate that the disulfide bond does not significantly influence the degradation of bhet’s ester linkage. experimental section materials luria bertani (lb) broth, calcium chloride, ampicillin, lysogeny broth, tris buffer, hcl, phenylmethylsulfonyl fluoride (pmsf), hi-trap columns, and nacl were procured from sigma-aldrich. isopropyl βd-1-thiogalactopyranoside (iptg) and bichionic acid assay were obtained from fisher scientific. a refractometer was purchased from hanna 92 i fine focus instruments. bl21 e. coli cells were obtained from dr. clark gedney’s lab in biological science department at purdue university. petase recombinant plasmid containing the gene to produce petase with a disulfide bond (referred hereto as a2) was purchased from integrated dna technologies (idt). amino acid sequence of the modified petase without a disulfide bond (referred here to as a1) was modeled using pymol™ software (schrödinger, inc. version 3). the sequence was sent to idt to synthesize the modified petase. both the organism containing/expressing the plasmid were cultured for extracting proteins by following similar procedures as described below. petase sequence modification the software pymol™ was utilized in order to model a1 and a2. a2, which contains a disulfide bond, was produced by isolating the active site of petase. its sequence is shown in figure 1c. to produce the sequence of a1 (figure 1b), disulfide bond (red colored portion) of the sequence in figure 1c was removed. rather than using the entire petase sequence, the active site was isolated to enable a focused investigation into the disulfide bond’s effect on petase’s active site. transformation of e. coli cells frozen bl21 cells were grown on petri dishes of luriabertani broth media until 20 colonies were visible on each plate. approximately 20 colonies of e. coli were transferred to 250 μl of 0.1m calcium chloride solution in a sterile microcentrifuge tube and mixed. to another microcentrifuge tube, 20 colonies of e. coli were added to 250 μl of calcium chloride solution and mixed. 10 μl of petase plasmid (e.g. a1) was added to one of these two tubes (tube 1) and vortexed for 5 seconds. the second tube without plasmids is referred here to as tube-2. then both tubes were placed in an ice bath for 10 minutes, followed by 90 seconds in a heating block preset at 42 °c, and finally submerged in an ice bath for an additional 2 minutes. 500 μl of super optimal (so) broth was added to both tubes and vortexed, and then the tubes were placed in a heating block at 37 °c for 30 minutes. the contents of tube-1 and tube-2 were added to two separate flasks, each of which contained 1 l of lysogeny broth medium and 200 mg ampicillin, and they were cultured in a shaker at 37 ℃ until an optical density of 0.6 at 600 nm was reached. to confirm transformation, the cells from flask-1 were checked against the cells from flask 2. flask-1 was cloudy due to the addition of the plasmids, whereas flask 2 was the negative control and remained clear. after this control test, the content of flask 2 was discarded. 0.1 mm iptg was added to flask-1 at this stage, and the cells of flask-1 were further incubated for 24 h at 37 ℃ in the shaker at rpm of 250.(yoshida et al., 2016) the content of flask-1 was transferred to several 15 ml centrifuge tubes and they were centrifuged at 4000 rpm for 20 minutes on an allegra centrifuge instrument. supernatant from all of the centrifuged tubes was stored in a glass bottle. the cell pellets from all centrifuged tubes were collected and resuspended in 50 ml of 40 mm ph 7.4 tris-hcl buffer containing 0.5m nacl (buffer 1). the resuspended cells were vortexed and divided equally into two centrifuge tubes of size 30 ml. to one of the tubes, 0.1m pmsf was added to test the effect of this protease inhibitor on enzyme activity. it is used for protein purification during cell lysis to prevent the proteases from degrading the protein. lysis the cells from both 30 ml size tubes were separately added to a mortar and pestle containing liquid nitrogen in it to allow the content of each tube to solidify and ground until fine powders were formed, which were then stored at -20 ℃. 5 grams of these lysed cells were resuspended in 20 ml of 20 mm phosphate buffer at ph 7.4 (buffer 2) containing 20 mm imidazole. they were centrifuged at 4300 rpm for 30 minutes. the pellet was discarded and the supernatant, which contained protein expressed from the transformed cells, was syringe-filtered through a 0.45 micrometer syringe. protein purification a histidine column (1-ml hitrap syringe) was used to purify the protein in the supernatant. the elution buffer consisted of 500 mm imidazole in buffer 1, and the binding buffer consisted of 20 mm imidazole in buffer 1. the hi-trap column was first prepared by eluting the binding buffer twice through the column at a rate of approximately 1 ml/min. vol 8 i 93 the supernatant containing the expressed protein was then eluted through the column. the elution buffer was run through the column in batches of 4 ml each time to elute the expressed protein which was collected separately in 15 ml centrifuge tubes. a total of 5 samples of each containing 4 ml eluant was collected. these 5 aliquots or samples were used for studying degradation experiments of pet model compound written below. plasmid containing petase enzyme with disulfide bond (a2) was similarly processed to obtain expressed protein for degradation study written below. a bichionic (bca) assay of each aliquot was done to measure total protein concentration. for this assay, each sample was mixed with bichionic acid obtained from sigma and the mixture was kept on a microplate for 30 minute at 37 °c before measuring its color change at 526 nm using a tecan microplate reader. bhet degradation 1m of bis(2-hydroxyethyl) terephthalate (bhet) was added to each eluted protein samples and the mixtures were allowed to continuously shake at 250 rpm and 37 °c for 72 hours. after 72 hours of reaction, the tubes were centrifuged, and 1 ml supernatant from each tube was collected for analysis by a refractometer and a uplc instrument. the reaction was terminated by heating the solution in tubes at 85 °c for 15 minutes .(austin et al., 2018) a total of 5 a1 samples, 5 a2 samples and 2 buffer solutions (control) were tested for bhet degradation. product analysis. analysis of eg production in each sample collected at 72 hours was done using a uplc equipped with a c18 column. a mixture of water and acetonitrile (45/55 wt/ wt) was used as a mobile phase at a flow rate of 50 μl/min. the sample injection volume was 1 μl. the yield of eg was calculated from uplc peak area using a pre-calibrated plot of peak area versus eg concentrations ranging between 10 µg/ml to 1000 µg/ml. a refractometer was also used to measure eg production. for refractometer, a standard calibration plot for eg solutions of known concentrations was prepared to assess the accuracy in measurements of eg percentage for known solutions. then, the percentage of eg yields in the bhet degraded solutions were measured. a good correlation in the yield of eg from uplc and refractometry techniques was observed. the conversion of bhet from the yield of eg was calculated using the following equation: bhet conversion bhet (g) = ((% eg)(1097 g/l eg)(254.238 g/mol bhet) (0.5))/((100)(62.07 g/mol eg)) the yield of terephthalic acid was not measured because it precipitates in water. results and discussion modeling modified petase plasmid sequence it has been reported that e. coli is a compatible host of petase.(seo et al., 2019) bl21 e. coli cells were utilized as vectors of the plasmids. using pymol™ software (schrödinger, inc. version 3), amino acid sequence of petase was modeled. figure 1a shows the complete petase structure, with the active site highlighted in yellow and red. in figure 1b, the active site of petase was isolated and modified, and the region which contains disulfide bond sequence was removed. this was done to test the effect of this disulfide bond on the activity of petase. removal of the disulfide bond sequence produced a much shorter protein with two helices and three beta strands (figure 1b). the sequence of the modified active site (a1) was gvmgwsmggggslisaannpslkaa apqapwdsstnfssvtvptlifacendsiapvn. the sequence of the modified petase was sent to idt to synthesize it. the unmodified petase active site sequence (a2) consisted of three alpha helices and four beta strands (figure 1c). the portion highlighted in red displays the section of protein which was not included in a1. the sequence of a2 was gvmgwsmgggg slisaannpslkaaapqapwdsstnfssvtvpt lifacendsiapvnssalpiydsmsrnakqflei nggshsc. busca software,(savojardo et al., 2018) which is a server through which a protein sequence can be analyzed, was used to predict expression of the petase active site. this program determined that unmodified petase 94 i fine focus is an extracellular protein while the modified petase is intracellularly expressed in the cytoplasm. 6-histidine tags were attached to the ends of the modified petase to enable purification with a histidine syringe column. bhet degradation efficiency using a1 and a2 petase active sites figure 2 shows protein concentrations, which is equivalent to enzyme concentrations of a1 and a2 petase, in the absence and presence of pmsf. the error bars show standard deviation from five measurements. protein concentrations of a1 petase in the absence and presence of pmsf are 31.57 ± 3.09 μg/ml and 37.52 ±2. 59 μg/ml, respectively, while these values for a2 petase are 33.82 ± 3.52 μg/ml and 33.82 ± 3.48 μg/ml respectively. the results indicate that protein concentration of a2 petase, which contains disulfide bond, did not change significantly in the presence of pmsf. there is a significant increase in protein concentration of a1 petase, which does not contain disulfide bond, in the presence of pmsf. pmsf is a serine protease inhibitor, which blocks the activity of proteases such figure 1a. structure of complete petase. figure 1b. modified petase sequence figure 1c. unmodified petase sequence without a disulfide bond (a1). with a disulfide bond (a2). as elastase that degrade petase. a1 petase is less stable in comparison to the a2 due to its lack of the disulfide bond. the disulfide bond makes proteins more globular, less likely to denature, and increases protein durability. removal of this disulfide bond from a1 made petase more susceptible to degradation by protease inhibitors, and the addition of pmsf blocked proteases from degrading a1. total protein concentration of a1 petase increases in the presence of pmsf. a2 is more stable due to its disulfide bond, so it was not impacted by proteases to the same extent as a1, and the addition of pmsf did not create a significant difference in protein concentration in it. bca assay of a control experiment without addition of petase shows no protein. bhet degradation by a1 and a2 petase demonstrate that both can efficiently degrade bhet to eg and tpa within 72 hours (figure 3). a control experiment containing only buffer solution showed no bhet degradation in terms of the yield of eg. a1 and a2 petase produced similar amount of eg (14 – 15%). the error bars in figure 3 represent standard deviation from 5 replicates. the presence of pmsf with a1 or a2 petase showed insignificant changes in the yield of eg. vol 8 i 95 0 10 20 30 40 50 control a1 a1+pmsf a2 a2+pmsf pr ot ei n c on ce nt ra tio n (μ g/ m l ) active site type figure 2. experimentally measured protein concentration of a1 and a2 petase in the presence and absence of pmsf. figure 4 shows total amount of bhet degraded in 72 hours. a1 and a2 petase resulted in 332.05 ± 14.44 g/l and 320.85 ± 12.58 g/l bhet conversion, respectively, in the absence of pmsf, which is quite high. bhet conversion by a1 petase is a little higher in the presence of pmsf, which is likely because of a slightly higher amount of protein in the solution as seen in figure 2. the activity of both proteins was further determined in terms of bhet degradation per unit (microgram) of each 0 4 8 12 16 20 co ntr ol a1 a1 +p ms f a2 a2 +p ms f e g p ro du ce d (% ) active site type 0 100 200 300 400 co ntr ol a1 a1 +p ms f a2 a2 +p ms fb h e t bi od eg ra de d (g /l ) active site type protein (figure 5). the error bars in figure 5 display standard deviation from 5 replicates. it shows that the activity of a1 petase (0.1073 ± 0.0168 g/μg enzyme) is slightly higher than that of a2 (0.0962 ± 0.0113 g/μg). the activity of a1 in the presence of pmsf is slightly lower (0.0843 ± 0.0191 g/μg) because of total measured protein concentration in the presence of pmsf was higher as discussed above. upon consideration of standard deviation, the activity of a1 and a2 petase appears to be similar, which ranges between figure 3. percentage of ethylene glycol produced by a1 and a2 petase with and without pmsf in 72 hours. figure 4. total bhet degraded by a1 and a2 petase with and without pmsf in 72 hours. 96 i fine focus 0.08-0.1 g bhet degradation/μg of protein. while absence of the disulfide bond in a1 structural sequence makes it less stable, owing to the lack of globular structure, and its activity was expected to be lower with reference to the activity of a2, the similar activity of a1 and a2 indicates that the active site disulfide bond does not have an impact on petase’s activity. the results shown in figure 5 demonstrate that the activity of a1 petase is significantly affected by the presence of pmsf. it indicates that the short structure of a1 is unstable in the presence of pmsf as the serine is blocked in the catalytic triad. in contrast, a2 exhibits a slight increase in activity upon the addition of pmsf because of the higher stability given by the disulfide bond, but it could be within the experimental uncertainty. thus, pmsf did not inhibit the activity of a2 petase’s active site as hypothesized from the fact that pmsf would block the serine at the catalytic triad and disrupt enzyme activity. the addition of pmsf prevents protease activity throughout the reaction, resulting in preserving the activity of enzyme a2. this work advances the understanding on the role of the disulfide bond of petase in the degradation of bhet as a model compound, which will enable future development of more active petase without this bond for degradation of pet to eg and tpa. conclusions this work described the cleavage of ester linkages, present in pet plastic, with petase enzymes in the presence and absence of a disulfide bond to elucidate the role of the disulfide bond on the enzyme’s activity. a modified petase enzyme without disulfide bond sequence was first modeled and synthesized. then, petase enzymes with and without the disulfide bond were cultured, purified, assayed, and used for cleavage of ester linkages of a pet surrogate substrate, bhet. typically the reaction between pet and petase must be run for several days, and the results are analyzed with cumbersome microscopic techniques to measure crystallinity loss of pet, which does not give a direct measurement of ester linkage cleavage. in contrast, bhet degradation forms eg and tpa monomers to quantitively measure the degree of ester linkage degradation and allows evaluation of enzyme’s activity more effectively. controlled experiments were also conducted in the presence and absence of pmsf as well as without an enzyme. the results showed that the total bhet degradation to eg and tpa was not significantly influenced by the disulfide bond of petase, which strongly conflicted prior works that predicted the disulfide bond increased protein activity. 0 0.03 0.06 0.09 0.12 0.15 control a1 a1 +pmsf a2 a2+pmsf b h e t /µµ g pr ot ei n (g /μ g) active site type figure 5. comparison of bhet degradation per microgram of enzymes in the presence and absence of pmsf. control experiment showed no degradation of bhet. vol 8 i 97 further research is necessary to determine the cause of this phenomenon. it is likely that a1’s short structure facilitates easy accessibility of its active site to bhet in the degradation process. although a2 may be more stable, a1 has the ability to degrade a greater amount of bhet in the same time period. the finding of this work that removal of the active site disulfide bond in petase does not have an impact on its activity is a stepping stone in designing a more effective version of the petase enzyme. acknowledgements k.s. greatly acknowledge dr. clark gedney for allowing to conduct this project in his laboratory, acting as a mentor, and providing necessary resources. dr. lavanya reddivari at purdue university is acknowledged for giving access to her uplc instrumentation facility for the purpose of analyzing samples. ms. brittany croy at west lafayette jr/sr. high school is acknowledged for acting as a mentor of k.s. in this project, which was submitted to the international science and engineering fair (isef). 98 i fine focus references 1. austin, h. p., allen, m. d., donohoe, b. s., rorrer, n. a., kearns, f. l., silveira, r. l., pollard, b. c., dominick, g., duman, r., & el omari, k. 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(2019). biodegradation of pet: current status and application aspects. acs catalysis, 9(5), 4089-4105. 21. thumarat, u., kawabata, t., nakajima, m., nakajima, h., sugiyama, a., yazaki, k., tada, t., waku, t., tanaka, n., & kawai, f. (2015). comparison of genetic structures and biochemical properties of tandem cutinase-type polyesterases from thermobifida alba ahk119. journal of bioscience and bioengineering, 120(5), 491-497. 22. tullo, a. h. (2019, october 6, 2019 ). plastic has a problem; is chemical recycling the solution? . c&en, 97(39). 23. yoshida, s., hiraga, k., takehana, t., taniguchi, i., yamaji, h., maeda, y., toyohara, k., miyamoto, k., kimura, y., & oda, k. (2016). a bacterium that degrades and assimilates poly (ethylene terephthalate). science, 351(6278), 1196-1199. 46 | fine focus effects of vermicompost and beneficial microbes on biomass and nutrient density in purple lady bok choy (brassica rapa var. chinensis) in a vertical hydroponic grow tower system chelsea pearson (temperance@rocketmail.com) rochelle gawel (gawelrochelle@gmail.com) donna m. maki (donmaki@nmu.edu) department of biology, northern michigan university marquette, mi vol 9 | 47 abstract indoor food production in vertical hydroponic systems can be done yearround in any climate, has less negative environmental impacts compared to industrial agriculture and is a way of increasing the nutritional value of crops. indoor hydroponic systems make it easy to control the precision of additives and amendments, such as fertilizers and vermicompost tea, to nutrient solution reservoirs. vermicompost tea is known to contain beneficial microorganisms and can help increase the biomass, nutrient density, and overall health of the plants. microorganisms and plants have co-evolved and is an essential relationship that deserves recognition and further research. the goal of the research presented in this paper was to better understand if vermicompost tea and its associated microorganisms, such as bacteria, fungi, nematodes, and protozoa, would increase biomass and nutrient density of purple lady bok choy (brassica rapa var. chinensis) in vertical hydroponic systems. statistical analyses were performed to compare the biomass and macroand micronutrients of three different treatments to one another and a control. there was a significant difference between the mean leaf and root biomass among varying concentrations of vermicompost tea solutions and added inorganic hydroponic fertilizer. the nutrient density for both macroand micronutrients also differed significantly due to different concentrations of vermicompost tea, suggesting that beneficial microbes may help plants uptake and absorb nutrients in a more efficient manner depending on the concentration of vermicompost tea and hydroponic fertilizer. 48 | fine focus introduction year-round growing of crops in a variety of climates is not possible using conventional agricultural practices. current industrial agricultural methods can also have a negative impact on the nutritional content of food and the health of the soil and the ecosphere (6). moreover, interest in consumption of locally grown food has risen with mounting evidence of many associated benefits. benefits of locally grown food include but are not limited to; the reduction in overall production costs due to eliminated long-distance transportation and the need for long-term storage, increased nutrient density, taste profiles, texture, and freshness (8). vertical hydroponic plant growth systems provide alternate means for food production and have potential to increase the amount of locally grown food in many communities. additionally, vertical hydroponic systems could easily be integrated into the average household and provide families in any climate with healthy food. vertical hydroponic systems demand less space, input of resources and are a more sustainable method of agriculture compared to industrial agricultural practices that are dominant in the united states (3, 7, 13). indoor agriculture and the use of vertical hydroponic grow towers, which is soilless by nature, provides solutions to problems surrounding food production. there are two fundamental systems for growing plants: in soil and soilless media. plants in common horticulture/ agricultural systems are typically fed with either inorganic/synthetic nutrients or organic/biological nutrients. the goal of using both inorganic and organic nutrients in horticultural/agricultural systems is to grow healthy plants with the least amount of input required and for as many months as possible. in systems in which biological nutrients are used, the nutrients are processed by living microorganisms which aids in the nutrient uptake by the plants (14). plants and microorganisms have co-evolved and have formed an essential symbiotic relationship, whereby microbes increase mineral availability and uptake by the plants thus increasing the nutrient density. the beneficial symbiotic relationship between plants and microorganisms was the pinnacle of the research presented, as living organisms were present in the vermicompost tea used for this research which had a variety of beneficial fungi, bacteria, protozoa (flagellates, amoebae, and ciliates), and nematodes (table 1). plants grown in hydroponic systems are typically isolated from their microbial partners, which would naturally be found in soil. isolation from symbiotic microbes may be a decided disadvantage to plant growth and health. results from animal studies have demonstrated the profound negative impact the absence of symbiotic gut microbes can have on animal health and development (9). the fraune and bosch study (2010) reflects the fact that symbiotic organisms, such as plants and microbes, could be negatively impacted when deprived of their symbiotic microbial partners. additionally, microbes help plants by producing nutrients (e.g., nitrate), plant hormones, and behaving as biocontrol agents. microbes are capable of chemically communicating with their plant host, stimulating the plant’s immune system, and aiding in the defense against pathogens (4, 5). vol 9 | 49 numerous studies of plants grown in soil demonstrate the positive impact microbes can have on influencing the plants’ physiology and health (12, 15, 18). “vermicomposting” (worm composting) is defined as a process in which earthworms play a major role in conjunction with microbes in the conversion of organic solid waste into a form of a stabilized soil conditioner. vermicompost is a nutrient-rich compost which has macro and micronutrients and contains high populations of diverse, beneficial microbes (1, 24). vermicompost tea is a ‘brewed’ leaching process for extracting key nutrients and microbes in worm castings into a water solution. in a soil-based study done by pant et al. (2009), they found the addition of vermicompost tea increased macro and micronutrient uptake, phenolics, and carotenoids in the plant’s tissue (20, 24). furthermore, vermicompost is available commercially and would be relatively easy for the indoor-grow enthusiast to incorporate into their ‘gardening’ practices. unfortunately, little evidence-based information exists regarding the addition of vermicompost tea and the associated benefits it may have on plant tissue in hydroponic systems. studies assessing the addition of beneficial microbes to hydroponic systems tend to focus on their use as biocontrol agents to combat plant pathogens (16). could microbes be harnessed to provide benefits beyond reducing disease? the goal of the research conducted for this study was to better understand whether vermicompost tea and its associated microorganisms would increase biomass and nutrient density of purple lady bok choy (brassica rapa var. chinensis) in vertical hydroponic systems. the rationale for choosing vermicompost as the source of beneficial microbes is due to its typically high populations and diversity of microbes, which has shown to benefit plants in a myriad of ways (1, 4, 15, 16, 20). purple lady bok choy was chosen due to its versatility, physiology, life cycle and stature. vermicompost detail summary organism result units desired level flagellates 106,214.36 number/g >10,000 amoebae 19,181.31 number/g >10,000 ciliates 32.28 number/g >1254 nematodes 28.31 number/g >10 bacterial 28.31 number/g >10 active bacteria 109.54 µg/g >3 total bacteria 2,118.75 µg/g >300 active fungi 87.76 µg/g >3 total fungi 3,478.17 µg/g >300 table 1: vermicompost detail summary analyzed by soil foodweb new york, analyzed june 17, 2019. 50 | fine focus it can be eaten fresh in salads, grilled, sautéed, fermented, or dehydrated. it is also known to have high levels of vitamins c, a, b6, calcium, iron, potassium, manganese, folate and is high in antioxidants due to an abundance of anthocyanins (24, 25, 26). not only are the anthocyanins responsible for the aesthetic purple coloration of the purple lady bok choy variety, but the pigments are also linked to the reduction of multiple diseases such as cardiovascular disease and cancer (24, 26). it has minimal heat and light requirements, was manageable within the given research space, has a quick germination rate and takes little time to reach full maturity, making this variety an ideal candidate for the indoor hydroponic research. the research tested and compared different concentrations and ratios of vermicompost teas and hydroponic fertilizer solutions. the prediction was that the addition of the vermicompost tea would increase the nutrient density and overall biomass of bok choy hydroponic systems. materials & methods seedling preparation: a total of one hundred-twenty rockwool cubes with a two-inch diameter were used per treatment. in each cube a total of three purple lady bok choy, brassica rapa var. chinensis seeds were planted surrounded by geolite (an irregularly shaped clay medium, hydrofarm llc), and nestled in two-inch net pots. the net pots were placed into trays, each tray associated with a specific tower, and covered with a clear plastic dome until the seeds were germinated. the purple lady variety of bok choy is a cool weather species, is shade tolerant, germinates in 2-4 days, grows 5-7” and can grow to maturity in 60 days. all the above factors made purple lady bok choy an ideal candidate for the indoor hydroponic research because it has minimal heat and light requirements, was manageable within the given research space, has a quick germination rate and takes little time to reach full maturity. for the first two weeks, the seedlings were watered with ph balanced tap water (ph 6.0 6.5). after approximately three weeks of growth, the seedlings were watered with a 1.25 grams per liter maxigro hydroponic nutrient solution (n-10, p-4, k-14). the germination took place under full spectrum leds on tiered “grow racks” on which the seedlings were grown for 30 days. after these 30 days of initial growth, the net pots were transferred to the vertical hydroponic grow towers (20 net pots per tower) and grew for an additional 30 days until they reached maturity and were ready to harvest. vermicompost tea preparation: the vermicompost used for the vermicompost tea was procured from carney’s crawlers llc, appleton, wisconsin. the microbial composition of the vermicompost was tested and verified by soil foodweb new york on june 17, 2019 (sample # 03-12178) using direct count and the differential interface contrast (dic) microscopy technique (table 1). dic microscopy was also used to determine the probable number of protozoa using four replications of a serial dilution of 10-1 to 10-6. the direct count of active bacteria and active fungal organisms was done using a fluorescein diacetate stain and counted under a fluorescent light. nematodes were identified based vol 9 | 51 on morphology and the population was determined by direct count method. the total bacterial count was determined using a fluorescein isothiocyanate stain and dic technique. the living organisms in the organic matter/soil particles were stained using fluorescein isothiocyanate, strained through a syringe filter and directly counted. the vermicompost tea solution was prepared by using 1.5 l of the vermicompost which was “brewed” in 16 l of ph balanced water (ph 7), in 5-gallon food-grade buckets. a paint strainer was used as a “tea bag” to separate the vermicompost from the water. the solution was constantly aerated with large (4 inches long and 2 inches in diameter) air stones (model asc-100, pawfly co), which helped to oxygenate the water and encouraged the growth of the beneficial bacteria and fungi. after the vermicompost tea brewed for 24 hours, two tablespoons of unsulfured pure blackstrap molasses (‘slow as’ brand) was added to feed the microorganisms that were extracted into the water from the vermicompost. the solution was aerated for an additional 24 hours before it was transferred to the vertical hydroponic towers. hydroponic tower design: there were three modular units, each supporting two grow towers (made from pvc pipes and pvc fence posts), for a total of six grow towers. two of the modules (with two towers each) were placed parallel to each other, while the third unit (with two towers) was placed perpendicular to the parallel modules which all together covered approximately 32 square feet (figure 1). one grow tower housed 20 plants, for a total of 120 plants for all three modular units. the towers were equipped with a water pump (bayite byt-7ao15 dc 12v solar hot water heater circulation pump with dc power supply adapter) that moved the solutions to the small reservoirs at the top of the towers. the solutions trickled down via gravity feed and subsequently covered (‘showered’) the roots of the plants in a consistent manner. pvc pipes were used as vertical support poles for each of the 6 led strips (t8 led grow lights, 4 feet, providing full-spectrum sunlight replacement at 400 nm 800 nm, monios-l co.) and provided overall support for the units. experimental treatments: each treatment was executed consecutively through implementation of all 6 towers at once. the utilization of all six towers for each treatment at the same time helped maintain efficiency and consistency of said treatment. the number of towers, 6, provided the minimum number of repetitions necessary for proper statistical analysis. each tower had a 16 l solution reservoir (5-gallon bucket). the control was prepared with 1.25 grams of mineral solution (general hydroponics, maxigro hydroponic nutrients) per liter of water. treatment one consisted of 0.625 grams of mineral nutrients per liter and ~25% by volume vermicompost tea. treatment two was made with 1.25 grams of mineral nutrients per liter and ~25% by volume vermicompost tea solution. treatment three was a solution composed of 0.625 grams of mineral nutrients per liter and ~50% by volume vermicompost tea. the temperature 52 | fine focus fig 1: design layout of the three modular units which housed the six vertical grow towers that were surrounded by six strips of leds each. the photo was taken in weston hall, room 1208, at northern michigan university, in marquette, michigan, where the experiment was conducted from september 2020 march 2021. vol 9 | 53 of the reservoir solutions was periodically measured and recorded. the solutions of each treatment were added alternately when the reservoirs were approximately 4-6 liters depleted. the reservoirs were completely emptied and refilled with fresh solutions approximately halfway through each growth cycle, which was ~15 days, except for treatment three. during treatment three an experimental error occurred where the reservoirs were refilled on day 28 rather than the 15-day time that had been previously done in all the other treatments. the ph was regularly checked and adjusted to ~6.0-6.5 by using general hydroponics ph adjustment chemicals. the plants were grown in the towers for 30 days, at which point they were mature and harvested. the plant biomass was dehydrated at 60°c (140°f) in dehydration ovens. the dehydrated biomass was used to determine the dry weights of the leaves/petioles and roots (the initial weight of the rockwool was deducted from the weight of the roots) which was used as a ‘dry weight method’ to perform proper statistical analysis. all equipment was cleaned with hydrogen peroxide solution (using ~2 tablespoons of 3% hydrogen peroxide per 1 gallon of water) and set up for the next treatment. plant nutritional analysis & sample preparation: before the plants were harvested, six leaves were taken from each tower at random (as per the laboratory technician’s instructions) and immediately sent to a & l great lakes laboratories (fort wayne, in) where the nutritional density of the plant tissue was analyzed. the samples were shipped via ups, unrefrigerated. once the plant tissue was received by the laboratory, the samples were dried overnight at 100-105°c. the dried tissue was ground with a wiley mill grinder and sieved through 20 mesh screens. the plant tissue was tested for a variety of macroand micronutrients (tables 2 and 3). total nitrogen was analyzed using the dumas method (using an elementar rapid-n cube), while mineral analysis was conducted using inductively coupled argon plasma (icap) run on a thermo icap 6500. environmental measurements: the windowless room in which the research was conducted was on the campus of northern michigan university in weston hall, room 1208. a govee bluetooth hygrometer thermometer tracked the room humidity (14-42.5 g kg-1) and ambient room temperature (68.36-72.86°f). there were no significant changes in room and reservoir temperature or humidity while the research was being conducted. the average temperature of the solutions in the tower reservoirs was 21-22°c (69.8-71.6°f) and was measured periodically with a submerged thermometer. statistical analysis: the variance among the towers was tested using a linear mixed model. the data was analyzed with r-studio using analysis of variance (one-way anova). first, the data was checked for normality with histograms and a shapiro-wilk test. homogeneity of variance was tested with levene’s test. 54 | fine focus mean macronutrients (%) nutrients: n s p k ca mg control 7.87 1.31 0.73 7.72 3.22 0.72 treatment 1 6.27(-) 2.07(+) 0.95(+) 7.07 3.02 0.66 treatment 2 7.02(-) 1.16 0.77 5.61(-) 2.71 0.39(-) treatment 3 5.85(-) 1.62(+) 0.93(+) 7.21 2.86(-) 0.53(-) table 2: mean macronutrient (%) for the control and each treatment with a (+) or (-) indicating a statistically significant increase or decrease in mean nutrient density compared to the control. no symbol indicates no significant change (n= 6). table 3: mean micronutrients (ppm) for the control and each treatment with a (+) or (-) indicating a statistically significant increase or decrease compared to the control. mean micronutrients (ppm) nutrients: fe b na zn cu al mn control 73.83 60.00 0.15 35.17 56.83 5.83 253.33 treatment 1 72.50(+) 89.50(+) 0.14 18.00(-) 5.50 8.83 277.50 treatment 2 85.50 59.50 0.08(-) 43.17(+) 5.17(-) 8.17 232.33 treatment 3 73.83 82.17 0.11(-) 38.83 9.00(+) 4.33 287.33 the normality and variance assumptions were met, and the anova was run. a post hoc dunnett’s and tukey’s test was used to determine if the leaf and root biomass of the treatments were significantly different from the control and each other. the nutritional analysis from the labs was also tested for significance using one-way anova and dunnett’s test, examining the overall mean macroand micro nutrient densities compared to the control. furthermore, each macroand micro mean nutrient density was comparatively analyzed among each treatment via a oneway anova. some macronutrients failed the levene’s test of homogeneity of variance (p> 0.05), thus a welch’s corrected one-way anova was run for those nutrients. results according to soil food wed new york lab, the vermicompost analysis resulted in above desired population levels for all organisms (table 1). importantly, the active bacterial and fungal populations were very high (109.54 µg/g, 87.76 µg/g respectfully). a linear mixed model showed that the variance among the towers was estimated at zero, meaning the growing conditions for each tower was the same. treatment two had the highest mean leaf biomass of 444 grams, followed by the control (404.8 g), treatment one (300.1 g), and treatment three (197 g). using a one-way anova, the mean leaf biomass was found to be significantly vol 9 | 55 different among the treatments (f= 235.4; df= 6,21; p< 2e-16; figure 2). in treatments one and three, the mean leaf biomass was significantly less compared to the control (dunnett’s: p= 0.0058 and p= 3.7e-06, respectively). treatment two showed no significant difference in leaf biomass compared to the control (p= 0.56). all treatments were significantly different from one another, excluding the control (tukey’s: treat1-treat2 p= 0.0005; treat1-treat3 p= 0.011; treat2-treat3 p= 3.0e-7). the mean root biomass was significantly increased in all treatments compared to the control (f= 5.932; df= 3,20; p= 0.00458; figure 3). the control had the lowest root biomass with a weight of 46 grams. treatment three had the highest root biomass of 79 grams, followed by treatment one (73.3 g), and treatment two (71.7 g). root mass did not differ significantly among treatments one, two and three (tukey’s: p> 0.05). the mean nutrient densities for microand macronutrients were tested separately with a one-way anova and dunnett’s post hoc test. these tests revealed that there were significant differences between nutrient levels among the treatments compared to the control for both macro(f= 107.5; df= 5; p= 9.27e-13; figure 4; table 2) and micronutrients (f= 235.4; df= 6; p< 2.16e16: figure 5; table 3). the macronutrients that were significantly greater than the control occurred in treatments one and three, which included sulfur (trmt1 p= 1.3e-08, trmt3 p= 0.002) and phosphorus (trmt1 p= 0.0007, trmt3 p= 0.002). the macronutrient, nitrogen, was significantly decreased compared to the control in all fig 2: results from anova, dunnett’s and tukey’s tests of the mean leaf biomass showed a significant difference among treatments (f= 235.4; df= 6, 21; n= 6; p< 2e-16). treatments one and three were significantly different from the control (dunnett’s: p= 0.0058 and p= 3.7e-06, respectively) and from each other (tukey’s: treat1-treat2 p= 0.0005; treat1-treat3 p= 0.011; treat2-treat3 p= 3.0e-7). letters indicate significant differences. bars indicate standard error. 56 | fine focus treatments (trmt1 p= 1.2e-07, trmt2 p= 0.0004, trmt3 p= 6.0e-12). treatment two had significantly decreased levels of potassium (p= 1.6e-07), calcium (p= 0.0008) and magnesium (p= 2.9e-11) compared to the control. treatment three had significantly decreased levels of calcium (p= 0.016) and magnesium (p= 3.0e-05) compared to the control. on the other hand, the micronutrients that were significantly greater than those in the control occurred in treatment one with boron (p= 4.7e-06), treatment two with iron (p= 0. 016), and zinc (p= 0.014), and treatment three with copper (p= 0.0018). the micronutrients which were significantly lower than the control occurred in treatment one with zinc (p= 5.8e-06), treatment two with sodium (p= 2.3e-08) and copper (p= 0.014), and treatment three with sodium (p= 0.0009). moreover, each individual nutrient underwent a one-way anova test, comparing the mean nutrient density of each treatment among the same nutrient in different treatments. macronutrients nitrogen, phosphorus, and magnesium failed the levene’s test for homogeneity of variance (p< 0.05), thus these macronutrients underwent a welch’s corrected anova instead, which doesn’t assume equal variances. the anovas resulted in all nutrients, both macro and micro, amidst each treatment to have a significant difference in mean nutrient density (p< 0.05; table 4; table 5). fig 3: results from anova, dunnett’s, and tukey’s tests of the mean root biomass showed a significant difference among the treatments (f= 5.932; df= 3, 20; n= 6; p= 0.00458). the three treatments were significantly increased compared to the control (dunnett’s: treat1 p= 0.023, treat2 p= 0.035, treat3 p= 0.004), but did not significantly differ among one another (tukey’s: p> 0.05). letters indicate significant differences. bars indicate standard error. vol 9 | 57 fig 5: the mean micronutrient (ppm) per treatment was significantly different compared to the control (f= 235.4; df= 6; n= 6; p< 2.16e-16). letters indicate significant differences. bars indicate standard error. fig 4: the mean macronutrient (%) per treatment was significantly different compared to the control (f= 107.5; df= 5; n= 6; p= 9.27e-13). letters indicate significant differences. bars indicate standard error. 58 | fine focus discussion in the presented research, treatment two (1.25 grams of mineral nutrients per liter and ~25% by volume vermicompost tea) had the highest leaf biomass (figure 2), the second highest root biomass (figure 3) and increased concentrations of micronutrients iron and zinc compared to the control (table 3). these two micronutrients are very important to human health yet are recognized to be deficient in many crop species and are part of the phenomenon of ‘hidden hunger’ that impacts significant portions of the human population (11, 21, 23). in contrast, treatment two had the greatest decrease in macronutrients compared to the other treatments, but the lab nutrient analysis showed that this decrease was not enough to drop the macronutrients into an unacceptable nutrient density level. there was no uniform gain or loss of nutrients among the treatments, although the lab analysis of leaf nutrient density showed that the control and all treatments were deficient in the micronutrient zinc except for treatment two, which had the highest level recorded. this may suggest that the concentrations of nutrient mineral solution and beneficial microbes in the vermicompost tea used in this treatment were the most effective in increasing overall health and biomass of bok choy (b. rapa var. chinensis). in abul-soud’s et al. study (2015), they found that optimal plant growth responses occurred with the addition of 10 20% vermicompost by volume, along with the addition of mineral nutrients (for the majority of crops in their research) (1). plants need nutrients to maximize their biomass in the form of ions (a positively or negatively charged compound), regardless of whether a system is synthetic or biological. the use of artificially based nutrients (inorganic salts/minerals) does not typically include living microorganisms, is unsustainable and is an attempt to mimic what natural biological systems have been successfully performing over eons of time (14). treatment one and three experienced a significant decrease in the mean leaf biomass compared to the control. these two treatments also had the least amount of mineral nutrient additives (0.625 grams per liter), which could have led to nutrient stress, reducing leaf biomass. in a similar study by aini et al. (2019), they found that the reduced nutrient content added to their hydroponic system led to smaller plants that took two weeks longer to reach maturity and had significantly less biomass due to nutrient stress (2). while treatment three did have the least amount of leaf biomass, it had the highest amount of root biomass. additionally, treatment three’s reservoirs were not emptied and refreshed with new solutions halfway through the growing process, which could have increased the bacterial concentration even further. the increase in root biomass and not leaf biomass may be due to a stress response caused by an overgrowth of microbes on the roots, resulting in an increase of root growth but limiting nutrient uptake to the rest of the plant (17). no visual evidence of plant disease was present in this treatment (or any of the other treatments). similar results were found in a study by li et al. (2018) where shoot growth was limited while root growth increased due to the lack of water micro vol 9 | 59 droplets and nutrient availability caused by aeroponic misting intervals and droplet size (17). comparatively, the increase in root growth found in treatment three and the li et al. research could reflect a stress response due to a lack of nutrients. the effects of treatment three may coincide with the study conducted by abul-soud et al. (2015), using vermicompost in soilless culture wherein they found increasing the vermicompost concentration to 15% was the plateau at which the plants received positive effects (1). beyond this limit, significant negative effects in yield, quality and nutrient density occurred with certain crops. though treatment three in the presented research had the greatest root biomass, the exact mechanisms for this result are not clear; it may be worth further investigation for applications when an increased root biomass is desired. increased root biomass in medicinal root crops cultivated in a greenhouse, using an aeroponic or hydroponic system, could make medicinal roots more readily available to the public (17, 19). according to a study conducted by pagliarulo et al. (2004), there is a multibillion-dollar market for medicinal botanical products, such as purple coneflower (echinacea purpurea), burdock (arctium lappa), ginseng (panax ginseng) and st. john’s wort (hypericum perforatum) (19). in their study, they found that e. purpurea had a significantly greater root biomass when grown in an aeroponic setting versus in soil (19). this suggests the use of indoor aeroponic systems can be very advantageous when operated properly. aeroponic and hydroponic systems function similarly one-way anova summary for macronutrients nutrients: n s p k ca mg df 3, 9.55* 3, 20 3, 10.17* 3, 20 3, 20 3, 8.72* f-value 57.56 55.78 42.43 23.38 7.10 89.17 p-value p<0.001 p<0.001 p<0.001 p<0.001 0.002 p<0.001 table 4: one-way anova summary outputs for macronutrients when assessed individually, resulting in all nutrients having significantly different mean nutrient densities among each treatment (p< 0.05, n =6). welch’s corrected anova was run on nutrients n, p, and mg due to unequal variances, hence the changes in the denominator df (*). table 5: one-way anova summary outputs for micronutrients when assessed individually, resulting in all nutrients having significantly different mean nutrient densities among each treatment (p< 0.05, n= 6). one-way anova summary for micronutrients nutrients: fe b na zn cu al mn df 3, 20 3, 20 3, 20 3, 20 3, 20 3, 20 3, 20 f-value 5.13 25.36 30.32 37.32 21.15 5.32 3.68 p-value 0.009 p<0.001 p<0.001 p<0.001 p<0.001 0.007 0.029 60 | fine focus and may have some of the same benefits. the experimental results of the research conducted for this paper showed that adding microorganisms via compost tea increased the root biomass of bok choy in a hydroponic system. the addition of beneficial microbes to indoor cultivation systems has great potential to increase biomass and reduce the need for synthetic fertilizers and pesticides conventionally used for crop production. a study done by giurgiu et. al (2018), showed that certain medicinal plants (hypericum perforatum) treated with trichoderma spp., (a known beneficial fungus) in rockwool substrate in a hydroponic system had a twofold increase in foliar biomass and enhanced root development compared to plants treated with a pathogenic microbe (10). their research highlights the complexity of plant-microbe interactions regarding the impact on plant growth and nutrient levels. the research conducted in this paper demonstrated variable plant responses with the different levels of inorganic fertilizer and vermicompost tea solutions. overall, research results indicate there may be no “one size fits all” when it comes to the implementation of amendments to hydroponic systems to reach the goals of improvements in plant biomass and nutrient profiles. similarly, sheridan et. al. (2017), investigated how plant growth promoting microorganisms (pgpms) affect the root zone microbiome of four common food crops (durum, potato, bread wheat and soybean) in recirculating hydroponic cultivation systems for the whole life cycle of each plant (22). they concluded crops inoculated with a mixture of commercial pgpms (with a composition of microbial communities associated with root rhizosphere, rhizoplane, and endosphere with a recirculating nutrient solution) were more stable in a plant based biological life support system over time, depending on the specific crop (22). however, more research is needed to better understand this dynamic and to fine tune experimental protocols to achieve desired results. vermicompost is a pgpm, is available commercially and would be relatively easy for the indoor-grow enthusiast to incorporate into ‘gardening’ practices. based on the results of the experimental study, the addition of vermicompost tea in a hydroponic system can result in an enhancement to the plant biomass and nutrient density of bok choy when applied at specific concentrations. a thorough analysis of the microorganism species which were present, persisted, or died off, on plant roots at the beginning and end of the experiment would provide more insight as to how the species of plants, amendments and microorganisms interacted and would enhance the continuation of the presented research. modifying the current experiment to include a second control consisting of 0.625 g of mineral nutrients in future studies may give further insight to how the beneficial microorganisms in the vermicompost tea influences the growth and nutrient density of bok choy. investigating the specific species of microbes’ and deciphering their impact on plant growth, biological and chemical properties, such as antioxidants and polyphenols, would be valuable. research conducted by chandra et vol 9 | 61 al. (2014) suggested that aeroponically grown crops may provide an environment in which plants produce higher levels of antioxidant activity (10). moreover, hydroponic systems might offer a higher level of reproducibility for influencing the concentrations of vitamins and phenolic compounds within a crop (10). these findings should be studied further using larger data sets and total environmental and nutrient input controls. with a greater understanding of the specific microbes and their impacts on plants, it would provide better insight as to how to adjust and fine-tune the percent solutions to achieve the specific desired results for certain plant species. the incorporation of beneficial microbes into hydroponic systems holds promise for improving plant growth and nutritional characteristics and could play an important role in the transition from conventional agriculture to indoor hydroponic systems which may lead to significant advancements in the future of farming. acknowledgements we would like to extend our sincerest gratitude to dr. donna maki for her unrelenting support and help with the overall execution of this research. we would also like to thank dr. alan rebertus for his generosity and abundant knowledge regarding r-studio and statistical analysis. much thanks to regan doran for his expertise and build of the vertical towers. additionally, we would like to thank sarah kunister for her help throughout the research and michael fishman for his assistance with the initial installation of the modular units. we would also like to extend our gratitude to mrs. marilyn scheidemantel, as our research was supported in part by a grant from the scheidemantel research fund for undergraduate students. 62 | fine focus references 1. abul-soud, m. a., emam, m. s. a., & abd el-rahman, n. g. 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(2014). anthocyanin accumulation and transcriptional regulation of anthocyanin biosynthesis in purple bok choy (brassica rapa var. chinensis). journal of agricultural and food chemistry, 62(51), 12366-12376. publisher corrections: table 3: an incorrect title and description for table 3 were originally published with the article, due to a publisher error. the corrected title and description were published on 12/4/2023. figure 2: the incorrect image for figure 2 was originally published with the article, due to a publisher error. the corrected figure was published on 12/4/2023. © 2025 datta. fine focus, 11(1), 5-11. doi: 10.33043/yrxbqqaa shared with cc-by-nc-nd 4.0 license. neelabh datta asutosh college affiliated with the university of calcutta, west bengal, india abstract a new coronavirus variation known as jn.1 first appeared in luxembourg in august 2023 and has since spread to a number of nations. it is distinguished by a high transmissibility, and it may outpace earlier omicron waves in terms of infections, hospitalizations, icu admissions, and fatalities. more research is required to establish the projected continued efficacy of current vaccinations, treatments, and testing. an outline of jn.1’s epidemiological, clinical, and virological characteristics as well as its effects on world health are given in this brief communication. keywords: jn.1, coronavirus, variant, outbreak, global health corresponding author email: neelabhdatta@gmail.com manuscript received 24 march 2024; accepted 28 may 2024. ojn.1: a new threat to global health https://creativecommons.org/licenses/by-nc-nd/4.0/ mailto:neelabhdatta%40gmail.com?subject= fine focus | volume 116 the jn.1 variant of covid-19 is a new sub-variant that has emerged in late 2023 and has become the dominant and most transmissible strain of the virus in the world. it has currently affected more than 41 countries, and is expected to cause a bigger wave of infections and deaths than the previous ones, according to global experts. the world health organisation (who) has classified jn.1 as a variant of interest (voi) because of its rapid spread and its ability to evade the immune system, which is causing a global surge of infections, hospitalisations, and icu admissions (1). jn.1 was first detected in luxembourg in august 2023 and is now present in many countries. it is derived from another voi, ba.2.86, which was first identified in the same month. ba.2.86 is a lineage of sars-cov-2 that is phylogenetically distinct from the omicron xbb variant lineages that were circulating before, such as eg.5.1 and hk.3. the genomic sequence of sars-cov-2 is known to code for a total of 29 distinct proteins. this set of proteins is categorized into three groups: 16 non-structural proteins that are crucial for the viral replication cycle, 4 structural proteins that form the virus particle, and 9 accessory proteins that modulate the host’s cellular environment. as part of the global scientific community’s response to the pandemic, researchers have determined the three-dimensional structures of these viral proteins (2). so far, over 1,228 sars-cov-2 experimental structures have been added to the protein data bank (pdb). these entries include two different functional domain structures of the nucleocapsid (n) protein and the structures for all 16 of the non-structural proteins. additionally, numerous structures of various protein complexes have been deposited. when these structures are compared to those of the closely related sars-cov, which caused the sars outbreak in 2003, a high degree of structural similarity is observed. (2) ba.2.86 has more than 30 mutations in the spike (s) protein, which is the part of the virus that attaches to human cells and is targeted by vaccines and antibodies. these mutations indicate that ba.2.86 has a high potential to escape the immune response and infect new hosts (3). jn.1 is similar to ba.2.86, but has one additional mutation in the spike protein, l455s, and three other mutations in non-s proteins. the leu455ser (l455s) mutation is a hallmark of jn.1 and is also found in hk.3 and other variants that are called “flip” variants (3). these variants have the leu455phe (l455f) mutation, which is similar to l455s, but with a different amino acid change. an infection assay was conducted by tamura er al. (4) using hiv-1-based pseudoviruses to evaluate the infectivity of different spike (s) protein mutations. the findings indicated that pseudoviruses carrying the s proteins from the b.1.1 or eg.5.1 variants demonstrated a marked increase in infectivity compared to those with the ba.2 s protein (figure 1). however, the infectivity of pseudoviruses with the ba.2.86 s protein was found to be on par with those bearing the ba.2 s protein (4). in order to analyse the effects of individual mutations on infectivity in more detail, tamura er al. (4) created 33 different ba.2 derivatives, each of which included a different set of mutations identified in the ba.2.86 variant. the infectivity of the ba.2 s pseudoviruses was not significantly affected by the bulk of these alterations. however, it was shown that two mutations in the receptor-binding domain (rbd), n460k and f486p, increased infectivity. notably, infectivity was significantly boosted by three unique mutations— f157s, n211del, and a264d—that were found in the n-terminal region of the ba.2.86 s protein. six mutations specific to ba.2.86 increased the infectivity of ba.2, whereas sixteen mutations had the reverse impact, according to the mutagenesis assays, which revealed a complex interaction of mutations influencing infectivity. in spite of this, ba.2.86’s overall infectivity was comparable to that of ba.2. this points to a possible evolutionary path for ba.2.86, in which the s gene may have mutated to avoid the humoral immunological response of the host, then acquired enhancing mutations to compensate for any infectivity lost as a result of immune evasion. in addition to infectivity assays, the cleavage efficiency of the s protein was assessed through datta | ojn.1: a new threat to global health 7 figure 1 comparative analysis reveals mutations in the spike (s) proteins of omicron subvariants ba.2, ba.5, xbb.1.5, xbb.1.16, ch.1.1, eg.5, ba.2.86, and jn.1, with reference to the ancestral sars-cov-2 caption continues on page 8 fine focus | volume 118 western blot analysis of cells used for pseudovirus production (4). the results revealed that cells expressing the ba.2.86 s protein exhibited a higher band intensity for the s2 subunit compared to those expressing the ba.2 s protein. analysis of point mutants based on the ba.2 s protein indicated that several mutations contributed to an increased cleavage efficiency of the s protein. moreover, the level of the s2 subunit within virions pseudotyped with the ba.2.86 s protein was greater than that in virions with the ba.2 s protein. among the ba.2-based point mutants, the three mutants—f157s, a264d, and n460k— showed not only increased cleavage efficiency in cells but also a higher incorporation of s2 protein into the viral particles released, suggesting a correlation between cellular cleavage efficiency and virion incorporation levels. the presence of a four amino acids insertion (17mplf) in the spike protein of jn.1 was reported by chakraborty, (6). this insertion might enhance the transmissibility of jn.1 by compensating for the loss of eight amino acids (24lpp, 69hv, 145y, 211n and 483v) in the spike. jn.1 was first detected in south india in december, 2023, and it was warned by scientists that it could become the dominant variant with 20–30% of all omicron infections worldwide in 2024. lower affinity for the ace-2 receptor but higher immune evasion than ba.2.86.1, was shown by jn.1 (6). a driving force for the higher transmission, compensating for the eight amino acids deletions, was jn1 17mplf spike insertion. a more compact spike was predicted by swiss-model by chakraborty, (6), although the surface amino acids for interaction with ace-2 receptor were changed. the side chains of basic amino acid might interact with viral rna for quick complete inclusion of virus into lung cells and thus might be involved in rapid spread among the people whose immunity to covid-19 vaccines was lost considerably. more mutations and deletions and less protection to covaxin and covishild vaccines were shown by spike protein of jn.1 along with more resistance to antibody of previously coronavirus-infected individual (6). involvement in interaction with the ace-2 receptor was indicated by the data for amino acids glu484, phe486, gln474, lys417, tyr453 and asn501 in the rbd of spike. but amino acids his442, pro482, lys480, lys478 might be important in this aspect, as suggested by chakraborty, (6) with jn.1 spike model. the first to interact were arg346, phe486, lys444, gly446, val445 and tyr449 (figure 2). a role for m-protein 3-d structure in the higher transmission of ba.2.86 and jn.1 subvariants was speculated by the finding of conserved m-protein mutations (6). a high degree of structural rigidity in a simple lipid bilayer was shown by molecular dynamics simulations and a role for m homodimers in scaffolding viral assembly was supported. an important electropositive cytosolic surface that might be important for interactions with n, s, and viral rna was displayed by m. the charges in its surface should be changed by the d3h, a63t, a104v mutations in the m protein. ntd, tmi, tmii, tmiii and ctd were designated as the m protein domains aa 1–19, aas 20–40, aas 51–71 and aas 80–100 and aas 101–222 respectively. c-terminal phe103, arg107, met109, trp110, arg131, and glu135 were suggested as the common interacting residues of the m-protein with s and n proteins. however, an interacting role with ser186 of n protein changing m-n interaction in jn.1 and ba.2.86 variants was found for ala104 of m-protein (6). caption continued from page 7 strain wa1. the rbd of ba.2.86 exhibits specific amino acid changes when compared to both the original sars-cov-2 and ba.2. these mutations are highlighted in structural models with pdb ids 7xiw (ba.2) and 6zp5 (wild type). a phylogenetic tree of rbd sequences illustrates the evolutionary relationships and genetic divergence among sars-cov-2 wa1, its variants, and subvariants, including ba.2.86 and jn.1, with corresponding genebank accession numbers provided for their s protein sequences. image credit to liu et al. (5) datta | ojn.1: a new threat to global health 9 jn.1 is believed to be associated with the second-biggest wave of infections in the us and unprecedented levels of wastewater viral load in several european countries (8). global experts predict that jn.1 may result in a bigger wave than the previous ones, possibly exceeding the omicron waves in 2022. professor christina pagel of university college london (ucl) stated that the jn.1 wave has not yet reached its peak and may do so in mid-january, either next week or the week after. she expressed her certainty that this wave will rival or surpass the first two omicron waves in 2022 (9). professor eric j. topol of scripps research wrote in an opinion piece in the los angeles times that jn.1 is now the main cause of infections in the us, based on the wastewater levels of the virus (8). he explained that the actual number of infections is difficult to track since most people either test at home or do not test at all, but he estimated that about 2 million americans are getting infected each day. it was noted that several countries in europe have reached unprecedented levels of wastewater viral load, exceeding omicron. this allowed him to conclude that this virus variant, with its plethora of new mutations, has continued its evolution with mutations adapted for infecting or reinfecting us (10). professor peter openshaw, a virus expert at imperial college london, was quoted as saying to the sun health that the coming weeks could see a major surge in infections and that the wave could be bigger than anything we have seen before. a recent study by qu et al. showed that ba.2.86 can infect cells in the lower lung and can enter cell membranes more efficiently than other versions of omicron, raising the concern that jn.1 may have a similar or higher tendency to infect human lung epithelial cells, which could pose a serious threat to public health (11). the current vaccines, treatments and testing are expected to remain effective figure 2 figure 2 shows the various muations in variants of sars-cov-2. the orange cube shows the amino acid difference at the site between each variant and the reference strain wuhan-hu-1 (genbank accession no. nc_045512.2) for the key mutations in the spike protein of ba.2.86, hk.3 and jn.1 variants, which are marked on the top. image credit to hang et al. (7) fine focus | volume 1110 against jn.1, but more rigorous and comprehensive studies are needed to confirm this (12). the experts advised people to take covid-19 booster and wear masks in public again to help curb the spread of jn.1. the us centre for disease control and prevention (cdc) stated that there was only a single change between jn.1 and ba.2.86 in the spike protein, which is the part that existing vaccines target. another recent study by jeworowski et al. investigated the immune escape potential of the two sars-cov-2 variants, ba.2.86 and jn.1 that have emerged and become dominant in different regions of the world (13). the study revealed that both variants can evade the neutralizing antibodies generated by prior infection or vaccination with earlier variants of sars-cov-2, such as xbb.1.5 and eg.5.1. the immune escape ability of ba.2.86 and jn.1 is comparable and significantly higher than that of the previous variants which could account for the rapid spread and prevalence of these variants in the global population. however, the study also suggested that immune escape alone may not be sufficient to explain the recent surge in jn.1 cases, as this variant does not exhibit any enhanced immune escape relative to ba.2.86 (13). therefore, other factors, such as viral fitness, host susceptibility, and environmental conditions, may also contribute to the increased transmissibility and infectivity of jn.1.the study also compares its results with two other studies that used a different cohort of participants, who had a higher exposure to xbb variants through infection or vaccination (13). it was found that the results were not consistent with those studies, as it observes a lower level of neutralizing activity against the newer variants, including xbb.1.5, eg.5.1, ba.2.86, and jn.1, in its cohort. this discrepancy may indicate a decline in vaccineor infection-induced immunity over time, which could pose a risk of re-infection in the coming winter months in the northern hemisphere. the study’s implications are relevant for the public health response to the covid-19 pandemic, as the emergence and spread of new variants pose a challenge for the effectiveness of vaccines, treatments, and testing (13). because the jn.1 variation may avoid the immune system, non-immunologic preventive measures may be the best way to stop its spread. this implies that in addition to making sure that public spaces are properly ventilated and sanitized, individuals should also practice excellent personal hygiene, which includes often washing their hands, using masks, and maintaining a safe distance from others (7). to combat the spread of the jn.1 strain, viral testing and self-isolation protocols should be reinforced in order to promptly identify and contain affected persons. developing vaccinations or booster tactics tailored to the jn.1 strain and its distinct features will strengthen immunity against this new danger. according to the cdc, the new covid-19 vaccinations for 2023–2024 can help the immune system block ba.2.86 and that they expect jn.1 to have a similar response and that the current treatments and testing methods remain effective against these variants. this is based on the analysis conducted by the sars-cov-2 interagency group (14) the world health organization (who) has emphasized that the current immunizations continue to protect against serious illness and death from jn.1 and other circulating variations. the who has also recommended individuals to maintain current vaccination records, particularly those who are at high risk of illness. in order to lessen the spread of the virus and its variations, the who has also advised individuals to keep up with preventative practices including mask use, social distance, and hand cleanliness. preliminary tests on a revised monovalent vaccination targeting xbb.1.5, subvariant, provide cross-protection against jn.1 (12). targeting the xbb.1.5 spike protein, a novel covid-19 vaccine has been created (12). the vaccine’s ability to protect people against other newly developing viruses is presently being tested and reported. interestingly, the vaccine can also generate strong immune responses against jn.1. a preprint study by wang et al. shows that the vaccine can produce high levels of antibodies that can neutralize jn.1, which may help prevent serious complications of covid-19, such as hospitalizations and deaths (15).since sars-cov-2 continues to mutate and transmit, it datta | ojn.1: a new threat to global health 11 is critical that worldwide communities collaborate in sharing data, exchanging experiences, and undertaking scientific study to acquire a more thorough knowledge of the emerging forms. this collaborative approach will allow for faster and better judgments in implementing effective public health initiatives. references 1. looi m. k. (2023). covid-19: who adds jn.1 as new variant of interest. bmj (clinical research ed.), 383, 2975. https://doi.org/10.1136/bmj.p2975 2. bai, c., zhong, q., & gao, g. f. (2022). overview of sars-cov-2 genome-encoded proteins. science china. life sciences, 65(2), 280–294. https://doi.org/10.1007/s11427-021-1964-4 3. kosugi, y., plianchaisuk, a., putri, o., uriu, k., kaku, y., hinay, a. a., jr., chen, l., kuramochi, j., sadamasu, k., yoshimura, k., asakura, h., nagashima, m., the genotype to phenotype japan (g2p-japan) consortium, ito, j., & sato, k. (2023). virological characteristics of the sars-cov-2 omicron hk.3 variant harboring the “flip” substitution. biorxiv. https://doi.org/10.1101/2023.11.14.566985 4. tamura, t., mizuma, k., nasser, h., deguchi, s., padilla-blanco, m., oda, y., uriu, k., tolentino, j. e. m., tsujino, s., suzuki, r., kojima, i., nao, n., shimizu, r., wang, l., tsuda, m., jonathan, m., kosugi, y., guo, z., hinay, a. a., jr, putri, o., … sato, k. (2024). virological characteristics of the sars-cov-2 ba.2.86 variant. cell host & microbe, 32(2), 170–180.e12. https://doi.org/10.1016/j.chom.2024.01.001 5. liu, z., zhou, j., wang, w., zhang, g., xing, l., zhang, k., wang, y., xu, w., wang, q., man, q., wang, q., ying, t., zhu, y., jiang, s., & lu, l. (2024). neutralization of sars-cov-2 ba.2.86 and jn.1 by cf501 adjuvant-enhanced immune responses targeting the conserved epitopes in ancestral rbd. cell reports medicine, 5. 6. asit kumar chakraborty. higher omicron jn.1 coronavirus transmission due to unique 17mplf spike insertion compensating 24lpp, 69hv, 145y, 211n and 483v deletions in the spike, 04 january 2024, preprint (version 1) available at research square [https://doi.org/10.21203/rs.3.rs-3830998/v1] 7. hang fan, si qin and yujun cui. emergence and characterization of the sars-cov-2 jn.1 variant: global prevalence and implications for public health. zoonoses. 2024. vol. 4(1). doi: 10.15212/zoonoses-2023-0061 8. topol, e. (2024, january 4). covid-2024: flu virus vaccine. los angeles times https://doi.org/10.1136/bmj.p2975 https://doi.org/10.1007/s11427-021-1964-4 https://doi.org/10.1101/2023.11.14.566985 https://doi.org/10.1016/j.chom.2024.01.001 https://doi.org/10.21203/rs.3.rs-3830998/v1 ojn.1: a new threat to global health abstract figure 1 figure 2 references © 2025 swain, colburn, de andrade, rosales, addorisio, stuart, galvao, hubisz, ouellette, hamssa, laranjo, pina. fine focus, 11(1), 12-23. doi: 10.33043/rq7a84ax shared with cc-by-nc-nd 4.0 license. analyzing selected fda approved drugs for effects on template switch mutagenesis in e. coli april swain1 emily colburn1 0009-0001-6905-2381 kamila de andrade2 pamela rosales2 sydney addorisio2 0009-0001-3776-5870 taylor stuart2 angelina galvao1 leigha hubisz2 margaret ouellette1 rana hamssa1 laura laranjo2 0000-0002-1225-8821 cara pina1 0000-0002-8098-8148 1framingham state university framingham, ma usa 2 salem state university salem, ma, usa https://creativecommons.org/licenses/by-nc-nd/4.0/ https://orcid.org/0009-0001-6905-2381 https://orcid.org/0009-0001-3776-5870 https://orcid.org/0000-0002-1225-8821 https://orcid.org/0000-0002-8098-8148 swain et al. | analyzing selected fda approved drugs for effects on template switch mutagenesis in e. coli 13 introduction dna mutations, changes that occur during dna replication, can induce genetic instability. among various mutation classes, this study investigates mutations caused by dna secondary structures able to form quasi palindromes (qps). qps are imperfect inverted repeats of dna that can give rise to hairpins and cruciform structures. dna replication and gene expression are fundamental processes shared by all living organisms, including human cells. e. coli have simpler systems than human cells, making them ideal for preliminary studies. their ease of genetic manipulation and rapid growth in the lab further enhance their utility. the thya hotspot in e. coli was the first natural qp hotspot studied for mutagenesis (1). these structures have been correlated with a specific type of mutation referred to as a template-switch mutation (tsm). tsms are associated with several human diseases as reviewed (2), including hereditary angioneurotic edema (3), osteogenesis imperfecta (4), and biotinidase deficiency (5). lynn ripley was first to propose the two mechanisms for qp-induced tsm, intramolecular and intermolecular (figure 1), both of which can involve a modification of the quasi-palindromic region into a perfected palindrome proposed to be after a momentary fork stalling event. (6) while tsm occurs on both leading and lagging strands during dna replication, the leading strand exhibits a bias towards tsm in both reversion (7) and tsm-specific assays (8). this bias is nullified in the absence of exonucleases (exoi and exovii) (8), which are known to prevent tsm (1, 9) with exoi playing the larger role (1). this suggests that both strands can undergo mutations, abstract quasipalindromes (qps) are dna sequences that are imperfectly mirrored, known to form secondary structures like hairpins and cruciforms. these sites have been linked with a specific type of mutation called template-switch mutation (tsm). certain drugs like 5-azacytidine, azidothymidine (azt), and ciprofloxacin are known to induce tsm. this study aims to assess the impact of five fda-approved drugs—three antitumor drugs (cpt-11, doxorubicin hydrochloride, and gemcitabine hydrochloride) and two anti-inflammatories (ibuprofen and dexamethasone)—on template-switch mutagenesis. studying fda-approved drugs for their impact on tsm is important, as they are associated with diseases like hereditary angioneurotic edema, osteogenesis imperfecta, and biotinidase deficiency. the findings reveal no statistically significant effects on frequency of mutations after treatment with gemcitabine hydrochloride, ibuprofen, dexamethasone, or doxorubicin hydrochloride. however, cpt-11 treatment showed a notable decrease in tsm, suggesting a potential role in disrupting the template-switching process. keywords: quasi-palindrome; mutagenesis; dna replication inhibitors; dna repair corresponding author email: aswain@student.framingham.edu manuscript received 24 april 2024; accepted 30 january 2025 mailto:aswain@student.framingham.edu fine focus | volume 1114 but the lagging strand is more adept at averting and correcting them (8). additionally, research has demonstrated that exposure of qp sites to chemicals can influence the rate of tsm (10, 11). azidothymidine (azt), a thymidine analogue used as an antiretroviral medication to prevent and treat hiv/aids, has been seen to increase tsm rates (11). other drugs found to increase rates of tsm include 5-azacytidine (5-azac), the antibiotic ciprofloxacin, and formaldehyde (10). 5-azac interacts with cytosine methyltransferase (12) to cause dna/protein crosslinks that stall replication (13), the antibiotic ciprofloxacin is a topoisomerase type ii inhibitor, like dna gyrase (14) and the chemical formaldehyde acts as a general mutagen, including creating dna/ protein crosslinks in human cells (15). this study focuses on testing additional fda-approved drugs, described below, for their impact on tsm frequency in e. coli. a mutational reporter in the lacz gene of e. coli has been used to identify the rate of quasi-palindrome mutations after exposure to various fda-approved drugs. these mutations become apparent when lacz alleles revert to lac+, a phenomenon observed only if a template-switch event occurs. additionally, since most studies on the influence of drug exposure on tsm have also used this mutational reporter in e. coli, continuing to use this system allows for consistent and fair comparisons across research. all the fda-approved therapeutics selected have at least one mechanism of action capable of causing replication fork stalling, a necessary condition for both intraand intermolecular tsm. it is expected that these drugs are either inhibiting a process during dna replication or disrupting dna through intercalation and will lead to an increase in tsm frequency. figure 1. quasi-palindrome template-switch mutation mechanisms two template-switch mutation models involving a temporary displacement of the nascent strand and relocation of the polymerase onto another strand different from the original template. in the intramolecular model, the nascent strand becomes the template, in the intermolecular model, the homologous strand becomes the template. both mechanisms created a situation that can perfect an imperfect palindrome. (created in https://biorender.com) https://biorender.com swain et al. | analyzing selected fda approved drugs for effects on template switch mutagenesis in e. coli 15 gemcitabine hydrochloride (dfdc) gemcitabine hydrochloride (dfdc) is an fda-approved drug used in cancer treatment. once inside cells, deoxycytidine kinase (dck) phosphorylates dfdc, and subsequent phosphorylation steps lead to its active forms, dfdcdp and dfdtp. dfdcdp inhibits ribonucleotide reductase, reducing the pool of deoxynucleotides, mostly dctps, and enhancing the concentration of dfdcdp. this self-potentiation mechanism promotes masked chain termination that inhibits dna synthesis and causes a fork stalling event (16). although there are no previous studies focusing on gemcitabine's chain termination effects on tsm, studies conducted with the azt chain terminator drug led to the hypothesis that gemcitabine might similarly be able to enhance tsm frequency (11). ibuprofen ibuprofen is an fda-approved drug that treats inflammation and is used mostly to treat mild forms of pain and reduce fevers. a previous study found that the reactive oxygen species (ros) generated by ibuprofen caused dna strand scission and could cause dna degradation (17). furthermore, ibuprofen was identified to have intercalative binding with the dna helix. this is where structures resembling base pairs will be stationed in the double helix rather than the appropriate base pairs and thereby distort the structure. in this sense, dna replication is prevented from operating regularly. we have chosen to test ibuprofen due to the potential role it has in genetic instability. dexamethasone dexamethasone is an fda-approved hormonal corticosteroid commonly prescribed to treat inflammation and swelling. dexamethasone is comprised of multiple aromatics and hydroxy, methyl, and oxo groups, making it similar in structure to other corticosteroids such as hydrocortisone and prednisolone. further studies examining the interaction between the dexamethasone-glucocorticoid receptor complex, and dna have suggested the potential for dexamethasone to behave as an intercalating agent (18), giving it the potential to cause template switch mutations. doxorubicin hydrochloride doxorubicin hydrochloride (doxo) is a chemical capable of inhibiting topoisomerase ii (19), generating reactive oxygen species (20), and intercalating dna (21). here we evaluate the potential role of doxo in tsm due to inhibiting topoisomerases and doxo mediated dna-protein crosslinks, similarly to how 5-azac and ciprofloxacin have been shown to promote tsm by stalling replication (10) implicating the dcm-dna covalent complex trapped by 5-azac as the initiator for mutagenesis. the leading strand of replication is more mutable than the lagging strand, which can be explained by blocks to the replicative helicase and/or fork regression. we find that template-switch mutagenesis induced by 5-azac does not require double strand break repair via recabcd; the ability to induce the sos response is anti-mutagenic. mutants in recb, but not reca, exhibit high constitutive rates of template-switching, and we suggest that recbcd-mediated dna degradation prevents template-switching associated with fork regression. a mutation in the dnab fork helicase also promotes high levels of template-switching. we also find that other dpc-inducers, formaldehyde (a non-specific crosslinker. irinotecan hydrochloride (cpt-11) irinotecan hydrochloride (cpt-11), derived from camptothecin, is a chemotherapeutic drug that inhibits topoisomerase i (22, 23). the inhibition of topoisomerase i by cpt-11 results in the formation of highly cytotoxic topoisomerase i-dna cleavage complexes, importantly triggering various cellular responses, including proteinlinked dna breaks 23 and replication fork arrest24. due to the similarities of properties with other tsm causing drugs, we have included the evaluation of cpt-11. fine focus | volume 1116 figure 2. mutational assay methods a. single colonies were selected, inoculated, and cultured overnight. b. cultures were diluted and incubated for 2 hours before pelleting via centrifugation. c. cultures were divided into treated and control tubes before adding drug or control at the provided concentrations. cultures were then incubated for 2 hours. d. cultures were pelleted via centrifugation and the cell pellets were washed before resuspending the cells and performing serial dilutions. dilutions were plated on lb and minimum lactose plates and incubated overnight. e. total colony forming units (cfus)/ml were determined and percentage of reverting cfus were calculated. (created in https://biorender.com) https://biorender.com swain et al. | analyzing selected fda approved drugs for effects on template switch mutagenesis in e. coli 17 methods bacterial strains, growth conditions, and media a lacz mutational reporter strain of escherichia coli, qp5, that reverts to lac+ when a 4 base pair deletion occurs due to a template switch mutation was acquired from dr. susan t. lovett (used in laranjo et al. 2017) 25. standard growth medium was luria broth (lb) medium, comprised of 1% bacto-tryptone, 0.5% yeast extract, and 0.5% sodium chloride (fisher scientific, waltham, ma, usa) and minimum lactose solid medium, comprised of 1x m9 salts, 0.2% lactose, 1mm magnesium sulfate, and 0.1mm calcium chloride (alpha teknova, hollister, ca, usa). plates were also comprised of 1.5% bacto-agar. mutational assay single colonies were inoculated and cultured in 1.5 ml of luria broth (lb) medium and grown overnight at 37°c with aeration (figure 2a). the culture underwent a 1:100 dilution in fresh 1.5 ml lb medium, followed by a 2-hour incubation period at 37°c with aeration. following incubation, the entire culture was pelleted by centrifugation at approximately 7000 rcf for two minutes so that the latent medium could be removed, and the pellet was resuspended in 150 µl of fresh lb (figure 2b). the resuspended culture was divided evenly (75 μl in each) among two tubes followed figure 3. mutation frequency of gemcitabine at varying concentrations gemcitabine was tested at 0.3 (n=1), 0.5 (n=1), 0.7 (n=1), and 0.9 μm (n=2) and mutation frequency were calculated. the fold increase of the samples was 0.67, 1.14, 0.93, and 1.14 respectively compared to the control showing no statistical difference. error bars represent 95% ci based on the average mutation frequency. fine focus | volume 1118 by an addition of 1.5 ml of fresh lb to each tube (figure 2c). the specified concentration of the selected drug – cpt-11 (0.5 μm, 1 μm), dexamethasone (60 μm), ibuprofen (150 μm), doxorubicin (0.025 μm, 0.4 μm, 0.5 μm) – was added to one tube while an equal amount of water was added to the other tube. both tubes were placed back in the incubator for an additional 2 hours to grow at 37°c with aeration. subsequently, both cultures were pelleted by centrifugation once more and the cell pellet was washed twice with 1 ml lb, followed by resuspension in 200, 400, or 600 μl lb. cultures were then subject to serial dilutions in a 96-well plate to obtain 10-fold dilutions up to a 1:100,000 dilution, spotted onto lb and minimum lactose plates and grown overnight at 37°c to determine the cfu (colony forming units)/ml (figure 2d). data collection and analysis the percentage of reverting cfus of the total was calculated for each drug by dividing the number of total cfu/ml on the lac plates by the number of total cfu/ml on the lb plates (figure 2e). all corresponding data points were averaged to obtain the average mutation frequency of each drug. statistical analysis was done using an unpaired t-test that does not assume equal standard deviation among populations (welch’s t-test) using graphpad prism version 10.2.0 for windows, graphpad software, boston, massachusetts usa, www.graphpad.com. figure 4. mutation frequency of ibuprofen at 150 μm ibuprofen demonstrated a fold increase of 0.74 compared to the control (n=3). this was not statistically different than the control group. error bars represent 95% ci based on the average mutation frequency. http://www.graphpad.com swain et al. | analyzing selected fda approved drugs for effects on template switch mutagenesis in e. coli 19 results of the four concentrations of gemcitabine hydrochloride tested (0.3 μm, 0.5 μm, 0.7 μm, and 0.9 μm), the fold change was only between 0.67 and 1.14 (figure 3) showing no statistical difference from the control group. similarly, ibuprofen showed a fold change of 0.74 and no statistical difference from the control (figure 4). when compared to the control group, dexamethasone resulted in a lower average rate of templateswitch mutations, though not statistically different from the control. dexamethasone was tested at a concentration of 60 μm and resulted in a 2.37-fold change in mutation frequency, with the average rate of mutation 1.56e-06 compared to the control of 3.71e-06 (figure 5). this was similar to the results of doxorubicin hydrochloride at a concentration of 0.4 μm which had a fold change of 2.41 and an average rate of mutation of 1.54e-06 (figure 5). testing done with cpt-11 showed a statistically significant (p=0.0383) change in reported template-switch mutation rate, producing a 3.08-fold decrease when tested at a concentration of 0.5 μm with an average rate of mutation of 1.20e-06 (figure 5). figure 5. comparison of mutation frequency of cpt-11, dexamethasone, and doxorubicin cpt-11 was tested at a concentration of 0.5 μm (n=17) and resulted in a statistically significant fold change of 3.08 compared to the control with a p-value of 0.0383. dexamethasone, at a concentration of 60 μm (n=5), showed a statistically insignificant fold change of 2.37 compared to the control. doxorubicin was also statistically insignificant, tested at a concentration of 0.4 μm (n=24) and displaying a fold change of 2.41 compared to the control. error bars represent 95% ci based on the average mutation frequency. fine focus | volume 1120 discussion this study aims to expand our understanding of which fda-approved drugs contribute to tsm. it is important to examine how these therapeutics, including some commonly used over-the-counter drugs, may affect mutation frequency, as tsm have been linked to the development of various diseases. there is still a significant gap in understanding tsm, their causes, and the mechanisms behind these mutations. the expected outcomes for these experimental drugs were that they would either inhibit a process during dna replication or act as intercalating agents to disrupt dna, leading to an increase in tsm frequency. however, for most of the drugs tested (ibuprofen, dexamethasone, doxorubicin hydrochloride, and gemcitabine hydrochloride), no statistically significant change in mutation frequency was observed. our preliminary investigation into the effects of gemcitabine hydrochloride on tsm frequency suggests no significant impact on the leading strand. contrary to the initial hypothesis, due to gemcitabine hydrochloride's chain termination effects during dna replication, our results from limited trials do not support this notion. instead, it appears that gemcitabine hydrochloride may have no effect on tsm frequency, aligning with its primary role in inhibiting dna synthesis rather than causing replication fork stalling 26. similarly, although ibuprofen's potential to intercalate within dna 17 could theoretically stall the replication machinery, our trials did not show a statistical increase in tsm frequency. ibuprofen, hypothesized to act as an intercalating agent, likely failed to intercalate the dna or caused excessive damage due to its relatively large size. this could explain the lack of change in mutation frequency, as the dna either remained unchanged or was too damaged to synthesize properly. both dexamethasone and doxorubicin hydrochloride did not result in a statistically significant change in tsm rate when compared to the control group. dexamethasone and doxorubicin hydrochloride, also predicted to intercalate dna and stall the replication fork, may have caused enough damage to prevent successful replication, leading to no observed change in tsm frequency, though additional trials may provide further insights. in contrast, cpt-11 showed a statistically significant decrease in tsm frequency. this supports the hypothesis that cpt-11 inhibits topoisomerase i and induces dna strand breaks. these breaks likely prevent the displacement of the nascent dna strand and instead promote dna damage repair through resection and homology search rather than quasi-palindrome hairpin formation, thereby reducing polymerase displacement onto the opposite strand. this highlights its potential role in mitigating tsm occurrence. we plan to further explore the potential impact of fda-approved drugs on tsm using our extensive fda-approved drug library. this ongoing investigation aims to investigate on the mechanisms underlying tsm, including both natural occurrences and drug-induced cases. additional research on the impact of drug exposure on tsm should be conducted in mammalian cells. while this experiment focused on e. coli and provided valuable insights, the effects on mammalian cells remain unknown. testing these drugs in mammalian cells would allow for a more direct evaluation of the conclusions drawn from this study in a more complex system. this approach would also enable observations in cells that are susceptible to tsm-associated diseases. such findings would be particularly compelling given that the drugs tested here are also used to 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(1989). arrest of replication forks by drug-stabilized topoisomerase i-dna cleavable complexes as a mechanism of cell killing by camptothecin1. cancer research, 49(18), 5077–5082. 25. laranjo, l. t., gross, s. j., zeiger, d. m., & lovett, s. t. (2017). ssb recruitment of exonuclease i aborts template-switching in escherichia coli. dna repair, 57, 12–16. h ttps://doi.org/10.1016/j.dnarep.2017.05.007 26. de sousa cavalcante, l., & monteiro, g. (2014). gemcitabine: metabolism and molecular mechanisms of action, sensitivity and chemoresistance in pancreatic cancer. european journal of pharmacology, 741, 8–16. https://doi.org/10.1016/j.ejphar.2014.07.041 https://doi.org/10.1016/j.dnarep.2017.05.007 https://doi.org/10.1016/j.dnarep.2017.05.007 https://doi.org/10.1016/j.ejphar.2014.07.041 analyzing selected fda approved drugs for effects on template switch mutagenesis in e. coli abstract introduction figure 1. quasi-palindrome template-switch mutation mechanisms methods figure 2. mutational assay methods figure 3. mutation frequency of gemcitabine at varying concentrations results figure 4. mutation frequency of ibuprofen at 150 μm discussion figure 5. comparison of mutation frequency of cpt-11, dexamethasone, and doxorubicin references © 2025 paxton. fine focus, 11(1), 48-50. doi: 10.33043/c3rcq358 shared with cc-by-nc-nd 4.0 license. a universal tongue? kaikeyi paxton ball state university, muncie, in usa did you know that, as of 2023, there are more than 7,000 known spoken languages in the world (eberhard et al., 2024)? did you know that the population of the world is more than 7 billion, and increases every day? did you know that out of that 7 billion, only about 1.5 billion speak english (statistics and data, 2024)? to most, those facts are common knowledge. to everyone, those facts are only a google search away. however, here is a fact you may not know-english is widely considered to be the language of science. this means that nearly all journals with national or international impact accept submissions in english only (malayil, 2024). this initially may make sense, but i would like to take some time to dig deeper and fully understand some of the history behind this, the implications of it, and how we as a community might work to create a more informed, inclusive environment. the english language began in europe with the angles, saxons, and jutes, who ultimately settled in britain. over time, the language underwent modifications due to common differences in dialect and, ultimately, became what we know it to be today. however, beginning in the 16th century, english spread throughout the world through british colonialism (encyclopaedia britannica, 2021). the british established occupation of north america and the indies and brought with them their language and culture. deeming their ways “superior” over the culture of the native inhabitants of these regions, the british imposed these ways upon the native people of their colonies. soon, english became the common tongue in most of north america, the indies, and in some places in every time zone of the world (crystal & potter, 2025). this process happened repeatedly, until the british empire reached its https://creativecommons.org/licenses/by-nc-nd/4.0/ paxton | a universal tongue? 49 end in the 1960s (encyclopaedia britannica, 2020). the 1960s were not all that long ago, and our world today still sees the effects of the spread of the british empire. in most schools across the world, english is taught in addition to the native language. i am from india, and i can say that anyone who went to school there even briefly had to learn english while they were in school. many indians who stayed in school for a while are fluent in english, while those who did not get the privilege to finish school either do not speak english at all or are nowhere near fluent. this is the case in many non-western countries around the world. it is in this light that i would like to challenge the thought that english should be the common language. it is true that it is convenient to have some kind of common spoken language in case of travel, but does it make sense? when people come to america or go to london, they are generally expected to be at least semi-fluent in english. non-english speakers are constantly required to accommodate english speakers, so why do we not accommodate them? however, let’s look even deeper into this, and examine it from within the light of our community of scientists. if a scientist wanted to publish their research in an accredited journal, it would need to be submitted in english. the only exception to this would be a regional journal, where only regionally relevant research could be published. for example, a botanist in kashmir could find something in the agricultural system there that affects the growth of kashmiri chilies, they could publish their research in hindi in a regional journal that would only reach farmers in kashmir. this makes sense. however, what happens if that same botanist found a chemical in kashmiri chilies that could be a component in a cure for a worldwide illness? what if that research was so important that pharmacologists and doctors everywhere needed to know that information to effectively treat their patients? what if that botanist didn’t speak english? how would that research get published? would we then expect them to learn english just to publish their paper? why can only english-speaking scientists get their work out there? science and research are two fields in which diversity is vital to our mission. we cannot grow in knowledge and understand the world around us if we remain a uniform community. this is something we know and can understand well. in our constant fight for diversity and open access to information, language should not be a barrier we are unwilling to overcome. restricting the language of information to one means that the amount of information out there is reduced and restricted. in today’s world, we have access to translators for almost every language. we certainly have access to translators for all the most common languages. if a paper is submitted in a different language, what’s the harm in hiring translators for that paper? if journals are worried about mistranslation or misleading information being inserted, then a process similar to peer reviewing could be used, where multiple translators work on the translation and check each other’s work. in our beautifully diverse world, there is so much learning that can happen. as scientists, our mission and our purpose is to find as much information as possible about the world around us, and language should not be a barrier to sharing this information with each other. while a common tongue can be helpful and convenient in some situations, sticking exclusively to that convenience is not always the right way to do things, and is not at all conducive to our mission. a person does not need to speak english to have accurate information or to share their research. if research is accurate, tested, original, and includes all the aspects we look for in articles that are read or published, it shouldn’t matter where the person is from or what language they speak. fine focus | volume 1150 sources 1. eberhard, david m., gary f. simons, and charles d. fennig (eds.). 2024. ethnologue: languages of the world. twenty-seventh edition. dallas, texas: sil international. 2. the most spoken languages 2024. home page english statistics and data. (2023, november 24). https://statisticsanddata.org/data/the-most-spoken-languages-1900-2024/ 3. malayil, j. (2024, march 24). prestigious journals hinder non-english speaking scientists, study finds. interesting engineering. https://interestingengineering.com/science/prestigious-journals-hinder-non-english-speaking-scientists-study-finds 4. crystal, d., & potter, s. (2025, january 8). english language-old english, middle english, modern english. encyclopædia britannica. https://www.britannica.com/topic/english-language/restoration-period 5. britannica, t. editors of encyclopaedia (2020, october 12). decline of the british empire. encyclopedia britannica. https://www.britannica.com/summary/decline-of-the-british-empire 6. britannica, t. editors of encyclopaedia (2021, october 22). when did the british empire begin?. encyclopedia britannica. https://www.britannica.com/question/when-did-the-british-empire-begin https://statisticsanddata.org/data/the-most-spoken-languages-1900-2024/ https://interestingengineering.com/science/prestigious-journals-hinder-non-english-speaking-scientists-study-finds https://interestingengineering.com/science/prestigious-journals-hinder-non-english-speaking-scientists-study-finds https://www.britannica.com/topic/english-language/restoration-period https://www.britannica.com/topic/english-language/restoration-period https://www.britannica.com/summary/decline-of-the-british-empire https://www.britannica.com/summary/decline-of-the-british-empire https://www.britannica.com/question/when-did-the-british-empire-begin https://www.britannica.com/question/when-did-the-british-empire-begin 76 | fine focus undergraduate student perspective irene yi linguistics undergrad student uc berkley i recently saw a humorous post online, poking fun at society’s expectations of younger generations to have life all figured out. it said, “it just hit me that five years ago, i was in high school and they were asking ‘where do you see yourself in five years?’ and i want you all to know [that] i was very wrong.” i relate to this deeply, but in the best way possible and completely unironically. five years ago, i could have never imagined all the wonderful paths this life would take me down. thanks to my incredible family, friends, and department, i have been presented with and supported in endless opportunities that have helped me grow as a person, student, and linguist. before i write more about what i’ve learned from undergraduate research, i’ll begin with a very brief introduction of myself. i am, in fact, not a microbiology student--i’m a linguistics undergraduate student at uc berkeley, finishing up my last semester of college. my passions lie in the linguistics subfields of sociolinguistics, historical linguistics, and indigenous language revitalization; specifically, i am fascinated by the way that language is used to construct and express identity, on both individual and community levels. i believe that linguistics, as a field, has so much potential for research that directly helps and empowers individuals and communities in their linguistic (and general) identities. in my time at berkeley, i’ve had the privilege of working with incredible people on a number of fascinating and important research projects, and i hope to continue working in linguistics academia for the rest of my life. after graduation, i’ll be moving across the country to work as a post-baccalaureate researcher in the linguistics department at yale, with a focus on historical linguistics and language revitalization. i am wholly and absolutely in love with the research i do, and the very existence of linguistics is what makes me get out of bed every morning with a smile on my face. coming to college, i knew i wanted to study linguistics in some capacity, though i had limited understanding of the wide variety of linguistics research, including methodologies, subfields, and interdisciplinary possibilities. fortunately, the linguistics department at berkeley not only has a strong representation of all the subfields of linguistics, they also greatly support undergraduate students’ opportunities to do all different types of research. the constant and consistent encouragement to make original contributions to the general linguistics research literature helped fuel my classmates and me to explore the vast expanse of research methods and ultimately find our favorite ones. while my work isn’t in microbiology, i think research contains many universal experiences and teaches us many of the same lessons. for any undergraduate students who are thinking about doing research, my biggest piece of advice is simply this: do it!! research is a beautiful, rewarding, and sometimes grueling process, and it is so, so worth it. more specifically, volume seven | 77 though, here are some things that i have learned throughout the years: 1) be patient with yourself and your research process. research is a long, consistent process; it’s not something you can cram at the last minute. not only would it be extremely stressful to try and get everything done in one day (or even one week… or one month), it is realistically impossible. because there are so many factors outside of one’s control during a research project, it’s also futile (and unsustainable) to try and speed the whole process up by working yourself to exhaustion every day. there’s no “if i just work double the amount and time that i do every day, i can halve the timeline of the process.” often, you actually need months to get approval, collect data, analyze data, and put it all together. plus, when spread out over months, you give yourself time to make mistakes, space to try new solutions and approaches, and--most importantly--freedom to take a day off and rest. i’m currently in the process of writing my senior thesis, and i always have to remind myself to be patient with the process. it took a few months’ time just to write a proposal to the institutional review board (irb), edit and improve the proposal based on feedback, and ultimately get approval from the irb to start collecting data. then, it took months of daily work to collect, transcribe, clean, and analyze the data. even though it took a long time, that doesn’t mean that i wasn’t working diligently at it during the entire process. all this is to say that a research project is inherently going to take time, and rather than trying to shorten the timeline by trying to “work harder” (because chances are, you are already working incredibly hard and maybe even at the limits of your capacity), you might as enjoy the process of watching your research develop and grow. 2) it’s okay if you realize that the research you’re doing-whether it be the methodology, data, or general topic--isn’t right for you long term. it’s great to explore different types of research, but you don’t have to feel guilty about finding out that a certain methodology isn’t what you want to do. in linguistics, there are endless ways of conducting research, gathering data, and analyzing it (depending on your subfield, research question, etc.)--and these methods vary quite a bit. exploring different research methods is for the purpose of finding what works for you. for example, i’ve done a handful of research projects where i realized quickly that certain ways of gathering data and analyzing it wasn’t anything i would want to do in the future. after the conclusion of said projects, i stopped doing those specific types of research. rather than looking at these experiences negatively as “wasted time,” i saw them as experiences that helped me learn about myself as a researcher, and it helped guide me to develop my ideas for future projects. knowing what research methods i liked and didn’t like allowed me to choose projects whose processes i knew would be interesting and enjoyable the whole way through. namely, when i was choosing my topic for my senior thesis, i knew that this project would be all-consuming, and i would be in this process for a whole year. i decided to pick a topic and a few methodologies that i would never get tired of (after having experiences that i learned from on research projects where i would get tired of a certain method or not enjoy the methods to begin with), and it made all the difference. 3) there is no shame in creating your own opportunities for research by yourself! if i could star, bold, and underline one important piece of advice, it would be this one. even without institutional funding or structure, there is still so much independent research that you have the capacity to do! in my freshman year of college, before i discovered the official program(s) for undergraduate research opportunities within the linguistics department, i became interested in the topic of gender in language and did my own digging into it. eventually, my own little pet project turned into a threeyear long personal research passion, opportunities to present my work at research conferences and be involved in related (and more official) research, and the foundation of the future research i want to do. the premise is this: in grammatically gendered languages (e.g. spanish, french, etc.), there are words that fall into the feminine gender or masculine gender (and in some languages, there is a neuter gender too). these genders are marked on the words, often in the form of a suffix. my interest in this topic began with the feminization of french profession nouns--specifically how a grammatically gendered language like french can reflect a history of misogynistic norms in the way the language is structured. it started with reading a simple footnote in my french textbook about how there were no officially recognized grammatically feminine forms for certain profession nouns like “author,” 78 | fine focus “professor,” or “doctor” because those were not jobs traditionally held by women in the history of french society. i then started looking into the history of the french language and other sociolinguistic literature on grammatically gendered languages. additionally, i reached out to some linguists in the french department at berkeley and asked about some of the issues surrounding this topic. finally, i wrote my findings into a “blog” of sorts, which later turned into a more academicallywritten paper, which then took me to presenting this work at conferences all over the world. as the feminization of french profession nouns is an ongoing debate in france, i updated my work with what was happening in france with feminist linguists and oppressive language institutions. i also realized during this personal research project that gender in language went beyond the gender binary, and that many language activists across the world (and across many languages) are now working on ways of making grammatically gendered languages more gender inclusive (e.g. the term “latinx” in spanish is used as a gender inclusive term, often used by nonbinary speakers to transcend the binary gender reflected in the grammar of the language). this semester, i was able to expand my interest in language and gender through a research project in berkeley’s linguistics department. in the department, there is a research program called the linguistics research apprentice practicum (lrap) that graduate students, undergraduate students, and sometimes even faculty are involved in. recently, a gender in language project was created through lrap, and i now have the chance to use what i learned through my french feminization pet project in research of how gender is marked in mandarin--and the social and historical implications of linguistic gender. specifically, we put together a nonbinary grammar and lexicon reference of mandarin, based on many gender-inclusive movements that are already going on in china, hong kong, and taiwan. had i never embarked on this personal research journey that began with just a simple interest, i would have never known just how passionate i am about the topic of gender in language--so much so that in the future, i want research the intersection of gender and sociophonetics (which is the subfield of linguistics that deals with how individuals construct, index, and express their identity with the qualities of the sounds in the way they speak). 4) if a personal pet research project isn’t for you, you can also cold-email supervisors and ask for their help in creating opportunities! sometimes, the research that you want to explore might require technical skills or knowledge that you don’t quite have yet--and the process to learn what you need might be out of your own control or grasp. in these cases, don’t be afraid to reach out to researchers in your department, or even at other schools, to ask for help! often, researchers are more than happy to chat or send you resources that can help you develop the skills and knowledge base that you are seeking. if you’re lucky, they might even have an official or unofficial research position for you (or a project that you can collaborate on). in my own research processes and research exploration, i cold-emailed a good handful of professors and researchers at other universities to ask for insight on various issues, and i received such great resources and help. specifically, while writing code for my thesis, i had to consider and try out many different computational models on my linguistic data, and talking with researchers whose previous computational work i had read helped me significantly in formatting and analyzing my own data (and of course, in writing code). in one case, one of my cold-emails from a while ago now actually led to me working remotely with a lab at a different university for many months on a very interesting and engaging project. through that project, i gained computational skills that i’m using not only for my thesis, but for work i want to do in the future. 5) criticism of your research isn’t criticism of you as a person (or as a researcher). in fact, critical responses can often be really helpful in making your research better. in academia, there is an immense (and sometimes unhealthy) amount of pressure on every researcher to continually produce work. while this is actually just a reflection of the toxic internalized capitalism present in academia, the best an individual can do is just to compartmentalize your work and you as a person into separate boxes of your identity. as much as you can be passionate about your work and have your time consumed by it, it’s very dangerous to start equating your worth as a person to how well your work does or how much work you produce (again, that’s what internalized capitalism will do to you). instead, you can use criticism of your work as something to improve your research with, because the process of research volume seven | 79 in academia will inevitably include critical feedback (on your writing, on the actual content of your work, on your very research question itself, etc.), and your mental health will greatly thank you if you learn to remove your self worth from your role in the institution of academia. 6) take your time in figuring out who you are and what research you want to do--and when do you figure it out, embrace it! ultimately, research should be something that brings you joy. at the very least, it should be something that you feel like is worth it, whether that be because you enjoy the specific topic, because you believe in the bigger purpose and impact of your work, or both. as obvious as it sounds, it took me my entire life to know myself as well as i do now, and i know that i’ll continue to grow as a person and researcher--and continue to get to know myself better-for the rest of my life. in the process of exploring different types of research in college, i had the misconception that there were certain methodologies that were more “expected,” or certain skills that were more “desirable” to have. i thought that i would have to check off a certain list of boxes to be academically marketable as a researcher, when in reality, the most important and “marketable” quality is that one is genuinely passionate about their research, and that they get up after every time they fall down to consistently pursue these passions. any skill you need along the way of your research, you will no doubt learn and develop as you go. finally, in the process of learning about yourself, you’ll also realize that your growth as a researcher doesn’t take away from anyone else’s-and similarly, someone else’s growth and success will never take away from yours. in a cutthroat, capitalistic institution like academia, it’s so, so important to support one another and value compassion and collaboration over competition. additionally, academia is often inaccessible as an institution, and many communities are excluded in research, discourse, and representation. this needs to change, and personally, i try to use the tools within and related to linguistics to dismantle the inaccessibility in academic spaces. i have come to realize that who i am as an academic is inextricably linked to centering and uplifting communities, such that whatever academic work and research i do will always be for the good of the community i am serving. this, then, becomes reflected in the subfields of linguistics that my research is focused on: sociolinguistics, historical linguistics, and indigenous language revitalization. while some of these subfields of linguistics is sometimes disregarded by traditional, elitist parts of academia as “not real research” because it serves communities rather than research literature production, i wholeheartedly believe that not only is it real research, it is even more important research because of the greater community implications it has beyond writing an academic paper. looking back, i have learned so much through all of the good and bad that comes with research. academia is not a perfect institution by any means, but i hope that through the researchers who go into this field to make the world a better place, the institution can become a little less flawed as well. i have gained so much from my experiences in undergraduate research, and i would highly encourage everyone to dip their toes in it as well. who knows? you might just fall in love with it. i will forever be grateful for the linguistics department at berkeley--and for everyone i have been lucky enough to cross paths with-for making me who i am today and bringing me the best years of my life so far. i want to give a million thank-yous to my family, who have always supported me and believed in me, even when i didn’t believe in myself. finally, i want to thank lauren andrews for reaching out to me and giving me this opportunity and platform to share my experiences; it honestly means so much to me, and i’m so humbled to have this space to reflect on the things i’ve learned. while that sarcastic post i saw online was meant to be a joke, i think about how lucky i am all the time to be in love with my major, research, and work. truly, i never could have seen it coming; my reality today is beyond high school me’s wildest dreams, and five years ago me wouldn’t believe you if you told her all the unseen ways she would learn through undergraduate research and grow into a version of herself who has never been happier. i can’t even begin to imagine what the next five years will bring. polymicrobial conditions affect antibiotic susceptibility in clinically relevant bacterial species william little1 andrea j. lopez1 eleanna carris1 allie clinton smith1 1 honors college, texas tech university, lubbock tx keywords: minimum inhibotry concentration (mic), antimicrobial susceptibility testing (ast), wound, diagnostics, synergism © 2024 little, lopez, carris, smith. fine focus, 10(1), 74-89. doi: 10.33043/ff.10.1.74-89. shared with cc-by-nc-nd 4.0 license. manuscript received 22 april 2023; accepted 26 september 2023 abstract chronic wounds, defined as those which remain open and inflamed for greater than six weeks, are a major area of clinical concern. resulting in thousands of amputations per year and billions of dollars spent globally in treatment, chronic wounds are notoriously difficult to successfully treat. two hallmarks of chronic wounds are that they are thought to harbor biofilm-associated bacteria and tend to be polymicrobial. while the research literature has repeatedly demonstrated the effects of biofilms on wound persistence and the changes to the efficacy of antibiotics, few studies have demonstrated what effect the polymicrobial condition has on the antibiotic tolerance of bacteria. to further explore this, four species of clinically relevant wound pathogens (pseudomonas aeruginosa, acinetobacter baumanii, staphylococcus aureus, and enterococcus faecalis) were tested in monoand polymicrobial conditions using the current gold-standard methods for determining antibiotic susceptibility. noticeable differences in antibiotic tolerance were observed in the polymicrobial condition, including both increased and decreased susceptibility, depending on the antibiotic used. our data demonstrate that the current clinical methods used for testing antibiotic susceptibility can generate results that are not representative of the infection environment, which may contribute to treatment failure and persistence of polymicrobial infections. https://creativecommons.org/licenses/by-nc-nd/4.0/ little et al. | polymicrobial conditions affect antibiotic susceptibility in clinically relevant bacterial species 75 introduction while a variety of chronic conditions affect americans each year, less prominent, yet still clinically-impactful conditions manage to slip under the radar of the popular consciousness; one of these is chronic wounds. often occurring in diabetic patients, the bed-ridden, and those with vascular diseases, chronic wounds are currently an area of major clinical concerncosting billions of dollars every year to treat and resulting in thousands of amputations in the united states alone.1, 2 the most common definition of these wounds is those which remain open and in a prolonged state of inflammation for greater than six weeks.3 standard treatment protocols do exist for the management of chronic wounds, including the use of oral antibiotics, cleaning of the wound via physical means (commonly called debridement), and even the use of strong, topical antibiotics, yet as seen in the treatment costs listed above, those protocols remain of mixed efficacy.4 to illustrate how these wounds occur, the case of a proto-typical diabetic patient will be used. for patients with diabetes, a gradual loss of sensation in the limbs, termed diabetic peripheral neuropathy, can often occur.5 when combined with decreased circulation to the extremities, such a patient may receive a wound on their foot, and due to the lack of sensation, the patient will remain largely unaware of the existence of the damage, continuing with their normal activities of daily life. 5,6 as the wound progresses in its infection and tissue necrosis, it may become apparent to the patient or to caregivers who are assisting them, and the patient will be brought in for treatment. the open wound will be treated with antibiotics, but the wound will often persist, and refuse to heal or reduce its inflammatory condition.5 at that point, debridement, specialized wound dressings, and further antibiotic treatment will be attempted, but the wound will often continue to remain infected, and amputation of the affected digit, appendage, or limb may be necessary to ensure the patient’s survival.5 as mentioned above, many thousands of patients receive amputations on the basis of chronic wound treatment failure every year, and so this area of study is a critical one to improving health outcomes in the united states.1, 2 a question remains, though. why do the current clinical best-practices in the treatment of these wounds fail so often? while it may be obvious that there exists some gap in the understanding of these wounds, it is by no means clear where that gap exists. in order to elucidate these gaps, it is imperative to first understand what is currently known by both the scientific and clinical communities around the structure and composition of these wounds. it is widely understood that two major features characterize chronic wounds.4 the first is that the bacteria within the wounds form biofilms. biofilms are defined by the secreted extracellular polysaccharide (eps) matrices that bacteria form to protect against dislodgement, to better control their microenvironment, and to serve as a mechanical barrier to external conditions.7 more simply put, biofilms are a structure by which bacteria protect themselves and adhere to their infection site.7, 8 for example, the necessity of brushing one’s teeth with an abrasive compound such as a toothbrush arises from the biofilms that oral bacteria form as they grow; this can be observed from the off-white fine focus | volume 1076 plaque that is physically removed from teeth during this cleaning. without the abrasive removal of biofilm, the bacteria would continue to adhere to the surface of the teeth, with poor outcomes for one’s oral health.9 the second well known characteristic of chronic wounds is that they tend to be polymicrobialmeaning that an infected wound is colonized by multiple species of bacteria, and almost without exception contain multiple species of pathogenic bacteria.10, 11 it is more commonly thought that infections consist of a single species of bacteria or infectious agent (fulfilling koch’s postulates), but the research literature has shown extensively that, once within a biofilm, numerous species of bacteria can happily coexist. next-generation sequencing (an advanced method of genetic analysis) of wound samples has revealed that up to several hundred different species of bacteria may infect chronic wounds, though not necessarily all simultaneously (demonstrating a dynamic and ever-changing nature of a chronic wound).12 this polymicrobial environment can also promote synergistic interactions among bacteria within the wound. synergistic interactions in bacteria are those in which multiple bacterial species inhabiting the same environment creates differences in behavior from a single species condition.13, 14 this often occurs by the upregulation of virulence factors among bacteria in those communities, and is associated with poorer patient outcomes.15 the synergistic interactions within the polymicrobial biofilm environment are also known to increase both the antimicrobial tolerance and resistance in constituent microbes through increased rates of horizontal gene transfer and differential gene expression.8, 16-19 tolerance to antibiotics is characterized by transient changes in a bacterial population in response to their environment, usually via differential gene expression (dge), which allows them to survive when exposed to antibiotics (fig. 1).20 this change in metabolism as a result of the synergistic interaction-caused dge often decreases the efficacy of antibiotics by changing the availability of target sites or processes, which leads to bacteria surviving a normally lethal treatment.20 resistance, on the other hand, involves the acquisition of specific genes that allow the bacteria to counter the effect of the antibiotic (fig. 1), and is a ‘permanent’ change in their genetic makeup, often associated with horizontal gene transfer (hgt).21 hgt is the method by which antimicrobial resistance genes are shared within a mature bacterial population, and the polymicrobial environment favors these interactions.12 using in vitro research models, biofilms have also been shown to play a major role in the antibiotic tolerance of pathogens via the mechanical barrier that the biofilm forms, as well as the increased dge that affects the efficacy of therapeutic compounds of the bacteria within the biofilm. the effect of biofilm formation alone can result in a one thousand fold decrease in antibiotic susceptibility due to the conditions within that microenvironment.20, 22, 23 while this has been extensively demonstrated in the literature, most studies investigating the contribution of a biofilm to changes in susceptibility have focused on monomicrobial, or single-species, bacterial suspensions.24-32 because of this, our aim was to determine what effects a polymicrobial condition has on the antimicrobial susceptibility of a group of bacteriaan effect independent of biofilm formationand one that is currently a gap in the research knowledge, especially little et al. | polymicrobial conditions affect antibiotic susceptibility in clinically relevant bacterial species 77 when compared to the large body of research on the effects of biofilm formation on antibiotic susceptibility. methods and materials species and strains  four species were chosen for use in the polymicrobial culture: acinetobacter baumanii (ab), pseudomonas aeruginosa (pa), staphylococcus aureus (sa), and enterococcus faecalis (ef). each of these species is both a common infector of chronic wounds and a member of the eskape pathogen family, which are a leading cause of nosocomial infections.17, 37 clinical and laboratory standards institute (clsi) -recommended quality control strains of a. baumanii (atcc® 19606), p. aeruginosa (atcc® 27853), e. faecalis (atcc® 29212), and s. aureus (atcc® 29213) were used.38 these strains are pan-susceptible and were used to ensure the reliability of the established mic breakpoints provided by clsi while eliminating any possibility of antibiotic resistance genes affecting the results of the experiments. figure 1 antibiotic resistance versus tolerance. (taken from 21) note. a microbial population (confined by a light-grey ellipse) initially consists of mainly antibiotic-sensitive cells (dark-grey). (a) in addition, the population may also contain resistant cells (black), resulting from a permanent change at the genetic level. after antibiotic treatment (+ab), only resistant cells remain. upon regrowth (-ab), the entire population is composed of resistant individuals. (b) alternatively, the population may contain persister cells (black), resulting from a reversible phenotypic switch to a tolerant state. after antibiotic treatment, only persister cells remain. upon regrowth, the population will exhibit the same sensitivity as the original population. fine focus | volume 1078 minimum inhibitory concentration (mic) our experiments were conducted in accordance with the clsi m100 and m7 guidelines for determining antibiotic susceptibility.38, 39 mics were determined by first conducting mono-species mics in 96-well plates and the results were cross-checked with the clsi’s established mic breakpoints for each species used stratified by drug. bacterial cryo-stocks were grown overnight in lysogeny (lb) broth (thermo fisher scientific, hampton, nh) , then the above clsi protocol was followed. mono-species mics were conducted first to ensure that the results were in accordance with published guidelines as an internal control. then, each of the four species was grown in lysogeny (lb) broth (thermo fisher scientific, hampton, nh) overnight separately, then combined in a 1:1:1:1 ratio inoculating dose, which ensured that total colony forming units (cfus) of bacteria and volume added was equivalent between the mono and poly-microbial conditions. antibiotic concentration was diluted across the 96-well figure 2 an example of broth microdilution technique as directed by clsi. note. a brief visual outline of the current diagnostic mic protocol, as used via clsi guidelines. taken from 42 little et al. | polymicrobial conditions affect antibiotic susceptibility in clinically relevant bacterial species 79 plate in accordance with clsi m7 and m100 guidelines, with the highest concentration at 128ug/ml; concentrations were serially halved across the 12 wells in a row down to 0.06ug/ ml. cation-adjusted mueller hinton broth (camhb) (thermo fisher scientific, hampton, nh) was used as the media, and after inoculation with the bacterial dose, the plate was sealed and incubated at 37⁰c. after 18-24 hours of incubation, the plates were removed and mics were determined visually via turbidity, as described as best-practice by the clsi (fig. 2).39 all experiments were conducted in triplicate with biological replicates. the results were recorded and compared with the mono-species results for that antibiotic to determine if any notable differences were observed. it has previously been observed that polymicrobial cultures gave results not in accordance with clsi breakpoint guidelines, but since those assays were conducted with the disk diffusion method, their results cannot directly compared to the broth microdilution methods commonly used in us clinical laboratories.40, 41 given the lack of consistency in experimental conditions from previous in vitro research, we aimed to use the current clinical method of broth microdilution to determine what effect a polymicrobial community had on individual mics among the species in a polymicrobial suspension. using the current clinical model allowed for effective comparison both across antibiotics and species in the polymicrobial environment and ensured the validity of results in the current clinical standards. limitations to this method do exist, however. given that the bacteria in a polymicrobial condition cannot be differentiated visually, it is not possible to establish changes for individual species’ mic in this condition on the basis of turbidity alone. because of this, an additional viability method was used to assess changes in antibiotic tolerance for each individual species (described below). viability two antibiotics penicillin and ceftazidime were tested with a modification of the mic protocol. after following the previously described mic protocol, the bacterial suspensions were extracted from the wells, diluted to the first order in phosphate buffered saline (1xpbs), then 10µl volume was spot-plated on selective and differential media for each bacteria (pseudomonas isolation agar for the recovery of pa (thermo fisher scientific, hampton, nh), mannitol salt agar (thermo fisher scientific, hampton, nh) for the recovery of sa, bile esculin agar for the recovery of ef, and leeds agar (thermo fisher scientific, hampton, nh) for the recovery of ab, to observe any possible differences in viability of those cells in monoand polymicrobial conditions. selective and differential agars were used to allow for the differentiation of individual species within the polymicrobial condition, though the results of these assays are not directly comparable to the mics for either of those antibiotics as they do not rely on the turbidity assessment used by the clsi. in addition, the scientific literature has demonstrated that the visual assessment of turbidity corresponds only to an approximately 50 percent decrease in od reading, so viable bacteria will still be present in wells above the visually assessed mic.43 fine focus | volume 1080 results our results are demonstrated in the following figures, which are divided into two parts. the first covers our mic data, and the second shows our viability data. for the mic charts, the values listed show the individual mics for each bacterium per antibiotic treatment, then the mic polymicrobial value from our data, when read in accordance with clsi guidelines. the viability data shows the comparison of spot plated viability values between the individual and polymicrobial conditions for a particular antibiotic treatment. mic values these data reflect comparisons of results obtained following the clsi gold standard broth microdilution method for individual suspensions versus a polymicrobial planktonic suspension. mic results were obtained for gentamicin, tobramycin, penicillin, tetracycline, doxycycline, and ceftazidime (fig. 3). for gentamicin (fig. 3a) and penicillin (fig. 3c) the polymicrobial mic was the same as the highest individual mic result. this demonstrates that there was no observable change in the polymicrobial community mic determinable by turbidity alone. for tetracycline (fig. 3b), the highest observable individual mic was e. faecalis at 32 µg/ml; however, the polymicrobial mic result was 128 µg/ml. this demonstrates that mixing the four species together changes antimicrobial susceptibility of the population by 4-fold for at least one species in the suspension. for ceftazidime (fig. 3d), the highest individual mic value was e. faecalis at >128 µg/ml; however, the polymicrobial mic result was 16 µg/ml. this demonstrates that the polymicrobial condition increases the susceptibility of e. faecalis by 4-fold. it is worth noting that any changes to individual mics within the predetermined minimum and maximum ranges cannot be evaluated via turbidity and mic alone in a polymicrobial condition. viability values  the viability assays were run in order to determine changes in viability of the individual and polymicrobial conditions following antimicrobial challenge. all viability assays were conducted in triplicate with biological replicates. these results are not comparable to conventional mics because the turbidity reduction observed in mics correlates to an estimated 50% reduction in viable cells, whereas these data relate to the total elimination of viable bacteria.43 for penicillin (fig. 4a), both sa and ab observed decreases to antimicrobial susceptibility, at 64 and 8-fold respectively, in the polymicrobial versus individual conditions. for ceftazidime (fig. 4b), ab observed a reduction in susceptibility by 2.86-fold, while sa observed an increase in susceptibility by 3-fold in the polymicrobial versus individual conditions. pa and ef do not exhibit any observable changes in susceptibility. discussion our data across the two methods mic and viability demonstrate several notable results. in the first case, gentamicin, tobramycin, penicillin, and doxycycline did not show a difference in the observable mic in the polymicrobial condition over the given values for the individual. in other words, the range of possible mic values of the individual condition overlapped with that of the polymicrobial. little et al. | polymicrobial conditions affect antibiotic susceptibility in clinically relevant bacterial species 81 because of this, and because the samples were assessed visually, it is not possible to determine the contribution of each individual species to the turbidity observed in the polymicrobial condition. for instance, in the case of gentamicin, the polymicrobial mic is 8µg/ml. given that the mic value in the individual condition for a. baumannii is 4µg/ml, it is not possible note. monomicrobial versus polymicrobial mic results for gentamicin (a), tetracycline (b), penicillin (c), and ceftazidime (d). † denotes that the value is within the published guidelines from the clinical laboratory science institute’s m7 and m100 manuals for mic breakpoints (clsi, 2018a, 2018b). mic values greater than 128 µl have been calculated as that number. clsi does not publish established breakpoint values for p. aeruginosa and a. baumanii when treated with penicillin and for e. faecalis when treated with ceftazidime. n=3. figure 3 comparison of minimum inhibitory concentrations (mic) in monoand polymicrobial conditions. for us to determine if any changes in mic for that species occurred, because the individual mic result for e. faecalis at 8µg/ml is the same as the polymicrobial result. put another way, in the case of gentamicin, the polymicrobial condition having the same mic as one of the individual mics (in this case e. faecalis) might represent that the individual species had no fine focus | volume 1082 change in mic, or it could represent that one or more of the species increased in mic up to the polymicrobial mic, but that change cannot be determined because the contribution of each individual species in the polymicrobial condition is not observable via an assessment of turbidity. two notable differences in mic were observed in the polymicrobial condition. with tetracycline, a decrease in susceptibility was observed in the polymicrobial condition, which correlates to a decreased antibiotic efficacy, presumably via tolerance as all strains of bacteria used are pan-susceptible. as the polymicrobial mic is substantially greater than any of the component individual mics, it is not possible to determine which bacterial species’ tolerance was increased, or which combination of those species was affected. with ceftazidime, however, figure 4 comparison of viability in monoand polymicrobial conditions. note. monomicrobial versus polymicrobial viability results for penicillin (a) and ceftazidime (b). † denotes that the value is within the published guidelines from the clinical laboratory science institute’s m7 and m100 manuals for mic breakpoints (clsi, 2018a, 2018b). viability values greater than 128 µl have been calculated as that number. clsi does not publish established breakpoint values for p. aeruginosa and a. baumanii when treated with penicillin and for e. faecalis when treated with ceftazidime. n=3. an opposite effect was observed, where a sensitization interaction occurred. in this case, e. faecalis can be identified as the bacteria whose tolerance decreased, as its individual mic is substantially higher than the polymicrobial value, whereas p. aeruginosa, s. aureus, and a. baumanii’s individual mic values are equal to or lower than the polymicrobial mic. as tetracycline and ceftazidime are both clinically important antibiotics, these results demonstrate a notable and potentially clinically significant change.44, 45 for the viability experiments, bacteria treated with penicillin and ceftazidime both showed notable differences in antibiotic susceptibility in the polymicrobial condition. when treated with penicillin in the polymicrobial environment, both s. aureus and a. baumanii showed notable increases in tolerance to the antibilittle et al. | polymicrobial conditions affect antibiotic susceptibility in clinically relevant bacterial species 83 otic challenge. for s. aureus, this difference amounts to a 64-fold increase in concentration necessary to successfully kill the bacteria, and for a. baumanii, an 8-fold increase. however, when treated with ceftazidime, s. aureus demonstrated a decreased tolerance of 3-fold, but a. baumanii showed an almost three-fold increase in tolerance to the compound. as penicillin and ceftazidime are both clinically important, oft-prescribed antibiotics, this shift in tolerances is highly relevant for care practitioners in the wound and lab environments. given that our results are consistent with both the existing literature around the polymicrobial effect on antibiotic susceptibility and uses the current clinical model, these data demonstrate that there exists a gap in the current clinical diagnostic schema for determining the antimicrobial susceptibility of polymicrobial infections. as it has also been repeatedly demonstrated in the literature that synergistic interactions among bacteria within a wound produce more negative outcomes clinically, and that chronic wounds harbor polymicrobial infections, our data is consistent both internally and externally with the observations found in clinical practice.10, 11, 15 since both tolerance and resistance can play important roles in the success of infection treatment, and as our data demonstrates, tolerance alone can notably change the susceptibility of bacteria to antibiotic treatments, it is critical that the clinical models be adapted to allow for the presence of polymicrobial cultures during the assessment process. this change could potentially result in more accurate assessments of antibiotic susceptibility across the clinical spectrum, and may be of particular benefit to the treatment of the notoriously intractable chronic wounds. though the rise of sequencing technologies in the clinical laboratory is no doubt of great value for clinical microbiologists and provides valuable data in the diagnostic process (particularly for microbial identification), it is important to note that because the above data assesses transient changes to antibiotic tolerance rather than antibiotic resistance, the former of which is the result of phenotypic rather than genotypic differences, methods such as 16s next-generation sequencing (ngs) or rapid qpcr will not be able to determine these changes in tolerance, since those technologies rely on and assess for the presence of antibiotic resistance genes.46 it is noteworthy that the differing combinations of bacterial species and antimicrobial drug yield different results. in some instances, the susceptibility is decreased, and in other instances the susceptibility of one or more species is increased. this implies that there is no ‘one size fits all’ approach to changes in susceptibility due to the polymicrobial condition, and that more research is necessary to understand why different bacterial consortia respond differently to antimicrobial challenges in clinical settings. it is also noteworthy that most of the existing literature, including this study, focus on either the contribution of either the polymicrobial or biofilm condition to changes in susceptibility, when in the clinical setting both of these conditions commonly exist simultaneously. this implies that the effects seen in these studies might be compounded when combined in the clinical setting, and more research needs to be done to understand both the cumulative effect of those conditions in the clinical wound setting and how the entirety of the microbial fine focus | volume 1084 environment can be taken into account when considering antimicrobial susceptibility in clinical diagnostic procedures. in conclusion, antibiotic susceptibility testing is a crucial part of the chronic wound treatment process. healthcare practitioners rely on the information that clinical microbiologists provide to determine the most appropriate antibiotic treatment, and it is critical that the information the clinical laboratory generates is representative of the infection, accurate, and applicable to the patient. using clinically-relevant models based upon the current gold-standard guidelines, our results show that the polymicrobial condition of wounds may be modifying their response to antimicrobial chemotherapy, and therefore the results from the current method may not be an accurate reflection of susceptibility in the chronic wound infection environment. in adapting the method of assessment to better reflect the wound environment by including polymicrobial cultures, it is entirely possible that the costs, both physical and economic, of chronic wound care might be minimized, leading to improved patient care and outcomes. little et al. | polymicrobial conditions affect antibiotic susceptibility in clinically relevant bacterial species 85 references 1. järbrink, k., ni, g., sönnergren, h., schmidtchen, a., 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(2017). next-generation sequencing applications in clinical bacteriology. biomolecular detection and quantification, 14, 1–6. https://doi.org/10.1016/j. bdq.2017.10.002 https://doi.org/10.1128/aac.46.8.2477-2481.2002 https://doi.org/10.1128/mmbr.65.2.232-260.2001 https://doi.org/10.2165/00003495-198529020-00002 https://doi.org/10.2165/00003495-198529020-00002 https://doi.org/10.1016/j.bdq.2017.10.002 https://doi.org/10.1016/j.bdq.2017.10.002 54 | fine focus anti-proliferation of melanoma cells and immune stimulation by the cyanobacterial indole-alkaloid scytonemin jadon evans (evanjm05@pfw.edu) aaron jones (joneaa@iu.edu) elliott blumenthal (blumenth@pfw.edu) *tanya soule (soulet@pfw.edu) department of biology, purdue university fort wayne *corresponding author department of biology, purdue university fort wayne, 2101 e coliseum blvd., fort wayne, indiana 46845 manuscript received 29 january, 2021; accepted 15 june, 2021 keywords: scytonemin, melanoma, spleen cells, anti-proliferative, natural products volume seven | 55 abstract under the stress of ultraviolet radiation some cyanobacteria synthesize scytonemin, a protective pigment against dna photodamage. in addition to photoprotection, scytonemin has been shown to have an anti-proliferative effect on various types of malignant cells. in this study the effect of scytonemin on melanoma and spleen cells was assessed both in vitro using tissue cultures and in vivo in mice models. melanoma and spleen cells were exposed to 0.08 to 10 µm of scytonemin, and cell proliferation was measured using tritiated thymidine uptake. the data suggest that scytonemin acts as an inhibitor for melanoma cells in a concentration-dependent manner while enhancing the proliferation of spleen cells, suggesting that it can potentially augment the immune response. furthermore, mice injected with melanoma cells and scytonemin produced fewer tumors than mice that did not receive scytonemin, although the data were not significant. this study adds to the growing body of research that scytonemin may be beneficial as a future anticancer agent to prevent tumor cell growth. 56 | fine focus introduction according to the centers for disease control, skin cancer is the most common type of cancer in the world, with 85,868 people in the united states diagnosed with melanomas of the skin in 2017 (3). consequently, the demand for a product that is both effective in killing tumor cells and safe for an individual to take is as great as it has ever been. since ultraviolet radiation (uvr) plays a major role in skin cancer, potential treatments could explore photoprotective effects of various compounds against solar uvr. longwavelength (uva) in the range of 320-400 nm, plays a role in longterm skin damage contributing to aging skin due to its deep penetration of the epidermis and dermis. uva is known to damage keratinocytes, which are found in the basal layer of the epidermis where most skin cancers occur (8). shortwavelength uvb in the range of 280320 nm is the major contributor in sunburns and contributes to skin cancer alongside uva by directly damaging dna and proteins (17). all living cells, including bacteria, can be harmed by uvr (8). as photosynthetic bacteria regularly exposed to uvr, cyanobacteria have developed several mechanisms to defend themselves against its harmful effects. these include physical migration away from uvr (1), synthesis of uv shock proteins (4), up-regulation of antioxidant defenses (14), and down-regulation of uvrsensitive proteins (7). of particular interest is the ability of some cyanobacteria to synthesize scytonemin, a photoprotective sheath pigment that protects primarily against uva radiation (5). scytonemin is a lipophilic, yellow-brown, indole-alkaloid pigment (fig. 1) (10) that efficiently absorbs uva in vivo at 370 nm (6). it is produced by certain species of cyanobacteria where it is induced upon exposure to uva, and is then deposited into the sheath surrounding the cells (5). in addition to its photoprotective properties, scytonemin demonstrates antiinflammatory and anti-proliferative qualities. tissue hyperplasia is a hallmark feature of hyperproliferative and inflammatory pathologies, such as rheumatoid arthritis, psoriasis, asthma, and cancer (15). scytonemin has been shown to exhibit anti-inflammatory properties that could potentially treat these diseases (11, 15, 16). for example, the topical application of scytonemin on mouse ear edema reduced swelling compared to mice receiving no treatment (16). furthermore, several studies argue that scytonemin inhibits cell proliferation through mechanisms of cell cycle arrest (15, 16, 18, 19). scytonemin has also been shown to interrupt hyperproliferation of renal cancer cells (18) and slow the proliferation of multiple myeloma cells (19). possibly the most thorough study on the anti-proliferative properties of scytonemin was that it could hinder actively proliferating cells, including malignant jurkat t cells, rheumatoid synovial fibroblasts implicated in arthritis, human lung fibroblasts, and human umbilical vein endothelial cells. this study also demonstrated that scytonemin was not cytotoxic towards non-proliferative human monocytes (15). these results convey the possibility for scytonemin to halt malignant cell growth without harming other body cells, which is a quality particularly sought after in cancer treatment research. figure 1. figure 1. structure of scytonemin (proteau et. al. 1993). volume seven | 57 since the literature suggests that the uvprotective agent scytonemin is implicated in cell cycle arrest, this study seeks to determine whether scytonemin will have anti-proliferative effects against melanoma cell growth in animals. therefore, the objectives of this study were to examine both the in vitro melanoma and spleen cell proliferation in the presence of different concentrations of scytonemin, as well as the in vivo effect of scytonemin on tumor growth in mice. if scytonemin inhibits melanoma cell proliferation without equally inhibiting spleen cell proliferation, then it has potential as a future therapy for malignant cells and should be further explored as an anti-tumor therapy. however, if scytonemin affects melanoma and spleen cells alike, then it could potentially harm tissue and might not result in the production of an effective therapy. while the findings of other studies already indicate that scytonemin slows malignant proliferation (15, 16, 18, 19), this study takes a slightly different angle by comparing the effects of scytonemin on cancerous cell proliferation (i.e., melanomas) to healthy noncancerous cell growth (i.e., spleen cells) from other tissues in the same organism. spleen cells were chosen over skin cells, for example, as the healthy non-cancerous control cells because they are more likely to interact with a therapeutic agent that enters the bloodstream. furthermore, the spleen plays a critical role in activating immune cells in response to bloodborne antigens. therefore, the hypothesis of this study is that in the presence of scytonemin, melanoma growth will decrease in tissues and animals while spleen cell proliferation will increase. methods melanoma and spleen cell assays b16-f1 melanoma cells were purchased from the american type culture collection (atcc© crl-6323) (2). spleen cells were extracted from male c57bl/6 mice obtained from charles river labs that were 4-6 months old at the time of extraction. for each assay, a final concentration of 5 x 104 melanoma or 1 x 106 spleen cells were placed in each well of a 96-well plate and assayed in triplicate for each condition. samples treated with scytonemin received 10 μl hplcpurified scytonemin (a gift from benjamin philmus, oregon state university) diluted in dmso with final concentrations of 10 to 0.08 µm. to better determine the effect of scytonemin on spleen cell growth, the mitogen concanavalin a (con a), which stimulates t lymphocyte proliferation, was added at a concentration of 0.1 µg per well in a volume of 200 ul of rpmi-1640 media plus 10% fbs, to determine whether scytonemin inhibited rapidly proliferating spleen cells (12). spleen cell plates were incubated for 48 hours, then 3h-thymidine was added followed by a second incubation for 24 hours, all of which took place at 37 °c under 10% co2. a cell harvester was then used to transfer the cells onto filters, which were washed approximately ten times using phosphate buffered saline (pbs). filters were then placed into counting tubes with ecolume™ scintillation cocktail (mp biomedicals, solon, ohio) to count the incorporated h3-thymidine using a beckman scintillation counter. all assays were done in triplicate and controls received no scytonemin or dmso treatment. previous experiments in our lab have demonstrated that dmso does not have an inhibitory effect on melanoma cell proliferation (unpublished data). cell counts were averaged and compared to control groups to determine the percent proliferation relative to the control groups. all statistical tests were done using anova and tukeyhsd post-hoc comparisons in r studio v1.2.1335 (13). in vivo assays for in vivo scytonemin experiments against melanoma tumor cells, 18 male c57/bl6 mice were obtained from charles river labs that were approximately 14 weeks old at the time of experimentation. each mouse was weighed before the initial intraperitoneal (ip) injection of scytonemin diluted in dmso to a final concentration of 3.5 µm g-1. scytonemin was then administered at 3.5 µm g-1 daily for two weeks through ip injections into nine treated mice, and the other nine mice received the same volume of sterile saline that had an equal concentration of dmso as the scytonemin treated animals. after two weeks a single tail vein injection of melanoma cells was performed. melanoma cells were diluted to 5 x 106 melanoma cells ml-1 in sterile saline, and 0.1 ml of this preparation was injected into the tail vein of all 18 mice. ip injections of scytonemin were continued for an additional week and the mice were monitored for any health changes 58 | fine focus for two additional weeks while the tumors grew. after two weeks of no injections eight mice in each group survived and were weighed once more and sacrified. when injected into the tail vein of syngeneic mice, melanoma cells migrate to the lungs and produce dark colonies. upon sacrifice the lungs were teased apart and the melanoma tumor cell colonies were counted on and within the lung tissue and the counts were compared against the control mice that did not receive scytonemin using an anova with tukeyhsd post-hoc analysis. all animal experiments were performed according to iacuc protocol #1111000244. results melanoma and spleen cell assays the melanoma cell assays showed that in the presence of 10 μm scytonemin, the percent inhibition compared to the control was highest at 87.60% ± 1.86 while in the presence of 2 μm scytonemin, the inhibition decreased to 52.03% ± 1.76 (fig. 2). the percent inhibition compared to the control was significant only at 10 μm and 2 μm scytonemin (p < 0.0001). the percent inhibition generally increased as the concentration of scytonemin increased, indicating a positive relationship among these variables. for the spleen cell assays, in all treatments except the 10 μm scytonemin dilution without con a (65.28% ± 5.34), the percentage proliferation relative to the control was over 100% (fig. 3). for the spleen cells receiving the mitogen con a, the percentage proliferation relative to the control was 211.14% ± 53.24 for the most dilute concentration of scytonemin, 0.08 μm, peaking at 387.00% ± 79.34 with 2 μm scytonemin. the percent stimulation compared to the control was significant only at 2 μm scytonemin, with p = 0.0012 regardless of whether con a was used. the results of the assays with and without con a display the same general trends, suggesting that the presence of the mitogen was not a confounding variable. scytonemin did not demonstrate any inhibition of these spleen tissue cultures while the 2 μm and 10 μm levels showed significant inhibition of melanoma cell growth. in vivo assays after treatment, there was no significant difference in the weights of untreated mice (28.44 ± 0.49 g) and those treated with scytonemin (29.63 ± 0.48 g). there were also no significant differences in the number of melanoma tumors in untreated mice (205.5 ± 51.29) versus those treated with scytonemin (142.50 ± 30.65). discussion mechanistically, scytonemin inhibits the hyperproliferation of cells by targeting multiple enzymes implicated primarily in cell cycle regulation. for instance, one study examined the effect on several kinases, including plk1, cdk1/cyclin b, checkpoint kinase 1, protein kinase a, protein kinase c, myt1 kinase, and tie2 kinase (16). of these enzymes, it was determined that scytonemin acted as an inhibitor for plk1, cdk1/cyclin b, checkpoint kinase 1, protein kinase c, and myt1 kinase, but did not inhibit protein kinase a or tie2 kinase (16). these results suggest that scytonemin binds nonspecifically to a variety of enzymes to prevent phosphorylation steps critical to cell cycle progression. other experiments explored the relationship between scytonemin and plk1, finding that scytonemin acts as a mixed inhibitor for plk1 and functions in a concentration-dependent, time-independent manner to induce cell cycle arrest (15, 16, 18). by looking at molecular signals in the cell, the specific mechanism of action of scytonemin appears to be g2 to m phase cell cycle arrest (15, 19). in light of these prior findings, the results of this study support the anti-proliferative potential of scytonemin as an inhibitor of important cell cycle enzymes implicated in cancer growth. scytonemin at 2 μm and 10 μm significantly inhibited melanoma cell proliferation by greater than 50% compared to controls in a somewhat concentration dependent manner. this is consistent with other studies that explore the effect of scytonemin on malignant cells (15, 18, 19). in addition, the results of the spleen cell assay demonstrates that 2 μm scytonemin significantly stimulated proliferation volume seven | 59 compared to the controls, which was an unexpected outcome. since scytonemin typically inhibits actively proliferating cell types (15), there was some concern that it would inhibit spleen and melanoma cell growth alike. however, this was not the case, and was especially true for spleen cells receiving con a. in these cells scytonemin appears to have enhanced proliferation, ranging from 164.79% to 387.00% relative to the control group. however, unlike the melanoma cell assays, the spleen cells were not affected by scytonemin in a concentration-dependent manner through 10 μm of scytonemin. this result could be due to high standard errors or data resolution, which does not show the trend between 0.4 to 2 μm of scytonemin. nonetheless, the data suggests that scytonemin inhibits melanoma cells without hindering spleen cell proliferation. given that scytonemin inhibits melanoma cell proliferation and potentially increases spleen cell proliferation, it is a promising therapeutic agent for melanoma treatment because it can slow cell growth while figure 2. figure 2. percent inhibition of melanoma cells compared to the untreated control for various concentrations of scytonemin. error bars represent the standard error of nine replicates. significant inhibition compared to the control is marked with an asterisk, for both the 2 µm and 10 µm samples, p < 0.0001. figure 4. 60 | fine focus simultaneously augmenting the immune system. the in vivo experiments on mice evaluating the number of tumors formed with and without the presence of scytonemin favored the inhibitory effects of scytonemin. even though there were no significant differences in the number of melanoma tumors in untreated mice versus those treated with scytonemin, the averages themselves indicate that with additional studies there may be some inhibitory effect of scytonemin on melanoma tumor cells. these studies could benefit by a dose-dependent analysis. the concentration of scytonemin used was based on a study using jurkat t cells (15) and it may have not been ideal to use the same concentration in a study on melanoma tumor development. to better compare the inhibitory effects of scytonemin with the previous body of knowledge, the inhibitory concentration at 50% inhibition (ic50) was determined. this value represents the concentration of scytonemin at which cell proliferation is stunted by 50% relative to the control. in a regression analysis of scytonemin concentration versus percent inhibition of melanoma proliferation, a positive correlation (r2 = 0.89) was identified (fig. 4). using this regression plot, the ic50 for scytonemin on melanoma cells was determined to be approximately 1.66 ± 0.34 μm. in a similar study measuring the effects of scytonemin on the cancerous jurkat t cell line, the ic50 was determined to be 2.5 ± 0.6 μm (15). these relatively close values could help inform future research studies exploring the benefits of scytonemin as an antiproliferative therapeutic. the results of this study support that scytonemin inhibits melanoma cell proliferation and enhances the immune response via spleen cell proliferation. as a result, future research should examine the role of scytonemin on in vivo tumor growth beyond our studies to determine whether scytonemin functionally inhibits malignant growth in body systems without cytotoxicity to healthy cells. further, in vivo systems can be used to examine the response of the immune system to scytonemin, to potentially affirm enhanced spleen cell proliferation and examine other cytokines and immune figure 3. figure 3. percent proliferation of spleen cells compared to the control for various concentrations of scytonemin in the absence (white bars) and presence (black bars) of con a. error bars represent the standard error of nine replicates. significant inhibition compared to the control is marked with an asterisk, at 2 µm scytonemin, p = 0.0012 for cells with and without con a. biorender.com. volume seven | 61 cells. research could also focus on experimental cancer therapies using scytonemin to prevent tumors from growing and metastasizing. since a growing body of literature suggests that scytonemin acts as an inhibitor for plk1 (15, 16, 18, 19), the role of plk1 in melanoma and spleen cell proliferation should be further studied. in addition, since dimethoxyscytonemin binds to plk1 with high affinity (9), it would be interesting to study how this scytonemin derivative affects proliferation in melanoma and spleen cell assays compared to purified scytonemin. overall this study, along with prior research, provides evidence that the therapeutic potential of scytonemin should be further explored. acknowledgements we wish to thank dr. benjamin philmus for the purified scytonemin extract used in this study. we also thank the purdue fort wayne office of university research and innovation for support through the undergraduate summer research support program. this research is in compliance with federal regulations and institutional policies relating to animal care and use according to iacuc protocol #1111000244. references 1. bebout bm, garcia-pichel f. 1995. uv-b-induced vertical migrations of cyanobacteria in a microbial mat. appl environ microbiol 61:4215-4222. 2. briles eb, kornfeld s. 1978. isolation and metastatic properties of detachment variants of b16 melanoma cells. j natl cancer inst 60:1217-1222. 3. cdc. 2020. skin cancer. https://www.cdc.gov/cancer/skin/statistics/index.htm. accessed 4. ehling-schulz m, bilger w, scherer s. 1997. uv-b-induced synthesis of photoprotective pigments and extracellular polysaccharides in the terrestrial cyanobacterium nostoc commune. j bacteriol 179:1940-1945. 5. garcia-pichel f, castenholz rw. 1991. characterization and biological implications of scytonemin, a cyanobacterial sheath pigment. j phycol 27:395-409. 6. garcia-pichel f, sherry nd, castenholz rw. 1992. evidence for an ultraviolet sunscreen role of the extracellular pigment scytonemin in the terrestrial cyanobacterium chlorogloeopsis sp. photochem photobiol 56:17-23. 7. huang l, mccluskey mp, ni h, larossa ra. 2002. global gene expression profiles of the cyanobacterium synechocystis sp. strain pcc 6803 in response to irradiation with uv-b and white light. j bacteriol 184:6845-6858. 8. jagger j. 1985. solar-uv actions on living cells. praeger, new york. 9. pathak j, mondal s, ahmed h, rajneesh, singh sp, rp s. 2019. in silico study on interaction between human polo-like kinase 1 and cyanobacterial sheath pigment scytonemin by molecular docking approach. biointerface res appl chem 9:4374-4378. 10. proteau pj, gerwick wh, garcia-pichel f, castenholz rw. 1993. the structure of scytonemin, an ultraviolet sunscreen pigment from the sheaths of cyanobacteria. experientia 49:825829. 11. rastogi r, sonani r, madamwar d. 2015. cyanobacterial sunscreen scytonemin: role in photoprotection and biomedical research. appl biochem biotechnol 176:1551-1563. 62 | fine focus 12. reeke gn, jr., becker jw, cunningham ba, wang jl, yahara i, edelman gm. 1975. structure and function of concanavalin a. adv exp med biol 55:13-33. 13. rstudio. 2018. rstudio: integrated development for r. rstudio, inc., boston, ma. http:// www.rstudio.com/. 14. shibata h, baba k, ochiai h. 1991. near-uv induces shock proteins in anacystis nidulans r-2, possible role of active oxygen. plant cell physiol 32:771-776. 15. stevenson cs, capper ea, roshak ak, marquez b, eichman c, jackson jr, mattern m, gerwick wh, jacobs rs, marshall la. 2002. the identification and characterization of the marine natural product scytonemin as a novel antiproliferative pharmacophore. j pharmacol exp ther 303:858-866. 16. stevenson cs, capper ea, roshak ak, marquez b, grace k, gerwick wh, jacobs rs, marshall la. 2002. scytonemin, a marine natural product inhibitor of kinases key in hyperproliferative inflammatory diseases. inflammation res 51:112114. 17. urbach f. 1997. ultraviolet radiation and skin cancer of humans. j photochem photobiol b: biology 40:3-7. 18. zhang g, zhang z, liu z. 2013. polo-like kinase 1 is overexpressed in renal cancer and participates in the proliferation and invasion of renal cancer cells. tumor biol 34:1887-1894. 19. zhang g, zhang z, liu z. 2013. scytonemin inhibits cell proliferation and arrests cell cycle through downregulating plk1 activity in multiple myeloma cells. tumor biol 34:2241-2247. volume seven | 63 ’ during the first 30 years of the 20th century, many scholars agree that cora wilson stewart was the most widely known authority of adult literacy in the world. by establishing the moonlight schools in rowan county kentucky in 1911, she rose to prominence as a well-regarded advocate for promoting literacy and adult education. stewart frequently held viewpoints which ran counter to the mindset of educators in the 1920s and 1930s. wilson was ahead of her time in many aspects of promoting literacy in an era largely before adult education was widely recognized (fig. 1). wilson is remembered for being an eloquent and colorful speaker, and grew up believing that furthering one’s education would open opportunities beyond the traditional molds that shaped rural appalachia over 100 years ago (1). cora wilson moved beyond a series of failed marriages and domestic abuse to devote most of her time to public affairs, eventually adopting adult literacy as her primary mission. as the daughter of a physician and schoolteacher, wilson would frequently go along on rounds with her father and help read or interpret medical advice, documents, and other communications to adult patients and families who could not read or write. eventually earning her teaching credentials from morehead (ky) normal school (later morehead state university), wilson later was the first woman to be elected as president of the kentucky education association, an advocacy group dedicated, then and now, to improved funding for schools, safety measures, smaller class sizes, and empowerment of employees and parents (2). shortly thereafter, stewart, as a superintendent for rowan county schools, created the first moonlight school in september 1911 (figs. 2-4). adults would attend wilson’s classes in the evenings, after children were home and chores were completed, and only on moonlit nights, to © 2024 mckillip. fine focus, 10(1), 2-5. doi: 10.33043/ff.10.1.2-5. shared with cc-by-nc-nd 4.0 license. objective lens: cora wilson as a pioneer in education in rural kentucky & beyond john l. mckillip, fine focus managing editor https://creativecommons.org/licenses/by-nc-nd/4.0/ objective lens: cora wilson as a pioneer in education in rural kentucky & beyond 3 figure 1 cora stewart wilson (1875-1958) in a photograph from january 1916, from press reference book of prominent kentuckians, 1916, p. 224 (royalty-free image). be guided to and from the school safely. wilson asked teachers to volunteer to teach in the evenings as the program grew. in 1914, stewart successfully urged the kentucky governor to create a state illiteracy commission, and expand the moonlight schools statewide, with the goal of eliminating illiteracy by 1920. regional communities held competitions, and various women’s groups became involved to further the cause. after a few years, wilson established the moonlight school institute, the first of its kind in america. eventually, over 1,600 students were enrolled, 350 of whom had learned to read and write. wilson also saw that similar schools be established to serve black students, and by 1915, fifteen moonlight schools served these adults (3). along this progressive path, stewart published numerous books specifically for adults who had limited or no literacy, to encourage them to read, as well as one entitled the soldier’s first book, aimed at military recruits for world war i. alabama and mississippi began moonlight schools shortly after the wilson schools in kentucky became successful. by 1916, eighteen states had moonlight schools. stewart went on to be chairperson of the illiteracy commission of the national education association. wilson was a pioneer and tireless advocate for education of underserved her entire adult life, and doubtless left a legacy which still remains underappreciated today. another similar journey is included in this, our tenth anniversary issue of fine focus; that of parisa (page 6), an international stem scholar who writes about her first tenure-track faculty position at an hbcu, and the necessary cross-cultural literacy to be explored and appreciated when navigating challenges in a unique but also rewarding academic climate. in many ways, parisa’s journey draws many parallels to that of cora wilson stewart – combining empathy and passion with determination and vision to achieve lifelong learning. fine focus | volume 104 figures 2, 3, & 4 the original cora wilson steward moonlight school located near the campus of morehead state university in kentucky, usa. photo credits: david nickell, hillsboro, ky. objective lens: cora wilson as a pioneer in education in rural kentucky & beyond 5 references 1. nelms, willie. cora wilson stewart: crusader against illiteracy. mcfarland & co, 1997. print. 2. kentucky education association: https://www.kea.org/about_kea.uspx accessed 3.4.2024 3. https://nkaa.uky.edu/nkaa/items/show/2669 accessed 3.3.2024 https://www.kea.org/about_kea.uspx https://nkaa.uky.edu/nkaa/items/show/2669 6 | fine focus objective lens: the pasteur act – bipartisan legislation to address the mdr crisis john l. mckillip managing editor, fine focus, professor, microbiology, ball state university vol 9 | 7 infectious disease is currently the 2nd leading killer in the world, and 4th in the u.s. globally, 17 million people die annually from bacterial infections (1). yet relatively few have even heard of antibiotic-resistant bacteria or understand the implications for global health. as i mentioned in this contribution in our spring 2020 issue (fine focus 6:6-8), no new classes of antibiotics have been developed to treat microbial infections in over 30 years, as pharmaceutical companies have instead pursued research and development of more lucrative drugs for non-infectious diseases. most large pharmaceutical companies have ceased natural product (np) discovery, leaving academic labs and small startup companies to explore antimicrobial therapeutics (2). this crisis is well-known among experts but rarely brought to the attention of the general public. alexander fleming’s discovery of penicillin in 1928 was met with his own prediction that bacterial resistance to this “miracle drug” would soon be documented (3,4). in the 70+ years since penicillin was introduced, overuse and misuse of antibiotics have contributed to the problem of mdr bacterial infections, as has the widespread use of antibiotics in agriculture for prophylaxis and growth promotion (5). in fact, the cdc has reported that over 70% of antibiotics used in the u.s. are in production animal environments (6,7). clinically, antibiotic stewardship and surveillance programs have shown limited success in addressing the mdr crisis (8). in recent years, both the cdc and the white house have outlined clear goals and objectives for directly addressing antibiotic resistance in order to slow the spread of mdr bacteria, while offering a timeline on collaborative international efforts required to make this happen by 2020 (5,6). unfortunately, this executive order signed by president barack obama (#13676) has not been addressed, during which time mdr bacterial infections have worsened and become more frequently diagnosed (9,10). incentivizing large pharmaceutical companies to resume new product discovery and development is an overdue action item; therefore, many scientists and members of the concerned general public may be wondering what, if any, initiatives are in play for operationalizing a solution to this pending crisis. one solution being explored in our teaching and research laboratories also addresses another international need. in 2015, singapore led in the global program for international student assessment (pisa) rankings in math and science; the us ranked 38th in math and 24th in science, well behind developing countries including china and india (15,16). addressing this dire need must come immediately by training new scientists with novel creative and authentic approaches. we are working to improve the quality and effectiveness of undergraduate education in stem by transforming the focus of the laboratory section of our undergraduate microbiology class from a traditional model to a coursebased undergraduate research experience (cure) design, wherein students would isolate antibiotic-producing bacteria while learning the scientific method. the concept of implementing the cure structure in laboratory science, as well as non8 | fine focus science classes, has been well documented and successful in recent years (11-14). at their best, cure models offer three distinct features: discovery, iteration, and collaboration, all of which are tied together into the goal of teaching undergraduates the process of doing science while working on an original research problem (14) – in our case, isolating and identifying antibioticproducing bacteria. using resources, expertise, and protocols modified from the small world initiative (swi) (17), our goal is to isolate and identify multiple metabolites from naturally occurring bacteria in soil, freshwater, and agricultural specimens. transforming the undergraduate microbiology curriculum in this way is a completely new teaching and research direction for us, and will blend pedagogy and original research into an entirely different direction than at present. we offer that our cure model epitomizes engaged student learning, focuses on students as future scientists, and involves curricular innovations to increase student retention in stem. this pedagogical approach is only part of an effective solution, to the global mdr crisis, however (18). additional efforts must be realized, and incentives are needed by elected officials for long-term substantive changes to directly address the crisis in the short-term future. in the summer of 2021, bipartisan legislation was introduced in the u.s. senate and house of representatives intended to jump-start the development of urgently needed new antibiotics, using a creative model that should efficiently incentivize interested pharmaceutical companies to begin antibiotic development once again, after a decades-long hiatus. this legislation is called the pioneering antimicrobial subscriptions to end upsurging resistance (pasteur) act of 2021 (19). introduced by co-sponsors senators michael bennet (dco) and todd young (r-in), this legislation is unique in a few ways. the pasteur act is designed help to revive and restart new product development within the antibiotic pipeline by providing sizable, subscriptionbased financial incentives for broad public access to innovative, high-priority antibiotics. traditionally, pharmaceutical profit margins are typically tied to how much of a drug is sold; however, pasteur contracts would instead pay for new antibiotics based on their exceptional value to public health. this approach solves the core problem facing antibiotic drug development: the pressing need for innovative drugs that don’t have high sales potential in the private drug market. as a result, pasteur will not only encourage the development of new types of antibiotics, but also help ensure patients have access to these drugs once they come to market. currently, s. 2076 awaits consideration by the senate committee on health, education, labor, and pensions. put simply, the pasteur act outlines an innovative way for the government to enter into contracts with pharmaceutical companies which would direct existing personnel and resources for new product development. under the pasteur act, the federal government would create market incentives for the development of lifesaving antimicrobial drugs. developers would be paid contractually agreed-upon amounts annually, for a duration ranging from five vol 9 | 9 years up to the antimicrobial’s patent life. the subscription contract eligibility and value would be based on the clinical need and novelty of the drug. thus, waste of funds is minimized because the subscription model allows for profit and funds accrued to be proportional to the need expressed by the company at the outset. the bill also provides additional resources to support education and implementation of well-directed antibiotic stewardship efforts. for example, these actions would include educating health care providers and the lay community on how to avoid overuse or misuse of these life-saving medications in order to slow the emergence of antibioticresistant pathogens to address this global crisis effectively, a cmprehensive multifaceted approach is needed. many experts agree that no action will be taken by pharmaceutical companies unless incentivized by federal intervention. put simply, there is no national solution to an international problem. the co-sponsors of the pasteur act feel that drug-resistant bacterial infections do not recognize borders, necessitating a coordinated, international approach to protect global health. moreover, these bacterial infections will disproportionately affect underserved people who lack access to quality care – displaced refugees, families suffering from natural disasters, impoverished individuals, and the immunocompromised, among many others. all are affected directly or indirectly. immediate federal action, through the pasteur act, will address a fundamentally broken market for antibiotics so that patients will have access to novel antibiotics they need to treat increasingly resistant diseases. what is the status of this legislation? at the time of this writing, senate and house approval is pending before it goes forward to the president for a signature and becomes law. currently, s. 2076 awaits consideration by the senate committee on health, education, labor, and pensions. the process is moving slowly but with input from engaged stakeholders, lawmakers may be reminded of the urgency of this initiative. you are encouraged to contact the offices of the cosponsors of this legislation and encourage them to devote their efforts to seeing this legislation through to final approval. then the real work must begin – augmenting this reinvigorated new antibiotic development with education for healthcare providers and the lay community on appropriate and effective antibiotic stewardship practices. 10 | fine focus references 1. martens, e., & demain, a.i. 2017. the antibiotic resistance crisis, with a focus on the united states. j. antibiotics 70:520-526. 2. hutchings, m.i., truman, a.w., & wilkinson, b. 2019. antibiotics: past, present, and future. curr. opin. microbiol. 51:72-80. 3. khardori, n., stevaux, c., & ripley, k. 2020. antibiotics: from the beginning to the future: part i. ind. j. ped. 87:39-42. 4. tan, s. y., & tatsumura, y. 2015. alexander fleming (1881-1955): discoverer of penicillin. singapore med. j. 56:366-367. 5. venter, h., henningsen, m.s., & begg, s.l. 2017. antimicrobial resistance in healthcare, agriculture, and the environment: the biochemistry behind the headlines. essays biochem. 61:1-10. 6. abadi, a.t.b., rizvanov, a.a., haertlé, t., & blatt, n.l. 2019. world health organization report: current crisis of antibiotic resistance. bionanoscience 9:778788. 7. michael, c.a., dominey-howes, d., & labbate, m. 2014. the antimicrobial resistance crisis: causes, consequences, and management. front. public health 2:1-8. 8. romo, a.l., & quiros. 2019. 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(2009). the benefits of undergraduate research, scholarship, and creative activity. in (m.k. boyd & j.l. wesemann, eds) broadening participation in undergraduate research: fostering excellence and enhancing the impact. council on undergraduate research, washington, d.c. 14. linn, m.c., palmer, e., baranger, a., gerard, e., & stone, e. 2015. undergraduate research experiences: impacts and opportunities. science 347:1261757 15. https://www.pewresearch.org/fact-tank/2017/02/15/u-s-students-internationallymath-science/ accessed 8.2.2022. 16. united states congress joint economic committee. (2012). stem education: preparing for the jobs of the future. washington, dc: author. retrieved from: http://www.jec.senate.gov/public//index.cfm?a=files.serve&file_id=6aaa7e1f9586-47be-82e7-326f47658320 accessed 9.2.2022. 17. http://www.smallworldinitiative.org/about accessed 9.28.2022 18. fluhler-thornburg, g., & mckillip, j.l. 2023. course-based undergraduate research experiences for laboratory learning in the life sciences. j. college science teach., in press. 19. https://www.congress.gov/bill/117th-congress/house-bill/3932 accessed 9.30.2022 staphylococci on hospital curtains • 53 the persistence of staphylococcus aureus on hospital privacy curtains. sarah e. cole1 brittany j. gasper* 1 current address 11200 sw 8th st, miami, fl 33199 * center for teaching and learning, kent state university, kent, ohio copyright 2019, fine focus all rights reserved manuscript recieved 19 march 2019; accepted 30 july 2019 corresponding authors sarah e. cole sarahecole95@yahoo.com brittany j. gasper bgasper@flsouthern.edu abstract while healthcare professionals are working in hospitals, they will often manipulate the privacy curtains during the care of their patients. studies have shown that the transfer of bacteria from hands to the curtains and vice versa is possible. despite the possibility of hospital curtains being a mode of infection transmission, studies have shown that 53% of hospitals surveyed did not have a policy for cleaning or changing their curtains. the question that this study focused on was whether curtain material affects the persistence of staphylococcus aureus. in this study, five different curtain types were inoculated with overnight, diluted, and finger imprint cultures of s. aureus. they were incubated at room temperature and were sampled for growth regularly onto mannitol salt agar plates. the colonies were counted, and one-way anova statistical analysis was completed on the data. the statistical analysis showed that the length of persistence of liquid cultures of s. aureus on the curtains was not dependent upon initial concentration. finger imprint inoculations of four curtain varieties had statistically significant longer persistence times than the liquid cultures. only the curtain type composed of 100% antimicrobial polyester with water repellant had significantly lower persistence times for the finger imprint culture than the other four curtains. the results suggest that the 100% inherently flame resistant antimicrobial polyester curtain material reduces s. aureus persistence times and that it may benefit hospitals to use this type of curtain. keywords introduction • persistence • health-care • associated infection • nosocomial infection • hospital curtain the environment of the hospital has an important role in the transmission of health-care associated pathogens which cause nosocomial infections. studies have found that hospital privacy curtains are quickly contaminated by microorganisms that can be transferred to the hands of healthcare workers, the patient, and the surrounding environment (21, 30). in 1988, there was an outbreak of multiple-antibiotic-resistant acinteobacter baumannii in the queen elizabeth hospital in birmingham, uk (7). after an investigation, carbapenem-resistant a. baumannii was isolated from surfaces of equipment, beds, mops, and curtains. it was determined that the curtains were the main source of the outbreak because they had the highest number of the organism present on them (7). in addition to the curtains being easily contaminated with pathogens that can be transferred via the hands to people or other surfaces, some bacteria can persist on hospital surfaces, including curtains, for weeks and even months (15, 19, 20). persistence in this case refers to the ability of an organism to survive and be cultured from a surface (15). a study by deangelis and khakoo (8) indicated that these lengthy persistence times are problematic. their study included surveys completed by hospitals on curtain cleaning policies. over half of the hospitals surveyed (53%) said they do not have a policy for cleaning the hospital curtains, 37% said they only clean their curtains when visibly soiled, 13% clean the curtains every year, 13% clean their curtains every three months, and only 13% clean their curtains every month. in other words, the most frequently some hospitals change their curtains is every month. since only 13% of the total hospitals surveyed clean their curtains that often, this means most of the hospitals either do not have a cleaning policy or change the curtains every three months or greater. mitchell et al. (2015) noted that the attention 54 • fine focus, vol 5 2019 materials and methods put into cleaning porous, soft surfaces such as privacy curtains and room furnishings is far less than that given towards disinfection of porous surfaces (18). as there are nosocomial pathogens such as staphylococcus aureus, klebsiella quasipneumoniae subsp. similipneumoniae (formerly known as k. pneumoniae) (10), and candida albicans that can last on hospital surfaces for longer than four months (15), this three month cleaning interval may not be frequent enough. this suggests there needs to be more frequent cleaning and a change of policy. despite the importance of the topic, there has not been a large amount of research completed on the topic of hospital curtains and bacteria or bacterial infections. the studies completed so far have varied substantially in their inoculation technique and their analytical methods. some have taken finger imprints from curtains actively hanging in hospital rooms (3, 14, 16, 17, 21, 22, 24). others have inoculated known concentrations of bacteria onto specified areas of curtain swatches (13, 27) or hospital fabric (20). these studies have also differed in duration from as short as seven days (17) to as long as six months (3, 14, 25). there are different varieties of hospital curtains available including those containing antimicrobial fabric and some coated with water repellant. however, most studies only used one type of curtain and this varied from vinyl (21) to flame resistant (fr) (22), propylene (14), disposable sporicidal (13), and polyester (16, 24), amongst others. only two studies (20, 24) were shown to compare more than one type of curtain, and only three studies used statistical tests to analyze their results (3, 17, 24). contradictory results of previous studies include some that have showed a high rate of methicillin resistant s. aureus (mrsa) on curtains (21) and those that have not (16). kurashige (2016) mentions that curtain type may influence these results and should be further examined. this study intended to expand on the existing research. the focus was on s. aureus, a species of pathogenic bacteria that causes nosocomial infections and has been shown to be capable of developing antibiotic resistance to become hospital-associated mrsa (9). this study combined aspects of previous findings by measuring the persistence times of s. aureus inoculated both directly with a known concentration or by fingerprint on five different curtain types. these curtain types included three different combinations of fr and non-fr polyester with no antimicrobial properties and two antimicrobial polyester curtains, one with and one without water repellant. we hypothesize that the composition of the different curtain types, specifically the presence of antimicrobial properties, will influence the persistence of s. aureus. curtain varieties and codes five different curtain varieties were used in this study. each curtain was given a code for ease of designation and to prevent bias on the part of the experimenter when counting colony-forming units (cfus) and persistence times. the curtain varieties and codes used in this study can be found in table 1. table 1: composition and codes of the five different curtains used in this study. inoculation of curtains each curtain variety was cut into 2 x 2 cm squares that were sterilized by autoclaving. sterile curtain squares were inoculated with s. aureus by three different methods. for each method, six squares of each curtain type were inoculated. all overnight (o/n) cultures of s. aureus (atcc 12600) were grown in liquid tryptic soy broth (tsb) media at 37°c for 24 hours. method one (concentrated liquid culture technique) – curtain squares were inoculated with 20 μl of a liquid o/n culture of s. aureus. this was the most concentrated solution inoculated and was intended to simulate an extreme contamination event such as staphylococci on hospital curtains • 55 direct contact of a contaminated fluid sample with the curtain. measurement of the od600 on a genesys 10s uv-vis spectrophotometer (thermo scientific) indicated this was equivalent to a total bacterial load of 2.0 x 107 cfu or a density of 5.0 x 106 cfu/cm2. method two (diluted liquid culture technique) – curtain squares were inoculated with 20 μl of a liquid o/n culture of s. aureus diluted to an od600 of 0.08 in a sterile 0.9% (w/v) sodium chloride solution. this was equivalent to a total bacteria load of 1.3 x 106 cfu or a density of 2.7 x 104 cfu/cm2. this od600 was chosen as the diluted culture standard as it closely represents the od¬600 value of the 0.5 mcfarland standard used for inoculated samples during antimicrobial susceptibility testing (6). a diluted sample was included to see if the persistence of s. aureus was dependent on the initial concentration inoculated and to represent direct contact between curtains and diluted fluid samples. method three (colony finger imprint technique) – curtain squares were inoculated with a colony of s. aureus by touching the colony with a gloved finger and pressing it onto the curtain piece, making sure to equally touch each area of the curtain. the colonies of s. aureus used for this step were similar in size and obtained from a quadrant streaked mannitol salt agar (msa) plate that was incubated at 37°c for 48 hours. curtain e was the only curtain where the overnight and diluted cultures were not inoculated onto the curtain. only the finger imprint technique was used to inoculate this curtain because it is water repellant and was not able to absorb the liquid cultures. after the curtain squares were inoculated, they were left to dry in separate sterile petri dishes for 20 minutes. measurement of s. aureus presistence results after inoculation and drying, an initial measure of bacterial growth was aseptically taken from each curtain square. a flame sterilized pair of tweezers was used to hold the curtain square steady while a sterile cotton swab dipped in a sterile 0.9% (w/v) sodium chloride solution was used to evenly sample each curtain with 10 strokes (five down and five up). this cotton swab was then used to evenly, continuously streak onto an msa plate that was incubated at 37°c for 48 hours. after incubation, the number of cfu on each plate was counted. if more than 300 cfu were present on the plate, it was recorded as too numerous to count. curtain squares were maintained at room temperature in sterile lidded petri dishes to represent the equivalent environment of a hospital setting. swabbed samples of s. aureus were taken from the squares at a maximum of three day intervals until growth of five cfu or less was seen on an msa plate incubated at 37°c for 48 hours for two consecutive samplings. to conserve resources, this was modified from neely and maley (2000), who sampled until no cfu were seen for two consecutive samplings, as five or fewer cfu was considered sufficient decline to end the measurement of persistence. once all of the data were collected, the number of days that the s. aureus persisted on each sample of each curtain type was counted. the day that the curtain pieces were inoculated and initially swabbed was considered day zero. all of this data were compiled and analyzed using one-way anova statistics with the tukey honestly significant difference test. the persistence time of s. aureus was measured (in days) and compared for the three different inoculation methods on curtain varieties a, b, c, and d. the comparisons of the mean persistence times from the three inoculation techniques for each type of curtain are shown in fig. 1. all curtain varieties in fig. 1 showed significantly longer persistence times after inoculation of a colony using the finger imprint technique compared to inoculation of a concentrated or diluted o/n liquid culture. curtains a, b, and c showed a p-value of < 0.0001 while the p-value for d was less than 0.05. there was no significant difference in the persistence times between inoculation of a concentrated or diluted o/n culture for any of these curtain types. the mean persistence times for s. aureus inoculated using concentrated o/n liquid cultures and diluted liquid cultures was compared for the four curtain varieties (a, b, c, and d). these mean persistence times are shown in figs. 2 and 3, respectively. the persistence time of s. aureus on curtain c was significantly longer than the other curtain types (p-value < 0.01). the mean persistence time for curtain varieties a, b, and d ranged from 7.76 to 10.0 days while the 56 • fine focus, vol 5 2019 discussion mean persistence time for curtain c was 14.5 days. interestingly, there was no statistically significant difference between the mean persistence times of s. aureus on these four curtain varieties when inoculated with a diluted o/n culture. the mean persistence times ranged from 9.83 to 13.3 days. a comparison of the mean persistence times for s. aureus inoculated using the colony finger imprint technique on all curtain varieties is shown in fig. 4. the mean persistence time of s. aureus inoculated onto curtain variety e was significantly less than the four other curtain varieties (p-value < 0.0001). the mean persistence time for the four other curtain varieties ranged from 22.0 days to 27.0 days while it was only 4.0 days for curtain e. there was no significant difference in the number of s. aureus cfus recovered over time from each curtain type (data not shown). regardless of the inoculation method, all curtain types a-e showed an initial high number of cfus that decreased at a steady pace before the bacteria were no longer able to persist. great variation independent of curtain type was seen in the specific numbers of cfus at any given time point, but the general decline was consistent. when comparing the mean persistence times of the o/n and diluted cultures of each of the curtains a, b, c, and d, there was no difference between the o/n and diluted cultures. in other words, the persistence following liquid inoculation was not dependent on initial concentration. on the other hand, there was a statistically significant difference between mean persistence times of the finger imprint cultures and both the overnight and diluted cultures for all of these curtains. in addition, mean persistence time of s. aureus inoculated by the finger imprint lasted much longer than the other cultures curtain varieties a, b, c, and d, implying that the finger imprint inoculation method did have an effect on persistence times, lasting more than 10 days longer than the o/n or diluted cultures. there was no significant difference in the initial cfu counts for any of the inoculation methods (data not shown). these data suggested that the solid inoculation of a bacterial colony increased the persistence of s. aureus compared to a liquid inoculation. to our knowledge, no research comparing the persistence times of bacteria inoculated from both liquid and solid cultures has been published. kotsanas et al. (2012) inoculated curtain samples with liquid cultures and took finger imprints at specified time points to culture from the curtain and measure persistence (13). they found s. aureus to persist for 10 days but show no growth at 2 months. this is consistent with the range of mean persistence times of 8.50 to 14.5 days we measured from our liquid inoculations. interestingly, they found < 10 cfu after one day of incubation when culturing by the finger imprint method and incubating at 37°c on horse blood agar, an enriched media (11). by comparison, our cfu values from curtains incubated for one day following liquid inoculation and cultured by swabbing and incubating at 37°c on msa were consistently higher than 10 cfus for all curtain types (data not shown). the counts ranged from a low of 13 cfus to a countable high of 123 cfus. of the 33 samples for which the cfu count after one day is available, 15 were found to be too numerous to count. the curtains in korvath et al. (2012) were inoculated with 4 x 105 cfu/cm2 of culture, comparable to the 5.0 x 106 and 2.7 x 104 cfu/ cm2 in the o/n and diluted samples, respectively. these data suggested the swabbing technique may be a more thorough method for culturing from the entire curtain sample than taking a finger imprint from the center of the curtain (13). with the comparison of the persistence time from s. aureus inoculated from concentrated o/n cultures on curtains a, b, c, and d, there was statistical significance between curtain c and a, b, and d (p < 0.01). c had the longest persistence time of all of the overnight cultures with an average of 14.5 days as opposed to the averages of 8.50, 10.0, and 7.67 days for curtains a, b, and d, respectively. this statistical significance suggested that the curtain material of c had an effect on the persistence of the s. aureus concentrated o/n culture. interestingly, these results were not reproducible when comparing persistence of s. aureus grown from diluted o/n cultures. this suggests a possible concentration-dependency on the persistence of s. aureus on this particular curtain material though the lack of statistical difference between the persistence of s. aureus from concentrated and diluted o/n cultures would conflict this. additional research is needed to address these conflicting results. the main distinction in curtain material between curtain c and the others is that it had the highest percentage of post-consumer recycled fr polyester staphylococci on hospital curtains • 57 figure 1: mean persistence times of s. aureus from different inoculation of techniques: the mean persistence times in days for curtain varieties a (panel a), b (panel b), c (panel c), and d (panel d) are shown after inoculation by concentrated o/n culture, diluted o/n culture (dil), or colony finger imprint technique (fp). the o/n and d cultures contained 2.0 x 107 and 1.3 x 106 cfu, respectively. each bar represents the mean persistence time ± standard deviation of n = 6 inoculated samples. statistical analysis was completed using a one-way anova (* indicates statistical significance with a p-value < 0.05). figure 2 – mean persistence times of s. aureus inoculated from a concentrated liquid o/n culture: the mean persistence times in days for curtain varieties a, b, c, and d inoculated with a concentrated o/n culture of s. aureus are shown. the o/n culture contained 2.0 x 107 cfu. each bar represents the mean persistence time ± standard deviation of n = 6 inoculated samples. statistical analysis was completed using a one-way anova (* indicates statistical significance with a p-value < 0.05). 58 • fine focus, vol 5 2019 figure 3 – mean persistence times of s. aureus inoculated from a diluted liquid o/n culture: the mean persistence times in days for curtain varieties a, b, c, and d inoculated with a diluted (dil) o/n culture of s. aureus are shown. the d culture contained 1.3 x 106 cfu. each bar represents the mean persistence time ± standard deviation of n = 6 inoculated samples. statistical analysis was completed using a one-way anova. figure 4 – mean persistence times of s. aureus inoculated by finger imprint of a bacterial colony: the mean persistence times in days for curtain varieties a, b, c, d, and e inoculated with a colony of s. aureus by the finger imprint technique (fp) are shown. each bar represents the mean persistence time ± standard deviation of n = 6 inoculated samples. statistical analysis was completed using a one-way anova (* indicates statistical significance with a p-value < 0.05). at 52%, higher than the 27% found in curtain a, the only other curtain containing this material. since curtain a is composed of the greatest percentage of total recycled polyester (54% including post-consumer and post-industrial), it seems unlikely that recycled polyester alone could account for the longer persistence time seen with inoculation of o/n cultures. further research will compare the persistence times of s. aureus inoculated from a range of dilutions onto curtains a, b, and c, composed of mixed recycled, no recycled, and post-consumer recycled polyester, respectively. most studies have only used one type of curtain. neely and maley (2000) compared the persistence of staphylococcus and enterococcus on multiple types of hospital fabrics (20). their values for persistence of methicillin sensitive s. aureus after liquid inoculation onto 100% polyester range from 10 – 56 days, comparable on the lower end to the range of liquid culture persistence from this study. compared to other fabrics, polyester and polyethylene provided the longest persistence times for s. aureus while inoculation onto 100% cotton, terry cloth, and a 60-40% cotton-polyester blend resulted in comparably shorter persistence times (20). while curtain varieties a, b, c, and d all showed increased persistence of s. aureus inoculated by the finger imprint method, curtain variety e had significantly decreased persistence times by comparison (p < 0.01). the mean persistence of s. aureus on curtain e (4.0 days) is more than 15 days shorter than the averages of the other four curtains, suggesting the material of this curtain did have an effect on the persistence time. curtain e is the only variety to contain 100% inherently antimicrobial fr polyester with a water repellant. curtain d also has antimicrobial polyester (56.6%), but it does not contain a water repellant. interestingly, the lower antimicrobial composition of curtain d did not seem to have an effect on persistence times as it showed no significant difference from curtains a or b that contain no antimicrobial composition. it is not clear if the significantly decreased persistence of curtain e is due to the 100% antimicrobial polyester, the water repellant nature, or both. in order to determine how the water repellant affected the persistence times from the finger imprint inoculation, additional experimentation would need to be completed comparing 100% fr antimicrobial polyester with water repellant materials to the same curtain material without the water repellant treatment. in addition, to determine how this curtain material affects the liquid cultures, experimentation would be staphylococci on hospital curtains • 59 needed comparing the persistence times of curtains a, b, c, and d to the 100% inherently fr antimicrobial polyester without water repellant. these results have an important implication in the hospital setting because the finger imprint cultures are a better representation of the inoculation method that would take place in the hospital. for example, in the hospital, healthcare professionals might touch the source of infection with their gloved hands and then touch the curtain. it may benefit hospitals to use this type of high percentage antimicrobial water repellant curtain to reduce persistence times within the hospital, especially in areas like the emergency department where contamination can occur quickly and frequently with high patient turnover rates. however, as curtain e was the only water repellant curtain examined in this study, more investigation with water repellant curtains both with and without antimicrobial composition would need to be done to confirm this. for the purposes of resources, the persistence times of s. aureus alone were investigated in this study, and samples from the curtains could only be taken every 1-3 days instead of every day. while s. aureus is important to study because it is a type of mrsa, it is also a commensal organism that colonizes up to 30% of the human population (32) and is a leading cause of bacteremia, infectious endocarditis, and device-related infections (5, 28). additionally, there are other important nosocomial infection-causing bacteria to study, including k. quasipneumoniae subsp. similipneumoniae, acinetobacter baumannii, and pseudomonas aeruginosa (1, 2, 4, 23, 29). these other bacteria may have different persistence times on these curtains than s. aureus and may be affected differently by the curtain materials. the persistence times of a variety of bacteria on various curtain types should be measured to determine how different curtain material can affect persistence. ideally these persistence times would be measured by culturing from the curtain samples every day. future studies will specifically measure the persistence times of enterococcus faecalis, escherichia coli, and p. aeruginosa on these different curtain types as these represent three common causes of nosocomial infections (12, 26, 31) and exemplify three species that contain multi-drug resistant strains including vancomycin-resistant enterococcus for e. faecalis, extended-spectrum cephalosporin-resistant e. coli, and carbapenem-resistant p. aeruginosa (26). acknowledgments the authors would like to thank laura cole for providing the curtain samples used in the testing. 60 • fine focus, vol 5 2019 references 1. agaba p, tumukunde j, tindimwebwa 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leeuwen w, van belkum a, et al. 2005. the role of nasal carriage in staphylococcus aureus infections. lancet infec. dis. 5:751-62. 62 • fine focus, vol 5 2019 128 | fine focus undergraduate student perspective steminists: an exploration of the impact of female educators in stem fields devon popson m.s. student, miami university, oxford, oh volume six | 129 abstract with a growing need for skilled stem workers in the us, it is necessary to examine why minority groups such as women continue to participate in stem at far lower rates. the historical exclusion of women from education created a system of stem education that was formed for and by men. despite changes in the education system that have allowed women to gain traction in almost every other content area, evaluation of the current status of the field reveals stem subjects remain dominated by men. additionally, there appears to be a new set of barriers that exclude women at all levels of stem education. changes to address these new barriers need to be made — and educators who are at the forefront of this occurrence have a unique opportunity to inspire change for future generations. specifically, female stem educators acting as mentors and role models have been shown to help female students overcome the obstacles they face in pursuing a stem education and career. in order to fully realize the potential of female educators, the treatment of female educators in academia needs to be evaluated to ensure they are properly supported by the schools they work for. 130 | fine focus introduction within the united states, there has been a large push to increase the number of skilled science, technology, education, and mathematics (stem) workers in order to keep up with the constant advancements being made in these fields. at the current rate, the u.s. is falling behind on the world stage in these areas (hossain and robinson, 2012). this lack of stem workers is likely due to a multitude of factors, including the fact that many minorities, including women, are still represented at low proportionate rates in these fields (burke and mattis, 2007, pg 5). addressing this underrepresentation is not only beneficial in increasing skilled stem workers, but also in allowing innovation to continue in these fields by providing diverse perspectives. the incorporation of women in stem has had a long, complex history. it is this history and the progression into the current era that provides the key to addressing what barriers are preventing female persistence in stem. women have been faced with a multitude of barriers to access and success within stem education and careers. access to the necessary education is a particularly important factor in ensuring an individual is capable of succeeding in any field, and stem is no exception. the first step in increasing female participation in stem careers is ensuring they have the necessary education. while stem subjects are generally viewed through the lens of higher education, participation in these subjects begins much earlier. children are likely first introduced to stem subjects in elementary school. developing the talent of these young students and maintaining their interests within the subject is an integral part of growing the stem field (hossain and robinson, 2012). it is with this perspective that it is necessary to evaluate how science is being presented to young learners and how the presentation can be adapted to be more inclusive to girls in and outside of the classroom. getting girls involved in stem from a young age is a good first step but is somewhat arbitrary if the institutions in higher education dissuade women from pursuing careers in these fields. for nearly the entire existence of higher education, women have struggled to gain equal access, especially in the fields of stem, which were primarily established by and for men. while the outright discrimination within stem education may not be a major factor in the lack of female participation today, the programs and institutions of colleges and universities still seem to be unwelcoming to female participation. with all of these issues within stem education, attention is frequently turned to educators at all levels. educators exist to facilitate learning and in doing so have the opportunity to serve as role models and supporters to students. it is this potential and their proximity to the issues with gender in stem that gives educators the opportunity to make a difference. by examining the evolving relationship between women, stem education, and the role educators play, it is possible to consider options for making improvements in the future. the historical role of women in stem the first step in addressing the lack of women in stem fields today is to look at past interactions. this approach allows the examination of past establishments that led to inequality, as well as an opportunity to evaluate the success or failure of any actions that may have been taken to address the issue. historically, stem education and employment opportunities have been dominated by men (nsb, 2018). this discrepancy has been theorized to have developed due to varying social barriers that prohibited women from being equal contributors volume six | 131 to stem fields. several of the main contributors to this inequality are traditional gender roles, access to education, and access to equal employment opportunities (bystydzieński, 2006, pg. 6). while these are not necessarily the only contributors, these factors have had a significant impact on women’s ability to be accepted in stem and, thus, need to be explored. in order to participate in any field, there are certain requirements for entry. in some cases, those requirements may actually serve as barriers. it is necessary to have the background, knowledge, and training associated with the field. for the majority of jobs within the stem fields, access to formal education is necessary to gain entry into the workforce. access to higher education in these fields, like many other fields, has historically been restricted to men. throughout the journey towards inclusivity, the argument for allowing women access to education has evolved. initially, the role of mothers in educating their sons was the strongest argument for expanding educational opportunities to women (solomon, 1985, pg. 2). over time, educational opportunities, especially at the collegiate level, expanded for women, but these opportunities were frequently unequal to those available to men (solomon, 1985 pg. 43). colleges and universities were commonly divided by sex, with 59% of them being open only to men in 1890 (solomon, 1985, pg. 44). this restriction to the necessary education to join stem fields acted as one of the largest barriers to inclusion for women. from 1890 to 1928, the number of coeducational, degree-granting institutions increased from 29% to 92% (solomon, 1985, pg. 44). this huge jump in the availability of education marked a change in attitudes about the abilities of women and their roles in society. this increase shows a marked improvement in educational opportunity for women and is credited in large part to growing feminist movements of the time. despite this increase in access to education, there still seemed to be large gaps between men and women in most stem fields. going into the 1970s, only 13% of phds in life sciences were awarded to females (ceci and williams, 2010). these shockingly low numbers show that, while the opportunity to receive the necessary education might be available, there were other factors that were holding women back from contributing fully to these fields. opportunity varied within stem fields as well. bystydzienski notes that engineering schools were particularly resistant to admitting women and were structured in such a way as to be unwelcoming to the few women who did manage to get in (2006, pg. 27). one reason women were frequently denied access to stem education was that it was thought to interfere with the traditional roles assigned to women by society. marriage, raising a family, and the work necessary for success in those endeavors, have traditionally been ascribed to women (wharton, 2005, pg. 134). because of this, men were viewed as better suited to filling positions that required a higher time commitment. family matters were not considered to be an infringement of a man’s time. the married family structure does not inherently limit women’s career opportunities and, in some cases, can provide a support system for it. alternatively, these expectations can conflict with women’s abilities to commit their full time and attention to such work. bystydzienski noted this phenomenon, stating that “research and personal memoirs also suggest how positive situations provided support and established safe havens during the sometimes tumultuous early years when a career is established, and, alternatively, how the family and community obligations that women are expected to assume could compromise or even curtail a promising career” (2006, pg. 27). all of this seems to show that women were more welcome in higher education and stem fields as long as it did not inhibit their ability to fulfill their familial duties. 132 | fine focus looking at the structure of society throughout history, one of the largest barriers to women in stem is the distinction between male and female work. there has been a longstanding belief that men and women are better suited for different jobs (wharton, 2005, pg. 167). this institutional ideology has acted as a barrier for women entering many job fields including those associated with stem. circumstances such as world war i and ii, however, allowed women to fill jobs left by men that they would not have had access to previously. the shift in the workplace demographic resulted in that which “has previously been viewed as quintessentially masculine were suddenly endowed with femininity and glamour for the duration” (wharton, 2005, pg. 168). the change in the availability of jobs also shifted attitudes about jobs that were suitable for women. wharton notes, “masculine jobs that had been filled by men prior to the war were relabeled as appropriate for women” (2005, pg. 168). this subsequent shift in attitudes opened up new opportunities in the lives of women. gender cannot be assumed to be the only factor that influenced acceptance into education and, beyond that, into stem fields. many other minorities were restricted in their access to education and had compounding effects with the experience of women. specifically, race has played an interesting role in its relationship with women in stem. while women overall were kept away from work and discovery opportunities in stem, there were even more discrepancies between women of different races and ethnicities. award-winning journalist rona cherry offers more of a look into this in her book, woman in the year 2000: however, in stem fields, a lower percentage of bachelor’s degrees were awarded to females than to males (36 vs. 64 percent). this pattern—in which females received higher percentages of bachelor’s degrees overall but lower percentages of bachelor’s degrees in stem fields—was observed across all racial/ ethnic groups. the gap between the percentage of stem bachelor’s degrees awarded to males and the percentage awarded to females was largest among white students (33 percentage points), followed by pacific islander (28 percentage points), hispanic (25 percentage points), american indian/alaska native (23 percentage points), asian students (21 percentage points), and students of two or more races (21 percentage points). black students (11 percentage points) had the smallest gap between the percentage of stem bachelor’s degrees awarded to males and the percentage awarded to females. while gender differences in stem may be smaller within specific racial groups, these numbers show unequal representation across all listed groups. this is possibly influenced by the fact that household labor is generally more equally shared between men and women in non-white families, allowing for a more equal representation of women in the workforce and in stem (wharton, 2005, pg. 134). additionally, with the combination of both racism and sexism, women of minority race groups were less involved in the stem work fields and educational fields than even women of white descent (solomon, 1985, pg. 76). women of color had to fight even harder to be acknowledged and accepted in the world of stem with less of a resulting outcome.  these results are contradictory to the statistics on women’s pursuit of entering the workforce. volume six | 133 white women listed “balancing work with family responsibilities” as the number one significant issue/challenge female scientists face in their careers (bystydzieński, pg. 75). however, african american families see more balance and equality when it comes to women entering the workforce and dividing labor between genders.  there were again significant differences that contradicted the participation in the stem fields, according to an nels (national educational longitudinal survey) study comparing african american females to white females. in 1988, 59% of african american females were looking forward to science classes and a whopping 70% believed science would be useful in their future, compared to 55% and 65% of white females, respectively. however, in 1988, african americans scored an average 45.12 on their science standardized testing versus white females’ average of 52.73, resulting in an average difference of 7.61 in scores (1990 was even greater with an average difference of 9.08, favoring white females) (nces, n.d.). so why is there such an extreme difference between statistics about women in terms of stem fields and education and breaking down the statistics to different races and ethnicities of women in terms of stem fields and education? as bystydzieński expressed, “this [nels study] examination suggested that simple assumptions about the mismatch between women and science are often based on the experiences of white women. in fact, in the african american community, gender is constructed in a very different way and many of the characteristics that are considered appropriate for females (e.g. high selfesteem, independence, and assertiveness, as well as high educational occupational expectations) are not inconsistent with characteristics that contribute to success in science” (2006, pg. 136). this follows bystydzienski’s findings of african american females not finding as high a disparity between work and family responsibilities as white females. yet despite this desire for more equality in the stem fields, women of color have been overlooked and ignored, even by those one would assume would be on their side. as kimberle crenshaw, lawyer and civil rights advocate, stated in her publication for the stanford law review on intersectionality, “for example, racism as experienced by people of color who are of a particular gender male tends to determine the parameters of antiracist strategies, just as sexism as experienced by women who are of a particular race — white — tends to ground the women’s movement. the problem is not simply that both discourses fail women of color by not acknowledging the ‘additional’ issue of race or of patriarchy but that the discourses are often inadequate even to the discrete tasks of articulating the full dimensions of racism and sexism” (1991, pg. 1252). women of color have found racism amongst those of the same race or the same sex, meaning that there is even a lack of understanding and support among their peers for their desire to pursue  stem careers, let alone acknowledgment for their efforts to enter said career fields. women of color and their voices have been erased (crenshaw, 1991, pg. 1253).  the differences in the numbers cannot be chalked up to or blamed on different attitudes. it comes down to the availability, access, and support different women had when pursuing stem ideas and education. yes, white women were not given the same access to stem fields and acknowledgment for their contributions, but even less opportunity was given to females of 134 | fine focus minority races and ethnicities. they weren’t provided the example of females that looked like them, nor were they held to the same expectations as others in their teachers’ eyes. women want to learn and want to be involved more than society gives them credit for, but without access to support and opportunities, their wants are left abandoned and disregarded and their voices are left erased. women in stem: the current picture historical inequality between men and women’s roles in society has led to a disparity in who was capable of partaking in stem education. this resulted in a staggering underrepresentation of women in stem fields. women had to push past these barriers to show that they were capable of succeeding in these fields. all of this activism has had a clear impact on the involvement of women in stem education. the number of stem doctorates awarded to women was six times higher in 1995 than in 1970 (burke and mattis, 2007, pg. 29). this marked improvement shows that some barriers that previously existed were overcome and we as a society have made great advancements towards closing this gender gap. while women have made great strides across history in efforts to improve the gender gaps in stem education, inequality still exists. despite an increase in collegiate attendance as a whole, women hold a disproportionately low number of undergraduate stem degrees (beede et al, 2011). this suggests that while traditional barriers limiting women’s access to education may have been overcome or removed, there are still barriers specific to stem fields that are inhibiting participation. the discrepancies of female involvement are not limited to education, but rather, also carry forward into the workforce. in a study entitled “women in stem: a gender gap to innovation,” beede et al. found that “although women fill close to half of all jobs in the u.s. economy, they hold less than 25 percent of stem jobs” (2011). this disparity more clearly shows that the gender gap appears more noticeable within stem fields than in other areas. beede et al. went on to show that the disparity in employment in stem fields exists regardless of education level, with the doctorate level having the closest to even proportions where men are still employed at almost double the rate of women (2011). while access to the necessary education was a major contributor to this gender gap in the past, this study shows that new barriers have emerged to take the place of those that limited women previously. this drastic statistic is explained at least partially by the fact that those women that hold stem degrees are less likely to obtain jobs within that stem field than men (beede et al. 2011). in order to remedy this gap, these new restrictions must be identified and novel solutions must be implemented. educational institutions are not the only organizations concerned about the gender discrepancy in stem. several private organizations have already taken steps to address the issue. in 2018, girl scouts of america released thirty new badges for girls ages five to eighteen to earn and two new “journey” programs for girls to participate in — all of which were stem-related. the thirty badges are given to girls for their advocacy and exploration in topics such as cybersecurity, robotics, computer science, space exploration, and more (kelly, 2018). girls in elementary school can earn badges for designing their own robot, creating algorithms for computer games, creating models of the solar system, and even creating their own home experiments to represent ideas such as static electricity or density. sylvia acevedo, ceo of girl scouts of america, stated that “girl scouts are learning how to proactively address some of the foremost challenges of today while also building skills that will set them up for a lifetime of leadership” (kelly, 2018). earning these new badges volume six | 135 encourages girls to take charge of their own stem learning and learn skills, such as problem-solving, the scientific method, computational thinking, and how to receive feedback to expand knowledge and understanding. these badges are the first step into a lifetime of learning, leadership, and stem appreciation. as of 2017, 1.7 million girls across the nation are involved in girl scouts. for many elementary school girls, it is a rite of passage to belong to this organization — earning badges, swapping stories, creating and exploring the world with their female peers and led by female role models in their community. for over 100 years, girl scouts of america has focused on developing young women to be strong ambassadors for their peers, promoting self-confidence, and making the world a better place to live. so why is the addition of thirty new stemrelated badges so significant? the numbers speak for themselves. according to the girl scouts of america, between the years 2012 and 2015, the interest in stem fields has skyrocketed in their young members. 71% of female participants agree that women can be good engineers as well as 58% disagreeing with the idea that engineering is better suited for men than women. participants in the girls steam ahead program across the country (which includes art with the original science, technology, engineering, and mathematics acronym) have grown in their appreciation for the subjects. “between 42% and 56% liked science more and between 39% and 42% liked math more after the program than they had at its start.” as girls learn skills associated with the stem badges and earn and participate in programs that encourage the growth of skills and knowledge, they are understanding the importance of stem in one’s daily lives. with the support of girls and women around them, they are not focused on the gender aspect of stem careers but are focused more on whether or not they have the tools and drive to reach for stem careers. the girl scouts of america have seen an increase in interest beyond just the stem badges and programs they now offer. girls steam ahead participants are increasingly interested in stem careers, with 77% to 90% now wanting more information about careers in math, science, and the arts. between 79% and 83% of those that focused on winning robotics badges are now interested in majoring in a science or engineeringrelated subject in college. girls are becoming stronger leaders in their schools and communities, they are getting more involved with stem in class, and are recognizing that stem careers are not boring or dull, but filled with advancements, discoveries, and revelations that they could be a part of later in life. with the implementation of these new stem badges and programs, girls are more likely to want to pursue college careers in these fields.  girl scouts of america isn’t stopping there. they pledge to “add 2.5 million girls to the stem pipeline by 2025” (2019). programs aimed at young girls are helping promote interest in stem fields. just these past few years have seen an increase in interest due to these new badges. girls are joining these programs for the badges but leaving with so much more: an appreciation for the work that comes with stem as well as a realization that stem isn’t just for men anymore. girl scouts of america hope that these kernels of interest, intrigue, support, and fun that are found in programs related to stem that are designed for young girls will carry with them through high school and into their future careers. 136 | fine focus while programs such as the one offered by the girl scouts provide an optimistic outlook for the future of women in stem, female involvement in stem education tells a different story. the lack of female participation in stem education is seen at almost all levels: more females than males opt out of science and math courses, some as early as sixth grade; fewer women than men pursue degrees in stem fields; and higher rates of women switch out of stem majors than men (burke and mattis, 2007). thus, the interest stops shortly after entering middle school and continues to drop for the next two decades. in fact, although girls at the age of 11 ½ show an interest in stem subjects, they begin losing interest at age 15 (trotman, 2017). a study done by microsoft and krc research found that in the midwest, only 46% of middle school females and 46% of high school females feel powerful doing stem despite 51% of middle school females and 62% of high school females understanding how to pursue a stem career (choney, 2018). and “while the u.s. bureau of labor statistics predicts that technology professionals will experience the highest growth in job numbers between now and 2030, only a fraction of girls and women are likely to pursue degrees that enable them to fulfill these new jobs” (choney, 2018). these high attrition rates could indicate several things, including a lack of motivation to pursue degrees in these fields or the presence of factors that impact the willingness of females to continue their stem interests. the numbers don’t add up. based on the excitement and the stories that girls shared after participation in the new gsa stem program, there should be an increase in the number of girls seeking stem careers. elementary school girls express interest in stem fields, they promise to pursue stem-related careers in the future, and they share a belief that women fit the mold for scientists, engineers, and mathematicians as much as men. and still, the numbers are discouraging. in regard to motivation to join these fields, this lack of participation and high attrition rates are somewhat surprising considering that women in stem jobs earn 33% more than women in comparable nonstem positions (beede et al, 2011). this financial incentive to pursue an education and career in stem would seem to suggest that more women would be following this path. it also suggests that there are other factors within these areas that are discouraging female participation. all of this culminates in stem careers that are lacking in the potential contributions of female minds and suggests that today’s society has placed new barriers to the inclusion of women within stem fields. the attention now turns to why the numbers don’t increase, but rather decrease as time continues. the national survey of science and mathematics education (nssme) from december 2018 provides information that might lead to an answer. only 34% of the elementary teachers surveyed have had courses in earth, life, and physical science. 59% have only had one or two courses in these three areas. in reference to elementary teachers’ preparations for mathematics, only 7% have taken courses in the following subject areas: algebra, geometry, number and operations, probability, and statistics. the majority group, 53%, have only had courses in one or two of these subject areas — all subject areas that the national council of teachers of mathematics recommends elementary mathematics teachers take in their college careers.  this is reflected in the preparedness the surveyed elementary teachers have reported in each subject. 77% report feeling very well prepared for reading/ language arts subject matter with mathematics following behind with 73% describing themselves as very well prepared. however, only 31% report feeling very well prepared for teaching science volume six | 137 curriculum. while 58% of high school teachers feel very well prepared to develop students’ conceptual understanding of science ideas, only 23% of elementary teachers feel very well prepared and only 9% of elementary teachers feel very well prepared to develop student’s awareness of stem careers. this means that elementary school-aged girls must find their intrigue in stem-related career fields outside of the school. they are not getting proper support in developing their own understanding of stem subjects or getting resources about stem careers from their teachers and from their schools. if teachers are not properly prepared for teaching stem subjects at the elementary level, they are less likely to promote the preparation in their students. the lack of understanding when it comes to science and math leads to a lack of conceptual understanding in students and, consequently, leads to a lack of interest in these subject fields further down the line, despite continuous reports of secondary level teachers having more understanding and preparedness in their respective subjects. this could be a major source of the lack in pursuit of stem field careers by women.  when asked how often elementary teachers spend on science and mathematics instruction, only 17% of k-3 grades taught science all/most days of the week and only 35% of 4-6 grades as well. the majority fall into the category of three or fewer days of the week. the average number of minutes spent on reading/language arts was 82-89 minutes per day. mathematics averages an hour a day and science takes less than half an hour in grades k-3. students are clearly not getting a proportional amount of exposure to stem subjects. this discrepancy could be caused by a multitude of factors. one probable cause is that elementary teachers are feeling unprepared or inadequately prepared for teaching science and mathematics and this is leading to a lack of focus on the subject matter in the daily curriculum. it could, however, be more than just a lack of teacher preparedness and pursuit of stem subjects in the classroom. there isn’t just one cause and one effect at play here. media plays a significant role in the lives of young people across the country and how women are presented in various media is a major influencer as well. how women are presented in the media, and the psychological messages it brings, could be a contributing factor. according to jocelyn steinke, “a study focused specifically on television programs likely to be watched by adolescent viewers and broadcast in 2006 found... female scientists characters were outnumbered by male scientist characters by 2 to 1, appeared in fewer scenes, and were less likely to be shown as independent and dominant” (2017). just a small depiction of women in stem fields in media, such as television and film, feeds into the stereotypes that male stem career holders are the heroes while female stem career holders are less formidable. take the award-winning television show, the big bang theory, which has been known to showcase one of the most diverse casts in televised scientific history. but even dr. amy farrah fowler (played by mayim bialik, who has a ph.d. in neuroscience from ucla) is seen continuously making decisions on what to sacrifice: her work or her personal life, and is often the butt of the jokes on the hit sitcom. 138 | fine focus media is often seen as a reflection of society, of culture, of truth, but “the lens through which we receive these images is not neutral but evinces the power and point of view of the political and economic elites who operate and focus it” (gamson et al., 1992). it is not a reflection of the truth at all, rather what those behind the camera want the audience to believe is the truth, and their version of the truth leads to the lack of strong, independent, formidable female characters in stem-related activities or careers. “thus, exposure throughout childhood and into the adolescent years to media portrayals that support and perpetuate a socially constructed masculine image of these fields may elicit gender biases that directly and indirectly affect adolescent girls’ views of who belongs in stem” (steinke, 2017). sixty percent of girls are more likely to pursue a career in stem fields if they knew for certain that men and women were equally employed in these professions (trotman, 2017). the media doesn’t portray equal employment in stem professions and, sadly, this is indeed the truth. despite women making up half of the college-educated workforce in the united states, only 28% of women make up the science and engineering workforce, including the fields of computer science and physical science (national girls collaborative project, 2018). further surveys indicate only 9% of the 2015 mechanical engineering workforce were women (nsb, 2018). despite the significant increase in female careers in stem fields since the early 1990s, the numbers don’t indicate equal employment rates between men and women. this doesn’t bode well for the 60% of girls found in trotman’s study.  unsurprisingly, this workforce gender disparity is seen at highly variable levels across the diverse areas of stem. by taking a closer look at female participation in these fields, it might be easier to speculate what factors could be causing this inequality to occur. figure 1 shows females’ participation by field as of 2008 (hill et al, 2010, pg. 14). in order to evaluate this further, these fields have been broken down into three broad categories: life science, computer science, and mathematics and engineering. certain stem areas, such as those within the life sciences, have seen a marked improvement in female participation and, in some cases, approximately equal participation to men. burke and mattis found that roughly 53% of bachelor’s degrees in biology are awarded to females (2007). this improvement is a testament to the initiative of past movements to incorporate women into stem and shows that equality is possible within stem fields. having seen the nearly equal proportion of women and men in this area, it is hardly shocking that 57% of females in stem jobs are in life and physical sciences (beede et al., 2011). both of these statistics may be indicative of the importance of representation within the classroom and the field. despite these relatively high numbers, the rates at which these degrees are being used for field-related jobs are significantly lower. one study found that “women earned 31.3% of chemistry ph.d. degrees between 1993 and 2003 but in 2002 were hired for only 21.5% of assistant professorships” (ceci, williams, and barnett, 2009, pg. 218). these statistics show that regardless of academic achievement, there are societal and cultural factors that are impacting women’s participation in stem. the improvements in life and physical sciences are balanced out by the acute inequality in the proportion of women in fields such as engineering and computer sciences. in the case of engineering, it is “the secondlargest stem occupational group, but only about volume six | 139 figure 1. women in selected stem occupations, 2008. all occupations are self-reported. source: u.s. department of labor, bureau of labor statistics, 2009, women in the labor force: a databook (report 1018) (washington, dc), table 11. 140 | fine focus one out of every seven engineers is female” (beede et al, 2011). one major contributor to this low rate of participation in the workforce is the gap that exists in education. unlike life sciences, where the percentage of degrees earned is fairly equal between the sexes, only 20% of engineering degrees are earned by women (burke and mattis, 2007). low rates of females earning degrees in engineering seems to indicate that the major issues limiting equal participation in this field stem more from educational systems rather than hiring or workplace institutions. while these percentages are concerningly low, there has actually been an improvement in engineering degrees awarded to women and participation in the field. in 1970, only 1% of engineering degrees were awarded to women, and this statistic rose to 9% by 1996 (christie et al. 2017). this continual growth of women within the field provides a basis for a positive outlook for women’s participation in engineering in the future. this rise in degrees earned was accompanied by an increase in female educators in the field. in fact, by 2006, the number of female faculty in engineering programs had risen from 1% in 1976 to 16-25% (ceci, williams and barnett, 2009, pg. 218). these improvements indicate that strides towards inclusion are making an impact. they may also indicate a connection between the role of diverse representation among faculty, and minority participation. despite these vast improvements, the increases in faculty hiring are not proportional to the increase in the pool of female applicants for these positions (ceci, williams and barnett, 2009, pg. 218). this mimics the same patterns that were seen in the life and physical sciences of increases in earned degrees not translating into a movement towards gender equality in hiring. in computer sciences, female participation has actually been declining in recent years. in 1984, women represented 30% of computer science undergraduates in 1984 but that number decreased to only 20% in 1999 (burke and mattis, 2007). this trend does not follow with those established within the life sciences and engineering fields. this decline in female participation is not just limited to education but is seen in the job sector as well. within the workforce, women are almost twice as likely as men to leave jobs in computer science fields (burke and mattis, 2007). this unusual pattern seems to indicate that the barriers to women within these fields may exist in different forms, or present themselves at different levels. these factors indicate that while traditional barriers, such as the outright barring of women from a formal education, may not be present anymore, there is still something preventing inclusivity in these fields. there are many factors that have been proposed to have contributed to or have caused this inequality. these factors include social and cultural pressures, perceived competence, intrinsic bias, and establishments incorporated in stem being exclusionary to women. while these factors may vary in their impact on this issue, it is necessary to consider as many contributors as possible in proposing potential solutions. while past analysis of this gender gap in stem has heavily considered potential biological differences, experts trying to understand the gender gap are now focusing on the impact of social pressures. ceci, williams, and barnett addressed this shift in their paper “women’s underrepresentation in science: sociocultural and biological considerations” by stating that increases in female participation in stem fields over the past 30 years “are evidence of the strength of cultural factors in determining such outcomes, because biology has not changed over this period” (2009). instead, it is more likely that these gender differences arise from differing expectations based on biological sex (ceci, williams, and barnett, 2009). despite the fact that biological differences volume six | 141 between men and women have been shown to be fewer and of less magnitude than had been previously assumed, the implications of these assumptions are still present in our society (wharton, 2005, pg. 24) another social factor that may impact women’s desire to join stem fields is the idea of an identity threat. this encompasses the fear of being marginalized within a field or the fear of fulfilling negative stereotypes of being in that field (cheryan and plaut, 2010). in this vein, just the possibility of being discriminated against or being perceived negatively acts as a deterrent to those who might otherwise be interested in joining different stem fields. even for women who choose to pursue this path, feelings of a social identity threat “can discourage women from persisting in these fields” (cheryan and plaut, 2010). not only does this factor potentially impact enrollment rates, but it could also contribute to the higher attrition rates seen among women in stem. individual cultures may also impact the achievement of women in stem fields. studies have found that “large cross-national variation in sex differences… suggests that culture rather than biology is involved because the observed patterns are not otherwise explicable” (ceci, williams, and barnett, 2009, pg. 225). this variation between the achievement of women in stem fields across cultures hints that this may be a factor that needs to be considered further. ceci, williams, and barnett stated that all of the data regarding sex differences suggests that “culture may play a major, though poorly understood, role in creating proximal differences that lead to differences in stem fields” (2009, pg. 226). the institution of education is almost undeniably influenced by these societal constructions of gender. this can be seen in the application of expectations associated with various skills and predispositions. this differentiation between gender can be explained by gender polarization, which states that males and females have distinct roles and interests and the crossing of those boundaries is somehow wrong (wharton, 2005, pg. 34). these schemas can influence the path individuals choose to pursue. hill et al stated that “children — girls especially — develop beliefs that they cannot pursue particular occupations because they perceive them as inappropriate for their gender” (2010, pg. 22). in addition to impacting students choices, these roles and expectations can result in differential treatment based on sex. in an educational context, studies have found that teachers in stem fields “provided boys with more formal and informal reward and support, and a good effective environment in which to learn” (ceci, williams, and barnett, 2009, pg. 228). in comparison “girls were largely ignored” within the same classrooms (ceci, williams, and barnett, 2009, pg. 228). this neglect in the classroom can leave females with a sense they lack belonging in stem fields and discourage them from continuing within a given field. institutional factors such as the gender makeup of the classroom and faculty may also influence participation within a field. in fact, wharton found that the “sex composition of the faculty and the type of institution a student attends are related (2005, pg. 67). while this refers to colleges and universities as a whole, the same principle can be applied within specific fields such as those in stem. this lack of representation within collegiate faculty only worsens at higher levels. “male and female students attending the most elite colleges and universities are least likely to be taught by female faculty members” (wharton, 2005, pg. 67). this lack of diversity within stem education can lead to students feeling as though they do not belong in the field. a study by cheryan and plaut explores the impact of the perception of the stereotypical student within a field on other’s willingness and desire to join that field. they found “the extent to which a person’s own perceived traits and attributes overlaps with 142 | fine focus these academic prototypes are related to improved attitudes toward the field” (2010). based on this and the knowledge that men have historically dominated stem fields, it is logical that women may be less inclined to pursue an education and career in these fields. many girls choosing their educational path state that they are not interested in pursuing stem (hill et al, 2010, pg. 21). this lack of interest is likely impacted by several factors, including a lower perception of their own aptitude for the area. in assessing one’s own ability, “girls assess their mathematical ability lower than do boys with equivalent past mathematical achievement” (hill et al, 2010, pg. 21). this lower assessment of ability may impact females’ perceptions of their ability to succeed in stem and, in turn, diminish their interest in exploring a stem education. this perception of being capable of succeeding in stem can be influenced by whether an individual perceives these skills as being learned or innate. hill et al. found that “when a girl believes that she can become smarter and learn what she needs to know in stem subjects — opposed to believing that a person is either born with science and math ability or not — she is more likely to succeed in a stem field” (2010). this information suggests that the way stem is presented to students matters in their willingness and interest in pursuing it at the higher education level. in a similar vein to underrating their abilities in stem topics, females also have lower expectations compared to men that they will succeed in stem as a whole (cheryan and plaut, 2010). the undervaluing of their skill and their potential achievement have the power to take a great toll on women’s interest in continuing in stem education or into a stem career. as girls venture more and more into what were traditionally male-dominated career fields, they still find barriers placed upon them at every turn. yes, the stereotypes that were previously attached to stem careers are starting to fade away as girls and women discover the benefits to pursuits in stem, but the retention rates are lacking; the numbers still don’t match. we must look to the future to see how the number of women in stem careers can grow if it’s not too late. “we have to rethink the way we raise our girls,” ceo and founder of the program girls who code, reshma saujani, states. “boys are pushed to take risks; girls are not. in fact, they feel like they have to be perfect at everything they do; they see getting a ‘b’ in math class as bad. we have to teach girls to be imperfect” (choney, 2018). the way girls approach their school work is much different than boys on a cultural, social, and cognitive standpoint. men and women’s brains are different, there is no denying, but the problem comes from outside forces as well, not just internal emotions or cognitive development. there needs to be a wall of support surrounding girls in order for them to consider approaching male-dominated career paths, especially stem. the future of women in stem the failure of the current system in stem education to encourage equal participation of women necessitates actions to diversify the individuals pursuing education in these fields. the ever-present question as to what can be done to mediate this gender inequality within stem fields is one that has brought about an array of potential causes and solutions. along this vein, a variety of programs have been enacted in recent years with the hopes of increasing female participation in stem higher education, as well as increasing retention rates within the programs. these preliminary steps in addressing this issue have had varying degrees of success. nevertheless, they lay volume six | 143 the groundwork for improvements in the future. the specialized high school admissions test given to high school students in new york city found that ninth-grade girls were on average 4.2 points higher on a 100-point scale than their male counterparts (barshay, 2019). when looking at exam scores, females taking the entrance exam made up 40 percent of the top 3 percent, meaning they were less than half of those receiving high marks on standardized exams for stem. looking at the ninth-grade math and science classes, however, females were half of the grades listed as 95 percent and above (barshay, 2019). despite what the admissions test may have led admissions boards to believe, girls were excelling at their classes such as geometry, biology, physical science, and algebra.  this goes completely against former harvard university president larry summers’s statement about how “one reason there are relatively few women in top positions in science may be ‘issues of intrinsic aptitude’” (jaschik, 2005). females are capable of making the cut — the grades and statistics prove it plainly. maybe one reason there are relatively few women in top positions in science (and technology, and engineering, and math) is that women aren’t granted the same amount of access into these programs. university of michigan physicist timothy mckay elaborates that “even the brightest women may not perform at their best when they feel that they are in a stressful environment where women don’t traditionally succeed” (barshay, 2019). women feel this extra pressure to succeed when it comes to their schooling, especially compared to their male counterparts. there is less room for error in women’s mindsets, just as saujani was stating. women “have” to be perfect at everything they do, so much so that even a decent grade, an above-average grade, is seen as failing and an embarrassment. women are in no way suffering from “issues of intrinsic aptitude”, but rather this idea that if they do not succeed completely the first time, then they are failing and the pressure to be perfect is winning.  a step to rectifying this inequity would be to further develop the opportunities to encourage women to join stem programs, jobs, research, and more. the first step to getting women’s feet through the door is to create acceptance of women in stem career paths. however, this is a drastic change that will not happen overnight. the acceptance of new is not something easily accomplished by the typical person, much less an entire professional career integrated into the daily workings of the world. it must start smaller. young girls need to see a path forward in stem. they need to see the opportunities and support that will meet them down the road. as the microsoft survey found, young girls stated that one of the main reasons for their decision not to join stem fields was because of a lack of female role models (choney, 2018). thus, mentors in universities would help ensure young women already interested can move from academia to career. by fostering the pursuit of a degree in the young women who are already interested in stem, universities can bridge the gap between men and women receiving stem degrees. the relationship between faculty and student can be implemented in institutions of higher education through mentorship programs. mentorship programs allow women to not only see themselves represented in stem but also to engage with educators to develop a support system. while these programs can be implemented in many different ways, they allow 144 | fine focus students to gain greater insight into stem topics and culture. lopatto said on this topic “mentors are teachers, but they are also coaches, gatekeepers to a community of scholars, and conduits for passing on the culture of science” (2010). programs with the aim of increasing female participation in stem have been implemented in various universities across the united states, as well as internationally (barabino et al, 2019). these programs allow students to engage in professional relationships to gain knowledge and skills from female educators throughout their time at the institution. these preliminary programs have had promising levels of success, especially when female students were paired with female educators as mentors. barabino et al found that “men could not act as role models in the same way women could” (2019). this information, coupled with the fact that the lack of role models serves as a deterrent to women considering stem education and careers, is evidence of the integral role female educators play in ensuring a future with equal opportunities for all individuals regardless of sex. the importance of having a mentor who could serve as a role model can be seen in a wide variety of improvements for the female students who took part in these programs. higher levels of professional success were shown in women who were mentored by female educators in their field rather than male mentors (barabino et al, 2019). these results suggest that mentorship in the education system carries through into future efforts within the workforce. mentorship programs offer a promising solution to the women pursuing and obtaining positions within stem fields, regardless of their degree earned. in addition to higher professional success, female students with mentors were more self-assured in their abilities and skills within their field (barabino et al. 2019). this is an important finding in addressing the lower evaluation of knowledge and skills seen in female stem students that have been cited as a contributing factor to the gender gap. providing female students with female educators as mentors increased the retention rates of these students by providing them with a stronger sense of belonging in stem (barabino et al, 2019). this increased sense of belonging could overcome the perception of scientists and mathematicians being primarily male that dissuades some from pursuing these fields in higher education. with the overwhelming success of mentorship programs within stem, it is unsurprising that organizations like achieving xxcellence in science (axxs) are considering it as a necessary tool in combating inequality in stem fields at educational and professional levels (burke and mattis, 2007, pg. 38). axxs even includes mentorship programs in a list of concrete steps professional organizations can take to promote leadership, visibility and recognition of women in stem (burke and mattis, 2007, pg. 39). in regards to mentorship, axxs calls professional organizations to 1. establish a national mentorship system for women, 2. establish mentoring as a core activity of professional societies, 3. develop effective mentoring programs, and 4. create a networking website for scientists. all of these steps, while meant for a professional setting, can also be applied in an educational setting. establishing mentorship programs and building networks of support for women on a national scale could be essential to overcoming many of the barriers that dissuade women from continuing in stem education. volume six | 145 as successful as these programs seem to be, they are difficult to implement largely due to the low numbers of female faculty in stem areas. currently, females make up only 4% of the full professors and 6% of associate professor positions in u.s. medical schools (burke and mattis, 2007, pg. 246). this occurrence is not isolated to the graduate school level. in the top 50 chemistry departments in the u.s., women make up only 12% of tenure track positions (burke and mattis, 2007, pg. 246). clearly the teaching profession is not excluded from the disproportionate representation seen in the rest of the field. without the female faculty to enact these mentorship programs, the opportunities these programs offer in mediating gender inequality in stem are lost. thus, to offer a better educational experience to women in stem and increase the success of these students, the treatment and hiring of female faculty need to be considered. while hiring more female faculty seems like a simple solution, the women considering these positions face the same deterrents as students considering stem studies. to address this, women need to be incentivized to consider these jobs or, better yet, the barriers to pursuing a career as a female stem educator need to be removed. smith et al addressed this challenge directly in their attempt to implement a program that addresses the needs of female stem faculty (2018). they primarily focused on the areas of autonomy, competence, and relatedness as a means to improve overall job satisfaction in those faculty (smith et al, 2018). within the timeframe of this study, the percentage of tenure track faculty who were female increased from 32% to 38% largely due to increased job satisfaction (smith et al, 2018). not only did the satisfaction of the female faculty increase, but these diversity programs increased the satisfaction of all individuals involved, regardless of whether or not they directly benefited from the program (smith et al, 2018). the results of this study provide hope for solving issues of gender inequality among stem faculty. an increase in hiring and retention of female faculty members in stem has the potential to increase female participation in stem higher education and in stem careers. implementing programs to increase the experience of female faculty and students in stem higher education is something all colleges and universities should consider as a means of diversifying participation in these fields. these programs take resources to implement, which is why programs such as advance are so important. the nsf advance program seeks to broaden the implementation of evidence-based systemic change strategies that promote equity for stem faculty in academic workplaces and the academic profession (nsf, 2019). this is accomplished by providing grants for work that is being done in accordance with that goal (nsf, 2019). the existence of funding like this and the strong focus that is being cast on this topic make now the perfect opportunity for higher education institutions to evaluate what they are doing to promote female participation in stem. the programs initiated under advance and other similar initiatives have produced data that give insight into some key characteristics found in successful programs. responses from the national conference for women in stem disciplines, which was funded by advance, clearly outlined several of these factors. the survey found respondents sought more flexible hiring practices for stem faculty and promoting a healthy work-life balance (burke and mattis, 2007, pg. 35). these may seem like broad goals that would benefit all faculty regardless of gender and, in fact, that may be the better approach in these programs. in general, the consensus at this conference was that “these policies should be available to both women and men equally in the department; the best departments have almost no measures targeted solely 146 | fine focus at women” (burke and mattis, 2007, pg. 35). while this may seem counterintuitive, these initiatives act to make stem faculty positions more accessible to everyone, especially women who have more pressure on them from society to perform housework and raise children. because of this additional workload, factors such as pausing the time clock for tenure track for women who are on maternity leave are critical for providing women with the same possibility of success in following this career path (burke and mattis, 2007, pg. 35). additionally, they addressed hiring factors that may result in women being perceived as less qualified applicants. the information addresses materials for female applicants such as recommendations where women are seen to be described differently and often less enthusiastically than men with the same level of qualification (burke and mattis, 2007, pg. 35). acknowledging these differences allows faculty selection committees to adjust for them and potentially hire more equally qualified women into positions where they can mentor and inspire future women entering stem higher education. mentorship programs have huge potential in promoting enrollment and retention of women in stem higher education. it is this potential that causes organizations such as the nsf, association for women in science (awis), and achieving xxcellence in science (axxs) all to promote mentorship as a means to combatting the current issues that exist as barriers (burke and mattis, 2007; nfs, 2019). in order for these kinds of programs to be feasible, higher education institutions need to prioritize hiring and supporting women educators in stem. creating an atmosphere that is inclusive for female educators paves the path for the inclusion and support of the future contributors to stem. unfortunately, it can be hard to change older voices and minds. it may be easier to start young when children are first learning, growing, and discovering themselves. this is why it is equally as important to change how stem is approached at the elementary level. the rest of this section will be devoted to the changes needed in elementary stem in order for further growth and inclusion of women in stem career fields. the girl scouts of america are on the right track, with their stem pledge to “galvanize a new generation of girls to explore stem and become confident stem leaders”. they started this initiative with the release of thirty stem-related badges for girls ages five through eighteen to earn and two new stem programs that will integrate girls into new stem projects. this is an initiative that is needed for young girls — an initiative that changes the game and evens out the playing field. it gives girls the opportunities they might be denied later down the line in their careers, or even opportunities not granted by their teachers in their everyday classrooms. as stated before, programs and opportunities like this are encouraging females who want to pursue stem career fields. but it’s not just the girl scouts of america’s responsibility to promote new generations of girls as stem leaders. if they don’t have support, their efforts will not amount to anything and the cycle will only continue. stem needs to be more predominant in the classroom, and this starts with teachers being ready and wanting to include stem in the everyday classroom. teachers don’t want to teach the unfamiliar. they want to stick to what they know, a natural human instinct. in order for teachers to want to teach stem subjects, they must feel confident in their abilities volume six | 147 and understanding of said subjects. as stated earlier, the national survey of science and mathematics education reported that only 34% of teachers have taken collegiate level courses in all three of the recommended sciences: life, physical, and earth. a simple switch would be improving this number and making sure 100% of teachers have taken at least one course of all three recommended science subjects. just that alone can help improve teachers’ confidence in teaching science. the same can be said about mathematics. universities could follow the nssme’s recommendation of five mathematical courses for teachers: algebra, geometry, number and operations, probability, and statistics. having at least one course in engineering and another in technology would allow preservice teachers the familiarity of the topics and help improve their confidence in teaching said subjects in their classrooms. once teachers are adequately prepared to teach stem subjects, the next step is for teachers to do just that: teach stem. however, stem is an entirely different world than english/language arts and thus cannot be approached in the same way. there are two helpful guides to teaching stem: student-centered instruction and inquiry-based learning. student-centered instruction is rooted in the constructivist theory of learning and is a newer idea in the educational world. according to edglossary. org, student-centered learning includes the following important characteristics: 1. teaching and learning are “personalized,” meaning that it addresses the distinct learning needs, interests, aspirations, or cultural backgrounds of individual students. 2. students advance in their education when they demonstrate they have learned the knowledge and skills they are expected to learn (for a more detailed discussion, see proficiency-based learning). 3. students have the flexibility to learn “anytime and anywhere,” meaning that student learning can take place outside of a traditional classroom and schoolbased settings, such as through work-study programs or online courses, or during non-traditional times, such as on nights and weekends. 4. students are given opportunities to make choices about their own learning and contribute to the design of learning experiences. allowing students the flexibility and personalization of learning will give students the opportunity to be more involved with the subjects (particularly stem subjects as they tend to be more hands-on) as well as feeling a larger responsibility for their success in learning. it also allows teachers the opportunity to understand where each of their students is personally and allows them to develop content and instruction that helps students exactly where they need help. it takes the focus off of the teacher and places it on the students, as the name suggests and as the learning should be. the second teaching strategy that is important to adopt in stem subject teaching is inquiry-based teaching. according to the center for inspired teaching, “inquiry-based teaching is a pedagogical approach that invites students to explore academic content by posing, investigating, and answering questions” (pg. 1). inquiry-based teaching is designed for all subjects at all ages and pairs nicely with studentcentered learning as neither includes direct instruction from teachers to students. inquiry-based teaching allows students to generate their own questions and explore possible answers on their own with guidance from their teacher instead of direct explanation and 148 | fine focus lecturing. inquiry-based classrooms are designed to help students develop skills beyond just understanding of instruction. “it teaches students to pose difficult questions and fosters the desire and skills to acquire knowledge about the world. students are given opportunities to take ownership of their own learning, a skill necessary for one to succeed in college and in most professional settings” (pg. 1).  having student-centered teaching and inquiry-based learning allows students the opportunity to dig deeper with their learning, to understand the importance of struggling to find a solution, to acknowledge that there isn’t always one right answer, and to feel empowered by their own learning. developing these approaches to stem subjects in the classroom will allow students, especially girls, a deeper appreciation of the subjects and what they are learning than they would should they just be lectured and told the answers.  this is exactly what the girl scouts of america are encouraging with their stem badges and programs: the enjoyment of stem subjects by allowing girls the opportunities to take charge of their own learning. having student-centered teaching and inquiry-based learning can help support this promotion of girls learning and enjoying stem beyond just what their textbooks teach them. having this support both in and out of the elementary classroom can help girls recognize their strengths in the stem subjects and set them up for a future career in the stem fields. conclusion girls deserve to explore stem. women deserve to be involved in stem. the voice is lacking, and the inclusion of women cannot and will not come overnight. but there are many tiny steps that can set up women for success from the beginning and implementing these ideas for girls as early as kindergarten will allow for the future growth of women in stem career fields. “i was taught that the way of progress was neither swift nor easy,” marie curie, first female nobel prize winner, once said. women’s voices are missing in all elements of science, technology, education, and mathematics. for now, the wheels of progress for the inclusion of women in stem education fields are slowly turning. women in the past fought and advocated for their inclusion in stem. the struggle started with a lack of access for women to higher education (solomon, 1985, pg 44). yes, more women were allowed into colleges and universities starting in the 1920s but welcomed may not be the right verb to express their inclusion.  despite their attendance in higher education institutions, in the 1970s less than 15% of life science ph.d.’s were awarded to women (ceci & williams, 2010). if that was not enough, marriage and family came into play as a barrier between women and stem fields, as women were seen better fit for jobs that focused on family life. these were just a few of the issues in history, not to include the major barriers faced by women who were also part of another minority group. scores in science and mathematics tended to favor white men the most, but even white women were seen to outscore women of color (nces, n.d.). women of color and their voices have definitely been erased (crenshaw, 1991, pg. 1253).  these barriers have shifted and evolved to lead to the current situation of women in stem education fields. currently, women are receiving much more recognition for their work than historically speaking, and they are receiving degrees at significantly higher rates than previously seen. despite this, women hold less than 25% of stem jobs as careers despite making volume six | 149 up over half of those in the current workforce (beede et al., 2011). this means that men are being employed at double the rate of women in stem career fields. this imbalance indicates issues within the field that need to be mediated on many levels. there are current stances to help level out the playing field, especially at a younger age. an in-depth look at the girl scouts of america reveals that their thirty new stem-related badges and two new stem programs have geared more girls into seeing themselves as future scientists, engineers, mathematicians, and technicians (kelly, 2018). up to 90% of girls who participated in one of the new stem programs agreed that they wanted more information about careers in math, science, and the arts (girl scouts of america, 2018).  despite the efforts currently being made, there is still a lack of female voice in stem education and careers. girls as early as sixth grade are losing interest in stem, with less than half of middle school females and high school females feeling capable of doing stem (choney, 2018). on top of the dwindling numbers still occurring, current elementary education teachers are not properly equipped to teach stem subjects in their general education classrooms, resulting in a lack of time on those subjects. in fact, 93% of teachers have not taken the 5 math classes at the collegiate level that the national council of teachers of mathematics recommends (nssme, 2018). another current issue involving women in stem career fields is the manner in which female scientists and mathematicians are represented in the media. female scientists on television and in movies are outnumbered by men 2 to 1 and are seen as more dependent and less capable than their male counterparts (steinke, 2017). this means that despite efforts of new programs and more acceptance of women at higher educational levels, girls are still not seeing stem careers as a viable option for themselves, nor are they seeing proper role models already in these fields for them to look up to. this is why we must look toward the future and take steps to start making changes today. women’s voices are lacking in stem education and stem careers. while an immediate solution will not just happen and women will not automatically be accepted and welcomed into stem fields, as marie curie pointed out about a century ago, small steps can be made toward a brighter future. first, there needs to be the understanding that women are put under more pressure to be perfect (barshay, 2019). this expectation of perfection leads girls to undervalue their own skills compared to equally qualified boys. the acknowledgment that women do not suffer from “issues of intrinsic aptitude” for this very reason is important as well (jaschik, 2005). women are capable of doing the work, they just are not always fully recognized for their successes due to the high pressures placed on them by themselves and others. next, there should be more mentoring involved in stem fields. a large reason behind girls and women not wanting to be a part of the stem career is the lack of role models they have to look up to. having mentoring programs in place at the collegiate level and higher educational institutions will help bring women together as students and teachers in recognition that we must all learn from one another. mentoring programs can lead to higher retention rates, especially women (beede et al., 2019).  150 | fine focus in addition, there should be efforts made to increase the number of female faculty at the collegiate level. this goal can be addressed by improving the working conditions of female faculty, who regularly face the same barriers that affect their students.. as of now, females are only 4% of the full-time professors in medical schools and only 12% of the tenure track (burke and mattis, 2007, pg. 256). having more women on the tenure track and as overall faculty members will help in the retention rate as well as encourage the mentorship programs that should be put into place. it will also help with diversifying colleges and universities and providing strong examples for students, the goal of this being the ultimate diversity of individuals working in stem fields. at the elementary level, there needs to be an increase in knowledge for teachers, starting with teachers taking the recommended mathematics and science classes. for example, 100% of the teachers should have all 5 mathematics courses at the collegiate level that are recommended by the national council of teachers of mathematics. having teachers more knowledgeable on stem subjects will increase their willingness to teach and confidence in teaching stem subjects in class. if teachers are more comfortable with the material they will be better able to translate it to their students and hopefully get more students interested in those topics. after this, teachers must take a student-centered learning approach and inquiries approach to teach stem at the elementary level. doing so will help young students, especially girls, feel more comfortable and confident in their abilities while spreading their understanding and knowledge of the topics (center for inspired teaching, pg. 1). improving girls confidence in their stem abilities has the potential to increase retention rates in higher levels of stem education. building on society’s seeming willingness to change, these are some of the many small steps that must be taken in order to bring more women into stem. as this world becomes more and more science and technology-based, this goal is more important than ever. as was stated in the research book, women and education, “i would like to see more women in science, not only for the sake of the many women who could do talented work but for the sake of science, because it would present a different face to society if the women in it were sufficiently numerous and confident not to follow the male models or definitions” (maccia, 1975, pg. 158). acknowledgements i would especially like to thank my co-author, kyra christiansen. this project would have been nearly impossible without her help and support. additionally, i would like to thank my thesis advisor, robbie williford, who acted as a supportive sounding board through this entire process. finally, stacy christiansen, lydia kotowski, chris harbeson, and rebecca conrad deserve thanks for their advice and assistance in editing. volume six | 151 152 | fine focus references 1. andersen, m.l. 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(2005). the sociology of gender: an introduction to theory and research malden. ma: blackwell. grace j. miller1 kaley necessary2 robert burchell 2 yui iwase2 nicole l. lautensack2 blake russell2 erik g. holder2 emma e. knee2 w. matthew sattley2 1 school of natural and applied sciences, taylor university, upland, in 2 division of natural sciences, indiana wesleyan university keywords: moringa oleifera, moringa stenopetala, antibacterial, plant extract, disk diffusion test, minimum inhibitory concentration, medicinal plant © 2024 miller, necessary, burchell, iwase, lautensack, russell, holder, knee, sattley. fine focus, 10(1), 58-73. doi: 10.33043/ff.10.1.58-73. shared with cc-by-nc-nd 4.0 license. manuscript received 19 february 2023; accepted 11 april 2023 positive effects of moringa oleifera and moringa stenopetala seed and leaf extracts against selected bacteria https://creativecommons.org/licenses/by-nc-nd/4.0/ miller et al. | positive antibacterial effects of moringa oleifera and moringa stenopetala seed and leaf extracts 59 59 abstract moringa oleifera is hailed as the “miracle tree” for its impressive catalog of nutritional, medicinal, and water purification benefits. a (sub)tropical plant with a rapid growth rate (3–5 m in a single season), moringa has proven beneficial in multiple ways in developing regions around the world. in addition to its high nutrient content and water clarifying properties, moringa seed and leaf extracts have shown potential as natural antibacterial agents. based on this, we anticipated that extracts from multiple species of moringa would exhibit potentially useful antibacterial properties against a range of bacterial species. to explore this, both disk diffusion and minimum inhibitory concentration (mic) culture techniques were employed to assess the inhibitory effects of seed and leaf extracts from m. oleifera and m. stenopetala against species of bacteria commonly used in research and teaching laboratories. aqueous seed extracts from both moringa species showed broad-spectrum activity but were especially effective at inhibiting the growth of gram-positive bacteria, including species of staphylococcus, streptococcus, and bacillus. moringa leaf extracts also exhibited antibacterial activity, with ethanolic leaf extracts showing greater efficacy than aqueous leaf extracts in disk-diffusion assays. temporary acidification (1 h at ph 2) of moringa seed and leaf extracts had a detrimental effect on their antibacterial activity. mic assays using moringa leaf extracts also showed more pronounced inhibition of gram-positive bacteria (mic = 12.5% v/v) versus gram-negative species (mic = 25% v/v). these results are of particular relevance in tropical areas where pharmaceutical drugs are scarce but moringa is widely available and often used as a nutritional supplement. moreover, the rising threat of multi-drug resistant pathogens lends greater importance to the study of antibacterial plant products that ultimately may find application in the clinical setting. fine focus | volume 1060 introduction recent explorations in herbal medicine among the scientific community have shown potential for the development of new antimicrobial products from various plant species for use in both developing regions having little access to pharmaceutical drugs and in developed areas that have experienced a rise in bacterial infections caused by antibiotic-resistant strains (2, 3, 4, 7, 17, 28, 30). one such medicinal plant that has experienced a degree of commercialization in recent years is moringa oleifera (also called the “horseradish tree,” “drumstick tree,” and, more recently, the “miracle tree”), a drought-resistant and rapidly growing tree species native to india but which is now widespread in tropical and subtropical regions around the world. growing to a maximum height of about 12 m but usually pruned yearly to ease harvesting, the use of m. oleifera leaves as a nutritional supplement and herbal medicine has been well documented (12, 15, 23, 24, 25, 27). m. oleifera leaf powder contains unusually high levels of protein, iron, calcium, magnesium, potassium, several vitamins (e.g., β-carotene and vitamins b2, b6, and c), and dietary fiber and other complex carbohydrates (15). because of this, m. oleifera products have been incorporated into feeding programs where malnutrition is prevalent. m. stenopetala also has an unusually high nutrient content, but as this species occurs only in northeastern tropical africa, the use of m. stenopetala as a nutritional supplement is limited mainly to regions in which the plant is indigenous (19). in addition to their nutritional benefits, the potential of moringa spp. as new sources of antimicrobial products is becoming more widely recognized (5, 8, 11, 20, 22, 27, 29). extracts of seeds and leaves of moringa spp. exhibit antibacterial properties through direct inhibition of growth in culture-based experiments. as has been shown using leaf extracts in experiments with xanthomonas campestris (13) and erwinia amylovora (14), this inhibition is primarily due to the production of compounds that compromise the structural integrity of the cytoplasmic membrane. several decades ago, eilert et al. (9) described both antibacterial and antifungal activity of moringa extracts and identified 4-(a-l-rhamnosyloxy) benzyl isothiocyanate as an active compound in aqueous preparations. however, other compounds, including phenolic acids, flavonoids, and alkaloids, likely also contribute to the antibacterial activity of these extracts (13). manilal et al. (20) recently showed antibacterial activity of moringa stenopetala extracts on par with clindamycin and vancomycin controls against clinical isolates of methicillin-resistant staphylococcus aureus (mrsa), and several other studies have shown inhibitory activity of moringa extracts against a variety of human pathogens, including proteus mirabilis, helicobacter pylori, and bacillus cereus (5, 11, 20, 27). although documentation of the antibacterial activity of moringa seed and leaf extracts is expanding, the clinical potential of these natural products is unknown, and further testing of these materials is both prudent and needed. based upon studies from other research groups and our own observations (21), we predicted that crude seed and leaf extracts from multiple species of moringa would exhibit broad-specmiller et al. | positive antibacterial effects of moringa oleifera and moringa stenopetala seed and leaf extracts 61 trum activity against a variety of bacterial species. to test this prediction, we compared the growth-inhibitory activity of aqueous seed extracts and both aqueous and ethanolic leaf extracts from two moringa species, m. oleifera and m. stenopetala, against selected species of gram-positive and gram-negative bacteria, some of which are known to cause opportunistic infections. in addition, the minimum inhibitory concentration (mic) of aqueous moringa leaf extracts was determined for several species of gram-positive and gram-negative bacteria. finally, as moringa leaves and seeds are commonly consumed as dietary supplements and could potentially have an inhibitory effect on susceptible members of the intestinal microbiota, we have simulated passage of these plant products through the stomach by testing the effect of a temporary shift to acid ph on the antibacterial activity of both aqueous and ethanolic moringa extracts. abolishment of the antibacterial efficacy of moringa extracts following exposure to low ph would suggest that these plant products can be consumed for nutritional purposes without concern that the composition of the gut microbiota would be detrimentally or otherwise affected. materials and methods bacterial cultures species of bacteria used in this study consisted of strains commonly employed in general microbiology teaching laboratories. gram-positive species included bacillus cereus (atcc 14579), staphylococcus aureus (atcc 29213), enterococcus durans (atcc 6056), corynebacterium xerosis (atcc 373), listeria monocytogenes (atcc 15313), streptococcus agalactiae (atcc 13813), and micrococcus luteus (atcc 4698). gram-negative test bacteria included proteus vulgaris (atcc 6380), yersinia kristensenii (atcc 33639), serratia liquefaciens (atcc 27592), and escherichia coli (atcc 25922). source cultures of all bacteria used to inoculate experimental media were routinely grown in bd bacto™ tryptic soy broth (becton, dickinson and company) at ph 7 and 37 °c. moringa seed and leaf extract preparation seeds of moringa oleifera and moringa stenopetala used in this study were obtained from educational concerns for hunger organization (echo; www.echonet.org, fort myers, florida, usa). leaves from each moringa species were obtained from plants propagated from seeds in our laboratory. to obtain the seed extracts, the dried moringa seed pods were husked by hand, revealing the small, white pit in each pod. the seeds were then ground into a fine powder using a mortar and pestle. for preparation of moringa seed extracts, 3 g of the powdered seed were combined with 15 ml of warm (60 °c) ddh2o and mixed by hand for 5 minutes using a glass rod. the slurry was then transferred to tubes and placed in a centrifuge at 3000 rpm for 10 minutes at room temperature. after centrifugation, the supernatant was extracted using a micropipette and vacuum filtered to remove any remaining suspended particles. the vacuum-filtered extract was then sterilized by pushing through a 0.45 µm cellulose acetate (ca) membrane filter into a sterile petri dish. fine focus | volume 1062 moringa leaves used for extract preparation were washed and dried thoroughly prior to processing. leaf extracts from moringa spp. were obtained by macerating 12 g of leaf tissue in 20 ml ddh2o. this slurry was then filtered through five layers of cheesecloth into two 15 ml conical tubes, which were placed in a centrifuge at 3000 rpm for 10 min. for each moringa species, the leaf extract supernatant was collected, and 10 ml were filtered through a 0.45 µm ca membrane filter into a sterile 17 ml test tube. preparation of the ethanolic leaf extracts was the same as water extraction except that the ddh2o was replaced with 20 ml of 70% ethanol. disk diffusion assays using ethanol-flamed forceps, pre-sterilized blank disks (6 mm in diameter; becton, dickinson and company) were aseptically placed in petri dishes that contained a single, sterile plant extract (either m. oleifera or m. stenopetala seed or leaf extract) in order to test for antibacterial activity using a modification of the disk-diffusion method described by bauer et al. (6). other petri dishes used for soaking blank disks contained either filter-sterilized ddh2o or 70% ethanol to be used as negative controls. all disks were soaked for 15 min before experimental use. to perform the disk diffusion plate assays, each species of bacteria was inoculated onto the entire surface of triplicate plates of difco™ mueller-hinton agar. plates were inoculated from fresh tryptic soy broth cultures (od600 of 0.05) of each bacterial species using pre-sterilized cotton swabs that had been inserted into the cultures and spread in multiple directions on the agar surface to produce a “lawn” of bacterial growth after incubation. disks containing either crude moringa extract or sterile solvent (either ddh2o or 70% ethanol, depending on the experiment) were placed on the inoculated plates in labeled quadrants using ethanol-flamed forceps. four disks were placed on each agar plate: 1) a disk impregnated with m. oleifera seed or leaf extract; 2) a disk impregnated with m. stenopetala seed or leaf extract; 3) a disk infused with 30 µg of the broad-spectrum antibiotic tetracycline (becton, dickinson and company) as a positive control; and 4) a disk containing only sterile solvent (i.e., no moringa extract) as a negative control. once in place, the disks were pressed gently into the agar surface with the forceps to ensure they would not move or become detached when inverted for incubation. all test plates were incubated at 37 °c for 24 h. following incubation, antibacterial activity was assayed by measuring the diameter (to the nearest mm) of zones of inhibition around the disks on each plate culture. measurements of zones of inhibition shown in each graph are averages of triplicate cultures for each bacterial species tested and are expressed as the mean ± standard error. statistical significance (p ≤ 0.05) determinations of growth inhibition among tested groups and controls were performed using one-way analysis of variance (anova) followed by tukey’s honest significant difference (hsd) test. to simulate and test the effect of oral ingestion of moringa leaf and seed products on antibacterial activity, we subjected both aqueous and ethanolic moringa extracts to temporary acidification. for this procedure, the ph of miller et al. | positive antibacterial effects of moringa oleifera and moringa stenopetala seed and leaf extracts 63 each extract was dropped to ph 2 using a sterile solution of 1 m hcl. after 1 h at room temperature (~23 °c), the ph of each extract was raised to neutral (ph 7) using sterile 1 m naoh. sterile blank disks were then soaked in the extracts, and disk diffusion experiments were conducted as described above. minimum inhibitory concentration (mic) assay mic assays were performed in triplicate using moringa oleifera aqueous leaf extract. ten test tubes were used for each mic experiment, with tube 1 initially containing 10 ml of 100% crude moringa leaf extract (obtained as described above) and tubes 2–10 containing 5 ml sterile tryptic soy broth (tsb). a serial dilution was then performed in which 5 ml of leaf extract from tube 1 was aseptically transferred to tube 2, producing a 1:2 dilution. after mixing well, 5 ml from tube 2 was transferred to tube 3 and mixed. dilutions continued in this way through tube 9. after mixing, 5 ml was removed from tube 9 and discarded. this resulted in all 10 tubes having a final volume of 5 ml, with tube 1 containing 100% moringa extract, tubes 2–9 containing two-fold decreasing concentrations of extract, and tube 10 serving as a control containing 100% tsb (i.e., no extract). all ten tubes were then inoculated with a test species of bacteria using a loop (a different 10-tube set was required for each species of bacteria tested for all three trials). after a 24-h incubation (unshaken) at 37 °c, growth in each of the tubes was assessed by visual inspection for turbidity, and the mic for each bacterial species was recorded as the tube containing the highest dilution of extract that showed no growth. the recorded mic was confirmed by transferring a portion of the contents of each tube onto separate plates of tryptic soy agar (tsa) by streaking for isolation and checking for growth after incubation (37 °c for 24 h). results and discussion antibacterial activity of aqueous moringa seed extracts in recent years, several studies have demonstrated the antibacterial activity of extracts from various species of moringa, especially moringa oleifera (5, 8, 11, 13, 14, 16, 18, 20, 21, 24, 26, 29). in agreement with these reports, most of which employed extracts from a single moringa species against one or a few species of bacteria, we observed antibacterial activity of seed extracts made from two different moringa species against a diversity of both gram-positive and gram-negative bacteria, some species of which, to our knowledge, have not been included in previous studies (e.g., enterococcus durans, yersinia kristensenii, serratia liquefaciens, streptococcus agalactiae, and corynebacterium xerosis). disk diffusion assays showed that aqueous seed extracts of both m. oleifera and m. stenopetala inhibited the growth of a wide variety of bacteria, with gram-positive bacteria being especially susceptible. zones of inhibition from moringa seed extracts against staphylococcus aureus were equal in size (~22 mm) to zones of inhibition produced by a tetracycline control disk (a zone of inhibition of >22 mm for staphylococcus spp. indicates susceptibility to tetracycline based on data from the european committee on antimicrobial susceptibility testing [10]). fine focus | volume 1064 growth inhibition by moringa extracts against other gram-positive cocci, including streptococcus agalactiae and micrococcus luteus, produced zones of inhibition that were of similar size (20–25 mm) to those observed with s. aureus (figure 1). for bacillus cereus, an endospore-forming gram-positive rod, the sizes of the observed zones of inhibition by moringa seed extracts indicated significant growth inhibition (p = 1.6 × 10-6) and actually exceeded that from tetracycline at about 20 mm versus 13 mm, respectively (figure 1). another opportunistic gram-positive rod, listeria monocytogenes, was also inhibited by seed extracts from both moringa species. however, with zones of inhibition having an average diameter of 16 mm versus 30 mm, the antibacterial effect of the moringa extracts was less than that observed with the tetracycline control (figure 1). although an inhibitory effect was still evident against them, gram-negative bacteria as a whole showed less sensitivity to aqueous moringa seed extracts than gram-positive bacteria. the four gram-negative bacteria tested were all of the family enterobacteriaceae. of these species, proteus vulgaris, an opportunistic pathogen often responsible for urinary tract infections, had the largest zones of inhibition (~15 mm) and was significantly affected (p = 4.6 × 10-6) by the moringa seed extracts (figure 1). by contrast, escherichia coli, figure 1 antibacterial activity of aqueous seed extracts of moringa oleifera and moringa stenopetala against selected mostly gram-positive bacteria, as compared to tetracycline and sterile water controls. note. the average sizes (from triplicate samples) of zones of inhibition on disk diffusion plate assays are shown. a measurement of 6 mm is a baseline representing the diameter of the disks themselves and equates to no visible zone of inhibition. miller et al. | positive antibacterial effects of moringa oleifera and moringa stenopetala seed and leaf extracts 65 serratia liquefaciens, and yersinia kristensenii exhibited no significant susceptibility (p > 0.05) to the moringa extracts over the sterile water control (data not shown). as was observed with the gram-positive bacteria, there was no significant difference (p > 0.05) between the antibacterial activity of moringa oleifera seed extract versus that of moringa stenopetala. antibacterial activity of moringa leaf extracts in addition to testing the antibacterial activity of moringa seed extracts, we also collected and tested the potential for bacterial growth inhibition using both aqueous and ethanolic leaf extracts. although a significant (p ≤ 0.05) effect was observed over sterile water controls (figure 2a), inhibition of bacterial growth by aqueous leaf extracts was less pronounced than that observed from aqueous seed extracts. this may be due to the antibacterial constituents being more concentrated in the seed extracts than in the leaf extracts, although other explanations, such as differences in the effectiveness of the extraction methods, are also possible. for the gram-positive bacteria tested, zones of inhibition around tetracycline control disks were approximately twice as large in diameter as zones of inhibition using moringa leaf extracts, which ranged from 12–16 mm in diameter (mean = 13 mm) for both moringa species (figure 2a). as was the case using seed extracts, the inhibitory effect of the leaf extracts was generally less pronounced against the gram-negative bacteria tested, which had zones of inhibition ranging from 7–11 mm in diameter (data not shown). to potentially augment the antibacterial effect of the moringa leaves, we repeated the test using ethanolic rather than water-based leaf extracts. against the same bacteria, ethanolic moringa leaf extracts produced larger zones of inhibition and, thus, a greater antibacterial effect than observed with water extraction. other studies have shown a similar increase in antibacterial activity of ethanolic over aqueous m. oleifera extracts, and hagos et al. observed greater antibacterial activity from methanol extraction over water extraction of m. stenopetala leaves (16, 26, 31). in the present study, sizes of the zones of inhibition using ethanolic leaf extracts from both moringa species were similar and had average diameters ranging from 12–21 mm (mean = 17 mm) (figure 2b), resulting in an approximately 30% increase in efficacy over aqueous leaf extracts. effect of acidification on the antibacterial activity of moringa seed and leaf extracts moringa seed pods and leaves can be an important nutritional supplement for peoples living in tropical and subtropical regions around the world (15, 23). although considerable work has been done to characterize the nutritive properties of moringa products when prepared in various ways for human consumption (15), to our knowledge, the potential effect of ingestion—and in particular the temporary acidification that occurs during passage through the stomach—on the antibacterial properties of moringa has not been investigated. in testing the effect of low ph on the antibacterial properties of moringa extracts, a significant decrease (p ≤ 0.05) in antibacterial activity occurred against most species and under most conditions as a result of temporary acidification. fine focus | volume 1066 figure 2 antibacterial activity of a) aqueous moringa leaf extracts, and b) ethanolic moringa leaf extracts against selected gram-positive bacteria, as compared to tetracycline and sterile solvent (ddh2o for part a; 70% ethanol for part b) controls. note. the average sizes (from triplicate samples) of zones of inhibition on disk diffusion plate assays are shown. a measurement of 6 mm is a baseline representing the diameter of the disks themselves and equates to no visible zone of inhibition. miller et al. | positive antibacterial effects of moringa oleifera and moringa stenopetala seed and leaf extracts 67 as aqueous seed extracts and ethanolic leaf extracts both showed strong antibacterial activity in previous experiments, we limited testing to these preparations from both m. oleifera and m. stenopetala against a selection of gram-positive and gram-negative bacteria. after 1 hour at ph 2 (23 °c), activity against the tested gram-positive bacteria was significantly (p ≤ 0.05) diminished, and in several cases entirely abolished, for aqueous seed extracts of both m. oleifera and m. stenopetala versus the untreated (nonacidified) control extract (figure 3a, b). the same trend was observed for the seed extracts against the tested gram-negative bacteria; in every case, antibacterial activity was severely diminished or entirely absent after the acidification treatment (figure 3c, d). similarly, with the exception of staphylococcus aureus among the gram-positive bacteria (figure 3e) and escherichia coli among the gram-negative bacteria (figure 3g), acidification of moringa ethanolic leaf extracts consistently produced smaller zones of inhibition around the test disks compared to the untreated controls (figure 3e–h). against the gram-negative bacteria in particular, zones of inhibition were entirely absent around most of the disks containing acid-treated extracts (figure 3g, h). these results suggest that antibacterial constituents occurring in seeds and leaves of moringa spp. are acid-labile and are likely broken down by digestive chemicals of the stomach when ingested (1, 9, 12). this brings into question the curative potential of ingested natural moringa seed and leaf products for the treatment of gastrointestinal bacterial infections, such as gastric ulcers caused by helicobacter pylori [even if antibacterial activity has been demonstrated in vitro (11)], and is a topic warranting further study. mic assays using moringa leaf extracts the results of minimum inhibitory concentration (mic) assays confirmed the antibacterial effect of moringa leaf extracts. crude aqueous leaf extracts from m. oleifera inhibited the growth of all bacteria tested to varying degrees. as with the disk diffusion test, the mic assays showed that gram-positive bacteria were generally more susceptible to the inhibitory effect of m. oleifera leaf extracts, with most species showing susceptibility at >50% dilution of crude extract (table 1). a representative mic preparation is shown in figure 4. only two of the four gram-negative bacteria tested (p. vulgaris and y. kristensenii) were inhibited beyond a 50% dilution of crude leaf extract, and no gram-negative species were inhibited beyond a 25% dilution (table 1). conclusions the results of this study and others cited in this report show that seed and leaf extracts of multiple species of the fast-growing, tropical to subtropical tree moringa exhibit clear antibacterial properties. both disk diffusion and mic assays indicated that among the species tested, gram-positive bacteria were more susceptible to moringa-derived antibacterial compounds than were gram-negative bacteria, possibly due to the presence of an outer membrane in the cell wall of the latter. moreover, this work presents evidence that the antibacterial components of crude moringa extracts are acid-labile and fine focus | volume 1068 figure 3 effect of acidification on the antibacterial activity of moringa seed and leaf extracts. note. the average sizes (from triplicate samples) of zones of inhibition on disk diffusion plate assays are shown. a measurement of 6 mm is a baseline representing the diameter of the disks themselves and equates to no visible zone of inhibition. a) moringa oleifera aqueous seed extract against selected continued on page 69 miller et al. | positive antibacterial effects of moringa oleifera and moringa stenopetala seed and leaf extracts 69 bacterial species gram reaction two-fold tube dilution series* 1 2 3 4 5 bacillus cereus positive + corynebacterium xerosis positive + + enterococcus durans positive + + + listeria monocytogenes positive + + micrococcus luteus positive + + + staphylococcus aureus positive + + streptococcus agalactiae positive + + + escherichia coli negative + + + proteus vulgaris negative + + serratia liquefaciens negative + + + yersinia kristensenii negative + + table 1 antibacterial minimum inhibitory concentration (mic) assay results for moringa oleifera aqueous crude leaf extract. note. *although the dilution series for all species tested contained 9 tubes, the results for only five tubes are shown because growth occurred for all species in tubes 6–9; growth also occurred in every case in tube 10, a positive control containing no moringa extract. the concentration (v/v) of total extract in each tube was as follows: tube 1, 100%; tube 2, 50%; tube 3, 25%; tube 4, 12.5%; tube 5, 6.25%. (+) growth present; (–) growth absent. gram-positive bacteria; b) moringa stenopetala aqueous seed extract against gram-positive bacteria; c) m. oleifera aqueous seed extract against selected gram-negative bacteria; d) m. stenopetala aqueous seed extract against gram-negative bacteria; e) m. oleifera ethanolic leaf extract against gram-positive bacteria; f) m. stenopetala ethanolic leaf extract against gram-positive bacteria; g) m. oleifera ethanolic leaf extract against gram-negative bacteria; h) m. stenopetala ethanolic leaf extract against gram-negative bacteria. controls are sterile ddh2o (a–d) or sterile 70% ethanol (e–h). continued from page 68 fine focus | volume 1070 subject to inactivation by conditions of low ph. thus, although still highly nutritive, it is likely that the antibacterial properties of moringa plant products are diminished upon ingestion. antibacterial compounds isolated from moringa seeds and/or leaves may prove beneficial and find application in the clinical setting, either in native form or by contributing to the development of highly effective semi-synthetic pharmaceuticals. the many nutritional benefits of moringa products have been well documented for some time and have gained considerable commercial promotion in recent years. with the rise in antibiotic resistance reaching alarming levels in the contemporary healthcare sector, the search for new antimicrobial drug options is of utmost importance. there could hardly be a more pertinent time than now to invest in the study of promising alternatives to traditional antibiotics, including those offered by species of moringa and other medicinal plants. acknowledgments the authors thank the iwu division of natural sciences and the iwu hodson research instifigure 4 representative minimum inhibitory concentration (mic) assay using crude aqueous leaf extract from m. oleifera. note. a two-fold mic serial dilution protocol was employed using 9 tubes, as well as a positive control containing no moringa extract; the numbers indicate the % dilution of the extract. miller et al. | positive antibacterial effects of moringa oleifera and moringa stenopetala seed and leaf extracts 71 references 1. adane, l., teshome, m., & tariku, y. 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(2019) in vitro antimicrobial activity of different solvent extracts from moringa stenopetala leaves. preventive nutrition and food science, 24, 70–74. https://doi. org/10.3746/pnf.2019.24.1.70 30. shalayel, m. h. f., asaad, a. m., qureshi, m. a., & elhussein, a. b. (2017) anti-bacterial activity of peppermint (mentha piperita) extracts against some emerging multi-drug resistant human bacterial pathogens. journal of herbal medicine, 7, 27–30. https://doi.org/10.1016/j. hermed.2016.08.003 31. vieira, g. h. f., mourão, j. a., ângelo, â. m., costa, r. a., & vieira, r. h. s. f. (2010) antibacterial effect (in vitro) of moringa oleifera and annona muricata against gram-positive and gram-negative bacteria. revista do instituto de medicina tropical de são paulo, 52, 129–132. https://doi.org/10.1590/s0036-46652010000300003 https://doi.org/10.47723/kcmj.v18i2.807 https://doi.org/10.1016/s1995-7645(11)60069-2 https://doi.org/10.1016/s1995-7645(11)60069-2 https://doi.org/10.1097/hnp.0b013e31820dbb27 https://www.thaiscience.info/journals/article/cmuj/10557966.pdf https://doi.org/10.7314/apjcp.2014.15.20.8571 https://doi.org/10.3389/fmicb.2013.00162 https://doi.org/10.3389/fmicb.2013.00162 https://doi.org/10.3746/pnf.2019.24.1.70 https://doi.org/10.3746/pnf.2019.24.1.70 https://doi.org/10.1016/j.hermed.2016.08.003 https://doi.org/10.1016/j.hermed.2016.08.003 https://doi.org/10.1590/s0036-46652010000300003 98 | fine focus preliminary studies on a novel antimicrobial compound producing bacterium discovered in the rio grande valley of texas julissa hernandez (juliehdz_3601@tamu.edu) anieto ugochukwu (uanieto@tamu.edu) texas a&m university higher education center in mcallen (hecm) vol 9 | 99 abstract we analyzed different soil samples collected across the rio grande valley (rgv) of texas for bacteria capable of producing antimicrobial compounds. of the more than 500 bacterial colonies tested, less than 1 percent gave any indication of the likelihood of producing antimicrobial compounds asdetermined by the diameter of the observed zones of inhibition created when tested on the cultures of safe eskape relatives. one of the soil isolates of interest was further studied with the 16s rrna gene sequenced and analyzed, and its antimicrobial compound was also extracted using the ethyl acetate and methanol extraction method. the results obtained suggest that the identified bacterium could be a novel specie or at least a sub-specie of the genus pseudomonas and the novel antimicrobial compound discovered possibly broad spectrum in nature considering that it is active against gram-positive and gram-negative bacteria, especially staphylococcus epidermidis, the safe relative of staphylococcus aureus. furthermore, positive inhibitory results were obtained when the antimicrobial compound was tested on bacillus subtilis, klebsiella pneumoniae, pseudomonas putida, enterococcus raffinosus, erwinia caratovora, enterobacter aerogenes and an unknown bacterium from an environmental sample etc. the newly discovered antimicrobial compound has been named “anietocin” and will be the subject of extensive studies in the coming research efforts. introduction the emergence of antimicrobial resistance in pathogenic bacteria has turned into a global phenomenon with the tremendous potential of being the leading cause of death if left unchallenged (aslam et al., 2018) . for this reason, many researchers across the globe have actively worked and are currently engaged in finding solutions to this problem. a number of scientific articles have been published, showcasing novel discoveries including one by darabpour et al. (2012)where they described an antibiotic producing pseudoalteromonas piscicida that was effective against most gram-positive bacteria. similarly, pishchany et al. (2018) described the successes of the novel antibiotic amycomicin on different bacteria including staphylococcus aureus. many of the discoveries have made it to patents as novel antibiotics as described by koulenti et al. (2019). such novel antibiotics that were described as efficient against multidrug-resistant gram-positive bacteria include ceftobiprole, ceftaroline, telavancin, oritavancin, dalbavancin etc. in addition to antimicrobial compounds derived from bacteria, a few studies have reported the use of other chemical compounds in our fight against multi-drug resistant bacteria, notably behroozian et al. (2016) reported the successful use of kisameet clay on eskape pathogens. similarly, hijazi et al. (2018) described the antimicrobial property of gallium compounds on eskape pathogens. perhaps to be considered an outlier to recent endeavors with novel antimicrobial compounds, is the article by nakonieczna et al.(2019) describing the latest photoinactivation method used on eskape pathogens. 100 | fine focus members of the eskape pathogens include enterococcus faecium, staphylococcus aureus, klebsiella pneumoniae, acinetobacter baumannii, pseudomonas aeruginosa and enterobacter spp. (mulani, et al., 2019). these bacteria are resistant to multiple drugs and are known to be virulent (mulaniet al., 2019). the mechanisms employed by these eskape pathogens to evade antibiotics include the alteration of the compounds, modification of the binding sites of drugs, intracellular decrease in the accumulation of the drug, loss of porin, the use of efflux pumps etc. (santajit and indrawattana, 2016). these mechanisms do not deviate at all from previously established mechanisms of evasion by all bacteria that are sensitive to antibiotics. in our efforts to contribute to slowing down the depletion of our potent antibiotics and the emergence of superbugs, we discovered a highly effective antimicrobial compound which has been named “anietocin” and is produced by a possibly novel specie or at least a sub-specie of the genus pseudomonas based on its determined 16s rrna gene sequence data. this novel pseudomonas spp. grows profusely in liquid and on solid tryptic soy media and creates a significant zone of inhibition when tested on different bacteria including a member of the eskape pathogens and safe eskape relatives. there are quite a number of reported studies where pseudomonas spp. have been researched for the possibility of producing antimicrobial compounds. for example raaijmakers et al. (1997) described the production of phanazine-1-carboxylic acid (pca) and 2,4-diacetlyphloroglucinol (phl) as it concerns the diseases of wheat while darabpour et al. (2010) described a p. aeruginosa strain pg-01 isolated from the persian gulf. the maximum zone of inhibition observed was 33 mm in a culture of methicillin-resistant staphylococcus aureus (mrsa). the possible pseudomonas spp. responsible for producing “anietocin” was isolated from a soil sample, and we hereby present the preliminary results obtained with the raw extracts of this novel antimicrobial compound on safe eskape relatives and klebsiella pneumoniae. materials and methods unless otherwise stated, all methods used were adapted with minor modifications from the small world initiative research protocols (4th edition) by broderick and kurt (2016) soil sample soil samples were collected in resealable bags from different locations across the rio grande valley of texas with particular focus in hidalgo county. the target soil samples were from the topsoil where organic matter is the most abundant. a soil sample data collection sheet as designed by the small world initiative (http:// www.smallworldinitiative.org/) was used to document the characteristics of the collected sample which included the general location, gps coordinates, dates and times collected, sample site descriptors, air temperature in celsius, humidity, depth, type of soil, soil temperature, ph of soil, soil water vol 9 | 101 content etc. soil samples were kept at room temperature and processed within 72 hours of collection. broth culture preparation 0.1 g of each collected soil sample was aseptically added to 10 ml of sterilized tryptic soy broth (tsb, bd bdl, 9046995, sparks md). this was thoroughly mixed using a bench top vortex machine, and thereafter 0.1 ml of this mix was used to inoculate another 9.9 ml of tsb to bring the final dilution to 10 ̄3. the broth cultures were incubated at 37 °c between 18-24 hours. solid media plating 10 μl of the 10 ̄3 dilution was taken and diluted in 90 μl of freshly prepared tsb broth and a final 100 μl of this dilution was taken and then spread plated on tryptic soy agar plates (tsa, becton dickinson bdl, 8346803, spars md) prepared with 25 μg/ml of 95% cycloheximide (acros organics, a0411477, china) to prevent fungal growth. the plates were incubated at 37 °c for 24-48 hours. colony selection after 24-48 hours, the incubated plates were observed for the formation of bacterial colonies, with particular attention paid to single colonies with unique characteristics such as size, color, smooth antimicrobial compounds production test the isolated pure colonies were collectively spotted grid style using sterile toothpicks on prepared tsa plates of cultures of safe eskape relatives and k. pneumoniae. the plates previously described were prepared using freezer stocks of eskape relatives and k. pneumoniae, where single colonies were re-inoculated into fresh broths before being transferred onto tsa plates using the spread plating techniques. these plates were spotted with the suspected antimicrobial compounds producing soil isolates and incubated at 37 °c for 24-48 hours thereafter they were removed and observed for any noticeable zones of inhibitions. antimicrobial compound screening the isolated colonies that showed noticeable zones of inhibition in the antimicrobial compounds production tests previously described were individually spotted on prepared plates of cultures of safe eskape relatives and k. pneumoniae and incubated as previously described. the plates were once again screened for evidence of antimicrobial activity by looking for evidence of “zones of inhibition” – a clearing on the bacterial lawn where the isolates were spotted using sterile toothpicks. this procedure is a confirmation of what was done prior in f. dna extraction, amplification, gel electrophoresis and sequencing the isolate of interest that demonstrated zones of inhibition against any of the safe eskape relatives and other tested bacteria had its dna extracted using 102 | fine focus the zymo quick-dna miniprep plus kit protocols (zymoresearch, 207601, usa). these isolates were grown in tsb broth overnight at 37 °c before the dna isolation. the gene sequence of the 16s rrna were amplified using 16s rrna forward (agagtttgatcctggctcag) and 16s rrna reverse (acggctaccttgttacgactt) sequences primers (integrated dna technologies, inc.) and thereafter, gel electrophoresis was performed to confirm the right size sequence at approximately 1500 base pairs. the amplified gene sequence was cleaned up using the monarch pcr & dna cleanup kit (new england biolabs) and sequenced for identification by eurofins genomics. organic extraction the antimicrobial producing isolate of interest was grown on solid media and was subjected to organic extraction as described in the small world initiative research protocols (4th edition) by broderick and kurt (2016). a spatula was used to cut 2-3 small pieces of agar (1 inch square) containing the antimicrobial producing culture isolate, which was then transferred into 100 ml glass bottle and placed in -20 °c freezer overnight, this procedure was replicated multiple times to increase the quantity of extract obtained.thereafter, 15 ml of 99% ethyl acetate (flint scientific, 273018, batavia, il) and 10 ml of deionized water were added to each bottle and shaken vigorously at room temperature for 20 30 minutes. a pipette was then used to transfer the entire liquid contents of the bottles to new bottles, avoiding the agar debris and allowed to sit for as long as needed for the two phases to separate. the separated ethyl acetate layer (top portion) up to 10 ml, was then transferred to new bottles and placed without caps inside the fume hood until all ethyl acetate had evaporated (usually between 96-120 hours). after the entire ethyl acetate had evaporated, leaving behind a white residue hereby referred to as the “the crude extract”, the white residue was dissolved by the addition of 80 μl of methanol (vwr, 0000200921, mississauga on) in each bottle and the solution kept at 4°c for further use, usually between 12-24 hours after extraction. screening for antimicrobial activity the solution obtained from the organic extraction (white residues dissolved in the methanol) was tested on prepared plates of safe eskape relatives and other bacteria. 30 μl each of the solution (crude extract dissolved in methanol) was dropped on the plates containing the prepared plates, another 30 μl of methanol dropped on a different part of the plate as negative control and in some cases different antibiotics disks (bd bbltm, sparks md) were tested against the solution for comparison. the plates were incubated at 37 °c for 24-48 hours. the zones of inhibition produced by the antimicrobial compound were measured and compared to zones of inhibition produced by the antibiotic disks. these steps were replicated several times and confirmed reproducible. vol 9 | 103 infra-red spectroscopy the antimicrobial compound solution was tested to determine the functional groups in the mixture using the fourier transform infrared spectrophotometer (shimadzu). resolution was set at 4 cm-1 and the happgenzel apodization was utilized for a total of 4 scans. results more than 100 unknown soil bacteria isolates were tested on eskape relatives for the ability to produce antimicrobial compounds. among them was an isolate with a yellow appearance that created a clear zone of inhibition when tested on b. subtilis and s. epidermidis and subsequent testing of the isolate of interest on k. pneumoniae, e. raffinosus and others (not shown) in figure 1 produced zones of inhibition of varying diameters. the culture of the suspected novel species or at least a sub-specie of pseudomonas was assumed to be producing an antimicrobial compound when spot tested on the 7 listed bacteria in table 1. this spot testing was replicated multiple times and the ability of the bacterium to produce clear zones of inhibition when spotted confirmed its antimicrobial producing ability. it was noted that some inconsistencies were observed when the antimicrobial compound producing bacterium was tested on cultures of e. coli, as some of the results were initially positive but came back negative on subsequent spot testing. cultures of e. coli sustained these variations and so were declared nonsensitive to the antimicrobial compound produced by the unknown bacterium. table 1 shows the results obtained when testing with the unknown compound on safe eskape relatives and k. pneumoniae. b. subtilis and s. epidermidis showed higher levels of sensitivity to the unknown bacterium, suggesting that perhaps the antimicrobial compound produced could have more effect on gram-positives species especially the non-sporeforming s. epidermidis. the rest of the test cultures showed varying degrees of sensitivity over many replicates, with e. coli, appearing to be the least sensitive. the unknown bacterium was used after incubation for 18 – 24 hours for best results. older cultures of this unknown bacterium showed less ability to inhibit the growth of the test cultures. 104 | fine focus fig 1: left to right. the unknown bacterium (pointed) on a culture of b. subtilis, the zone of inhibition can be clearly seen on the plates. table 1: tester bacteria are used to verify the antimicrobial compound produced by the unknown bacterium. = no zone of inhibition observed, + = zone of inhibition observed, ++ = large zone of inhibition observed tester bacterium result 1. enterococcus raffinosus + 2. escherichia coli 3. erwinia caratovora + 4. pseudomonas putida + 5. staphylococcus epidermidis ++ 6. enterobacter aerogenes + 7. bacillus subtilis ++ vol 9 | 105 the raw extract from the antimicrobial compound producing bacterium was obtained as described in the materials and methods section and was used on the test cultures to confirm potency. the results obtained showed consistency as earlier observed when the antimicrobial compound was directly used on the test cultures. s. epidermidis showed the highest level of sensitivity as can be observed from the zone of inhibition obtained. e. coli though, originally declared non-sensitive, appeared this time to show a slight degree of sensitivity, suggesting that perhaps much is left for us to understand on the culturing conditions of this unknown bacterium to produce the maximum and/or most potent form of the antimicrobial compound. for this testing phase, a bacterium obtained from an environmental sample was added to the line-up of test cultures and was sensitive to the crude extract of the antimicrobial compound being tested. 106 | fine focus fig 2: top to bottom. the antimicrobial compound zones of inhibition (pointed) on a culture of s. epidermidis, and an unknown environmental sample extract bacterium, methanol was used as the negative control and represents the alphabet “b” and written as methanol in some of the plates. vol 9 | 107 and none of the commercially available antibiotic discs has produced this result on any bacterium. this suggests a unique killing efficiency that may become the new and effective antibiotic against resistant pathogens. to determine this unknown antimicrobial compound’s effectiveness when compared with known antibiotics, we set up an experiment with s. epidermidis and k. pneumoniae, representing gram-positive and gram-negative bacteria respectively. for s. epidermidis, penicillin 10 iu (bd, bdl 9154628, sparks md) was used to compare to the unknown antimicrobial compound while 1.25 μg trimethoprim/ sulfamethoxazole (sxt, bd bdl, 9078838, sparks md) was used for the same comparison on k. pneumoniae. the choice for these two well-established antibiotics is hinged on their known efficacy when used on the aforementioned bacteria. for example, some strains of k. pneumoniae strains are susceptible to sxt as reported by ballén et al. (2021). similarly, it is common knowledge that penicillin is the oldest commercially viable antibiotic in modern medicine. its mode of action being to interfere with the synthesis of the bacterial cell walls as described by soares et al. (2012). this novel antimicrobial compound appeared more potent than penicillin (10 iu) on s. epidermidis and sxt (1.25 μg) on k. pneumoniae when measured by the observable zones of inhibition produced as can be seen in figure 3 below and this experiment was replicated many times with the same outcomes. going by the bd bbltm antimicrobial susceptibility tables, penicillin (10 iu) with values greater than 29 mm in diameter on staphylococcus species is the top range effectiveness but, we achieved a little more than 500 mm in diameter of zone of inhibition using the novel antimicrobial compound. as far as we know, no research article has ever reported this extremely large number with either crude or refined extract 108 | fine focus fig 3: top to bottom. the unknown antimicrobial compound (red arrow) on s. epidermidis and k. pneumoniae. p-10 and sxt are arrowed in blue. the zone of inhibition produced by the unknown antimicrobial compound on s. epidermidis was slightly above 500 mm in diameter. vol 9 | 109 cm-1. a very sharp peak at approximately 900 cm-1 suggests the presence of a dior trisubstituted alkane group. discussion not much is known about this likely pseudomonas sp. that produces the novel antimicrobial compound anietocin but the outcomes of the spot testing experiment across a battery of safe eskape relatives and including an eskape pathogen itself suggest that it could well become an effective antimicrobial compound. a much better outcome is seen when tested on grampositives than on gram-negatives, with s. epidermidis showing an outstanding level of sensitivity when exposed to anietocin. as s. epidermidis is the safe version of the eskape pathogen s. aureus, we are hopeful that this positive result will be replicated when tried on it. the preliminary results obtained show the superiority of anietocin on s. epidermidis by the observed and measured zones of inhibition using only the crude extract when compared to 10u penicillin. we hypothesize that a cleaned up and pure anietocin compound will effectively confirm this superiority by giving much wider zones of inhibition with lower minimal inhibitory concentration (mic) values. our next focus is to purify the anietocin antimicrobial compound, determine its chemical structure, minimal inhibitory concentration based on the european committee on antimicrobial susceptibility testing (eucast) guidelines, the full genome analyses of the suspected pseudomonas spp. and other studies that will assist in moving it from laboratory to the pharmacy. the result of 16s rrna genes sequencing analysis (genbank accession number on964909, seqidanieto), suggests the likelihood of a relationship with the genus pseudomonas, azotobacter, halopseudomonas, atopomonas, and azorhizophilus. the closest matches with a max score and total score of 723 and 86% percent identities suggest a possible closer relationship with p. aeruginosa strains ddm 50071, nbrc 12689 and atcc 10145 (genbank accession numbers nr_117678.1, nr_113599.1 and nr_114471.1 respectively). considering the unique nature of this isolate by its ability to produce an antimicrobial compound, previously undescribed as far as we are aware, we hypothesize that this suggested pseudomonas spp. is likely a new addition to the genus or a sub-specie at the minimum. its antimicrobial compound is equally novel based on the observed preliminary results and therefore referred to as “anietocin” for the purposes of proper identification. the results of the infra-red spectroscopy analyses, suggest the possible presence of alkenes (=c-h) and alkanes (c-h) functional groups with peaks seen at 3100-2900 cm-1 regions; however, this can be regarded as inconclusive because the peaks appear weak which does not correlate with the description of known peaks at the same region. this weak appearance could be a function of the crude nature of the extract, with numerous artifacts interfering with the analyses. a critical look at the fingerprint 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rapacka-zdonczyk, a., ogonowska, p. & grinholc, m. 2019. photoinactivation of eskape pathogens: overview of novel therapeutic strategy. future med chem, 11, 443-461. vol 9 | 111 10. pishchany, g., mevers, e., ndousse-fetter, s., horvath, d. j., jr., paludo, c. r., silva-junior, e. a., koren, s., skaar, e. p., clardy, j. & kolter, r. 2018. amycomicin is a potent and specific antibiotic discovered with a targeted interaction screen. proc natl acad sci u s a, 115, 10124-10129. 11. raaijmakers, j. m., weller, d. m. & thomashow, l. s. 1997. frequency of antibiotic-producing pseudomonas spp. in natural environments. appl environ microbiol, 63, 881-7. 12. santajit, s. & indrawattana, n. 2016. mechanisms of antimicrobial resistance in eskape pathogens. biomed res int, 2016, 2475067. 13. soares, g. m., figueiredo lc fau faveri, m., faveri m fau cortelli, s. c., cortelli sc fau duarte, p. m., duarte pm fau feres, m. & feres, m. mechanisms of action of systemic antibiotics used in periodontal treatment and mechanisms of bacterial resistance to these drugs © 2024 marble, mckillip. fine focus, 10(1), 38-57. doi: 10.33043/ff.10.1.38-57 shared with cc-by-nc-nd 4.0 license. lillia g. marble john l. mckillip department of biology, ball state university, muncie, in 47306 daily case trends of covid-19: a comparative analysis of indiana and washington state keywords: covid-19, statistical analysis, indiana, public health, washington manuscript received 11 december 2023; accepted 21 april 2024 abstract the covid-19 pandemic has caused immense and immeasurable disruption to billions of lives worldwide, and the strain on healthcare workers and facilities will undoubtedly be seen for years to come. many factors impact the incidence and prevalence of covid-19 in states, such as policies and legislation, funding, partisanship of the statehouse, vaccination rates, and rurality. the purpose of this paper is to analyze the differences in the daily positive cases between indiana and washington state and examine the respective ways each state tried to mitigate the morbidity and mortality of the virus. indiana and washington state were chosen as the states have similar populations in different geographical locations in the country and varying responses to the pandemic. data was obtained from the respective state health departments over a period of two and a half years from march 2020 to december 2022. independent t-tests were used for the analysis of the data between indiana and washington. overall, indiana had a higher daily positive case rate when compared to washington. indiana had a lower vaccination rate and had more hospitalizations and deaths compared to washington and the us population as a whole. the difference in the findings of each state could be attributed to the partisanship of the state and the ways in which partisanship influences the enacting of legislation and policies intended to mitigate disease, as well as public health funding allocated by the state. https://creativecommons.org/licenses/by-nc-nd/4.0/ marble & mckillip | daily case trends of covid-19: a comparative analysis of indiana and washington state 39 introduction & history the covid-19 pandemic is a global health crisis caused by the novel coronavirus sars-cov-2. the virus, which causes a pneumonia-like illness in humans, was first identified in wuhan, china in december 2019 originating from a “wet market” in which infected live animals were sold for food, becoming zoonotic when it started infecting humans. this viral illness started quickly spreading throughout china and other surrounding countries. in january 2020, the world health organization (who) declared this outbreak of the novel coronavirus a public health emergency, and by march 2020, the who declared it a worldwide pandemic, at which point there were more than 118,000 cases in 114 countries and more than 4,000 documented deaths globally (9). the first case of the novel coronavirus in the united states was reported on january 20th, 2020, in seattle, washington after family-related travel to wuhan, china (9). by april 10th, 2020, the united states had become a global hotspot for the sars-cov-2 virus, with more than 500,000 cases since january (9). as the only industrialized country without universal healthcare and equitable access to care, the united states was especially susceptible to a high rate of spread, quickly surpassing china to become the epicenter of cases in the world early in the pandemic (18). two weeks prior to the trump administration’s issuance of a nationwide emergency, governor jay inslee of washington state issued a statewide emergency on february 29th, 2020, making it the first state to implement public health interventions to slow the spread of the virus (12). by the end of march 2020, many state and local governments had issued similar statewide emergencies and implemented various public health measures to slow the spread of this novel virus, one of these states being indiana who declared a statewide public health emergency on march 6th, 2020. these public health measures included lockdowns/ travel restrictions, social distancing, and face covering mandates, recommended by the world health organization (who) and centers for disease control and prevention (cdc) based on evidence-based practices for mitigating airborne or droplet transmitted diseases. various states implemented much stricter mitigation measures, while other states were much laxer in the policies they implemented. since early 2020, the pandemic has had a profound impact on the global economy and healthcare systems. the hospitals and healthcare systems in many countries – including the united states – were overwhelmed, underprepared, and vastly understaffed for the influx of patients admitted with this virus. shortages of personal protective equipment and medical supplies became the norm after the pandemic caused severe disruptions in the global supply chain. vaccines were developed and approved for emergency use in record time, with the first vaccine doses administered to healthcare workers and first responders in december 2020. while vaccines have been effective in reducing the severity of illness and hospitalizations, new variants of the virus have emerged, and the pandemic continues to pose a significant public health threat. the purpose of this paper is to compare disease fine focus | volume 1040 morbidity and mortality between two states of similar populations – indiana and washington state – with varying degrees of public health policy and mitigation strategies. the two states are relatively similar in population (in: 6.7 million; wa: 7.6 million) (37) and have a few similar demographics; they both have the same median age of 37.8 years, they have a similar female population percentage (in: 50.4%; wa: 49.6%), and similar high school education levels (in: 89.8%; wa: 91.9%) (35,36). although these are a handful of the similarities between indiana and washington, there are some major differences; for example, indiana is located in the midwest region of the united states, while washington is located in the pacific northwest. these states also differ historically in their political outlooks. in the 2020 presidential election, 57% of voting-eligible residents in indiana voted republican, while only 38% of voting-eligible washington residents voted republican – compared to 41% and 58% voting democrat, respectively (45,46). historically, indiana has been republican-led since 2004, and washington has been democrat-led since 1988. analyzing two states in differing parts of the country with varying political views yet otherwise similar demographics allows for a diverse set of data to compare. daily testing data from indiana and washington state was collected and analyzed comparatively using independent samples t-tests. several sets of data were compared: each year of 2020, 2021, and 2022, respectively; the delta variant wave in mid-2021; the omicron variant wave from december 2021 to january 2022; the 2020-2021 winter/respiratory season; the 2021-2022 winter/respiratory season; and the entirety of the data from march 2020 to december 2022. the covid-19 delta and omicron variant waves were compared as they were the two largest covid-19 waves in the dataset and the most transmissible variants in the dataset, resulting in higher incidences (7,11,13, 20). the winter/respiratory seasons were also compared as respiratory illnesses tend to increase in the winter months in the peak influenza season from december to march. the difference in policies implemented by each state as well as the partisanship of the state ultimately played a major role in determining the course and outcome of the pandemic by impacting the incidences of cases. the differences in data could be attributed to a variety of factors including vaccination rates, public health initiatives & legislation, funding, and access to healthcare facilities. materials & methods study design the covid-19 incidences in indiana and washington state were compared using daily positive cases reported to each state’s respective department of health. the daily positive cases were collected for each state (appendix a) and analyzed using independent t-tests on spss. to control for differences in population, each daily data point was divided by each state’s population (in: 6.7 million; wa: 7.6 million), as reported by the 2020 census bureau (37) to standardize the values per 100,000 people. the statistical analyses were as follows: all time (march 2020 – december 2022); 2020 marble & mckillip | daily case trends of covid-19: a comparative analysis of indiana and washington state 41 calendar year; 2021 calendar year; 2022 calendar year; delta variant wave (july-september 2021); omicron variant wave (november 2021-january 2022); 2020-2021 winter/respiratory season; and 2021-2022 winter/respiratory season. other values such as vaccination rates, death rates, and hospitalization rates were also collected and compared. statistical analyses to analyze the statistical differences between the case rates of washington and indiana, independent t-tests were used. to control for differences in population, the daily case counts were divided by their respective state’s population, as reported by the 2020 us census bureau. independent t-tests were used to compare different data sets from the pandemic. the daily positive covid-19 case rates for the two states were compared from the period of march 2020 to december 2022 using independent t-tests. analysis was also done on the individual years of 2020, 2021, and 2022 using independent t-tests, respectively. the delta variant wave and the omicron variant wave were also compared using independent t-tests as they were the two largest covid-19 variant waves in the dataset. in addition, the covid19 cases during the yearly flu/respiratory season from october to may – with peaks from december to march – was also analyzed using independent t-tests as respiratory illnesses tends to increase in the winter months. the daily case values were the primary comparative analysis between indiana and washington, but other values were collected and compared as well. these values obtained from the state departments of health included hospitalization numbers, death counts, and vaccination rates. hospitalization and death rates were calculated by dividing the total counts of hospitalizations and deaths from covid-19 in the state by the total number of covid-19 cases. the hospitalization and death counts were divided by the population of each respective state to get the standardized value per 100,000 people. standard deviation was analyzed for each independent t-test performed and included in the subsequent data tables. standard error was also used in the graphs indicated by the error bars on each column. standard error was used in the graphs due to the sheer size of the data and because it does not follow a normal bell-curve distribution. results the general trend, after controlling for population, showed that indiana had a higher covid19 incidence with a p value of <0.001. indiana continued to have a higher incidence in all analyses, albeit not all results were statistically significant. the exceptions from the statistically significant p value are the daily case rates from the 2021-2022 respiratory/flu season (in>wa, p = 0.134) and the 2022 calendar year (in>wa, p = 0.419) (see tables 1 & 3). when compared to the total us data for hospitalization and death rates, indiana had higher rates (7.64% and 1.28%, respectively) and washington had lower rates (4.23% and 0.81%, respectively) than the us average (5.84% and 1.08% respectively) (table 5). similarly, for both the primary series and the updated booster, indiana had lower vaccination rates (57.1% and 10.4%, respectively) and washington had fine focus | volume 1042 higher vaccination rates (76.1% and 24.5%, respectively) than the total us vaccination rate (69.3% and 16.3%, respectively) (table 4). rurality was defined using the federal office of management and budget’s definition of a rural county which classifies a county as rural if the largest urban area in that county is less than 50,000 people (39). using this definition, indiana had a greater percentage of rural counties and a greater percentage of populates (78.3% & 34%, respectively) in those counties than the us average (65.2% & 14%, respectively), while washington had a lower percentage of rural counties and a lower percentage of populates (51.3% & 9.3%, respectively) compared to the us average (table 6). the mean and standard error are graphed for each of the tables below. in addition, the maximum and minimum values for each month (march 2020 – december 2022) were graphed for each state (figures 2 & 3). public health funding data is also included for each state (table 7). case rates table 1 daily case rates standardized per 100,000 people daily covid-19 case rates (per 100,000) time state mean sd p value all time (mar 2020 – dec 2022) indiana 29.13 37.69 < 0.001 washington 20.25 32.16 2020 (mar 2020 – dec 2020) indiana 25.94 31.36 < 0.001 washington 10.46 11.32 2021 (jan 2021 – dec 2021) indiana 30.67 26.53 < 0.001 washington 19.48 17.98 2022 (jan 2022 – dec 2022) indiana 30.21 49.91 0.419 washington 29.47 48.62 note. the mean and standard deviation for each state in each analysis is listed, with the corresponding p values of each analysis. marble & mckillip | daily case trends of covid-19: a comparative analysis of indiana and washington state 43 figure 1 mean daily cases per 100,000 people and standard error of indiana and washington state for the years of 2020, 2021, 2022, and march 2020-december 2022. note. the numerical results of the statistical analysis can be found in table 1. covid-19 variant waves case rates (per 100,000) time state mean sd p value delta (july 2021 – sept 2021) indiana 37.96 19.34 < 0.001 washington 28.55 11.05 omicron (dec 2021 – feb 2022) indiana 102.08 73.01 0.048 washington 82.52 81.56 table 2 daily case rates standardized per 100,000 for each of the delta and omicron variant waves. note. the mean, standard deviation, and p values are listed for each state in each of the analyses. fine focus | volume 1044 figure 4 mean daily cases and standard error for the omicron and delta variant waves standardized per 100,000 people. note. numerical results are shown in table 2. table 3 results from comparative analysis of daily cases for each of the winter/respiratory seasons standardized per 100,000 people winter/respiratory season daily covid-19 case rates (per 100,000) time period state mean sd p value flu season 2021 (dec 2020 – mar 2021) indiana 43.04 33.57 < 0.001 washington 18.49 12.29 flu season 2022 (dec 2021 – mar 2022) indiana 73.15 77.58 0.134 washington 62.01 77.62 note. the mean, standard deviation, and the corresponding p values for each analysis are listed. marble & mckillip | daily case trends of covid-19: a comparative analysis of indiana and washington state 45 figure 5 mean daily cases and standard error per 100,000 people reported for indiana and washington for the 2021-2022 and 2022-2023 winter/respiratory seasons. note. numerical results shown in table 3. fine focus | volume 1046 other factors table 4 vaccination rates for indiana, washington, and the total united states. covid-19 vaccination rates state primary series updated booster indiana 57.1% 10.4% washington 76.1% 24.5% united states (total) 69.3% 16.3% note. data is included for both the primary series and the updated booster. (10;17;43) table 5 hospitalization and death rates and total hospitalizations and deaths from covid-19 standardized per 100,000 people for indiana, washington, and the total united states. covid-19 hospitalizations & deaths state hospitalization rates hospitalizations (per 100,000) indiana 7.64% 2391 washington 4.23% 1070 united states (total) 5.84% 1860 state death rates deaths (per 100,00) indiana 1.28% 388 washington 0.81% 205 united states (total) 1.08% 337 note. the hospitalization rates for each state were calculated by dividing the total number of covid-19 hospitalizations by the total number of covid-19 cases reported. marble & mckillip | daily case trends of covid-19: a comparative analysis of indiana and washington state 47 table 6 rural county data for indiana, washington, and the total united states rural counties state % of rural counties % of population in rural counties indiana 78.3% 34% washington 51.3% 9.3% united states (total) 65.2% 14% note. a rural county is defined using the office of management and budget’s definition of rural counties as an urban area less than 50,000 people (39). the percentage of rural counties was calculated by dividing the number of rural counties by the total number of counties. the percentage of population in rural counties was calculated by dividing number of inhabitants in the rural counties by the total state population. table 7 public health funding data from indiana and washington state public health funding state funding per person (2019) funding per person (2021) public health ranking indiana $15 $15 40th washington $46 $89 9th note. data includes per person public health funding for 2019 and 2021 from state health compare (30 & 40). fine focus | volume 1048 discussion the covid-19 pandemic has had a severe impact on the global economy and on the lives of billions of people worldwide. the effects of this pandemic on healthcare systems and individuals will be seen for decades to come. a comparative analysis of daily cases from indiana and washington state and the effect of the implemented public health policies of each respective state were used to determine the best practices to mitigate disease for future public health emergencies. the mitigation strategies such as social distancing guidelines and face mask mandates implemented had a direct impact on the incidence and prevalence of covid-19 cases. the implementation of such policies is crucial in reducing the spread of covid-19 and mitigating the impact of the pandemic on society. health is multi-factorial, so it is difficult to pinpoint the exact cause of the differences in the cases between indiana and washington state; however, there are several factors that have been shown to impact the incidence and prevalence of disease which are directly applicable here. tables 1, 2, and 3 demonstrate the general trend that washington had a lower daily case incidence than indiana, albeit insignificantly during the 2021-2022 winter/respiratory season (table 3) and the 2022 calendar year (table graphs figure 2 maximum and minimum reported cases per 100,000 people per month from march 2020 to december 2022 in indiana. marble & mckillip | daily case trends of covid-19: a comparative analysis of indiana and washington state 49 figure 3 maximum and minimum reported cases per 100,000 people reported per month from january 2020 to december 2022 in washington state 1). in addition to generally having a smaller daily case rate, washington’s peaks tended to have a shorter duration than indiana’s peaks during the different variant waves (figure 4). the phrase “flattening the curve” was used in early 2020 to describe the intended effect of the mitigation strategies, and it is illustrated in the differences of the peaks (figures 2 & 3). these states vary in the legislation and implementation of public health policies designed to mitigate the spread of disease; without such measures, morbidity and mortality would have likely been much higher. the difference in the data could be largely attributed to the implementation of policies meant to reduce the transmission of disease (such as mask mandates and social distancing guidelines) and vaccination rates. other factors contributing to the differences in data include public health funding which impacts access to healthcare (including testing sites), and education initiatives which combat the misinformation crisis. the combination of these factors ultimately helped dictate the course of the pandemic in each state. immunizations are used to prevent severe illness and death from infectious diseases, and the covid-19 virus is no different. the first doses of the covid-19 vaccine were administered to healthcare workers and first responders fine focus | volume 1050 in december 2020, with the general population receiving doses based on a roll-out schedule in early 2021. washington had a primary series of vaccination rate of 76.1% (table 4) and indiana’s primary series vaccination rate was 57.1% (table 4). as vaccination prevents severe disease resulting in hospitalization or death, the hospitalization and death data was also analyzed (table 5). in washington, 4.23% of cases resulted in hospitalization and 0.81% of cases resulted in death, and 7.64% of cases in indiana resulted in hospitalization and 1.28% of cases resulted in death (table 4). washington had 1070 hospitalizations per 100,000 people (table 5) and indiana had over twice that number with 2391 hospitalizations per 100,000 (table 5). the higher rates of hospitalization and deaths are inversely correlated with vaccination rates; the greater percentage of the population that is vaccinated against the virus, the lower the hospitalization and death rates are. public health policies such as mask mandates, social distancing, and lockdowns were implemented to slow the spread of the virus. although each individual policy is effective, the greatest outcome on the incidence and prevalence of cases was found to be a combination of such factors (23). in addition to the policies, the timing and duration of the policy implementation was also important – factors found to be notably influenced by political factors in the state, such as the partisanship of the state governor (1,2,5). generally, democratic governors tended to implement statewide mask mandates and stay-at-home orders sooner than their republican counterparts, and they also tended to continue having these policies in place for a longer period (5). states which were quicker to implement policies and leave them in place for a longer duration saw a reduced incidence and prevalence in covid-19 cases while states which were slower to implement policies and quicker to rescind them had the opposite effect (25). washington tended to have a reduced incidence and prevalence of cases compared to indiana (table 1). washington is democratic-led and was the first state to implement any mitigation policies, and yet was one of the last states to rescind its mask mandate in 2022 (44) and had a smaller case incidence. indiana is republican-led and was one of the first states to rescind its mask mandate in early 2021 (21), less than one year after implementing it – a factor the higher daily case incidence can likely be attributed to. similarly, public health funding – which impacts the mitigation strategies implemented – is also influenced by partisanship of state government; democratic-led states tend to allocate more funds toward public health and education, while republican-led states tend to allocate funds away from public health (6). public health funding is important in expanding access to healthcare facilities – including covid-19 testing locations – but it is also critical in educating the public on various health topics and combatting misinformation. when misinformation is combatted through education initiatives, it leads to higher levels of vaccination and higher rates of adherence to mitigation strategies such as masking and social distancing (29). these concepts are demonstrated in each respective state; washington has historically ranked high for public health, while indiana has historically ranked low – according to the cdc, marble & mckillip | daily case trends of covid-19: a comparative analysis of indiana and washington state 51 washington had a public health ranking of 9th in the country, while indiana had a ranking of 40th (40). furthermore, washington’s response to the pandemic included an 93% increase in state public health spending per person from 2019 to 2021 ($46/person and $89/person, respectively), while indiana’s state public health spending remained stagnant at $15 per person from 2019 to 2021 (30). these contrarieties in public health funding can be directly seen in the availability of covid-19 testing sites statewide; washington had over 1000 testing locations (43), while indiana had 554 testing sites (16). the lack of accessibility of healthcare sites and testing facilities disproportionately affects rural areas – communities that historically tend to be underfunded, lack healthcare infrastructure, and have higher rates of poverty; these discrepancies which have been further highlighted by the covid-19 pandemic (8). rural counties have been shown to have higher rates of covid-19 and lower vaccination rates – oftentimes due to misinformation and lack of education surrounding vaccines and public health (8). indiana’s low public health funding coupled with the state’s rurality (table 7) likely contribute to the higher incidence and illustrate the impact of accessibility of healthcare and education on case incidence and prevalence in a community. while the general trend of data across the two states is that indiana had a significantly higher daily case rate, the 2021-2022 winter/respiratory season (december 2021 – march 2022) (table 3 & figure 5), and the 2022 calendar year (table 1 & figure 1) are the exceptions with p values of 0.134 and 0.419, respectively. the commonality between these time frames is the peak of the omicron variant wave, in which states were reporting cases of nearly 23,000 cases per day. the omicron wave from early december 2021 to early february 2022 had the highest reported daily case counts throughout the course of the pandemic, with the mean (in: 102.08; wa: 82.52, table 2) being triple the mean of the delta wave in mid-2021 (in: 37.96; wa: 28.55, table 2), and four times the mean of the entire dataset from march 2020 to december 2022 (in: 29.13; wa: 20.25, table 1). the number of cases reported during the nine-week long omicron wave accounted for nearly thirty percent of all cases in the two-and-a-half-year duration of data, a significant percentage for such a short period of time. with the omicron variant resulting in many asymptomatic infections, these numbers are probably vastly underreported as pcr tests were in short supply especially in states with limited testing; in addition, countless self-administered tests were not reported to the state health department. the variation of the p values could be attributed to testing availability. washington has about twice the amount of testing locations in comparison to indiana, meaning that covid tests are more available and accessible to everyone, including those in rural counties who may typically lack access to healthcare services (16, 43). during the peak of the omicron wave, washington state also increased access to testing due to the increased incidence and prevalence of cases, which ultimately led to higher reported cases during the wave and throughout the rest of 2022; indiana presumably had similar case levels but due to lack of fine focus | volume 1052 testing accessibility, cases undoubtedly went untested, and therefore, unreported to the state. increased access to testing sites allows individuals who are exposed to the virus or present with symptoms to be able to test without having to travel long distances. in relation to this dataset, the omicron variant was the most transmissible variant, with an estimated basic reproduction number (r0) between 10 and 24 – almost triple that of the delta variant, which was estimated between 5 and 8 (11). the transmissibility of the omicron variant . the enhanced transmissibility and immune evasion cause rapid spread in communities; this sharp increase in cases is seen in figures 2 and 3 which detail the highest and lowest reported daily cases of each month. in addition to its immune evasion properties, the omicron variant also causes more asymptomatic infections compared to other variants (11). access to testing and healthcare facilities plays a critical role in catching these asymptomatic infections. despite the sociopolitical landscape of the state and the public perception of the disease, these policies – which are recommended by scientists and public health experts – should be implemented as soon as possible to curb new cases and prevent full-scale outbreaks, as well as mitigate the impact on society. the current trend of disseminating misinformation and rejecting evidence-based methods in government legislation is detrimental to the lives of countless individuals as well the effectiveness of mitigation strategies in future public health emergencies. if any lesson is to be learned from the death and disaster of the covid-19 pandemic, it is that adequate public health funding and appropriate evidence-based policies implemented quickly are vital and essential to effectively reducing the morbidity and mortality of disease. author correspondence correspondence concerning this article should be addressed to: lillia g. marble, lmarble@bsu.edu. marble & mckillip | daily case trends of covid-19: a comparative analysis of indiana and washington state 53 references 1. adolph, c., amano, k., bang-jensen, b., fullman, n., & wilkerson, j. 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(2014, august). rural indiana: a demographic and economic overview. indiana state university rural-urban entrepreneurship development institute. https://www2.indstate. edu/news/pdf/2014-08_in%20rural_counties_economic_overview.pdf 29. roozenbeek, j., schneider, c. r., dryhurst, s., kerr, j., freeman, a. l. j., recchia, g., van der bles, a. m., van der linden, s. (2020, july 6). susceptibility to misinformation about covid19 around the world. r. soc. open sci. 7: 201199. http://dx.doi.org/10.1098/rsos.201199 30. state health compare. (2021). per person state public health funding map. https://statehealthcompare.shadac.org/map/117/per-person-state-public-health-funding#a/37/154 31. substance abuse & mental health services administration. (2023, january 4). 2021 national survery on drug use and health. https://www.samhsa.gov/data/report/2021-nsduh-detailed-tables 32. thomas, l. (2021, february 22). an overview of spike protein antigen covid-19 vaccine candidates. medical news. https://www.news-medical.net/news/20210222/an-overview-of-spikeprotein-antigen-covid-19-vaccine-candidates.aspx https://doi.org/10.1002/jmv.26615 https://doi.org/10.1016/j.compbiomed.2021.104369 https://doi.org/10.1016/j.compbiomed.2021.104369 https://www.pewresearch.org/journalism/2020/06/29/americans-rate-cdc-highly-trump-and-his-administration-poorly-on-getting-the-facts-right-about-covid-19/ https://www.pewresearch.org/journalism/2020/06/29/americans-rate-cdc-highly-trump-and-his-administration-poorly-on-getting-the-facts-right-about-covid-19/ https://doi.org/10.1101/2020.10.08.20209619 https://doi.org/10.1016/s1473-3099(20)30484-9 https://doi.org/10.1016/s1473-3099(20)30484-9 https://www.pewresearch.org/short-reads/2020/08/27/more-americans-say-they-are-regularly-wearing-masks-in-stores-and-other-businesses/ft_2020-08-27_maskwearing_01a/ https://www.pewresearch.org/short-reads/2020/08/27/more-americans-say-they-are-regularly-wearing-masks-in-stores-and-other-businesses/ft_2020-08-27_maskwearing_01a/ https://www.pewresearch.org/short-reads/2020/08/27/more-americans-say-they-are-regularly-wearing-masks-in-stores-and-other-businesses/ft_2020-08-27_maskwearing_01a/ https://www2.indstate.edu/news/pdf/2014-08_in%20rural_counties_economic_overview.pdf https://www2.indstate.edu/news/pdf/2014-08_in%20rural_counties_economic_overview.pdf http://dx.doi.org/10.1098/rsos.201199 https://statehealthcompare.shadac.org/map/117/per-person-state-public-health-funding#a/37/154 https://statehealthcompare.shadac.org/map/117/per-person-state-public-health-funding#a/37/154 https://www.samhsa.gov/data/report/2021https://www.news-medical.net/news/20210222/an-overview-of-spike-protein-antigen-covid-19-vaccine-candidates.aspx https://www.news-medical.net/news/20210222/an-overview-of-spike-protein-antigen-covid-19-vaccine-candidates.aspx fine focus | volume 1056 33. trust for america’s health. (2022). state profile: indiana. https://www.tfah.org/state-details/ indiana/ 34. trust for america’s health. (2022). state profile: washington. https://www.tfah.org/state-details/ washington/ 35. us census bureau. (2020). quickfacts indiana. https://www.census.gov/quickfacts/in 36. us census bureau. (2020). quickfacts washington. https://www.census.gov/quickfacts/wa 37. us census bureau. (2020). 2020 census results. https://www.census.gov/programs-surveys/ decennial-census/decade/2020/2020-census-results.html 38. us department of agriculture. (2007, september 4). washington – rural definitions: state-level maps. https://www.ers.usda.gov/webdocs/datafiles/53180/25602_wa.pdf?v=0 39. us department of health & human services. 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(2020). washington. https://www.270towin.com/states/washington https://www.tfah.org/state-details/indiana/ https://www.tfah.org/state-details/indiana/ https://www.tfah.org/state-details/washington/ https://www.tfah.org/state-details/washington/ https://www.census.gov/quickfacts/in https://www.census.gov/quickfacts/wa https://www.census.gov/programs-surveys/decennial-census/decade/2020/2020-census-results.html https://www.census.gov/programs-surveys/decennial-census/decade/2020/2020-census-results.html https://www.ers.usda.gov/webdocs/datafiles/53180/25602_wa.pdf?v=0 https://www.usnews.com/news/best-states https://doi.org/10.2105/ajph.2020.305906 https://doi.org/10.1016/s0140-6736(21)02796-3 https://doh.wa.gov/data-and-statistical-reports/diseases-and-chronic-conditions/communicable-disease-surveillance-data/respiratory-illness-data-dashboard https://doh.wa.gov/data-and-statistical-reports/diseases-and-chronic-conditions/communicable-disease-surveillance-data/respiratory-illness-data-dashboard https://doh.wa.gov/data-and-statistical-reports/diseases-and-chronic-conditions/communicable-disease-surveillance-data/respiratory-illness-data-dashboard https://doh.wa.gov/newsroom/masking-requirements-healthcare-long-term-care-and-correctional-facilities-end-april-3#:~:text=olympia%20%2d%2d%20effective%20april%203,people%20age%205%20and%20older https://doh.wa.gov/newsroom/masking-requirements-healthcare-long-term-care-and-correctional-facilities-end-april-3#:~:text=olympia%20%2d%2d%20effective%20april%203,people%20age%205%20and%20older https://www.270towin.com/states/indiana https://www.270towin.com/states/washington marble & mckillip | daily case trends of covid-19: a comparative analysis of indiana and washington state 57 appendix indiana and washington dataset available at https://doi.org/10.33043/ff.10.1.38-57 fine focus 36 i fine focus oxidative stress response in bacteria: a review sydney r. addorisio, rebecca m. shteynberg, matheus s. dasilva, jacob m. mixon, kyla mucciarone, lily vu, kristina l. arsenault, vanessa briand, sarah parker, savannah l. smith, claudia e. vise, cara pina, laura t. laranjo department of biology, salem state university, salem, ma usa department of biology, framingham state university, framingham, ma usa manuscript received 30 august 2021; accepted 3 january 2022 vol 8 i 37 abstract oxidative stress response (osr) is a defense mechanism used to maintain redox homeostasis after an increase in levels of reactive oxygen species (ros). due to ros, cell components are vulnerable to damage including the membrane and dna which can impact essential functions and lead to cellular death. without repair, damages caused by ros have the potential to disrupt cell function in an irreparable manner. bacterial cells respond to ros using both endogenous and exogenous pathways depending on their method of metabolism and evolutionary ability. bacteria have developed regulatory mechanisms to contain damage and are also known to use antioxidants as defense. in this review we will cover the damage induced by ros to different cellular structures, and mechanisms of osr used by bacterial cells to promote survival. 38 i fine focus introduction the term oxidative stress (os) was coined by helmut sies et al, in 1985 [42], and is used to describe the stress an organism can experience when there is a disruption in its redox homeostasis due to reactive oxygen species (ros). ros are oxygen-containing chemicals that are highly reactive, such as hydrogen peroxide (h2o2), ozone (o3) and their radicals. when they interact with cellular components, those components can become oxidized. interactions with ros can result in damaged dna, rna, lipid membranes, and proteins which may result in cellular death. in contrast, though counter-intuitive, this stress can also act as a cellular signal to encourage proliferation or survival [35]. os affects all types of organisms, including bacteria. the presence of ros and oxidative stress in bacteria is connected to the evolution of microbes in an aerobic environment millions of years ago [25]. defenses against ros and os are complex, including genome regulation to reduce levels of ros, and cellular damage repair. the mechanisms by which bacteria experience, defend against, and repair oxidative damage provides crucial conceptual insight into an organism and its metabolism. the purpose of this review article is to outline both endogenous and exogenous oxidative stress causes and responses to that stress in bacteria. causes of oxidative stress the way different bacteria respond to oxygen is mostly defined by their method of metabolism and reflects how that microbe evolved to respond to oxygen and its potentially harmful byproducts. oxidative stress is created by the production and accumulation of reactive oxygen species and the production of ros and can be driven by numerous endogenous and exogenous factors [49]. bacteria may be exposed to many environmental stressors, which may lead to the production of ros and subsequent oxidative stress. the production of endogenous ros can result from a cell’s own metabolic processes. ros can be produced through the oxidation of respiratory enzymes during cellular respiration [40]. in the bacterium model escherichia coli, ros were confirmed to be produced as a byproduct of both aerobic and anaerobic cellular respiration with the simultaneous generation of both superoxide and hydrogen peroxide. this occurs when molecular oxygen collides with redox enzymes and flavoenzymes. nadh dehydrogenase ii, involved in bacterial respiratory metabolism, lipoamide dehydrogenase, involved in glycine catabolism, and fumarate reductase, involved in anaerobic bacterial respiration, are each known for the production of ros due to their particular ability to transfer their electrons to molecular oxygen [29, 30]. while healthy aerobic cells have evolved to efficiently scavenge the produced ros, obligate anaerobes may experience increased damage from ros produced if exposed to oxygen. exogenous factors also have the potential to produce ros within the cell. if bacteria are in an environment with a high concentration of oxygen, they will experience os (discussed in depth in a review by haugaard [14]). other environmental factors can include toxic chemicals and substances introduced into the environment inhabited by the bacteria, such as herbicides, industrial additives, and medications. paraquat, a lethal herbicide, as well as menadione and phenazines (industrial additives), are redox cycling compounds, molecules that can accept electrons as well as donate electrons to oxygen, producing ros. they are exogenous causes of bacterial oxidative stress due to this ability [28]. noticing how ros are generated exogenously is important to understand why prokaryotes face os. one of the first observations made by scientists when beginning to study the causes of ros was realizing that radiation usually preceded the formation of ros. radiation has been observed to break water down to hydroxyl radicals, which may then externally damage the cell [45, 55]. membrane damage reactive oxygen species can interact with structures found in the bacterial cell membrane. cellular viability, membrane selective permeability, and proton motive force, rely on an intact membrane structure [26]. ros-mediated stress can disrupt these by causing intracellular damage such as lipid peroxidation, which can be detrimental to the cell membrane structure [15]. lipid peroxidation occurs when ros takes away electrons from the lipids, disrupting associated functions of the membrane. vol 8 i 39 lipid peroxidation starts a cascade down the rest of the membrane until the entire membrane is affected. the created lipid radicals react with oxygen to form lipid peroxyl radicals. they can react with other lipids to form hydroperoxide which can be broken down into lipid peroxide. these reactions can affect the structure of the lipids by shortening their tails. these chemical mechanisms are covered in detail in the review by girotti [12]. it is thought that as the number of double bonds in the fatty acid increases, then the sensitivity to oxidation also increases [53]. the alterations of lipids in bacterial membranes cause morphological changes to bacteria. when campylobacter jejuni interacts with ros, the spiral-shaped bacteria change to a coccoid form. ros increases the cell membrane permeability, which results in the structural change seen in c. jejuni [57]. e. coli also experiences morphological changes from the bacillus form to a coccoid form [18]. these morphological changes occur when the bacterium tries to heal itself from the damage caused by ros by removing pieces of the membrane [58]. if this damage to the membrane is not repaired, it can lead to damage of the dna and eventually the death of the cell [18]. another damaging effect of oxidative stress is a decrease in proton motive force (pmf) from inhibition of transport across the cell membrane. the reduction of pmf due to oxidative damage of the membrane structure interferes with the ability of obligate aerobes to create atp [11]. as a result, when their membranes are damaged this can lead to an inhibition of cell growth and eventually cell death [8]. without a fully functional membrane, obligate aerobes cannot produce enough atp to carry out necessary cellular functions. protein damage proteins are also affected by ros due to their structure. amino acids can be modified by ros which can damage proteins. proteins containing cysteine and methionine are particularly susceptible to being oxidized. both can become oxidized to form sulfenic acids, while further reduction can become an irreversible modification [17, 56]. these modifications can affect the functionality of the associated protein. for example, when methionine is oxidized, proteins become denatured and the hydrophilic properties of methionine are lost, which results in structural alterations [2]. it is important to note that although methionine and cysteine are more readily oxidized, other amino acids can also become damaged by ros. amino acids such as arginine, lysine, proline, histidine, and threonine can be carbonylated [39, 50]. when amino acids are carbonylated, this is an irreversible sign of aging and oxidative damage as shown in e. coli [8]. these damages, unless repaired, ultimately lead to loss of function of proteins and cause protein misfolding. the degradation of proteins can be repaired by enzymes and chaperone proteins. heat shock proteins (hsp), a group of chaperone proteins, can aid in the refolding, repair, and recycling of damaged proteins. a redox regulated chaperone protein, hsp33, is specific to prokaryotes and is inactive under reduced conditions. this chaperone protein is activated when the environment becomes oxidized. upon exposure to protein oxidation, hsp33 also unfolds but instead of losing activity and aggregating, it uses the structural rearrangements to activate its chaperone function [16]. additional heat shock chaperone proteins that are not redox regulated can also refold proteins that have been affected by oxidative stress, thus helping restore protein function. dna damage overproduction of ros can lead to modification of nucleotides or the sugar phosphate backbone of the dna helix. damage to the dna can potentially result in mutations, or changes in the genetic sequence. when ros interacts with dna, it will oxidize the structure, generating damage to the dna in the form of strand breaks and base modifications [10]. similar to other endogenously caused os, oxidative dna damage is an inevitable consequence of aerobic cellular metabolism (discussed in reviews by storz & imlay and sigler et al. [43, 46]), and threatens the survival and growth of bacterial cells. ros can interact with both purine and pyrimidine bases, and the deoxyribose sugar backbone of the dna molecule. the likelihood of the oxidation of dna bases depends on the redox potential of the individual nucleotide. it is shown that purines are more likely to oxidize because of their low redox potential. when there is a single oxygen from ros reacting with a purine, it results in the addition of a carbonyl 40 i fine focus group this is the most common damage to purines. for example, guanine has a low oxidation potential and that makes it a prime target for ros, resulting in the formation of 8-oxo-7, 8-dihydroguanine (8-oxog) [22]. after hydroxyl radicals react with dna, the lesions are going to result in a damaged site that, when replicated, causes mutations. the oxidation product of guanine, 8-oxo-g, can base pair with either adenine or cytosine. the base pairing of 8-oxo-g with adenine in bacteria is seen to cause g:c to t:a base transversion mutations [31, 32]. if these transversion mutations happen in sites responsible for protein function, the mutations may decrease the bacterial cell’s survival. base excision repair (ber) is the most common dna repair mechanism to address oxidative damage. glycosylases are a ubiquitous family of enzymes that catalyze the removal of damaged bases from the dna strand in the ber pathway. dna repair mechanisms however, often overlap. oxidized bases can be excised through both the ber pathway, which removes a single lesion using glycosylase, and the nucleotide excision repair (ner) pathway, which removes a lesion-containing segment of the dna strand [7]. the state of the bacteria, however, may influence the use of these repair pathways. for example, in starving p. putida, 8-oxo-g repair is limited, increasing mutations for the purpose of adaptation [37]. regulatory modifications oxidative stress influences how much mrna from genes not required for stress response is transcribed. a study by muthukrishnan et al. performed in e. coli cells noted a decrease in the transcription rates specifically for genes not related to stress when the cells were experiencing os. the researchers noted that under oxidative stress, there was a 76% decrease in the cells ability to transcribe compared to cells under normal conditions. this is likely caused by a decrease in the number of available inducers, regulators, and transcriptional components, extending the time frame in which it takes transcription to occur and decreasing growth rate [33, 60]. translational control under oxidative stress conditions is focused on producing proteins necessary to negate os [3, 51]. oxidative stress negatively impacts the ability of e. coli cells to translate proteins in a timely manner. as concentrations of h2o2 increase, the rate of ribosome translational elongation decreases and the time it takes for the translation elongation rate to recover increases. the cause of this reduced translational elongation rate is the downregulation of trnas by degradation within e. coli under oxidative stress [60, 61]. demonstrated by zhong et al., translation elongation rates under continual experimental os conditions recovered by an increase in trna species 75 minutes after the initial drop [60]. in e. coli, small rnas (srnas) are shown to help cells adjust to environmental changes by controlling expression of key proteins, causing a competition for binding which can result in repressed translation. genome regulation under oxidative stress is also affected by the number of functional and available small rnas (as discussed in a review by van assche et al. [51]). for example, in salmonella enterica, small rnas ryhb-1 and ryhb-2 participate in the oxidative stress response and deletion of these srna lead to an increase of ros in the cell. these srnas are upregulated by oxyr, an important regulon later discussed, a result of oxyr interacting with the srna promoters [3]. small rnas help the cell adjust in response to oxidative stress. another example of srna involved in managing oxidative stress is found in the bacterium deinococcus radiodurans r1, where a specific srna, osia, is required for helping the bacterium handle varying levels of oxidative stress. when the srna is knocked out, the mutant bacteria are more sensitive to h2o2 and produce less catalase, an enzyme that breaks down h2o2. chen et al. show that in the osia mutant, the mrna of a catalase gene, kata, has a decreased half-life. the regulation of kata by osia helps d. radiodurans r1 to cope with oxidative stress, induced by h2o2 [4]. removal of ros in order to neutralize ros, bacteria are equipped with antioxidant molecules (aox) [1]. aox are responsible for protecting bacteria from and fighting against ros and include enzymatic and non-enzymatic antioxidant defenses. enzymatic defenses directly target ros molecules, inactivating them and converting them into molecules that are significantly less reactive [44]. non-enzymatic defenses such as vitamins c and e and thiols are molecules vol 8 i 41 that naturally behave as reducing agents. non-enzymatic antioxidants become oxidized in place of sensitive cellular components [38]. (discussed in reviews by j.g. scandalios and staerck et al. [38, 44].) bacteria use both enzymatic and non-enzymatic methods jointly to address ros and os [1]. enzymatic inactivation of ros includes enzymes such as dismutases, catalases, peroxidases, and reductases, commonly found in aerotolerant bacteria [44]. these enzymes, such as superoxide dismutase (sod), an enzymatic inactivator of superoxide, help to prevent varying types of damage from os. loss of these enzymes have been shown to coincide with increased oxidative damage [19, 47]. one example function of sod is in the periplasm of e. coli. sodc is thought to be a dismutase that detoxifies the superoxide anions that are released from the oxidative phosphorylation process [21]. this process allows for ros damage to be prevented in the membrane. another enzyme involved in ros inactivation is catalase which deactivates hydrogen peroxide into oxygen and water. catalase has been shown to increase survivability of bacteria in the presence of hydrogen peroxide [36]. these enzymatic defenses continue to evolve to help bacteria survive in the presence of ros. enzymatic antioxidants are often regulated by systems called regulons. regulons are groups of bacterial genes that are regulated together to control specific responses. a prominently known regulon, seen in many gram-negative and some grampositive bacteria is the oxyr regulon. an influx of hydrogen peroxide (h2o2) in the cell will initiate a response from the oxyr transcription factor, causing it to convert to its oxidized form. once oxidized, the transcription factor will positively regulate the genes associated with the oxyr regulon [54]. the rpos and perr regulons also respond to h2o2. rpos behaves as a sigma factor, recruiting rna polymerase, while perr is similar to oxyr and is more often found in grampositive cells [52, 9]. while oxyr, rpos, and perr regulons tend to respond to h2o2, the soxrs regulon functions to counteract an increase in superoxide radical anions. the ohrr regulon is another antioxidant regulon which functions to identify and destroy organic peroxides during oxidative stress [9]. although every antioxidant regulon is not present in all types of bacteria, more than one regulon can contribute to bacterial stress responses [9, 52]. non-enzymatic antioxidants can be acquired from the environment, synthesized biologically, or both. vitamin c, or ascorbic acid, is a water soluble, non-enzymatic scavenger of free radicals. ascorbic acid is taken from the environment, though some bacterium, such as streptomyces antibioticus and acetobacter suboxydans are able to produce it [59, 48]. additionally, vitamin c is capable of assisting in the regeneration of oxidized vitamin e, another important antioxidant [34]. vitamin e or alpha tocopherol is lipidsoluble and resides in the hydrophobic region of the cell membrane; it works to defend the membrane from oxidative stress injuries. vitamin e acts to reduce lipid peroxyl radicals and will eliminate the chances of lipid peroxidation of the cell [13, 20]. non-enzymatic defenses can also work jointly with enzymes. glutathione (gsh) is a thiol that can detoxify hydrogen peroxide and lipid peroxides in conjunction with glutathione peroxidase (gsh-px) and will reduce hydrogen peroxide into water and oxygen by donating an electron to hydrogen peroxide. this antioxidant will also protect the cell from lipid peroxidation and convert vitamins c and e back into their active forms [1, 6, 27]. thiols, such as glutathione, thioredoxin, and glutaredoxin, become oxidized to decrease os within the cell. gramnegative bacteria both synthesize and import active thiols like glutathione from the environment using dedicated transporter systems. in contrast, most gram-positive bacteria do not synthesize glutathione, but can import it [5]. once cellular thiols are inactivated through oxidation, they must be returned to their active, reduced form. this maintenance is performed by enzymes such as glutathione reductase, thioredoxin reductase, and glutaredoxin [5]. glutathione reductase helps to keep glutathione in its reduced form to respond to an increase of ros when the cell is under oxidative stress [5]. thioredoxin is reduced by thioredoxin reductase (discussed in this review by lu & holmgren [23]). the importance of thioredoxin reductase can be noted in a study by serrano et al. of lactobacillus plantarum where they found that overexpression of trxb1, a thioredoxin reductase, results in a high resistance to ros [41]. the thioredoxin system also plays a role in dna repair and is highly conserved among various bacteria including, e. coli, helicobacter pylori, bacillus subtilis, and mycobacterium tuberculosis [23]. 42 i fine focus glutaredoxin is important in that it will deactivate oxyr when h2o2 levels are reduced to a normal range [54]. thiol maintenance is vital for the survival of bacteria in oxidative stress environments. conclusion while oxidative stress can be an important signaling strategy for bacteria, it can also be a dangerous source of cellular damage [35]. ros of both endogenous and exogenous creation threaten to oxidize key components of the cell [49]. this review has discussed the range of damage that can be caused. ros can oxidize proteins resulting in both reversible and irreversible damage, lipid peroxidation can threaten the integrity of the cell membrane, and dna damage can induce mutations within the genome [17, 56, 15, 10]. fortunately, microbial evolution has provided bacteria with mechanisms to counteract os and its damage. although more abundant in aerobic bacteria, all bacteria have some mechanisms for addressing ros. regulatory modifications can be made to both transcription and translation to modulate the use of resources and the production of antioxidants [3, 33, 51, 60]. ros can then be neutralized using enzymatic and non-enzymatic mechanisms and the damage repaired [38, 44]. bacterial genomes have entire regulons dedicated to these responses. antioxidants and their maintenance enzymes are closely regulated to both evaluate the oxidative state of the cell and ensure bacterial survival. while bacteria have the capability to respond to oxidative damage, the process is not infallible if the stress outweighs the capacity of the cell to respond. vol 8 i 43 references 1. birben e, sahiner um, sackesen cs, erzurum s, kalayci o (2012) oxidative stress and antioxidant defense. 2. brot n, weissbach h (1983) biochemistry and physiological role of methionine sulfoxide residues in proteins. arch biochem biophys 223:271–281 3. calderón il, morales eh, collao b, calderón pf, chahuán ca, acuña lg, gil f, saavedra cp (2014) role of salmonella typhimurium small rnas ryhb-1 and ryhb-2 in the oxidative stress response. res microbiol 165:30–40 4. chen y, xue d, sun w, han j, li j, gao r, zhou z, zhang w, chen m, lin m, wang j, zuo k (2019) srna osia stabilizes catalase mrna during oxidative stress response of deincoccus radiodurans r1. microorganisms. doi: 10.3390/microorganisms7100422 5. couto n, wood j, barber j (2016) the role of glutathione reductase and related enzymes on cellular redox homoeostasis network. free radic biol med 95:27–42 6. curello s, ceconi c, bigoli c, ferrari r, albertini a, guarnieri c (1985) changes in the cardiac glutathione status after ischemia and reperfusion. experientia 41:42–43 7. demple b, halbrook j (1983) inducible repair of oxidative dna damage in escherichia coli. nature 304:466–468 8. desnues b, cuny c, grégori g, dukan s, aguilaniu h, nyström t (2003) differential oxidative damage and expression of stress defence regulons in culturable and non-culturable escherichia coli cells. embo rep 4:400–404 9. dubbs, j. m., & mongkolsuk, s. 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regulation, biodegradation, biomonitoring, soil vol 9 | 27 abstract crude oil is a major environmental pollutant in the niger delta region of nigeria due to heightened operations of the petroleum industry in this location. this comparative study of two different petroleum hydrocarbonpolluted soils from the bodo and tombia communities in rivers state was carried out to determine microbial abundance (distribution) in relation to the depth and soil texture of the polluted sites. soil samples were collected at 15 cm and 30 cm from each site. baseline physicochemical parameters and microbial counts were determined using standard methods. hydrocarbon utilizing bacteria were screened with 2,6-dichlorophenol indophenol (dcpip) for crude oil degradation efficiency. the average total petroleum hydrocarbons (tph) collected from the areas values are higher than the intervention value of 5000 mg/kg as stipulated by the department of petroleum resources (dpr) in the environmental guidelines and standards for the petroleum industry in nigeria (egaspin). microbial distribution patterns were not significantly different (p=0.08) between the topsoil and subsoil in the sites investigated. there was no significant variance in the relationship between soil texture and microorganism distribution (p=0.998). an interaction of the effects between the two independent variables produced an insignificant variance (p=0.411). average viable culturable heterotrophic and hydrocarbon utilizing microbial counts for the sites varied considerably between 105 and 106 colony forming units per gram of soil (cfu/g) at both depths. a consortium of pseudomonas spp., proteus spp., sphingobacterium spp., and bacillus spp. isolated from the sites efficiently degraded crude oil, showing a 100% decolourization of dcpip in bushnell-haas-crude oil broth. this study demonstrated that soil texture and depth affect microbial diversity and their function in crude oil contaminated sites. 28 | fine focus introduction crude oil, a hydrocarbon-containing material, is one of the most serious environmental contaminants. it contains n-alkanes, cycloalkanes, and polycyclic aromatic hydrocarbons (pahs), which are harmful to the environment and human health [4]. drilling, shipping, and processing of these products, among other operations, can cause crude oil pollution. saturated and aromatic hydrocarbons, asphaltenes, and resins make up the most crude petroleum oil [19]. oil spills are common occurrences because of manufacturing and transportation errors due to the widespread usage of oil and petroleum products in our everyday life [18]. oil is a liquid fossil fuel extracted from below the ground, formed over millions of years due to high pressure and heat. because of the time required for it to form, it is a nonrenewable energy source. the liquid components of oil accumulate in porous rock formations, and different types of crude, “sweet or sour ”, have varying sulphur content [2]. crude oil is refined and used as fuel for transportation or cooking in forms such as petrol, diesel, cooking gas, etc., with other derivatives such as bitumen used for binding asphalt, which is used in making roads [14, 2]. for over a century, crude oil has been extracted from subsurface reservoirs with devastating effects on communities and environments from which it is being extracted due to spillage. in ogoniland, nigeria, the contamination from spillage, vandalism and illegal refineries, killed large areas populous with mangroves. levels found in the more polluted sites are high enough to cause severe impacts on the ecosystem and on human health [24]. toxic effects may occur when the spill is new and has a substantial amount of light aromatic hydrocarbons. the oil can coat vegetation and water masses, suffocating them and causing death and affecting animal life in the area of contamination. most of the toxic hydrocarbons evaporate or are degraded by microbes within the soil quite quickly [24]. polycyclic aromatic hydrocarbons (pahs) build up quicker in soil, being strongly adsorbed onto sediment, making soil a noteworthy pah sink [26]. crude oil pollution may have effects on physical properties of soil such as decreased soil aeration, decreased soil macroporosity/ permeability, hydraulic conductivity, and increased bulk density, which could affect vegetation. one research found soil sampling beyond the depth of 15 cm a constraint due to hard pan formation from crude oil and soil [1]. crude oil contamination also inhibits the germination of food crops thus affecting the livelihoods of farmers in areas of spillage [23]. there is a need to recover soil and water polluted by crude oil, which is achieved by a process known as remediation; remediation can be physical, chemical, or biological (bioremediation). bioremediation is preferable due to its efficiency with both cost and safety. furthermore, it does not require the addition of foreign or toxic chemicals, something not true of chemical remediation. vol 9 | 29 bioremediation mainly uses fungi and bacteria as agents for remediation and can be combined with mechanical treatment and dispersants application. microorganisms in their metabolic activities produce energy that forms the key to the bioremediation process. the continued presence of desirable microbes with appropriate metabolic capacity is an important prerequisite for the bioremediation process [30, 12]. subsurface microorganisms tend to be capable of degrading hydrocarbons found in crude oil. bioremediation methods include bioaugmentation, which involves introducing microorganisms with known degradation ability for the contaminant to a polluted site followed by nutrient enrichment (biostimulation). biostimulation is when nutrients like nitrogen and phosphorus are added to stimulate microbe growth [36, 30, 8] or organic fertilizers such as poultry droppings, sewage slurry, and cow dung are also used [21]. to determine which microorganisms are best suited for bioremediation at a specific site, microorganisms already present in the polluted site are isolated and their effectiveness tested ex situ. any bacteria found in a contaminated site are likely to have hydrocarbon degrading abilities and the appropriate bacteria can be introduced in situ for remediation. methods of choice for bioremediation depend on the physicochemical properties of the polluted matrix, on the degree, and the age of the spill. some families of microorganisms found to be effective in bioremediation include proteobacteria, actinobacteria, firmicutes and fungi strains. for effective remediation, variable environmental conditions are controlled such as oxygen availability, hydrocarbon solubility, and nutrient balance [9]. colony forming units (cfus) are used to determine the abundance of various microbes in the soil. in order to identify the components of the crude oil, a total petroleum hydrocarbon (tph) test is needed. this is necessary for the bioremediation process. fig 1: scene of the oil spill in ogoniland showing the extent of damage to land and water. photo credit george osodi 30 | fine focus this study seeks to find the correlation between elevation gradient (soil depth), soil texture, and microbial distribution (abundance) in polluted soils in response to an observed conflict in the literature reviewed. some literature concluded that there is reduced microbial presence with soil depth in non-polluted soils, while others concluded that there is no variance in microbial distribution with soil depth in polluted soils. methodology sample collection: the polluted soil samples were collected from 15 cm and 30 cm depths from bodo, gokhana, rivers state, and tombia, degema, rivers state. the coordinates are 2.76nw, e90 and 4.79n, e6.90 respectively. soil samples were immediately placed on ice before being transported to the laboratory where they were stored at 4°c before handling. experimental setup: the soil samples were labeled as surface soil and subsurface soil depth respectively. four (4) for surface and four (4) for subsurface soils. for the tombia site, the two topsoil samples were labeled tsp i and tsp ii with the two subsurface soil samples from the same site labeled tssp i and tssp ii. for the site at bodo, topsoil samples were labeled bsp i and bsp ii, with the subsurface samples labeled bssp i and bssp ii. microbiological analysis: enumeration of total heterotrophic bacteria and total fungi to enumerate the total heterotrophic bacteria and fungi in the samples, soil samples were given a 10-fold serial dilution after separation in normal saline. samples were spread across plate count agar plates and potato dextrose agar (pda) plates in duplicates. the plates were incubated for 24 hours at 37°c while potato dextrose plates were incubated for 72 hours at 28°c. bacterial colonies and fungal spore forming units were thereafter counted and isolated. enumeration of total hydrocarbon utilizing bacteria and fungi to determine the hydrocarbon utilizing bacteria (hub), samples were inoculated onto bushnell haas agar in duplicates with crude oil placed in the plate cover via the vapour-phase transfer method. the duplicate samples were then incubated at 30 °c for 5 then plates with bacteria colonies between 30≤cfu≤300 were counted [7]. for fungi, the bushnell haas agar was supplemented with 0.05% chloramphenicol and incubated for 5-10 days at 30°c. discreet colonies were sub-cultured on pda. anaerobic plates were all incubated in a selfsetup candle jar and incubated under the above stated conditions [17]. identification of isolates using biochemical and microbiological tests the bacterial isolates recovered from this study were characterized using the following parameters: gram staining (gr) vol 9 | 31 and morphology. this was done using microscopy. the morphology of bacteria could either be round (cocci), rod-shaped (bacilli), spiral (spirilla), comma (vibrios) or pleomorphic etc. this is determined using the gram stain, catalase test [34], methyl red and voges-proskauer [33], indole test [16], motility test [37], citrate test, lactose test, sugar utilization test (glucose, sucrose & lactose) [20], triple sugar iron agar (tsia) test [3], and oxidase test [29]. fungal isolate identification procedure agar with fungi to be identified was placed on a grease free slide. lactophenol blue was applied to the cut agar. the slide was then covered with a coverslip and viewed under the 40x lens of a microscope [22]. based on these results, a most probable microorganism was determined for the samples based on morphology and biochemical activity. determination of total petroleum hydrocarbons (tph): polluted soil samples were analyzed using a gas-chromatography flame ionization detector (gc-fid). this method is designed to resolve a broad range of hydrocarbons (i.e., n-c8 to approximately n-c44) [20]. residual total petroleum hydrocarbon (tph) and polycyclic aromatic hydrocarbon (pah) contained in the polluted sample were extracted and then quantified using a gas chromatograph mass spectrophotometer (gc-ms). hydrocarbon degradation screening using spectrophotometry: bacteria degradative abilities were screened using the redox dye, 2, 6-dichlorophenol indophenol (dcpip). 1 g of dcpip was dissolved in 1 l of sterile water for preparation. organisms to be screened were calibrated with a mcfarland standard in normal saline to ensure parity in the amount of culture in all isolates. each tube was vortexed to homogenize the mixture\. on achieving identical quantity of organism for each tube, 1 ml of organism in normal saline suspension was transferred into 9 ml nutrient broth and incubated for 24 hours at 35 ˚c. bushnell-haas broth was prepared according to the manufacturers guides and 9 ml were dispensed into test tubes and supplemented with 0.3 ml of crude oil prior to sterilization. to each test tube, 0.4 ml of the isolates were transferred into the bushnell haas broth with sterile medium. 0.3 ml of the dye dcpip were then added. preparations were done in duplicates with appropriate controls. the first control comprised dcpip, bhb and crude oil. the second control contained only dcpip and bhb while the last control comprised dcpip, bhb and sterile water. the setup was monitored at an interval of 6 hours for 120 hours for colour and optical density changes using a spectrophotometer at au600 [7]. 32 | fine focus determination of soil texture: sieve method the sieve method was used in combination with the soil texture triangle to determine the clay, silt or sand content of the soil. in this method, sieves of different fineness are used. the soil sample is first weighed. the various sieves are stacked according to their fineness from top to bottom in order of least fine to finest. the weighed soil sample was placed in the sieve and the setup was placed in a shaker [15]. the soil was separated into its components based on fineness. each component was then weighed using a weighing machine accurate to the nearest 0.1 grams. percentages were assigned to each soil segment and comparison in accordance to the world reference base for soil resources (wrb) was performed. method of calculation: % fraction = [mass of fraction(g) / total mass of soil analyzed (g)] x 100% [31]. the table below shows the soil texture classification according to wrb and usda: table 1: soil texture classification. table 1 shows the diameter of soil particles in millimeters for each soil texture type in the usda and wrb measurements. it gives a range to use for measured weight for soil left in pan, giving the weightiest pan being the predominant texture for a soil. results physicochemical parameters the results for the total petroleum hydrocarbons (tph) and polycyclic aromatic hydrocarbons (pah) and other physical and chemical parameters such as the ph, conductivity and soil texture analysis are tabulated below. table 2: results of physicochemical analysis. these test measure different physical and chemical parameters that may affect microbial growth in the soil. ph as an example gives insight that the soil is more so acidified and flora may be adjusted to suit such environments. total petroleum hydrocarbon (tph) was above recommended amount by the dpr (5,000 mg/kg) which made a clear show of pollution in the studied areas. vol 9 | 33 tph is above the recommended limits of 5,000 mg/kg as prescribed by the department of petroleum resources (dpr) as well as pah for the bodo topsoil and tombia top and sub soils. ph for all soils were found to be more acidic and temperature was moderate within the soils. table 3: soil texture analysis. soil texture was classified using the fao’s relative method of classification. the tombia soil going by analysis was found to be more clayey (9.56% clay) in comparison to the bodo soil (0.13% clay) generally, more sandy. in table 3, the results for soil texture are shown. bodo soil was determined to be sandy soil going by the percentage of sand (99.87% sand, 0.13% clay/silt) while tombia soil was determined to be silt/clay (90.44% sand, 9.56% silt/clay) relative to bodo soil using relative classification of soil for agriculture [13]. 34 | fine focus microbiological and biochemical analysis heterotrophic bacteria were enumerated using nutrient agar while the fungi on potato dextrose agar (pda). bushnell haas agar was deployed for both bacterial and fungal enumeration (fungal enumeration differed by supplementing the agar with an antibiotic (chloramphenicol)). table 4: heterotrophic count. all bacteria and fungi numbers within the soil were counted using petri plates. this was to find the abundance of microbes at each strata in the soil sampled. table 5: hydrocarbon utilizers. bacteria and fungi that could utilize crude oil as a growth source were enumerated on a mineral medium. table 6: organisms identified and codes. bacteria discovered from samples identified through biochemical test and microscopic examination are listed out below. pseudomonas was the most abundant species found within all the soils. it is a well-known hydrocarbon utilizer. vol 9 | 35 biodegradation screening twelve bacterial isolates and four fungal isolates were screened for their degradative abilities on crude oil. this was done using the redox dye 2,6 –dichlorophenol indophenol based on the microorganism’s ability to decolourize the dye within a 120-hour period. this was observed using spectrophotometry at optical density au 600nm taking readings at intervals. 36 | fine focus figure 2 a and b: degradation rates of the isolates for crude oil. microorganisms ability to degrade crude oil based on the discolouration of the redox dye 2,6 –dichlorophenol indophenol was performed every 12 hours using spectrophotometry to measure. the organism for tssp ii 10-4, pseudomonas had one of the greatest powers and was identifiable from the greenish colour growing on nutrient agar. vol 9 | 37 table 7: two-way anova analysis with replication was carried out to determine the effects of soil texture and soil depth on how microorganisms were distributed throughout samples taken. this was done independently and dependently under each factor under consideration. 38 | fine focus statistical analysis a two-way analysis of variance was performed, shown in table 7, to determine the first objective of this study – the effect of soil depth and soil texture on the distribution of microorganisms. it tested 3 null hypotheses: h01: that soil depth has no significant effect on microorganism distribution h02: that soil texture has no significant effect on microorganism distribution h03: that the interaction between soil texture and soil depth has no effect on microorganism distribution the two-way anova revealed no significant difference (p=0.08) in the relationship between soil depth and microorganism distribution and no significant difference (p=0.998) in the relationship between soil texture and microorganism distribution. equally, there was no significant difference (p=0.411) when the effect of both factors (soil depth and soil texture) was combined in relationship to microorganism distribution. discussion two sites of known crude oil contamination, bodo and tombia, were selected for sample collection. two samples of topsoil and two samples of sub soil were collected from both sites and subjected to various analysis. the change in transmittance/absorbance in colorimetric tests [seen in figure 2] on inoculation with the various isolates, shows action of crude oil/hydrocarbon degrading microorganisms (bacteria) and proves that there was pollution by crude oil at the abovementioned sites. one such proof of crude oil degrading activity observed was the change in absorbance/transmittance values at 600nm, for sample bssp ii 10-4 er3, from 1.837 to 0.677 over a period of five days, which indicates degradation of crude oil. table 6 shows which biochemical and microbiological tests, combining microscopy and morphology, were used in the identification of some of the microorganisms present at the sites of crude oil pollution. from these tests, five (5) species of bacteria were identified – three were from the tombia site (sphingobacterium sp., pseudomonas sp. and bacillus sp.) and the remaining two (proteus sp. and staphylococcus sp.) were from the bodo site. pseudomonas sp. made up 70% of the species and sphingobacterium sp. made up 10%. at bodo, proteus sp. and staphylococcus sp. made up 50% each of the species identified. it is noteworthy that the bodo soil was determined to be sandy soil going by the percentage of sand (99.87% sand, 0.13% clay/silt) while tombia soil was determined to be silt/clay (90.44% sand, 9.56% silt/clay) relative to bodo soil using relative classification of soil for agriculture. this did not prevent cultures of bodo soil samples from yielding similar levels of viable counts for hydrocarbon degrading bacteria and fungi as seen in the results. a closer examination of the count figures in the mentioned tables, using figure 3a and 3b for rough analysis, shows there is an insignificant difference in prevalence of microorganisms between the two sites. this is confirmed by the statistical analysis. vol 9 | 39 figure 3a and 3b: effect of soil depth and soil texture on microbe abundance. the chart shows the abundance of all microorganisms found in bodo (fig. 3b) and tombia (fig. 3a) areas in log (cfu/g). the subsoil markedly in both soils has a greater abundance of microbial life. this shows the role texture and depth have on the presence of microorganisms to begin degradation intrinsically in the soil. 40 | fine focus heterogeneity (evenness of mixing of different fractions of the soil) and microbe diversity, and highlighted the affinity of certain classes of microbes to certain fractions of soil, which was dependent on the mineral constitution of those fractions of soil. this allows a possibility that the bodo soil was more heterogeneous or that its fractions supported a different taxon of microbes in relatively equal abundance with the tombia soil and could explain why the microbe distribution in our results showed no significant variance with soil texture between the two sites. heterogeneity and microbe composition were not explored in the tombia and bodo soil samples, which leaves room for this to be incorporated for study in further research. the average total petroleum hydrocarbons (tph) in each site were 15,642.5 mg/ kg in the topsoil and 11,342 mg/kg in the subsoil collected from the bodo community whereas polluted soil from the tombia site had an average of 14,684.9 mg/kg and 9,647 mg/kg of tph in the top and sub soils respectively. these values are higher than the intervention value of 5000 mg/kg of soil as stipulated by the department of petroleum resources (dpr) in the environmental guidelines and standards for the petroleum industry in nigeria (egaspin), indicating pollution [21]. to run the two-way anova, the serial dilution values of 10-5 were the only values used and were converted to log(cfu/ml) before use. in cases where one serial dilution showed tntc (too numerous to count) and another dilution had a valid value within the range 0≤ cfu≤300, a value of 300 cfus was adopted for the dilution that was tntc, in accordance with practices a two-way anova analysis, however, found the effect of the said factors (soil depth and soil texture) on the distribution of microorganisms in polluted soil to be insignificant. as shown in table 7, the relationship between elevation gradient and microorganism distribution was insignificant (p=0.08). the relationship between soil textures of samples, which differed based on the source site, and microorganism distribution was found to be insignificant (p=0.998) as well. the effect of a combination of the independent variables was equally found to be of no significant effect (p=0.411). thus, the null hypotheses were accepted. this result tallied with the conflicting outlier study of spina et al. [32], which stated the studied independent factors had no significant relationship with microbial abundance thus provoking this study. the soil texture was determined using the sieve method with guidance from the wrb soil texture classification found in table 1 [31, 5] and the texture triangle by the usda, which classifies all particles mm>0.063 as sand. still, looking at the varied structure of the two soils, the results in table 3 revealed that the bodo site samples had significantly less coarse sand than the tombia site, and significantly more medium sand than the tombia site, with a possibility existing that medium sand could hold sufficient moisture and organic matter. the higher percentage of silt/ clay in tombia could have compensated in moisture and organic matter content, which has been indicated as necessary to support microorganism growth [11, 35]. seaton et al. [28] analyzed a sample size of over 330 soils from varying habitats and found a relationship between soil textural vol 9 | 41 in research done by global alliance for tb development for tuberculosis (tb) patients under cohort study [6]. where the values of all serial dilutions for a sample were na, the results were omitted from the statistical computation as reported by parshionikar et al. [25] in a revised document prepared to serve as an internal guide to personnel of the u.s. environmental protection agency on microbiological methods of analysis. where all dilutions were tntc, a count value of 300 was adopted for the final dilution [25]. where both plates, at the same dilution, gave a count of tntc and a nonzero count, a value of 300 was assigned to the tntc plate and it was averaged with the count of the non-zero plate [6]. where plates on equal serial dilution levels had one non-zero count and a zero count, the counts were averaged to find the log (cfu/ ml) [6]. where a plate of the same dilution level had an na count and the other a nonna count, the log(cfu/ml) of the non-na plate was used [6]. to ensure equal number of rows for both independent variables in the analysis, data rows were deleted in order to match rows with unusable data (nu) in either of the groups. for instance, if tsp1 had an unusable dilution value at 10-5, tssp 1, bsp1 and bssp1 would also be deleted, and this resulted in a deletion of 8 out of 32 entries [6]. plates with na and 0 counts on the final serial dilution were marked nu and excluded from computations [6. 10]. conclusion soil depth and soil texture, when considered independently or concurrently, have no significant effect on the microorganism distribution/abundance in petroleum hydrocarbon polluted soil based on this study. soil depth appears not to be a determinant of microorganism’s abundance from this study, which could be attributed to the fact that different taxa of microbes thrive in different mineral conditions, that is, there are relatively equals number of microbes in top strata as there are at lower strata with the only difference being in their diversity. soil texture also proved to not be a determinant of microorganism abundance evidenced by the difference in soil texture between the tombia and bodo sites, with a surprisingly insignificant difference in microbe numbers. the force of mineral based selection or heterogeneity in soil texture seems a probable force here as well. another reason for the non-significant difference in texture could be because the tombia and bodo sites have relatively similar textures where sand to silt/clay content is concerned. from the presence of hydrocarbon degrading activity of the isolates observed, proteus sp. and staphylococcus sp. are suitable organisms for the bioremediation of the bodo site. sphingobacterium sp., pseudomonas sp. and bacillus sp. are potential microorganisms for bioremediation judging from their presence in soil samples from the tombia site of which the same samples have shown activity in degrading crude oil in vitro as observed using colorimetric studies. of course, pseudomonas sp. seemed to thrive best with a presence in both topsoil and subsoil and a seemingly high prevalence considering frequency of identification in the isolates. this may be attributed to the bacteria being a facultative anaerobe [27] and hence could have the best suitability for remediation at tombia. 42 | fine focus references 1. abosede, e. e. 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(n.d.). retrieved july 26, 2021, from https://en.m.wikipedia. org/wiki/soil_texture#cite_ref-6. 32. spina f., spini g., poli a., blieux a., regnier t., gramellini c., varese g. c., puglisi e. (2018). molecular and microbiological insights on the enrichment procedures for the isolation of petroleum degrading bacteria and fungi. frontiers in microbiology 9, 2543. 33. sylvia, m. (2009). methyl red and voges-proskauer test protocols. american society for microbiology, 1, 1–9. www.asmscience.org. 34. tankeshwar, a. (2021). gram staining: principle, procedure, results. microbe online. https://microbeonline.com/gram-staining-principle-procedure-results/. 35. turner, s., mikattla, r. (2017). microbial community dynamics in soil depth profiles over 120,000 years of ecosystem development. frontiers microbiology 2017, 8, 874. 36. ulrich a. c., brown l. d., cologgi d. l., gee k. f. (2017). bioremediation of oil spills on land. in oil spill science and technology: second edition, 699–729. https://doi. org/10.1016/b978-0-12-809413-6.00012-6. 37. watson r. (n.d). glucose broth and durham tubes or motility agar. general & medical microbiology molb/micr 2021. https://www.uwyo.edu/molb2210_lab/ info/biochemical_tests.htm#glucose. fine focus 12 i fine focus phage therapy: challenges and opportunities immadi siva ratnakar [sivaratnakar.immadi@gmail.com] department of biosciences, sri sathya sai institute of higher learning, vidvagiri puttaparthi, india manuscript recieved 31 august 2021; accepted 24 september 2021 vol 8 i 13 the development of antibiotic resistance in bacteria is a growing concern. this situation demands a search for antibiotic alternatives. bacteriophages—natural viral predators of bacteria—are viewed as a possible alternative to treat bacterial infections. many clinical trials today have not found phages effective as therapeutics. some of the major challenges regarding usage of bacteriophage as a therapeutic have been: horizontal evolution of bacteria, limited host range of bacteriophage, removal of endotoxins in preparations, the technical feasibility of isolation, mode of administration, rapid clearance and immune rejection. these issues have been addressed in this review. applications of genetic engineered phages and other remarkable nonhuman applications are also discussed. abstract 14 i fine focus introduction after the golden age of antibiotics (1950-60), use of antibiotics as the first line of defence has increased dramatically (1). inappropriate prescription of antibiotics by clinicians is a major problem today (2). seventy percent of antibiotic use in the usa is attributed to use on cattle (3). indiscriminate usage and misuse of antibiotics have accelerated the emergence of antimicrobial resistance. a growing list of infections is becoming harder to treat, as antibiotics are becoming less effective (4). sir alexander fleming expected the arrival of the antibiotic resistance era and was worried about the rise in antibiotic resistance by self-medication (5). it is estimated that by 2050, bacterial infections will cause 10 million deaths every year (6, 7). alternatives are urgently needed to effectively treat these infections and prevent the return of pre-antibiotic era. there is active research currently undertaken for the development of novel classes of antibiotics (8, 9). bacteriophages (also known as phages) might provide us with a promising alternative for antibiotics. these are the viruses that infect bacteria and are the most abundant living entities in the world. it is predicted that every millimeter of a natural sample has 107 phage particles (10). the application of phage as therapeutics against bacteria is called phage therapy (bacteriophage therapy). phage therapy is drawing global attention due to the rise in antimicrobial resistance. early studies on phage therapy were conducted in georgia (11). to date, only a few clinical trials have been conducted to modern standards (randomized, placebo-controlled, double-blinded) by the united states (us) food and drug administration (fda) as well as the european medical agency (ema) jurisdictions. it is necessary to re-evaluate the challenges involved in phage therapy. here, a review of the challenges, possible solutions, safety and concerns for therapeutic phage applications is presented. other potential applications of phages and their studies in humans are also discussed. why the forgotten magic? bacteriophages were first discovered independently by frederick twort in 1915 (12) and félix d’herelle in 1917 (13). d’herelle realized the potential of these devourers of bacteria as a therapeutic and conducted further research on phages. one of the early investigations by d’herelle was in india in 1927 (14). the mortality rates of cholera-infected study subjects decreased from 66.66% in control groups to 5.8% in phage treated groups. phages offered a great scope of enquiry, but the simplicity in the production of antibiotics gave antibiotic therapy a lead over phage therapy. the global spread of antibiotic-resistant bacteria and the comeback of the pre-antibiotic era alarmed the scientific community and warranted a search for antibiotic alternatives. phage therapy is a superior alternative to antibiotics with many theoretical advantages. while antibiotics kill bacteria broadly, phages bind and infect the bacteria specifically. high specificity is an important advantage, as it might minimally impact beneficial microflora, making phages safer than antibiotics. the specificity also limits the number of bacterial types gaining specific phage-resistance mechanisms (15). bacteriophages follow lytic and lysogenic pathways of infection. phages that follow the lysogenic pathway, integrate their genome into the bacterial genome, eventually lysing the cell. phages following the lytic pathway enter bacteria, reproduce within and lyse the cell (figure 1). these released phages infect other bacteria. in this process, the number of phages increases exponentially. this exponential growth of phage is advantageous, as theoretically, a smaller dose is needed. the exponential increase is seen specifically where hosts are present, making phages themselves contribute to the dosage at the required site (16). biofilm forming bacteria cause infections such as bacterial vaginosis, urinary tract infections, and middle-ear infections. biofilms are polymeric matrices produced by bacteria as a defence mechanism that allows them to adhere to surfaces (17). even when a bacterium is sensitive to an antibacterial agent, the antibiotic fails to penetrate through the biofilm matrix, increasing the resistance of the bacteria by 1000-fold (18). a phage has a distinctive capability of tackling biofilms efficiently (5) by encoding depolymerases that allow their direct penetration into the biofilm (19). phages are also vol 8 i 15 known for stimulating an immune response. receptorbinding proteins of a few phages display collagen motifs (20), which can co-stimulate the number and longevity of t cells (21). furthermore, van belleghem et al. (22) demonstrated that phages can induce reproducible immune responses from monocytes. recent studies have also shown that phages have antiviral properties and that phage therapy may also hold promise as a treatment for sars-82 cov-2 (23, 24). figure 1: phage attaches to the host cell and injects dna to initiate the infection. in lytic cycle, phage dna and proteins are synthesized and assembled into virions. these replicated phages lyse the cell wall and infect another host. lysogenic cycle involves an additional step of integrating their genetic material to form an endogenous prophage, which compromises with the safety of the therapy. 16 i fine focus challenges and potential solutions in phage therapy 1. host range challenge: bacteriophages selectively bind to specific receptors of bacteria, which confer a relatively narrow range of infectivity (25). this would narrow the infectivity range challenging the choice of phage for therapeutic use. as a minimum requirement, phages used in phage therapy should follow only the lytic pathway to ensure the safety of the patient (figure 1). bacterial infections, where the currently isolated phages lack a lytic cycle, are therefore not treatable with phages, and the bacteria include rickettsia, coxiella africanum, mycobacterium leprae, proteuspenneri, citrobacterkoseri, salmonella arizonae, porphyromonas spp, and hafniaalvei spp (26). potential solutions: antibiotics: at present, phage therapy is generally considered as a last resort when a single bacterial strain dominates. in such situations, the synergistic use of antibiotics like ciprofloxacin in combination with phages can reduce the bacterial load by 10,000 times (27, 28). this combined therapy can boost bactericidal activity with their different mechanisms of attack. however, the choice of the combination is crucial. antibiotics must not interfere with phage replication (29). phage cocktails: another possible solution is by employing a combination of phages (so-called phage cocktails), which cover a large spectrum of bacterial strains. bacterial isolates of a patient are screened against a library of lytic phages for infection susceptibility. the infectious phages are administered together as multivalent phages (30). d’herelle’s pyophage and intestiphage (11) are a few well known accessible commercial phage cocktails. genetic engineering: genetic engineering can be used to improve the host range by modifying tail f ibres (31). it can also improve the eff icacy of phage therapy by converting a temperate phage to a lytic phage by removing its repressor genes (32). 2. endotoxins challenge: in the recent phagoburn clinical trial, a team of doctors had to reduce the dosage of phage administration from expected 106 pfu/ml to 10-100 pfu/ml due to high endotoxin concentrations in the phage preparations (33). bacterial debris may remain in the phage preparations even after filtration. in the historic era (around 100 years ago) of phage therapy, not all the debris was removed, and the authors reported a few chemical contaminants which brought about death and illness (30, 34–36). the typical phage purification process (ultracentrifugation in cscl gradient) requires intensive labour, high expense and is time consuming (37). potential solutions: the bacterial debris, having pyrogens and toxins (38), can be cleared and high purity levels can be achieved with nanofibrillated filters (39). endotoxin removal proteins are now available commercially (40). for a large scale production, usage of surrogate hosts could offer a superior solution(37). challenge: in recent clinical trials, patients suffered many side effects due to increased concentrations of endotoxins among which abdominal pain, sudden fever and chills were common. many biologists attribute endotoxins as the prime cause for these side effects (41). expression of endotoxin genes in phages or rapid lysis of bacteria in patients can release toxins (38, 42). potential solutions: a therapeutic phage having a lysgene (endolysin-deficient phage) cannot lyse the peptidoglycan layer of bacteria after infecting the bacteria. phages attack the bacteria and do not lyse the host membrane, which does not lead to the release of endotoxins. the macrophages then eliminates these incapacitated bacteria (43). vol 8 i 17 3. immune rejection and rapid clearance challenge: large phage titers trigger the release of neutralizing antibodies in high amounts (44), which would hinder the action of phages. being in continuous exposure with phages, 81% of healthy individuals show antibodies to t4 phage, prior to the treatment itself (45). though the phage kinetics are much faster than the release of neutralizing antibodies (30, 46), the presence of such anti-phage antibodies before phage administration, and the release of anti-phage antibodies during the treatment brings concerns (47). potential solutions: liposomal delivery of phages can decrease the clearance rate of phages by guarding the phages against anti-phage antibodies (48). frequent administration of phages can reduce the rate of neutralizing antibodies (49). cell-mediated immunity can be combated by making the phage protein coat express polyethylene glycols, which increase the phage circulation time in the blood (50). challenge: geier et al. (51) first observed rapid clearance of phages when lambda phages were injected in high titers into transgenic mice lacking immune response. the administered phages are rapidly cleared by the reticuloendothelial system (res) and are not available for therapeutic use in the body (52, 53). potential solutions: longer circulating phages can be obtained by “serial passage” into the bloodstream of a mouse, and selecting phages with higher circulation time than the original wild-type phage (54). according to levin and bull (55), phage treatment should only decrease the pathogen to an extent where the immune system can successfully clear the bacterial load. phage engineering can help us generate phages that don’t replicate or proliferate (56, 57). these can make an immune safe therapeutic phage. 4. horizontal gene transfer once a phage infects a bacterium, the phage genome is replicated inside the host and eventually, phages assemble and lyse the bacterium (figure 1). while the phage genome gets packed in the phage capsids, accidentally 1 in 107 phages receive the bacterial genome (58). this phage is now called a transducing particle. infection of transducing particles in bacteria causes horizontal gene transfer (hgt) or transduction (59). transduction enhances hgt of virulent, resistant, metabolic and other fitness genes (60, 61), which enable the bacteria to rapidly adapt and evolve to changing environmental conditions. 4.1 development of phage resistance a complication of phage therapy is that the bacteria can gain resistance to the phage during the treatment (15, 62–64). among 12 phage therapy clinical trials, seven studies reported phage resistance (65). bacteria show phage resistance generally by modifying phage receptors (64). often, such changes would affect bacterial fitness and could reduce its virulence (66, 67) as seen in tom’s case (see human trialsbelow). the development of phage resistance is partially advantageous, but phages interfere with many cellular pathways, including translation, transcription, replication, and so, it is harder for bacteria to gain resistance against phages compared to antibiotics (68). while khawaldeh et al. (69) did not find any development of phage resistance after administering a phage cocktail to a patient detected with pseudomonas aeruginosa uti (urinary tract infection), zhvania et al. (70) did report phage resistance in staphylococcus aureus after phage administration to a patient with netherton syndrome. 5. intracellular treatment challenge: antibiotics can treat intracellular bacterial pathogens (like m. tuberculosis) as they have the capability of entering the 18 i fine focus cell (71). a phage requires bacterial receptors to bind and kill a pathogen. the inability of phages to enter macrophages brings concern in tackling intracellular pathogens using phage therapy. internalization of phages into the infected cells is a crucial step to treat intracellular pathogens. potential solutions: targeting extracellular stage: phage therapy was found to efficiently decrease pathology and prevent ulceration in mycobacterium ulcerans infection, where phages targeted a temporary extracellular stage of the bacterium (72). using cell penetrating peptides: the model phage m13 decorated with cell penetrating peptides was localized in the er, endosomes, and golgi within 6 hours of internalization in hela cells (73). however, phages displaying such peptides might circulate for a lesser time (74). liposomal internalization: liposomes with a positive charge fuses with the negatively charged cell membrane to deliver phages inside the cell membrane (75). using non-pathogenic host: tm4 phage was utilized for intracellular drug delivery into infected macrophages. non-pathogenic m. smegmatis was loaded with phages, and these were phagocytosed by macrophages. the phage lytic cycle then reduced intraphagosomal bacterial counts (76). 6. which route works best? 6.1 oral oral administration of a therapeutic is the most convenient and often desired mode of administration. oral delivery of phage poses two major challenges regarding viability and gut transit. the lower survival rates of phages (7%) meeting the hostile acidic environment of the stomach (77) presents a major challenge. even, administering phages with alkali could increase the risk of opportunistic infections (78, 79). administration of phages with yoghurt, or encapsulation of phages, are a possible solution to enhance the survival rate of phages (80, 81). genetically modified phages could offer a simplified and cheaper methodology than encapsulation (82). the gut transit of phages is a question without a clear understanding. there are examples of phages entering the bloodstream (83–85) and not entering the bloodstream (54, 86, 87) after oral administration. furthermore, majewska et al. (88) showed that phage-induced igg and iga hinder the gut transit of phages. experiments by międzybrodzki et al. (80) conclude that the phage entry into the bloodstream depends on the type of phage and the host. therefore, more studies need to be conducted to find specific phage strains which can easily enter the human gut. 6.2 other routes the most effective mode of phage administration is unclear yet. for pulmonary infections, phage delivery through inhalation seems a more convenient and effective administration (89, 90). contradicting this view, few studies show greater effectiveness through intraperitoneal and intravenous routes of administration for pulmonary infections (91, 92). although intravenous administration of phages strongly elicits an immune response, czaplewski et al. (93) believe that phage administration intravenously is also a promising alternative for antibiotics. the two famous success stories in phage therapy (see human trials) utilized systemic phage delivery. considering simplicity and effectiveness, topical administration is highly advisable for eye, ear, nose and skin infections; oral administration for gut infections; intrarectal for prostate infections; and intravenous for systemic infections (94). 7. side effects in phage therapy until recently, phages were considered safe for human use as they selectively bind to only bacteria. also, the abundance of phages in the human body indicates that phages are inherently safe since we are continuously exposed to them enterically and topically (34, 95). however, recent studies question this concept of phages “generally regarded as safe” (96). a systematic review conducted by steele et al. (97) concludes that there is limited evidence supporting the safety of phages. tetz et al. (98) goes on to call phages “potential mammalian pathogens.” vol 8 i 19 the complex interactions of phages with the human body can cause serious side effects in phage therapy. few such side effects could be chronic glomerulonephritis by the accumulation of antiphage-antibody complexes in the glomerular region (23, 99); increased concentration of endotoxins and inflammatory cytokines in the blood (84, 98); increased gut permeability, weight loss, messy hair (100); sudden fever and chills (101). another potential side effect could be the unpredictable consequences of the human microbiome by the introduction of phages (102). the most feared phenomenon by many phage biologists is the integration of virulence genes—like cholera toxin, staphylococcal enterotoxin and shiga toxin—from the phage that can enhance the virulence armoury of bacteria. this could lead to the “evolution of new human pathogens” (103). it is therefore necessary that the therapeutic phages must be fully sequenced to confirm the absence of undesirable genes such as toxins. 8. technical feasibility in a review by czaplewski et al. (93) about alternatives for antibiotics, phage enzymes were thought to have the highest potential to replace antibacterials, while phages were scored relatively lower for their technical feasibility. unlike antibiotics, phage (virus) preparation and storage is costlier. the shelf-life of phages must be long enough for laboratory study or commercial application (104). a therapeutic bacteriophage should not lose its activity before treating patients. attainment of stability is a crucial part of development (105). the phage should grow well under industrial and laboratory cultures and must be easy to store and maintain (37). since every phage strain has different optimal conditions (temperature, ph, buffer) for preservation, not all the diverse number of phages can presently be stored efficiently. lyophilization and spraydrying (107) are current methods available to obtain phage powders, and the optimal conditions of storage for different therapeutic phages are yet to be explored. zhang et al. (106) have successfully produced a freeze-dried phage powder of the model phage m13. 9. other challenges to date, bioethical theories regarding phages have not been published; intellectual property protection is limited in the case of natural phages (108); statistically evident double-blind clinical studies were not reported in adequate numbers. these factors create uncertainty in the development of a dedicated regulatory framework for phage therapy. the peculiar characteristics of phages have made their clinical assessment more complex, demanding further clinical research. regardless of whether there is abundant research performed, commercial implementation of phage therapy would pose a significant challenge. investment factors as well as profitability are exceedingly unknown (95), which hinders pharmaceutical investments (109). potential solutions: in-depth research and large-scale phage production is possible through industrial investments. phage producing centres and hospitals should have good collaboration (110) and be able to work together to provide positive evidence for the regulatory bodies which would grab the attention of pharmaceutical industries. human trials in vitro studies of phage therapy do not consider complex biological interactions, which influence the treatment. our knowledge of phages in vitro is exceptional, but in vivo behaviour of phages is less well-known (111). this demands a need to study phages in a biological system (preferably in humans) to evaluate the efficacy and side effects of the treatment. two major institutes conducting such investigations since the historical era (1917-1995) are eliava institute of bacteriophages, microbiology and virology (ibmv, georgia) and hirfeld institute of immunology and experimental therapy (itet, poland). these institutes, along with felix d’herelle reference center for bacterial viruses (canada), the leibniz institute dsmz (germany), and queen astrid military hospital (belgium) are empowered with huge phage banks to store therapeutic phage cocktails (112–114). eliava institute has treated around a hundred foreign patients since 2012, and the numbers are expanding every year (115). the us navy has also developed 20 i fine focus table 1: data from clinical trials evaluating the efficacy of phages as therapeutic agents year of study problem and etiologic agent route of administration success rate reference 1981 to 1986 suppurative infections by staphylococcus, pseudomonas, e. coli,, klebsiella, and salmonella various 92% (n=550) (41) 1987 to 1999 suppurative infections by escherichia, klebsiella, proteus, enterobacter, pseudomonas and staphylococcus oral, local, intraperitoneal, topical 86% (n=1307) (84) 1987 skin infections by pseudomonas, staphylococcus, klebsiella, proteus and e. coli oral and local 74% (n=31) (118) 1989 post operative wound infections by pseudomonas and staphylococcus local 82% (n=65) (119) a proprietary capacity to purify phage strains for specific infections (116). phage therapy is now being studied globally due to the development of antibacterial resistance. one of the earliest and well-designed controlled trials in georgia was during the 1960s (112). a total of 30,769 children less than 7 years were included. during the annual dysentery period, an anti-shigella phage cocktail targeting shigella boydi, s. newcastle, s. sonnei, s. flexenerei was administered to the children present on one side of a street. the children on the other side of the street received a placebo. a nurse reviewed the subjects once a week for 109 days. dysentery was encountered by 1.76 in 1000 children receiving phage treatment, while 6.7 in 1000 from the controlled group were affected. but, many such studies conducted in the historic era (1917-1995) needed methodological evidence, and the results are not reliable and reproducible (30). many phage biologists, therefore, believe these results to be spurious. a need for clinical trials set to modern standards—placebocontrolled, randomized, double-blind studies—are required to settle the debate on the efficiency of phage therapy. the first us-fda approved phase i clinical trial was executed in 2009 (117), and numerous other studies followed. most of the studies concluded phages as not a significant therapeutic (table 1). many of these studies failed to recruit statistically significant numbers, having small patient groups, which drastically limited the conclusions drawn (10). a recent phase ii clinical trial (phagoburn) approved by france, belgium, and switzerland national health regulators was terminated vol 8 i 21 year of study problem and etiologic agent route of administration success rate reference 1992 skin and nasal mucosal infections by k. ozaenae, k. rhinoscleromatis and k. pneumoniae intraperitoneal 100% (n=109) (120) 1995 urinogenital inflamation by various agents oral and local 92% (n=46) (8% more than antibiotic treated group) (121) 2009 (double blind) otitis by p. aeruginosa local 76% decrease in bacterial count (n=12) (122) 2009 (double blind) chronic venus leg ulcers by p. aerginosa, s. aureus, and e. coli local no significant difference from control. (n=39) (117) 2016 (double blind) diarrhea by e. coli oral no significant decrease in diarrhea in different groups (83) 2018 (double blind) urinary tract infection by staphylococcus aureus, e. coli, streptococcus spp., pseudomonas aaeruginosa, enterococcus spp. intravesical decrease of bacterial titers is 67% (n=9) (101) 2019 burn wounds by p. aeruginosa topical 69% cured, 23%-adverse, 1 person died (n=13) (33) 2020 systemic infection with staphylococcus aureus intravenous 61.5% cured within 7 days (n=13) (123) 22 i fine focus prematurely (several times before the trial had started), as the eligible patient recruitment was inadequate (33). in light of the wma-declaration of helsinki, which states “in the treatment of a patient, where proven prophylactic, diagnostic and therapeutic methods do not exist or have been ineffective, the physician, with informed consent from the patient, must be free to use unproven or new prophylactic, diagnostic and therapeutic measures, if in the physician’s judgment it offers hope of saving life, reestablishing health or alleviating suffering. where possible, these measures should be made the object of research, designed to evaluate their safety and efficacy. in all cases, new information should be recorded and, where appropriate, published.” (124), physicians offer phage therapy as the last resort. often this includes combined therapy with antibiotics and in many cases, the patients recovered.one such case is that of tom patterson who is a 68-year-old professor in the psychiatry department, university of california medical school. during his vacation to egypt, he contracted a systemic infection (initially thought to be food poisoning) by mdr (multi drug resistant) a. baumannii. all standard antibiotic treatments failed. his wife steffanie strathdee—associate dean of global health sciences, university of california— obtained an emergency authorization for treating her husband with phages. tom was administered phage cocktails intravenously. there was a change in antibiotic resistance profiles. bacteria had developed phage resistance. tom finally got treated. the team then received a $1.2 million grant over three years and became the directors of ipath (innovative phage applications and therapeutics) (79, 125, 126). another case study is that of isabelle holdaway, a 15-yearold girl who suffered from p. aeruginosa and mycobacterium abscessus infection. doctors performed a lung transplant, but the infection was still not cleared. one month post the transplant, mycobacterium abscessus was isolated and the patient was diagnosed with a mycobacterial infection. though her survival chance was predicted to be less than 1%, doctors gave a try for phage therapy. holdaway’s mycobacterial isolates were screened against more than 10,000 phages. two of the 3 selected phages were following temperate life cycle. bacteriophage recombineering of electroporated dna (bred) was used to prepare the lytic derivative of these phages. the cocktail of these phages was used for phage therapy. holdaway received the intravenous phage treatment without any significant side effects. she was discharged after 9 days of her treatment. after 11 months of her treatment, virtually all her lesions disappeared (32, 127). evaluation of clinical trials is necessary to evaluate the effectiveness of phage therapy (see table-1). phages are found to be safe in all the trials, but only a single double-blind clinical trial claims the efficacy of phage therapy. thus, well-designed clinical trials are highly warranted to further evaluate the efficacy of phage therapy. other applications bacteriophage has its application in varied arenas, from targeting mdr infections to targeting rot in harvested potatoes. phages also may be used in decontaminating the hospital environment, which would decrease the incidence of nosocomial infections. 1. agriculture and food safety bacteriophage application is becoming advanced in animal husbandry, food safety, and agriculture (128). it was in 2005 when for the first time, a bacteriophage product— agriphagetm—was formally approved by the regulatory agency of the us government to treat crop diseases (129). since then, many phage products have hit the commercial markets (table 2). the use of phages in agriculture was also exploited by omnilytics inc. when a customer sends in infected plant material, customized phage products are prepared and given to the customer (37). phages are also gaining popularity in the food industry to attack foodborne bacterial pathogens. in 2006, the fda approved listshieldtm, a phage cocktail targeting listeria monocytogenes (which contaminates ready-made food products) as safe for consumption. few other products include agriphage, biotector, ecoshield, finalise, listshield (130). other than phages, phage-derived enzymes are also attractive investments. the usage of phage lytic enzymes in food conservation (as an antibacterial) was first reviewed in 2005 (131). few commercially available enzymes are listextm & lmp-102 (from phages targeting listeria) (132), ecp-100 (from escherichia coli o157:h7 phage) vol 8 i 23 table 2: examples of commercially available phage products product name company targeted bacteria reference lmp 102 intralytix listeria monocytogenes (135) listshield intralytix listeria monocytogenes (136) ecoshield intralytix e. coli o157:h7 (137) salmofresh intralytix salmonella spp. (138) shiga shield intralytix shigella flexneri, s. sonnei, s. dysenteriae (139) listextm p100 micreos ltd listeria monocytogenes (132) salmopro phagelux inc salmonella enterica (140) agriphage certis usa llc xanthomanas campestris (141) phageguard micreos food safety llisteria monocytogene s salmonella ee. coli o157 (142) (133), and salmofreshtm (from phage targeting salmonella enterica) (134) are used in the food industry. 2. promising applications usage of phages is highly promising in eye drops and antiseptics, which follow topical administration. such commercial phage products are highly effective and can attract high investments from pharmaceutical companies. eye drops, for example, were found to be a statistically significant treatment for p. aeruginosa infection (143). regarding antiseptics, eliava institute in georgia has developed a commercial biopolymer bandage, with phage cocktails called “phagobioderm”. for the development of other such commercial phage therapy products, projects like phagoflow (144) and phagomed (145) are implemented. 24 i fine focus 3. genetic engineered phage the lack of efficiency and other challenges met by phages in the therapeutic domain in the modern synthetic biology era can now be met by genetic engineering of phages (113, 146, 147). genetically engineered phages have found their application in many other areas. phage engineering is used for the targeted delivery of phages (148) and is also being exploited for protein and gene delivery. tao p. et al. delivered proteins and genes in vitro and in vivo by using the t4 phage (149). przystal et al. used phages as vectors to target orthotopic glioblastoma and suppressed the growth of glioblastoma by a systemic combination of temozolomide and suicide gene therapy (150). folate-conjugated m13 coated by poly(caprolactone-b-2-vinylpyridine) which encapsulated hydrophobic antitumor drug doxorubicin acted as a nanosized drug delivery vehicle (151). phage coat protein can be modified by expressing immunogenic peptides that could deliver vaccines (152). phages are also exploited to edit the microbiota by artificially synthesizing phages and modifying their tail fibers (153). summary though phage therapy is a promising and attractive source of treatment for emerging bacterial infections, further understanding of phage biology is essential before reimplementation of phage therapy. in vivo studies are required on liposomal phage delivery or delivery of genetically modified phages. there is no consensus view on the most effective route of phage administration, dosage and pharmacokinetics. also, phage interaction with the immune system is not well known when compared to the knowledge we possess in regarding antibiotics. the efficacy of genetically modified phages is to be evaluated in vivo. there is a need to be careful with genetically modified phages used in therapeutics. if phage resistance arises, switching to new phages with different technology, achieving the same efficacy would be difficult. programs like the “science education alliance-phage hunters advancing genomics and evolutionary science” (sea-phages) trains students to isolate phages characterize genomes against a particular pathogen. such programs need to be conducted with increased rigour across the globe. the establishment of more phage banks to store such newly found isolates can lessen the challenges faced by the host range. since phage therapy deals with viruses, the high cost involved in phage isolation is an obvious hurdle for commercial production. for industrial-scale production of phages, a search for surrogate hosts is necessary, which might marginally reduce the production cost. despite these challenges, the author believes that bacteriophages can lead us into a progressive future with varied applications in agriculture, aquaculture, poultry, sewage treatment, and therapeutic use in humans aquaculture, poultry, sewage treatment, and therapeutic use in humans. aquaculture, poultry, sewage treatment, and therapeutic use in humans. acknowledgements i sincerely thank dr. sathyam narayana raju for his constant support and guidance. vol 8 i 25 references 1. davies j. 2006. where have all the antibiotics gone? canadian journal of infectious diseases and medical microbiology 17:287–290. 2. manyi-loh c, mamphweli s, meyer e, okoh a. 2018. antibiotic use in agriculture and its consequential resistance in environmental sources: potential public health implications. molecules 23:795. 3. landers tf, cohen b, wittum 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columbia, 6270 university blvd, vancouver, bc v6t 1z4, canada 3british columbia speleological federation, penticton, bc, canada ¥present address: department of genetics, faculty of natural and agricultural sciences, university of the free state, 205 nelson mandela dr, park west, bloemfontein, 9301, south africa (https://orcid.org/0000-0002-4945-3516) øfaculty of pharmaceutical sciences, the university of british columbia, pharmaceutical sciences building, 2405 westbrook mall, vancouver, bc v6t 1z3, canada #equally contributed as first authors *correspondence: ncheeptham@tru.ca https://orcid.org/0000-0002-1913-4151 vol 9 | 67 abstract caves offer a unique habitat for microorganisms, which allow the adaptation of exclusive metabolic pathways to the resources available. this environment could enable the production of primary and secondary metabolites with unique antimicrobial or enzymatic properties. white rabbit cave is located in the monashee mountain range in south-central british columbia, a metamorphic range not known for cave and karst development. the present study has recovered bacterial isolates from the white rabbit cave and assessed them for their antimicrobial properties by employing the agar plug assay. one hundred and six bacterial isolates were cultivated from the collected samples, among which, five bacterial isolates displayed antimicrobial properties against a methicillin-resistant staphylococcus aureus (mrsa) strain. furthermore, these five isolates were identified with 16s rrna gene sequencing and through phylogenetic analysis. it has been observed that three of them (b076, b053, and b079) were identified as streptomyces spp. (phylum actinobacteria) while the other two were recognized as paenibacillus spp. (b039) and paenibacillus terrae (b016) (phylum firmicutes). to the best of our knowledge, this is the first study that identifies bacterial species with antimicrobial properties from white rabbit cave in the monashee mountain range in british columbia, canada. introduction caves are unique resources that researchers have recently honed in on. they offer a biologically, chemically, physically, and geologically exceptional habitat for microorganisms to adapt, allowing them to possess unique properties (1-3). generally, caves have four distinct zones: the entrance where the surface and the underground meet; the twilight zone where there is little light and plants are unable to grow; the transition zone where there is no light but, environmental conditions like temperature are still sensed; and the deep zone where there is no light, it is very humid, and the temperature is constant (1). in the subsurface areas of caves, specifically in the transition zone and in the deep zone, there is considerable influence from specific rock surfaces (1, 4). each distinctive zone within a cave influences microhabitats for microbial communities. these microhabitats are dictated by microbial metabolic needs, abilities, tolerances, and rock chemistry (4). the twilight and transition zones also offer very constant environments (constant humidity and temperature, and the absence of light), allowing certain adaptable microbes to thrive here with few perturbations necessitating the microbial communities are relatively stable (1, 4). some cave characteristics, including high humidity, little to no light, and little airflow, make energy production challenging for typical microorganisms other characteristics such as low concentrations of organic nutrients, high concentrations of minerals, seasonally dependent air currents, air pressure changes to equalize with surface air pressure and water level changes, are also included for 68 | fine focus such influence (1-3). these conditions and unusual resources provide a unique niche where most microorganisms are unable to survive (3); however, certain adaptable microbes are able to thrive because of their distinctive physiologies and their abilities to exploit cave environments (4). the cave environment causes microorganisms to adapt their metabolic pathways to the resources available, which could permit the production of primary and secondary metabolites that have antimicrobial properties (1-4). for example, streptomyces tendae, strain hki 0179 that has been isolated from a cave in grotta dei cervi, italy was found to produce the antibacterial peptide cervimycins a-d effective against methicillin resistant staphylococcus aureus (mrsa) (5). in another study, athucy 3314, a strain of taxopsis calypsus, found in francthi cave, greece has been observed to produce an antibacterial lipid against mrsa, methicillin susceptible staphylococcus aureus, vancomycin resistant enterococci (vre) faecalis, vre faecium and vancomycin susceptible enterococcus faecalis (6). white rabbit cave is situated in the monashee mountain range in south-central british columbia, a metamorphic range not known for cave and karst development. this is an area of high annual precipitation, frequently referred to as the interior rainforest, occurring within the interior cedar hemlock and engelmann spruce subalpine biogeoclimatic zones. named after the white marble in which it is formed, white rabbit is one of the few known marble caves in western canada. the cave is formed in a narrow band of marble in the mt. grace formation of the shuswap metamorphic complex (7) in this study, we would like to understand whether there are some bacteria that live in this canadian white rabbit cave who can produce antimicrobial activity against some chosen multidrug resistant bacteria in the hope to explore and discovery more potential new antibiotic sources. here, we describe the isolation of the white rabbit cave bacteria and screen them for antimicrobial activity against the mdr and non-mdr bacterial strains using the agar well diffusion assay. the identification of the positive bacteria with capacities to produce antimicrobial activities were revealed by using the 16s rrna gene profiling. materials and methods sample collection sites and cave measurements the entrance of the white rabbit cave is in a polje (an extensive depression having a flat floor and steep walls) over 300 m in diameter at its base, 500 m wide at the upper edge and ranges from 60 to 250 m deep. it is among the largest surface karst features in the country. a stream flows year-round into the cave, and the suspected resurgence is over 2 km away as the crow flies and 400 m elevation below the main entrance. the first known record of the cave entrance is in a geological survey report from the late 1970’s, where the entrance and resurgence are identified on maps (8). first explored and surveyed in 2009, it now stands at 4213 m surveyed length and 398 m deep as of september 2015, currently the sixth deepest cave in canada with many leads still to be explored (figure 1). vol 9 | 69 figure 1. map of white rabbit cave in the monashee mountain range in south-central bc, canada. sample collection points are denoted as sample location 1 and sample location 2, which are indicated in fossil vadose passage. samples from location 1 consisted of sediment and calcite and samples from location 2 consisted of calcite only. distribution of bacterial isolates showing antimicrobial activities cultured from sample location 1 is also presented here. 70 | fine focus figure 2. location of sample collection in the white rabbit cave in the monashee mountain range in south-central bc, canada. a. fossil vadose passage (white arrow heads). b. sample location 1 for sediment sample collection. c. calcite popcorn structures. d. calcite mineralization. e. entire fossil passage (green arrow heads). the purple and the yellow arrow heads correspond to the sediments and yellow microbial mats respectively. sample collection and isolation of bacteria the samples collected from various locations in white rabbit cave consisted of sediments, calcite, and calcite mineralized with copper (figure 2). sediments were collected from location 1 (figure 2b), which is in fossil (i.e. inactive) vadose passage (figure 2a), consisting of undisturbed silts and sands on a ledge 1.5 m above the floor. calcite was collected at both locations 1 and 2, the latter being 10 m above location 1 in the fossil vadose passage in the same rift. furthermore, the samples are categorized as: 1 and 2 containing sandy sediments; 3 and 4 containing. small pieces of a blue-green mineral formation; and 5 and 6 consisting of white minerals. one hundred microliters of decimally diluted (10-1, 10-2, 10-3) samples were pour plated on hickeytresner (ht) (yeast extract 0.1%, beef extract 0.1%, n-z amine 0.2%, dextrin 1%, ph 7.3), r2a (teknova, hollister, ca, usa), difco™ actinomycete isolation agar media (thermo fisher scientific inc., waltham, ma, usa), and soil agar media plates in order to isolate the bacteria as per previous studies (2, 3). the plates were incubated at 12.5°c for 15 days. morphologically distinguishable colonies were selected and re-streaked for pure cultures. vol 9 | 71 antimicrobial activity screening the cave bacterial isolates were subjected to antimicrobial activities assay against the non-resistant strain of e. coli (e. coli-33) and mdr strains such as methicillin-resistant staphylococcus aureus (mrsa)-43300, e. coli (ndm type carbapenemase) 15-102, e. coli (oxa48 type carbapenemase) 15-124, and e. coli (new delhi strain) 15-318. the nonresistant bacterial strain serratia marcescens and yeast strain candida albicans were also included in the antimicrobial assays. all the strains were procured from our previous studies (2). the tested bacterial strains were inoculated in 3 ml of the nutrient broth (criterion™dehydrated culturemedia, hardy diagnostics, ca, usa) and incubated overnight on a shaking test tube rotator overnight at 37°c. the antimicrobial activities assay was conducted employing seeded agar method. all the tested bacterial strains were inoculated at a concentration of 106 cfu/ml in 250 ml molten agar media with a gentle shaking and poured in a nunc® bioassay dish (245 mm x 245 mm x 25 mm) (cole-parmer scientific experts, montreal, qc, canada). the agar plug assay for the antimicrobial activity screening was implemented in accordance with a previous study (2). the agar plugs (0.5 square centimeter) of each cave bacterial isolate after being properly incubated was aseptically cut using sterile scalpel. each of the agar plug that contained the bacterial isolate was then placed on to the tested microbe’s-seeded agar plates prepared. the antimicrobial assay plates were prepared in duplicates and incubated at 8°c for a period of 2-3 days. a disinfectant agent, vikron (vital environmental solutions, alberta, canada) and sterile water were served as positive and negative controls respectively. the antimicrobial activities were measured as the zone of inhibition around each bacterial colony. the diameter of the zone of inhibition was measured manually with electronic vernier caliper (guilin, guangxi, china) in accordance with previous studies (2, 3). furthermore, bacteria isolates that displayed inhibitory activity were selected to test their antimicrobial production in the broth culture. the selected bacterial plugs from the cultured plate (day 7) were inoculated in 75 ml of ht and r2a broth and incubated at 12.5°c on a rotatory shaker (150 rpm) for 15 days. the incubated culture was tested on four occasions (day 4, 6, 8, and 15) against three pathogenic bacteria (pseudomonas aeruginosa, s. aureus (mrsa)-43300, and e. coli (ndm type carbapenemase)15-102) using the kirbybauer disk diffusion test (9) as used in a previous study (10). the plates contained the incubated-culture-soaked disks were incubated for 24 hours at 37°c followed by the measurements of the zone of inhibition as indicated above. molecular identification of the positive candidates genomic dna extraction and sequencing the positive candidates exhibiting antimicrobial activities were inoculated in nutrient broth for 36 hours at 15°c. the 72 | fine focus bacterial genomic dna extraction was performed in accordance with previous studies (2, 3). the extracted genomic dna were sent to marcrogentm inc, seoul, south korea, for molecular identification of the bacterium through 16s rrna gene sequencing. the forward primer 27f (5’-agagtttgatcmtggctcag-3’) and the reverse primer 1492r (5’-tacggytaccttgttacgactt-3’) were used for the 16s pcr amplification reaction. the dna sequences obtained were analyzed using the blast (basic local alignment search tool) algorithm with the available sequences in genbank at the national center for biotechnology information (ncbi) http://www.ncbi.nih. nlm.nih.gov/genebank/index.html with the “limit to type” option active and inactive, respectively. 16s rrna gene sequences were identified with the >98% identity and coverage to the closest homologue in the genbank. the sequences were assigned with the dna data bank of japan (ddbj) gene accession numbers. sequence alignment and phylogenetic analysis the first five sequences of each blast search were downloaded from genbank and compiled into a dataset for phylogenetic analyses, with duplicates removed. sequence alignment and phylogenetic analyses were conducted in molecular evolutionary genetics analysis (mega) version 6 (11). the evolutionary history was inferred by using the maximum likelihood method based on the tamura-nei model (12). the tree with the highest log likelihood (-1444.9044) is shown. initial tree(s) for the heuristic search were obtained automatically by applying neighbor-join and bionj algorithms to a matrix of pairwise distances estimated using the maximum composite likelihood (mcl) approach, and then selecting the topology with superior log likelihood value. the tree is drawn to scale, with branch lengths measured in the number of substitutions per site. the analysis involved 51 nucleotide sequences. all positions containing gaps and missing data were eliminated. there were a total of 541 positions in the final dataset. results cultured bacterial diversity a total of one hundred six bacteria were isolated from cave sediments, calcite, and calcite mineralized with copper samples. forty-four isolates were isolated on ht media and thirty-six were isolated on r2a media. twenty and six bacterial isolates were cultivated on actinomycete isolation agar and soil agar media plates respectively. notably, ninety-four bacteria were isolated from cave sediments whereas the rest were from the mineralized samples. antimicrobial activities and molecular identification of positive candidates five bacteria from sample location 1 of the white rabbit cave revealed antimicrobial activities (table 1). the isolate b016 exhibited antagonistic effect against all the tested microbes. notably, b016 exhibited inconclusive results against e. coli 15-318. conversely, both b053 and b076 isolates vol 9 | 73 exhibited antagonistic effects against s. aureus mrsa 43300, serratia marcescens and candida albicans while b079 and b039 were only antagonistic against s. aureus mrsa 43300. 74 | fine focus table 1: antimicrobial activities. bacterial isolates cultivated from the white rabbit cave exhibited antibacterial activities against tested microorganisms. +/= inconclusive as the results could not be confirmed through further repetition of tests; + = positive inhibitory activity; = no inhibitory activity. vol 9 | 75 furthermore, the five positive candidates underwent further testing of antimicrobial production in the broth culture. the bacterial strain b053 displayed antibacterial activities against all tested microbes when cultivated both in ht and r2a broth media. the ht broth media cultivated b053 exhibited antibacterial activities on table 2: antibacterial activities of the six positive candidates cultivated in the broth culture of ht and r2a media. three test bacterial strains, s. aureus mrsa 43300, e. coli (ndm type carbapenemase) 15-102, and pseudomonas were used for the antimicrobial assay which was conducted on the 4th, 6th, 8th and 15th day of incubation. (+ = positive inhibitory activity; = no inhibitory activity; n/a = conditions that were not tested) all four days (day 4, 6 , 8 and 15), while in the r2a broth only on the 15th day of incubation when grown in the r2a broth. notably, the b076 exhibited an antagonistic effect against mrsa 43300 test bacteria on all four days of the assay, but only when cultivated in the ht media (table 2). 76 | fine focus 16s rrna gene identification exhibited three bacteria that belonged to the phyla actinobacteria (streptomyces spp.) and the other three belonged to firmicutes. the firmicutes bacteria were identified as paenibacillus terrae, paenibacillus spp. and bacillus safensis (figure 3). all sequenced bacteria exhibited >99% identity to their closest homologue. the dna data bank of japan (ddbj) accession numbers were assigned to each of the isolate’s sequences. the samples a (5ffbb08d3a01a5000f85e942.b016), b (5ffbb08d3a01a5000f85e942.b079), c (5ffbb08d3a01a5000f85e942.b053), d (5ffbb08d3a01a5000f85e942.b039), e (5ffbb08d3a01a5000f85e942.b076) were assigned with the ddbj accession numbers lc605904, lc605905, lc605906, lc605907 and lc605908, respectively. phylogenetic analysis the evolutionary relatedness of the antimicrobial bacterial isolates demonstrated two key phyla containing our bacterial isolates: actinobacteria and firmicutes. three bacteria are recognized from the genera streptomyces which falls in the actinobacteria clade while the remaining three, paenibacillus terrae, bacillus safensis, and paenibacillus spp. fall in the firmicutes clade. each of the six bacterial isolates from white rabbit cave are shown in figure 3 with an asterisk (*), visually indicating their evolutionary relatedness vol 9 | 77 figure 3. evolutionary relatedness of taxa displayed antimicrobial properties. 16s rrna sequences of the bacterial isolates b016, b039, b053, b076, and b079 were placed in the phylogenetic tree closest to their homologues obtained from the ncbi gene database. for each of the six bacterial isolates that showed antimicrobial properties the five closest homologues were chosen with % identity and % query cover >98%, this was repeated with typed material. these sequences were aligned by muscle with default parameters. the final construction was done using mega6 by the maximum composite likelihood method, with a bootstrap test of 1000 replicates. the bacterial isolates that showed antimicrobial activities are indicated with light blue shade. ‘t’ represents the typed isolates. 78 | fine focus antagonistic activities of streptomyces spp. 1492 against mrsa, vancomycin resistant enterobacter faecium (vre), and acinetobacter baumannii. (17). streptomyces spp. are prolific antimicrobial agent producers (18). for instance, a previous study that examined the whole genome sequences of two streptomyces spp., icc1 and icc4, revealed 37 and 35 biosynthetic gene clusters respectively (4). furthermore, uhpllc-hrms analysis of the synthesized secondary metabolites from these streptomyces spp. identified three diketopiperazines: cyclo (leupro), cyclo (pro-val) and cyclo (4-hydroxypro)leu]) (4) which are considered to possess antimicrobial agents in accordance to a previous study (19). the current study also recovered antimicrobial activities from the phylum firmicutes with paenibacillus (b016, b039) as the foremost genera. firmicutes have been frequently found in cave settings, particularly bacterial species from the bacillus and paenibacillus genera (3, 20, 21). for example, a former study has implemented roche 454 pyro-sequencing of the 16s rrna gene sequences that revealed the presence of firmicutes from the 400 years old ice strata from an alpine ice cave (21). the genus paenibacillus, especially paenibacillus terrae b016 exhibited close homology, has been widely studied for its wide range of production of bioactive molecules such as polymyxin, xylanases protease, amylase, and cellulase (22). these bioactive molecules could potentially be attributed for their antimicrobial properties (23-26). antimicrobial properties of paenibacillus have been observed in other habitats as well. in one of the previous studies, paenibacillus spp. ghs.8.nwyw.5 was found in active discussion white rabbit cave is a white marble, karstic cave found in the monashee mountain range in british columbia, canada. two locations were identified in fossil vadose passage for sample extraction: sample location 1 provided sediment and calcite samples, while sample location 2 provided only calcite samples. however, there was insufficient bacterial load collected from the calcite samples; therefore, further investigations could not be conducted. thus, all the bacterial isolates were obtained from the sediment samples from sample location 1. the current study has evaluated the antimicrobial activities of one hundred and six bacterial isolates cultured from white rabbit cave, among which only five have exhibited antimicrobial activities. 16s rrna gene sequencing revealed 50% of the positive candidates were from the actinomycetes phylum where the sole genus was streptomyces spp. (b053, b076, b079). these findings were consistent with previous studies where it has been shown that the limestone cave environment has been predominantly inhabited by actinobacteria (13-15) and streptomyces spp., both of which were found to display antimicrobial activities (1, 3, 4, 16). a previous study from the iron curtain cave, canada isolated two streptomyces spp., icc4 and icc1 where both isolates exhibited antimicrobial activities. icc4 exhibited antagonism against the regular non-resistant strains of e. coli, s. aureus and mdr strains of e. coli 15318, e. coli 15-102, e. coli 15-124 and p. aeruginosa while icc1 only showed activity against e. coli 15-102 and e. coli 15-124 (3, 4). similarly, another study from turkish karstic caves has specifically exhibited the vol 9 | 79 layer permafrost soil and in saline spring sediment in nunavut, canada, and exhibited antimicrobial activity against mrsa, listeria monocytogenes, salmonella enterica, and e. coli o157:h7 (27). similarly, in another study paenibacillus spp. smb1 isolated from a halo-alkaline lake in india exhibited antimicrobial properties against gram-positive bacteria via the production of bacitracin a (25). notably, neither of the isolates, ghs.8.nwyw.5 or smb1, were found in a cave environment; however, they were found in unique, yet somewhat extreme habitats showing the resilience and adaptability of paenibacillus as a genus, taking special notice of their ability to produce secondary metabolites to thrive and take advantage of each environment. furthermore, paenibacillus spp. was also known to possess an antibiotic resistance gene in their genome. a previous study has reported the isolation of paenibacillus spp. lc231 from lechuguilla cave. the whole genome sequencing, functional genomics, and biochemical analysis of the isolate revealed its resistance towards a wide array of antibiotics (28). the broth culture of the five positive candidates did not show any antimicrobial activities except for streptomyces spp., b053 and b076. it has been observed that both these isolates exhibited antagonistic effects only when subjected to specific ht and r2a broth culture media and on specific days of incubation which is in line with previous studies (3, 29). a previous study has shown that two streptomyces spp., icc1 and icc4, isolated from the iron curtain cave, canada exhibited antagonistic effect only when grown in r2a and v8 & ht media respectively (3). further investigations should emphasize whole genome sequencing, functional genomics, extraction and purification of potential bioactive compounds, optimization of seed media, production media, amount and age of seed inoculum, and growth and production condition to fully characterize these bacterial isolates for their antimicrobial activities. the present study attempted to shed light on the antimicrobial activities of the bacterial isolates cultivated from white rabbit cave, canada which has never been elucidated before. however, we believe that this preliminary approach shows the white rabbit cave habitat as a resource for potential bioactive molecules, but we recognize that this study is by no means a whole picture of the cave habitat. future studies should explore the details of microbial diversities by implementing high throughput metagenomic approaches while also bio-prospecting novel molecules such as antimicrobial sand and enzymes of industrial and pharmaceutical importance. additionally, studies should also look upon the potential microbial-mineral interactions in the formation of cave deposits in order to expand the present knowledge of biospeleology. acknowledgements authors thank mitacs globalink summer research internship (agp&nc), tru internal research grant (nc) and bat caver program (ks), the alberta speleological society (ks), and mountain equipment coop for financial support for this study. 80 | fine focus authorship statement nc applied for research grant, designed and directed the study. sg helped supervised undergraduate students in the lab. krs applied for research grants for sample collections and collected samples. cj, agp, gk, vy, tl performed lab experiments and analyzed the data. mw and sg prepared the manuscript with nc’s supervision. krs wrote the cave description section. cj, agp. krs and gk read the manuscripts and offered inputs. declaration the authors declare that there is no conflict of interests. vol 9 | 81 references 1. ghosh s, kuisiene n, cheeptham n. 2016. the cave microbiome as a source for drug discovery: 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icbiotech 20:843-847. 84 | fine focus genomic evolution in pseudomonas fluorescens as a result of gradual temperature changes spencer a. lee salee@tlu.edu or spenceralee@me.com robert m. jonas texas lutheran university department of biology, seguin, tx keywords: experimental evolution, climate change, microbiology, genomics, natural selection vol 9 | 85 abstract as climate change continues to affect global temperatures, organisms will need to not only adapt but evolve to survive the changing climate conditions. temperature selection experiments were performed on pseudomonas fluorescens to select for growth at lower temperatures. the p. fluorescens temperature selection experiment selected for cells that can grow at a new minimum temperature which is over 20˚c lower than the optimal growth temperature (25-30˚c). previous experiments established the low end of p. fluorescens’s growth temperature as 4˚c. the genomes of the newly selected and reference strains of p. fluorescens were sent for sequencing, and the results showed differences in protein sequence between the two strains. this experiment is a model for evolution as a result of gradual temperature change (similar to climate change) over generations, and the resulting genomic changes recorded show which protein families could evolve as an organism adapts to a gradually changing temperature. introduction climate change is a looming threat to ecosystems and life on earth. temperatures are predicted to increase by 4˚c across the world within the next 80 years (14). climate scientists predict that climate change could move ecological zones over 600 km north of their current locations (14). this shift will force organisms to either move with the ecological zone, evolve to survive in new conditions, or perish as a result of a changing climate (14). this data demonstrates that climate change and evolution due to climate change are unequivocally relevant to all species on earth. species living in the northern part of north america, europe, and asia, as well as species on mountains are most likely to be affected by the shifting of ecological zones due to climate change (14). in addition to average temperatures increasing, the range of temperatures is expected to change as well with extremes becoming more extreme (11). a dynamic model of drosophila survival and evolution at different temperatures explained that population collapse can arise when temperatures approach an organism’s heat tolerance (11). in addition, temperature selection can arise when an organism is at a temperature that is a few degrees away from its thermal limits (11). temperature selection experiments involve subjecting organisms to temperatures outside of their ideal temperature range and measuring the changes between the initial and final organisms. studies utilizing temperature selection may be instrumental in determining the genetic effects of climate change (2). bacteria are excellent candidates for temperature selection studies because of their rapid generation time, easy growth conditions, and simplicity (in comparison to multicellular organisms). a bacterial temperature selection experiment with escherichia coli showed that bacteria are capable of evolving to increase their fitness significantly at the set temperature within a period of fewer than two months (400 generations) (2). throughout the temperature selection process, the bacterial population gained fitness-improving mutations in their genomes (2). despite 86 | fine focus the addition of prevalent mutations in the bacteria’s genomes, the evolved bacteria did not lose their fitness in their ancestral environment (2). bacteria adapting to low temperatures use various methods including sensing the temperature, adapting the structure of their enzymes, changing the composition of their membrane, using heat shock proteins, and using cold shock proteins to allow themselves to survive at a low temperature (6). it can be hypothesized from this information that genes relating to these factors are the ones most likely to be affected as an organism undergoes a temperature selection experiment or climate change. while there is no general pattern for amino acid composition of cold-adapted enzymes when compared to others of the same function, it is commonly shown that the amino acid composition of enzymes is different in cold-adapted and non-cold adapted enzymes (6). enzymes are found in pathways relating to sensing environmental conditions and membrane composition maintenance as well as in metabolic pathways (6). the pseudomonas fluorescens species complex contains at least 52 species of gram-negative motile rods (12). p. fluorescens, a psychrophile, has an optimal growth temperature of 25-30 ˚c but has been shown previously to be able to grow at temperatures ranging from 4-37 ˚c (8,12). p. fluorescens contains a relatively large genome (between 6.1-7.1 million bp) when compared to other bacteria like e. coli (4.6 million bp) (3,7,10). genomic analysis of one strain of p. fluorescens showed that it had approximately 90 rrna/trnacoding genes and over 5,500 genes that coded for proteins (7). this is nearly 1,300 more protein-coding genes than e. coli k-12 (3,7). analysis of a different strain of p. fluorescens showed over 6,100 open reading frames within its genome (10). a large portion of its genome is devoted to genes dealing with motility, chemotaxis, and other environment-sensing and interacting genes (7). the large genome of other members of the genus pseudomonas are theorized to allow the bacteria to survive in a wide range of environmental conditions and arose as a result of evolutionary adaptations (13). it was also proven that as bacterial genome size increases, more genetic real estate is dedicated to regulatory proteins (10,13). its large genome and high number of regulatory genes allow p. fluorescens to be a plausible model organism for the environmental evolution of multicellular organisms. p. fluorescens has been the subject of both temperature adaptation and experimental evolution experiments. one experimental evolution experiment subjected the bacteria to media with varying amounts of carbon substrates and determined the evolved and original strains’ fitness in both the simple and complex media (1). experiments involving low temperatures consistently reached a minimum temperature of 4-5 ˚c, but not lower, in their procedures (5,9). it is unknown whether the temperatures chosen are the true lower limit of the bacteria’s growth range or whether this temperature is the lowest temperature suitable for laboratory experiments as the generation time reaches around 9 hours (5). in this experiment, the main possible mechanisms of genomic change are adaptation, mutation, and genetic drift. the purpose of this experiment was not only to determine the minimum growth temperature of p. vol 9 | 87 fluorescens but also to determine which genes evolve as an organism is subjected to extreme temperatures. materials and methods media nutrient agar plates were made using difco™ nutrient agar. 23 g/l of nutrient agar was suspended in deionized water. it was heated with frequent agitation and boiled for 1 minute to completely dissolve the powder. nutrient broth was made using difco™ nutrient broth. 8 g/l of nutrient broth powder was suspended in deionized water. it was heated with frequent agitation and boiled for 1 minute to completely dissolve the powder. approximately 5 ml was distributed into test tubes, which were sterilized by autoclaving. additional plates and test tubes were made as needed using the same methods. experimental methods a lab stock of pseudomonas fluorescens strain atcc® 13525 was grown at approximately 18 ˚c (the room temperature of the lab in february) and stored in a 4 ˚c refrigerator. glycerol stocks of the stock strain were created by putting 500 µl of 50% glycerol and 500 µl of bacterial culture in a cryovial. all stocks were stored at -70 ˚c. two test tubes containing 5 ml of sterile nutrient broth were each inoculated with one colony of p. fluorescens from the plate grown at 4 ˚c. the tubes were labeled, and a small stir bar was put into each tube. the temperature of the tubes was recorded every day until the end of the experiment. the tubes were placed into a beaker along with a thermometer. the beaker was placed into a styrofoam container on a magnetic stirrer. glycerol stocks were created of every other passaged culture and every four passages, the culture was streaked on a nutrient agar plate to check for uniformity and lack of contamination. p. fluorescens develops flocculent growth patterns creating small clusters that can be described as visible aggregates. all cultures were grown until they contained significant amounts of visible aggregates. new cultures were inoculated from the previous culture. the first culture was grown at the room temperature of the lab in may (~ 23 ˚c). the temperature was decreased by 1-1.5 ˚c daily until there was no more growth observed and the broth began to freeze despite agitation. the temperature of the culture of p. fluorescens was recorded multiple (3 or more) times daily at morning, noon, night, and other times the researchers were available. sequencing/basic bioinformatics samples of both the original and final cultures were sent to the sequencing center for dna extraction, whole-genome sequencing, and sequence alignment to the reference genome. the dna from both cultures was extracted using the illumina dna prep and illumina miniseq midoutput reagent kits. the dna was pairedend sequenced using 150 bp reads. the resulting sequences were aligned against the genome of p. fluorescens strain atcc® 13525, and variant/snp analysis were performed using the geneious prime application using default settings. the results were then obtained from the sequencing center (fort collins, co). 88 | fine focus additional bioinformatics the resulting variant/snp data from the initial and final cultures were compared against each other manually to determine where the sequences differed between the initial and final strains. the variant/snp data was compared manually due to the lack of available sequencing software. the genes containing an altered primary structure, location of nucleotide change within the genome, and the amino acid change were all recorded. the number of amino acids changed per protein (aa/p) was recorded and the overall results were analyzed. results fig 1: average daily temperature of each culture throughout the experiment (left to right: blue dots, culture 1; orange dots, culture 2; grey dots, culture 3; yellow dots, culture 4; cyan dots, culture 5; green dots) the average daily temperature of the culture of p. fluorescens was calculated. the day of the experiment, the average culture temperature on that day, and the number of the culture growing on that day are shown below in figure 1. the culture number indicates the number of passages since the original plated p. fluorescens. the equation of the trendline confirms that the temperature of the culture was decreased by 1-1.5 ˚c daily. growth of the culture was observed at temperatures below 4 ˚c. vol 9 | 89 the analyses from both the initial and final strains from the experiment were compared and a list of the genes that contained altered amino acid sequences was compiled. the altered genes were then grouped based on their function or the location of the resulting protein as shown in table 2. the altered genes list contained 53 enzyme-encoding genes, 17 transport or membrane affiliated genes, 17 transcription factor related genes, 8 motility or flagellum related genes, 7 stress related genes, 3 genes that produced products involved in signaling pathways, 2 environmental factor genes, and 31 genes that produced proteins with other or unknown functions with some falling into multiple categories. 90 | fine focus table 1: genes in which coding sequences (cds) differed between initial and final strains and their corresponding protein function or location (numbers in parentheses denote multiple protein cds in a family that have altered sequences) vol 9 | 91 the bioinformatics yielded the difference in protein primary structure between the initial and final bacterial strains. the number of proteins with an altered primary structure and the number of amino acids changed per protein (aa/p) were recorded. the relative number of proteins with altered primary structures and average amino acids changed per protein are shown by group in figure 2. in total, there were 2459 amino acids changes recorded across 138 genes that showed amino acid changes. fig 2: relative number of proteins and average amino acids changed 92 | fine focus the enzyme group was the most numerous with 53 proteins changed, but its average amino acid change per protein was one of the lower values at 12. the environmental factor related gene group had the fewest genes change (2) but they changed, on average, by the largest amount (63 aa/p). the amino acid changes per protein are shown by group in the box and whisker plot in figure 3. fig 3: proteins affected by temperature selection: [x = 17.85 aa/p, s = 24.22 aa/p (enzyme group mean = 12.26 aa/p, transport/membrane group mean = 10.82 aa/p, transcription factors group mean = 14.65 aa/p, motility/flagellum group mean = 26.13 aa/p, stress reaction group mean = 22.86 aa/p, signaling group mean = 3.33 aa/p, environmental factor related group mean = 63 aa/p, and other/unknown group mean = 28.10 aa/p)] vol 9 | 93 discussion this experiment determined a new minimum growth temperature of p. fluorescens, and genome sequencing and resulting analysis yielded informative data about the genomic changes resulting from evolution due to changing temperature. bacterial growth at less than 4-5 ˚c during this experiment (2-2.75 ˚c) establishes a new minimum growth temperature of p. fluorescens and overwrites the previously established lower limit of p. fluorescens’s growth (8,12). low-temperature adaptation by bacteria is accomplished by sensing the environment, altering enzymatic structure to retain functionality, modifying membrane composition to maintain proper fluidity, and using heat shock and cold shock proteins (6). the experimental data confirmed the prediction that genes with structural changes would fall into the categories of enzymatic genes, regulatory genes (transcription factors and signaling), genes affecting the membrane and associated proteins, and environmental related genes with additional categories of motility and unknown genes also being affected. while the exact mechanisms of change in this study are not known, they are believed to be adaptive. laboratory medium lacks the selective pressures of the nature, so some genetic changes could occur in that environment that may not necessarily correlate to evolution in nature. a more ideal experimental set-up would passage cells equivalently at different temperatures to ascertain which alterations in the genome are directly temperature dependent. due to the ubiquity of enzymes in a biological system and their need to change structure to retain their function at lower temperatures than optimal (6), numerous enzymatic genes contained changes in their structure as predicted. only subtle changes in primary structure were needed for the enzymes to function properly at a lower temperature, which is why the enzyme group was the most numerous but one of the groups with the fewest amino acid changes per protein (as shown in figures 2 and 3). the genes related to environmental factors contained the fewest genes changed, but they changed by the largest amounts (also shown in figures 2 and 3). this is hypothesized to be due to the evolution of the bacteria at their minimum growth temperature. the protein coded by an environmental response gene has a specific structure that allows it to respond to environmental changes. if the structure of many of the environmental response genes changed, the bacterium would not be able to react properly to its new environment. that is why we predict that category had fewer genes with amino acid changes. the median protein length in bacteria is 267 amino acids (4), so the average change of approximately 18 aa/p means that if the gene (and resulting protein primary structure) is altered, 6.7% of the amino acids in a protein are changed. the percent change was not calculated for specific genes as the bioinformatics largely listed the genes by their family and was not specific. as predicted, numerous genes relating to the membrane changed their amino acid sequences. for example, the mltg gene codes for a multifunctional fusion protein that functions as an endolytic peptidoglycan terminase and catalyzes the phosphorylation of dtmp to dtdp (15). this gene changed 94 | fine focus in 25 amino acid residues. as a gene coding for an integral membrane protein with enzymatic functions, mltg’s change in structure shows that membrane-related genes are affected by temperature-dependent evolution. motility is more difficult at lower temperatures. this difficulty is exacerbated close to, and at, the freezing point of water because of the aqueous nature of biological systems. because of this, it is understandable that nearly 10 genes relating to motility would change to alter the sequence of the resulting proteins. this also, explains why stress-related genes were also affected by the temperature selection. the original bacterial strain’s genome changed drastically over the course of the experiment, as shown by the sequencing and bioinformatics results. therefore, the final bacterial strain was deemed independent from the initial strain and was given the strain identifier “ds5683” with the full strain name being pseudomonas fluorescens ds5683. this experiment shows that an organism subjected to experimental evolution due to a gradual temperature change will accumulate mutations in enzymatic genes, membrane-related genes, regulatory genes, motility genes, stress-related genes, and environmental-related genes. because enzymes have an ideal temperature they function at, a change in environmental temperature requires a change in the primary structure to allow the enzyme to maintain its functionality. these results can be extrapolated to model genetic changes as a result of gradual temperature changes. as climate change increases global temperatures, it is hypothesized that the most numerous genes affected will be enzymatic genes and the most numerous structural changes will occur to environmental-related genes. in addition to those genes, regulatory genes, motility genes, and stress-related genes are also predicted to be altered due to climate change. even though the temperature change due to climate change is not expected to cause all species to go extinct, the results of this experiment predict that genomic changes are expected to become prevalent in species experiencing the temperature change acknowledgments we thank dr. danielle grove (texas lutheran university) for assisting in the development of this project and the sequencing center for providing genome sequencing and bioinformatics services. we also thank the tlu vpaa and department of biology for funding. declarations funding: this study was funded by the texas lutheran university vpaa and department of biology. competing interests: the authors declare no conflict of interest. ethics statement: no approval of research ethics committees was required to accomplish to this study because microbe use is not regulated by ethical committees. vol 9 | 95 references 1. barrett, r. d. h., maclean, r. c., & bell, g. 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(2021, september 29). multifunctional fusion protein. uniprot consortium european bioinformatics institute protein information resource sib swiss institute of bioinformatics. retrieved february 12, 2022, from https://www.uniprot.org/uniprot/a0a3d0k8l7 phillip c. betts jordan t. froese* department of chemistry, ball state university, muncie, in 47306 investigation of the effects of mutating iron-coordinating residues in rieske dioxygenases keywords: rieske dioxygenase, toluene dioxygenase, oxidative dearomatization, green chemistry, enzyme engineering, bioremediation © 2024 betts, froese. fine focus, 10(1), 90-108. doi: 10.33043/ff.10.1.90-108. shared with cc-by-nc-nd 4.0 license. manuscript received 8 april 2023; accepted 19 may 2023 abstract rieske dioxygenases are multi-component enzyme systems, naturally found in many soil bacteria, that have been widely applied in the production of fine chemicals, owing to the unique and valuable oxidative dearomatization reactions they catalyze. the range of practical applications for these enzymes in this context has historically been limited, however, due to their limited substrate scope and strict selectivity. to overcome these limitations, our research group has employed the tools of enzyme engineering to expand the substrate scope or improve the reactivity of these enzyme systems in specific contexts. traditionally, enzyme engineering campaigns targeting metalloenzymes have avoided mutations to metal-coordinating residues, based on the assumption that these residues are essential for enzyme activity. inspired by the success of other recent enzyme engineering reports, our research group investigated the potential to alter or improve the reactivity of rieske dioxygenases by altering or eliminating iron coordination in the active site of these enzymes. herein, we report the modification of all three iron-coordinating residues in the active site of toluene dioxygenase both to alternate residues capable of coordinating iron, and to a residue that would eliminate iron coordination. the enzyme variants produced in this way were tested for their activity in the cis-dihydroxylation of a small library of potential aromatic substrates. the results of these studies demonstrated that all three iron-coordinating residues, in their natural state, are essential for enzyme activity in toluene dioxygenase, as the introduction of any mutations at these sites resulted in a complete loss of cis-dihydroxylation activity for all substrates tested. https://creativecommons.org/licenses/by-nc-nd/4.0/ betts & froese | investigation of the effects of mutating iron-coordinating residues in rieske dioxygenases 91 introduction in recent years, the environmentally deleterious effects of the chemical industry have increasingly been recognized, leading to a concerted effort to employ more sustainable methods for the production of fine chemicals (19, 36). these environmentally sustainable chemical methods have collectively been referred to as “green chemistry”, which has been defined by a series of principles that serve to guide the application of sustainable chemistry (15, 34). owing to this increased focus on green chemistry, the application of enzymes as catalysts in the chemical industry has grown in popularity (7). enzymatic catalysts are entirely biodegradable, they operate in aqueous media as opposed to petroleum-derived solvents, they do not require high-temperature applications, and they are produced from renewable resources, complying with many of the principles of green chemistry (15, 34). one class of enzymes that have been widely applied as catalysts in the production of fine chemicals are the rieske dioxygenases (16, 23). rieske dioxygenases are non-heme iron dioxygenases that are commonly found in soil bacteria (39). these enzymes have frequently evolved as a means for soil bacteria to metabolize aromatic pollutants in their environment and to use these pollutants as carbon and energy sources (figure 1) (12, 42). the ability of these enzymes to catalyze the cis-dihydroxylation of aromatics to produce chemically versatile cis-diene-diol metabolites with high selectivity has made them very useful catalysts in the production of valuable compounds (16, 23). rieske dioxygenases are produced by a wide range of soil bacteria strains, including pseudomonas (42), burkholderia (6), nocardioides (32), and comamonas (22). in fact, metagenomic sequencing studies have revealed that aromatic dioxygenases are ubiquitous, particularly in contaminated environments (17). owing to their natural role in the remediation of aromatics in the soil and therefore the detoxification of these environments (12,42), these enzymes play crucial roles in soil ecology. because of this role for rieske dioxygenases, these enzymes also have tremendous potential for utility in the field of bioremediation, the use of microorganisms to degrade anthropogenic pollutants in an environment. aromatic pollutants such as benzene and toluene are ubiquitous in the environment and pose significant threats to human health, as these compounds are often figure 1 metabolic pathway by which soil organisms break down aromatic pollutants in their environment (12, 42). note. the role of rieske dioxygenases in this metabolic pathway is highlighted (red). fine focus | volume 1092 carcinogenic and endocrine disruptors (4). these aromatic pollutants are frequently introduced into the environment through fossil fuel combustion, oil spills, and the misuse of petroleum products and solvents, creating a need for a means to remove these toxic compounds from the environment (4). to this end, rieske dioxygenases have been employed as bioremediation catalysts, including applications in the degradation of polychlorinated biphenyls (pcbs), which remain a hazardous presence in many environments (14, 21). despite their applications in chemical synthesis and in bioremediation, the utility of rieske dioxygenases in these contexts has remained limited by their strict selectivity and by their finite substrate scopes. as many rieske dioxygenases have evolved to metabolize non-polar aromatics, their active sites are organized to effectively bind non-polar substrates and orient them for 2,3-dihydroxylation (9, 18, 28). this results in the substrate scopes and the activities of these enzymes being restricted by the size and by the electronics of any potential substrates (10, 35). to alleviate these restrictions on the utility of rieske dioxygenases, multiple research groups have applied the tools of enzyme engineering to improve their reactivity or to expand their substrate scopes (5, 3, 11, 20, 26, 33, 37, 38). this has included studies that have engineered rieske dioxygenases specifically to improve their utility in the bioremediation of aromatic pollutants in the soil (1, 21). our lab has recently reported the development of improved rieske dioxygenase variants through the application of rational enzyme engineering (27). these studies have led to the development of rieske dioxygenases with improved reactivity or expanded substrate scopes, increasing the practical utility of these green chemical tools (1, 3, 5, 11, 20, 21, 26, 27, 33, 37, 38). when engineering metalloenzymes, studies have traditionally avoided mutations to the residues responsible for coordinating metal ions, based upon the assumption that these residues are essential for enzyme activity. recently, however, studies have shown this assumption to be incorrect (29, 40). these studies have shown that mutations altering, or even eliminating, iron coordination in heme proteins can alter or improve the reactivity of these proteins in specific contexts (29, 40). our laboratory was inspired by these studies to investigate whether a similar strategy could result in the development of improved rieske dioxygenase variants. to our knowledge, no such study has been reported for rieske dioxygenases, thus this investigation would serve to inform future engineering studies performed with this enzyme class. based upon reported results with other metalloenzymes (29, 40), it is predicted that mutating the iron-coordinating residues of a rieske dioxygenase enzyme will result in altered enzyme activity and/or substrate scope. this strategy has the potential to improve the activity of rieske dioxygenases for specific substrates or expand their substrate scopes. materials and methods general experimental e. coli bl21 (de3) competent cells were obtained from thermofisher. plasmid isolation/purification was performed using new betts & froese | investigation of the effects of mutating iron-coordinating residues in rieske dioxygenases 93 table 1 sequences of mutagenic primers used. primer name primer sequence tdo h222a fwd cgacatgtacgcggccgggacgacctcgcatctgtctggcatcctg tdo h222a rev gtcgtcccggccgcgtacatgtcgctgcaaaactgctctgcggcg tdo h222c fwd cgacatgtactgcgccgggacgacctcgcatctgtctggcatcctg tdo h222c rev gtcgtcccggcgcagtacatgtcgctgcaaaactgctctgcggcg tdo h222d fwd cgacatgtacgatgccgggacgacctcgcatctgtctggcatcctg tdo h222d rev gtcgtcccggcatcgtacatgtcgctgcaaaactgctctgcggcg tdo h222e fwd cgacatgtacgaagccgggacgacctcgcatctgtctggcatcctg tdo h222e rev gtcgtcccggcttcgtacatgtcgctgcaaaactgctctgcggcg tdo h228a fwd gacgacctcggcgctgtctggcatcctggcaggcctgccagaagac tdo h228a rev gatgccagacagcgccgaggtcgtcccggcatggtacatgtcgctgc tdo h228c fwd gacgacctcgtgcctgtctggcatcctggcaggcctgccagaagac tdo h228c rev gatgccagacaggcacgaggtcgtcccggcatggtacatgtcgctgc tdo h228d fwd gacgacctcggatctgtctggcatcctggcaggcctgccagaagac tdo h228d rev gatgccagacagatccgaggtcgtcccggcatggtacatgtcgctgc tdo h228e fwd gacgacctcggaactgtctggcatcctggcaggcctgccagaagac tdo h228e rev gatgccagacagttccgaggtcgtcccggcatggtacatgtcgctgc tdo d376a fwd cgagcaggacgcgggggagaactgggtcgagatccagcacatcctg tdo d376a rev cagttctcccccgcgtcctgctcgaacacgccaccggcagagaagg tdo d376c fwd cgagcaggactgcggggagaactgggtcgagatccagcacatcctg tdo d376c rev cagttctccccgcagtcctgctcgaacacgccaccggcagagaagg tdo d376e fwd cgagcaggacgaaggggagaactgggtcgagatccagcacatcctg tdo d376e rev cagttctccccttcgtcctgctcgaacacgccaccggcagagaagg tdo d376h fwd cgagcaggaccatggggagaactgggtcgagatccagcacatcctg tdo d376h rev cagttctccccatggtcctgctcgaacacgccaccggcagagaagg fine focus | volume 1094 england biolabs monarch® miniprep kit. transformations of electrocompetent cells were performed on an eppendorf eporator®. whole-cell assay cultures were grown in greiner bio-one polystyrene clear, round-bottom 96-well plates. all cultures were incubated in a barnstead maxq 4000 digital orbital incubator shaker equipped with an enzyscreen universal clamp system unless otherwise stated. fluorescence analyses were performed using a biotek® synergy™ h1 monochromator-based multi-mode plate reader, using corning® polystyrene black, opaque, flat-bottom 96-well plates. all reagents were obtained from milliporesigma unless otherwise stated. media were made at ph 7.2 and streptomycin was added at 50 μg ml−1. all e. coli cultures were maintained at 37 °c unless otherwise stated. targeted mutagenesis protocol the pcp-02 expression system was used as the template for toluene dioxygenase mutant generation (30). saturation mutagenesis was performed following the polymerase chain reaction (pcr)-based procedure of liu and naismith (24, 41). amplification was performed using an abi geneamp® 9700 thermal cycler and q5® dna polymerase (new england biolabs). mutagenic primers were designed according to the procedure of liu and naismith (24, 41). primer sequences are shown below (table 1). parameters for the relevant pcr reactions are shown below (table 2). sequencing analyses were performed by eurofins genomics© (louisville, ky). whole-cell fermentation 96 well-plate assay protocol e. coli (bl21 (de3)) electrocompetent cells were transformed with isolated pcp-02 plasmids expressing toluene dioxygenase (parent and/or mutant libraries), and with isolated pcp-01 plasmids as negative controls (30). the transformation cultures were selected on lb + streptomycin plates overnight. single colonies were inoculated into 160 µl lb + streptomycin media with 0.3% glucose in a 96-well round bottom seed plate and incubated table 2 pcr reaction components and thermocycling protocol utilized for the generation of toluene dioxygenase mutants. pcr reaction components thermocycling protocol sterile h2o – 22.5 µl 98 °c – 30 s q5® reaction buffer – 10 µl 98 °c – 10 s }x20q5® high gc enhancer – 10 µl 72 °c – 4 min dntps (10 mm) – 1 µl 72 °c – 2 min forward primer (10 µm)2.5 µl reverse primer (10 µm)2.5 µl template dna (pcp-02) – 1 µl q5® dna polymerase – 0.5 µl total – 50 µl betts & froese | investigation of the effects of mutating iron-coordinating residues in rieske dioxygenases 95 with shaking overnight. all plates included 3 or more wells containing e. coli (bl21 (de3)) pcp-02 cells expressing the parent toluene dioxygenase enzyme, and 3 or more wells containing e. coli (bl21 (de3)) pcp-01 (negative control) (30). seed plates were used to inoculate 5 µl into 155 µl lb media containing streptomycin in a fresh 96-well round bottom assay plate, and the cultures were incubated with shaking for 2.75 h. the assay plates were then pelleted, and the supernatant discarded. cultures were resuspended in 150 µl minimal media (kh2po4 – 7.5 g l−1; citric acid – 2 g l−1; mgso4·7h2o – 5 g l−1; trace metal solution – 2 ml l−1 [na2so4 – 1 g l−1; mnso4 – 2 g l−1; zncl2 – 2 g l−1; cocl2·6h2o – 2 g l−1; cuso4·5h2o – 0.3 g l−1; feso4·7h2o – 10 g l−1; ph 1.0]; conc. h2so4 – 1.2 ml l−1; ferric ammonium citrate – 0.3 g l−1; glucose – 4 g l−1; thiamine – 0.034 g l−1; ph 7.2) (8) containing streptomycin and incubated for a 1 h recovery period. following this, the cultures were induced to a final concentration of 0.5 mm iptg and the incubation temperature was reduced to 30 °c. after a 2 h induction period, aromatic substrates were added as 68 mm stock solutions in dmso to a final concentration of 2 mm. cultures were incubated with aromatic substrates for 1.5 h at 30 °c, after which the cultures were pelleted. a 100 µl portion of supernatant from each well was transferred to 96-well black opaque assay plates. the reaction was initiated by adding a 50 µl of naio4 stock solution to each well to a final concentration of 10 mm, and the assay plates were incubated with shaking at room temperature for 30 min. cleaved diols were detected by adding 50 µl of fluoresceinamine stock solution (prepared with 3 µl conc. hcl (11.65 m)/1 ml fluoresceinamine solution) to each well to a final concentration of 0.1 mm (30). assay plates were incubated with shaking at room temperature for 5 h. the fluorescence response from each well was analyzed at 485 nm (ex), 520 nm (em), and normalized to the mean fluorescence response of the negative controls ([i − i0]/i0). results identification of iron-coordinating residues because our lab has extensive experience working with toluene dioxygenase (tdo) (27, 30), and because tdo has been one of the most widely used rieske dioxygenases in the field of organic synthesis (16), this enzyme was selected as the template for mutagenesis in this study. to identify the residues that would be targeted for mutagenesis, the reported crystal structure of toluene dioxygenase was analyzed using the molecular visualization software chimerax (9, 13). this analysis revealed that the catalytic iron atom of toluene dioxygenase is coordinated by two histidine residues (his222 and his228) and by one aspartate residue (asp376) (figure 1). these residues were therefore determined to be the appropriate targets for mutagenesis in this study. production of targeted toluene dioxygenase variants to test the hypothesis of this study, it was determined that each iron-coordinating residue of tdo would be mutated to three alternate residues capable of coordinating iron (aspartate, glutamate, and cysteine for native histidine residues; glutamate, cysteine, and histidine for the native aspartate residue) and to one residue that would eliminate iron-coordination at that fine focus | volume 1096 site (alanine). to generate the desired variants of toluene dioxygenase, targeted mutations were introduced through the pcr-based method of liu and naismith (24, 41). mutagenic primers were designed according to this well-established protocol (table 1), and the corresponding mutagenic pcr reactions were carried out as described. the successful introduction of the targeted mutations was confirmed through sequencing analysis performed by a third-party contractor. representative aligned sequencing data is shown for the h222 mutants in figure 3. upon completion of this work, expression systems had been developed for twelve novel toluene dioxygenase variants with mutations introduced at the iron-coordinating residues (h222a, h222c, h222d, h222e, h228a, h228c, h228d, h228e, d376a, d376c, d376e, d376h). figure 2 visualization of the tdo active site with bound substrate (toluene, purple) (9). note. the catalytic iron atom (orange) and iron-coordinating residues are highlighted (yellow). residues within 7 å of the substrate (toluene) are shown. image generated with chimerax software (13). betts & froese | investigation of the effects of mutating iron-coordinating residues in rieske dioxygenases 97 figure 3 multiple sequence alignment of sequencing data from tdo variants with single active site mutations. note. alignment performed with m-coffee (25). image generated with espript (31). fine focus | volume 1098 screening of toluene dioxygenase variants to test the cis-dihydroxylation activity of the tdo variants produced in this study, a fluorescence-based assay that had previously been reported by our laboratory was applied (30). this assay, performed in 96-well plates, detects the presence of cis-diol metabolites produced by active dioxygenases in the bacterial growth media through the conversion of the cis-diol metabolites to a corresponding dialdehyde, and subsequent conjugation of the dialdehyde to a fluorescent probe (30) (figure 4). in this way, the amount of fluorescence detected in each well provides information as to the amount of cis-diol metabolite produced by the enzyme variant present in the corresponding well. as the possibility existed that the introduced mutations would cause the structure of the tdo active site to be altered, while retaining some cis-dihydroxylation activity, it was determined that the enzyme variants should be tested on a small library of diverse aromatic substrates (figure 5). this would afford the opportunity to determine the effect of the introduced mutations on the enzyme’s activity for a wide range of substrates, including those the native enzyme has high activity for, those the native enzyme has low activity for, and those the native enzyme has no activity for. to test the cis-dihydroxylation activity of the designed tdo variants, vectors expressing each set of variants were separately transformed into e. coli (bl21 (de3)) alongside vectors expressing the parent enzyme (pcp-02) and a negative control (pcp-01) (30). six colonies expressing each variant were then inoculated into separate wells of a 96-well plate and cultured according to an optimized assay protocol (30), before being treated with the relevant aromatic substrate. following an incubation period in the presence of the relevant substrate, the cells were removed, and the growth media was carried through the fluorescence-based assay protocol (figure 4). analysis of the resultant data from these assays revealed that all twelve of the designed tdo variants (h222a, h222c, h222d, h222e, h228a, h228c, h228d, h228e, d376a, d376c, d376e, d376h) lacked any activity for all eight substrates used in the screens (figure 6). this included a figure 4 coupled reactions employed by the fluorescence-based assay to detect and quantify the cis-diol metabolites produced by active dioxygenases (30). betts & froese | investigation of the effects of mutating iron-coordinating residues in rieske dioxygenases 99 complete loss of activity for the native substrate (toluene), and other substrates for which the native enzyme possesses activity (benzyl alcohol, benzyl acetate, n-butyl benzene, t-butyl benzene, methyl benzoate) and no gain in activity for substrates that are not metabolized by the parent enzyme (benzyl acetamide and benzylamine) (figure 6). discussion to study the effect of mutating the iron-coordinating residues of rieske dioxygenases on the activity of these enzymes, the first step was to select a member of this class of enzymes to act as the template for mutagenesis. toluene dioxygenase (tdo) is a well-characterized member of the rieske dioxygenase enzyme family, which is derived from the soil bacterium pseudomonas putida f1 (42). due to the historic importance of tdo in the production of valuable compounds (16), and because of our laboratory’s experience working with this rieske dioxygenase system (27, 30), tdo was chosen as the engineering scaffold for this study. due to the structural similarity observed with many rieske dioxygenases (2), the results of this study would be expected to be applicable across many members of this enzyme family. the goals of this study were to investigate the effects of both altering and eliminating the iron-coordination of key residues in the active site of toluene dioxygenase. as tdo has been well characterized (9), the identification of the iron-coordinating residues of this enzyme (his222/his228/asp376) using chimerax (13) was trivial (figure 2). to effectively test the hypothesis of this study, it was determined figure 5 aromatic substrates used to screen for cis-dihydroxylation activity among the tdo mutants produced. fine focus | volume 10100 note. fluorescence response was normalized to the mean fluorescence response of the negative control (e. coli bl21 (de3) pcp-01) ([i – i0]/i0) (21). parent activity was determined from positive controls (e. coli bl21 (de3) pcp-02), with the fluorescence response normalized to the negative control ([i – i0]/ i0) (30). figure 6 cis-dihydroxylation activity of h222 tdo variants (a), h228 tdo variants (b) and d376 tdo variants (c) for representative aromatic substrates (n = 6). betts & froese | investigation of the effects of mutating iron-coordinating residues in rieske dioxygenases 101 that all three of the residues coordinating the catalytic iron would be mutated to three alternate residues capable of coordinating iron (aspartate, glutamate, and cysteine for native histidine residues; glutamate, cysteine, and histidine for the native aspartate residue) and to one residue that would eliminate iron-coordination at that site (alanine). this afforded the opportunity to evaluate the effects not only of eliminating iron coordination at specific sites, but also the potential beneficial effects of remodeling the active site and/or altering the electronics of the catalytic iron center by changing the iron coordinating residues while maintaining iron coordination. once the successful generation of the targeted tdo variants had been confirmed, the next step was to test the cis-dihydroxylation activity of these variants. as the possibility existed that the introduced mutations would cause the structure of the tdo active site to be altered, while retaining some cis-dihydroxylation activity, it was determined that the enzyme variants should be tested on a small library of diverse aromatic substrates. these aromatic substrates included the native substrate (toluene), one polar substrate for which the native enzyme possesses high activity (benzyl alcohol), one polar substrate for which the native enzyme possesses moderate activity (benzyl acetate), three polar substrates for which the native enzyme possess little or no activity (methyl benzoate, benzyl acetamide, and benzylamine), and two sterically bulky substrates for which the native enzyme possesses low activity (n-butyl benzene and t-butyl benzene) (figure 5). by testing the activity of the designed mutants across these substrates, it would be possible to determine whether the introduced mutations had advantageous or detrimental effects in the cis-dihydroxylation of the native substrate, as well as in the cis-dihydroxylation of both sterically bulky and polar classes of substrates. upon testing the activity of each designed tdo mutant for all the designated substrates, it was revealed that none of the mutants designed in this study possessed activity for any of the diverse substrates selected (figure 6). these results revealed the critical role played by the iron-coordinating residues of rieske dioxygenases in their native state, as any alteration of these residues, either to other residues with the potential for iron-coordination or to residues incapable of coordinating iron, resulted in complete ablation of enzyme activity. although the hypothesis that the mutations introduced in this study would significantly alter the activity and/or substrate scope of the enzyme was proven correct, these changes were shown not to be beneficial for enzyme activity. the loss of activity observed with mutations to non-coordinating alanine likely indicates that coordination by three residues is essential for iron to be bound to the active site of rieske dioxygenases in a catalytically active state. the loss of activity observed with mutations to alternate iron-coordinating resides may indicate that these alterations result in a significant remodeling of the active site that prevents iron from binding in a catalytically active state. alternately, these results may indicate that the active site iron of rieske dioxygenases must specifically be bound by two histidine residues and one aspartate residue to effectively participate in the catalytic mechanism. further studies, including enzyme structure elucidation/homology modeling, fine focus | volume 10102 docking analysis, and molecular dynamics simulations are required to precisely elucidate the cause of the loss of activity observed from these alterations to the iron-coordinating residues. these studies will be performed in due course. although this study did not succeed in producing novel tdo variants with improved or expanded activity, the results described will serve to guide future studies targeting the engineering of rieske dioxygenases. with the increasing recognition of the need to employ more sustainable techniques in the chemical industry, and to remove harmful pollutants in contaminated environments, the field of enzyme engineering will continue to increase in importance. this is reflected in the fact that the engineering of rieske dioxygenases to improve their utility either as green-chemical catalysts or as tools for the bioremediation of contaminated environments continues to be an active area of research (1, 3, 5, 20, 21, 26, 27, 33, 37, 38). metagenomics research has shown that rieske dioxygenases commonly evolve among soil bacteria (17), meaning that many unannotated rieske dioxygenases remain to be studied in the context of enzyme engineering. as the field of microbiology continues to discover more diverse organisms and the powerful natural catalysts they produce, these new catalysts will provide valuable templates for the field of enzyme engineering. in this way, the natural ecological role of soil organisms can be harnessed and enhanced to create a safer and more sustainable future. this study, by demonstrating the importance of preserving key active site residues in the generation of rieske dioxygenase mutant libraries in the pursuit of enzyme engineering, will inform and expedite these efforts. conclusions inspired by recent reports that have shown that the alteration of iron-coordinating residues in metalloenzymes can alter or even improve the activity of these enzymes in certain contexts (29, 40), the iron-coordinating residues of toluene dioxygenase (tdo) were comprehensively mutated in this study. these residues were mutated both to alternate residues that are capable of coordinating iron and to a residue that is incapable of iron coordination. following the confirmation of successful mutagenesis, these new tdo variants were tested for their ability to catalyze the cis-dihydroxylation of a diverse group of potential aromatic substrates through a fluorescence-based assay system (30). the results of this study demonstrated the critical role played by the iron-coordinating residues of rieske dioxygenases in their native state. these results revealed that any alteration of these residues, either to other residues with the potential for iron-coordination or to a residue incapable of coordinating iron, resulted in a complete loss of cis-dihydroxylation activity for any of the classes of substrates tested in this study. although this study did not produce any rieske dioxygenase variants with improved or altered cis-dihydroxylation activity, the findings of this study will serve to inform future engineering studies targeting these enzymes. based on these results, it is clear that any attempts to engineer novel variants of rieske dioxygenases should make every effort to preserve the iron-coordinating residues in their native state. betts & froese | investigation of the effects of mutating iron-coordinating residues in rieske dioxygenases 103 acknowledgements this material is based upon work supported by the national science foundation under grant no. 2147098 (research in undergraduate institutions) and by the ball state university junior faculty aspire grant program. this work was made possible in part by the ball state university provost start-up program. author correspondence correspondence concerning this article should be addressed to: jordan t. froese, jtfroese@ bsu.edu. mailto:jtfroese%40bsu.edu?subject= mailto:jtfroese%40bsu.edu?subject= fine focus | volume 10104 references 1. ang, e. l., obbard, j. p., & 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(1989) toluene degradation by pseudomonas putida f1. nucleotide sequence of the todc1c2bade genes and their expression in escherichia coli. journal of biological chemistry, 264, 14940-14946. https://doi.org/10.1016/s0021-9258(18)63793-7 https://doi.org/10.3390/molecules26113431 https://doi.org/10.3390/molecules26113431 https://doi.org/10.3390/molecules26113431 https://doi.org/10.3390/molecules26113431 fine focus 48 i fine focus the influence of infant formulae on the growth of commensal and pathogenic streptococcus species in the infant oral cavity geneva waynick*, mary jane carmichael department of biology, hollins university, roanoke, va usa *corresponding author contact information: waynickgb@hollins.edu phone: (757) 755-0185 pi contact information: carmichaelm@hollins.edu (336) 830-4041 keywords: dental caries, infant formula, microbiome, oral cavity, streptococcus mitis, streptococcus mutans manuscript received 9 july 2021; accepted 22 november 2021 vol 8 i 49 abstract the oral microbiome is a complex community of microorganisms that influences the health of the human host. a number of diseases are associated with dysbiotic oral microflora in infants and children, including dental and gastrointestinal diseases. a variety of factors can influence the composition of the oral microbial community in infants, including mode of delivery, feeding method, and diet. this study focuses on the effect of nutritional differences in infant formulae on the growth of a commensal species (streptococcus mitis) and a pathogenic species (streptococcus mutans) that are commonly found in the infant oral cavity. a culture-dependent model was utilized to test the effects of one infant formula (nutramigen enflora) supplemented with a probiotic (lactobacillus rhamnosus) and a similar infant formula without probiotic supplementation (enfamil neuropro) on the growth of each species. a snyder’s media test was used to assess acidogenic potential of each species. bacterial growth in each formula was assessed by measuring colony forming units (cfus) and by measuring the ph of the culture media over an 8 hour incubation. results indicate that the probiotic formula may selectively inhibit the growth of the pathogen and aid in producing more favorable conditions for the commensal. these findings may make nutramigen enflora the preferred infant formula for overall health. the results of this study may assist parents in selecting alternatives to breastmilk that will support the proper development of the infant oral microbiome by favoring the growth of commensal bacteria. 50 i fine focus introduction the human microbiome is defined as the totality of microorganisms which inhabit the human body (25). estimates indicate that there are about as many bacterial cells as there are human cells in the body (46). the gut microbiome alone harbors about 1,000 species of bacteria with a combined total of 2 million genes, which is 100× the number of genes in the human genome (55). our microbial symbionts are intimately intertwined with both systemic and specific bodily function (22), and each niche in the human body nurtures a unique community of microbes. the microbiome is inherently linked to the health of the human host. the effects that the microbiome exerts on the human body are directly linked to the state of the microbiome itself: changes in species composition and relative abundance can be characterized on a spectrum from health to dysbiosis. many endogenous and exogenous factors (e.g. immune function, body site, diet, antibiotic use, lifestyle) influence the environmental conditions of the niches within the human body (figure 1), which in turn influence microbial community composition and thus overall host health (22). dysbiosis, a state in which the microbial community composition becomes unbalanced, is triggered by alterations in factors such as diet, immune function, hygiene, and hormone levels (27). it is well known that a dysbiotic microbiome is associated with a multitude of disease states of the body and the mind, such as cancers, obesity, cardiovascular disease, psoriasis, and major depressive disorder (57, 20, 22). the impacts of the microbiome on health are plentiful and significant, and thus the ways in which we humans care for our microbiomes is of high importance. the oral cavity is a distinct niche within the overall human microbiome that is of interest due to its association with diseases in adults and children such as dental caries, periodontal disease, various cancers (i.e. oral, esophogeal, pancreatic, and colorectal) and gastrointestinal diseases (56). figure 1: internal and external factors that influence the human microbiome. factors that shape the human microbiome can be internal (a condition of the body that is not easily changed, such as age, genetics, and mode of delivery at birth) or external (including lifestyle choices such as diet, exercise, and drug use). vol 8 i 51 the process of acquiring the oral microbiome begins during gestation, continues through parturition and infancy, and is highly influenced by factors such as genetics, gestation length, delivery mode, and diet (59). following birth, the infant is exposed to the multitude of microbes present within their environment (27). the oral cavity is colonized in a sequential manner, first with pioneer colonizers attaching and enabling subsequent colonizers to adhere and form a biofilm (52). the most common pioneer colonizers of the oral cavity are gram-positive bacteria including streptococci species such as s. mitis and s. salivarius, because of their ability to adhere to previously uncolonized epithelial tissue and their presence in breastmilk. pathogenic species like s. mutans are less abundant than commensal species, but their overgrowth is linked with a dysbiotic oral microbiome and diseases of the oral cavity like dental caries (57). typically, pathogenic species such as s. mutans disrupt the normal balance of the oral microbiome by producing acids that lower the ph within the environment to a level that favors aciduric, pathogenic species. this triggers the overgrowth of the pathogenic species and the simultaneous inhibition of commensal species like streptococcus sanguinis that are less tolerant to low ph conditions (57, 59). the composition of the oral microbiome fluctuates preceding the first tooth eruption, after which it begins to stabilize. because of this, more research is needed to determine if the composition of the predentate oral microbiome influences the more permanent oral microbiome later in life (59). the acquisition and development of a healthy oral microbiome is important for a child’s overall health as several childhood diseases, as mentioned above, are associated with a dysbiotic oral microbiome (59). nutrition is an important factor that can influence the trajectory of the development of the early oral microbiome (59) and thus the health of the individual as a whole. during infancy, the primary source of nutrition and early microbial exposure is either breastmilk or infant formula. breastfeeding is widely considered to be the optimal nutrition source for infants (3; 54). however, there are many reasons why breastfeeding may not be a suitable dietary choice. for instance, preterm or low birth weight infants may need a more nutritionally dense food source such as infant formula to achieve a healthy weight (40). similarly, infant formulae are desirable for infants suffering from malnutrition due to famine (40). hiv-positive mothers also rely on infant formulae to avoid transmission of the virus to the infant (40), and the advent of soy-based formulae in the 1920’s provided a solution for lactose-intolerant infants (40). infant formula utilizes either cow’s milk, soy, amino acids, or goat’s milk as a replacement for breastmilk. some infant formulae containing probiotics tout claims of boosting the microbiome, including lactobacilli and bifidobacteria in the formulations (18). however, these claims have not been clinically supported, and the effects of these formulations on the composition of the oral microbiome have not yet been thoroughly investigated this study aims to elucidate the relationship between the nutritional profile of infant formulae with the growth of one commensal and one pathogenic constituent of the infant oral microbiome.. a commensal species, s. mitis, and a pathogenic species, s. mutans, were used to represent beneficial and pathogenic streptococcus species commonly found in the infant oral cavity. s. mitis is a gram-positive commensal species of the oral cavity and is understood to be one of the primary colonizers of the oral cavity (19). like s. mutans, s. mitis metabolizes carbohydrates such as lactose and sucrose into lactic acid (30, 38). s. mitis produces an enzyme known as neuraminidase in addition to a number of adhesins which may aid in the adherence and subsequent colonization of surfaces within the oral cavity (31). the results from some studies suggest that s. mitis may supplement host immunity through modulating the expression of various immune markers (61). additionally, one study has shown that s. mitis induced the expression of an antimicrobial peptide that not only aids in deterring pathogenic microbes, but that s. mitis itself is tolerant to (17, 42). the production of antimicrobials that target pathogenic microbes in turn may boost host health by limiting the growth of pathogens. s. mutans is a gram-positive facultative anaerobe that is known for its role in the development of dental caries, especially in infants and children (24). two characteristics of s. mutans that contributes to its pathogenicity are its acidogenity and acidurity: the ability to metabolize sugars 52 i fine focus (glucose, fructose, lactose, sucrose) to lactic acid and thrive in low ph conditions, respectively (36). this increase in acidity resulting from s. mutans’ metabolism erodes the dental enamel and leads to dental caries. therefore, a decrease in ph is indicative of the growth of s. mutans and the dysbiotic state that can be associated with its growth (36). these species were grown on media containing one of two infant formulae that were chosen for their similarity in nutritional composition to elucidate the effect of probiotic supplementation on the growth of the commensal and pathogenic species: a cow’s milk-based formula without probiotic supplementation (enfamil neuropro) and a cow’s milk based formula with probiotics (enfamil nutramigen with enflora lgg). the overall growth of the two species was assessed by counting cfus to determine the cell density of the culture media every hour throughout the duration of the experiment. the metabolic activity of acidogenic bacteria was measured by ph level of the culture media to determine the rate of converting sugars into lactic acid. metabolic responses indicative of commensals thriving suggest that infant formula is beneficial for the establishment of a microbiome on a healthy trajectory, while the opposite is true with the opportunistic pathogens. methods species descriptions two species of streptococcus-group bacteria were chosen for this study: one commensal (s. mitis) and one opportunistic pathogen (s. mutans) that are commonly found within the infant oral cavity (59). both cultures were sourced from the american type culture collection (atcc.org, manassas, va). s. mitis type strain atcc 49456 was the sole strain available from atcc. s. mutans type strain atcc 25175 was selected for its origin from dental caries. snyder’s media test a snyder’s media test was used to determine the cariogenic potential of both species in the study. snyder’s media (thomas scientific, swedesboro, nj) contains a ph indicator that is used to qualitatively assess a decline in ph over time, which is indicative of cariogenic potential as acid erodes dental enamel and causes dental caries. the faster the inoculated media changes color, the greater the cariogenic potential of a species (48). a change in media color from blue-green to yellow within 24 hours indicates high cariogenic potential, while a change in color to orange indicates moderate cariogenic potential and no color change indicates low cariogenic potential. in this study, the color of the media for each species was compared after 24 hours of incubation. both species were incubated at 37°c in snyder’s media in triplicate for 24 hours followed by comparisons of the media color to the uninoculated control. growth curves under baseline conditions growth curves were created for each species under baseline conditions to describe the general growth kinetics of both species. the approximate time of the mid-log phase (i.e. the midpoint of the logarithmic phase) was needed for this study. the experimental cultures were inoculated with mid-log starting cultures because the mid-log phase is indicative of the fastest growth rate (i.e. maximum slope). the amount of time needed to reach the plateau phase for each species was used to determine the length of time for incubation during the experiment. growth curves were produced by measuring the optical density of the culture grown in the control media (tryptic soy broth, thermo fisher scientific, waltham, ma) in triplicate until the plateau phase of growth was reached (i.e. until growth became steady). for each species, three fresh cultures were inoculated in 10 ml tsb and allowed to reach turbidity (24 hours). uninoculated media was placed in a cuvette to be used as a blank for the spectrophotometer (vernier spectrovis plus, vernier, beaverton, or). the batch culture was inoculated with a 1:20 dilution (ashley hawkins, personal communication) of turbid culture and media. the optical density of each batch culture was measured at 600 nm (51) every 30 minutes for the first 4 hours, then every hour until a plateau in the growth curve was reached. these measurements were plotted against time to create growth curves (figures 2 and 3) from which the mid-log and plateau phase could be determined. vol 8 i 53 preparation of culture media once baseline growth curves were established, both species were maintained in tryptic soy broth (tsb) at 37°c. tsb was chosen as a control media because it was an atcc recommended growth media for each species. two types of infant formula (table 1) were compared in this study: a cow’s milk-based formula (enfamil neuropro, mead johnson, chicago, il) and a cow’s milk-based formula supplemented with probiotics (nutramigen enflora, mead johnson, chicago, il). the nutramigen enflora formula is supplemented with lactobacillus rhamnosus, one of the most widely used probiotic strains utilized as a dietary supplement (26). a 1:5 (v/v) dilution of infant formula to tsb without dextrose was prepared for each formula following hinds et al. (2016). infant formulae were diluted with tsb without dextrose (thermo fisher scientific, waltham, ma) to ensure bacterial growth without adding another carbohydrate source. assessment of cell density under experimental conditions for both species, cultures in tsb at the mid-log phase were prepared for the experiment in triplicate. each of the infant formula media and the control media was inoculated with 100 µl of starting culture and vortexed to ensure homogeneity in replicates of three, for a total of 18 culture tubes including the control. plate counts were performed by diluting the starting culture with fresh tsb to dilutions of 10-4, 10-5, and 10-6. 100 µl of each of these dilutions were plated onto tryptic soy agar (tsa) plates in replicates of three. the tsa plates were incubated at 37°c for 24 hours, after which colony forming units were counted in plates that had between 30-300 colony forming units (cfus) (33). the following formula was used to calculate the original cell density of the starting culture: original cell density=(colony forming units)/(original sample volume) assessment of metabolic activity under experimental conditions every hour over the course of each incubation, the ph of the culture media was measured to monitor the change in acid production using a ysi ecosense ph 100a meter (ysi incorporated, yellow springs, oh). the meter was calibrated according to manufacturer’s instructions before use. a decrease in ph indicates acid production, which is related to the cariogenic potential of a culture. statistical analyses statistical analyses were conducted using jasp (version 0.11.1, jasp team 2019). shapiro-wilks tests were used to confirm a normal distribution of data. normally distributed data were analyzed using independent t-tests; when assumptions for parametric statistics were violated, data were analyzed using the non-parametric mann-whitney test. independent t-tests and mann-whitney tests were performed on ph data to compare the effect of each species on the ph of each media at a given time point. analysis of variance (anova) was used to assess the change in ph over time within the same media and species and to assess the change in ph between media types for a given species at a given point in time. tukey’s post-hoc tests were performed for all anovas with significant p-values (α = 0.05). the same tests were utilized on data reporting cfus, except for time points where one of the three replicate data points were missing due to inadequate growth during plate counts. in these cases, mean values will be used for data reporting, as there were not enough replicates at each time point to conduct statistical analyses. cfu and ph data were compared over time for observable trends between the two measurements for each species in each media type. data from hours 0 and 8 were used as time points in the analysis because they represented the start-point and plateau phase of each incubation, respectively. results growth curves under baseline conditions the growth curves produced for both s. mitis (figure 2) and s. mutans (figure 3) showed a logarithmic curve with the lag phase from hours ca. 0-3 hours, a log phase from ca. 3-6 hours, a plateau phase from ca.6-8 hours, and the mid-log point ca. hour 4.5. 54 i fine focus snyder’s media test the degree of color change in the inoculated snyder’s media was compared relative to the uninoculated test media (figure 4a). after 24 hours, a color change in the media inoculated with s. mutans (figure 4b) indicated a positive result and a lack of color change in the media inoculated with s. mitis (figure 4c) indicated a negative result. figure 2: the growth curve of s. mitis under baseline conditions. growth in tsb was quantified by optical density, measured by spectrophotometery, over 8 hours. error bars represent standard deviation of the mean (n= 3). figure 3: the growth curve of s. mutans under baseline conditions. growth in tsb was quantified by optical density, measured by spectrophotometery, over 8 hours. error bars represent standard deviation of the mean (n= 3). colony forming units the mean cfus of s. mutans increased over time in tsb, with a ca. 10x increase between hour 0 and 8 (table 2; figure 5a), whereas the mean cfus of s. mitis decreased over time in tsb, with a ca. 16x decrease between hour 0 and 8 (table 2, figure 5a). the mean cfus of s. mutans decreased over time in neuropro and enflora, by ca. 0.18x and 6.8x, vol 8 i 55 respectively. the mean cfus of s. mitis increased over time in neuropro and enflora, with an increase of ca. 3x and 2.1x respectively between hour 0 and 8 (table 2, figures 5b and 5c). at all time points in each media, s. mitis had greater cfus than s. mutans, except at hour 8 in tsb (table 2, figure 5). to show which media yielded the highest cell density for each species at a given time, the mean values for cfus in different media types were compared within a species (table 2, figure 6b). neuropro had the highest cfu count for s. mitis of ca. 2.59 x 107 compared to ca. 5.07 x 106 in tsb and ca. 2.40 x 107 in enflora at hour 0 (table 2, figure 6a). at the end of the incubation, cfus of s. mitis in neuropro was 347x and 2x higher than tsb and enflora respectively. for s. mutans at hour 0, tsb yielded the highest cfu count of ca. 4.31 x 106 compared to ca. 2.40 x 105 in neuropro and ca. 3.90 x 105 in enflora (table 2, figure 6b). at the end of the incubation, cfus of s. mutans in tsb were 208x and 841x higher than neuropro and enflora respectively. cfus of s. mutans decreased ca. 15% in neuropro and ca. 87% in enflora over the course of the incubation. figure 4: results of snyder’s media test. a change in the color of the media from dark green to orange within 24 hours indicates a positive result. cultures pictured are representative of all three replicates for each species: a) uninoculated media, b) media inoculated with s. mutans, c) media inoculated with s. mitis. ph the mean ph of s. mitis significantly decreased over time in all three media types (table 4, figure 7b). between hour 0 and hour 8, the ph decreased by ca. 2.6% in tsb, ca. 1.9% in neuropro, and ca. 1.2% in enflora. the mean ph of s. mutans significantly decreased over time in tsb but increased slightly over time in neuropro and enflora (table 3, figure 7a). between hour 0 and hour 8, the ph decreased by ca. 13.2% in tsb and increased by ca. 0.3% and ca. 0.4% in neuropro and enflora respectively. results of the independent t-tests comparing ph levels of s. mutans to s. mitis showed a significant difference in each of the media types at two time points (table 4, figure 7). none of the mann-whitney tests were significant (table s1). at hour 0 and 6, there was no significant difference in ph between the two species when cultured in all three of the media types (table 4, table s1). at hour 8, there was a significant difference in ph between the two species when cultured in all three of the media types. in neuropro and enflora, s. mutans had a higher ph than s. mitis. in tsb, s. mutans had a lower ph than s. mitis. 56 i fine focus figure 5: mean colony forming units (cfu) of s. mutans and s. mitis over time. a) in tsb, b) in neuropro, c) in enflora. error bars representing standard deviation of the mean are included when n = 3. the results of the anovas comparing the ph levels over the course of the incubation were significant (p < 0.001) for both species in all three media (figure 8, tables s2, s3, and s4), except for s. mutans in neuropro (p = 0.753). for s. mutans and s. mitis, there was a significant decline in the ph levels at 0, 4, and 8 hours when cultured in tsb. in neuropro, the ph levels at hours 0 and 8 significantly declined for s. mitis. in enflora, the ph levels at hours 0 and 8 were significantly different in s. mutans and s. mitis, with an increase in ph for s. mutans and a decrease in ph for s. mitis. for s. mutans, the ph increased by ca. 0.3% and 0.4% in neuropro and enflora, respectively, and dropped by ca. 15% in tsb. for s. mitis, the ph decreased by ca. 2.7%, 2%, and 1% in tsb, neuropro, and enflora, respectively. the relationship between cfus and ph for the species and media that resulted in increased bacterial growth over time, there were notable trends between cfus and ph over time. for s. mutans grown in tsb (figure 9), s. mitis grown in neuropro (figure 10), and s. mitis grown in vol 8 i 57 figure 6: mean colony forming units (cfu) of (a) s. mitis and (b) s. mutans in tsb, neuropro, and enflora over time. error bars representing standard deviation of the mean are included when n = 3. enflora (figure 11), the general trend is as the cfus increase, the ph also decreases. in optimal growth conditions (figure 6 & 7), there appears to be a steeper slope of the trendline relating cfus to ph as compared to less optimal growth conditions (figure 11). for the two conditions with the highest growth, s. mutans in tsb (figure 9) and s. mitis in neuropro (figure 10), the inflection point where cfus increase sharply and ph decreases sharply was around the 4.5 hour mark (ca. mid-log phase). figure 11 is representative of the remaining conditions: no inflection point in the slopes of the cfu and ph graphs was observed. the trends outlined above indicate that the high-growth conditions (s. mutans in tsb, s. mitis in neuropro and enflora) had a stronger relationship between an increase in cfus and decrease in ph than the low-growth conditions (s. mutans in neuropro and enflora, s. mitis in tsb). discussion snyder’s media test confirms the cariogenic potential of s. mutans the results of the snyder’s media test showed that s. mutans has a greater cariogenic potential than s. mitis. this reaffirms that s. mutans is an acidogenic and aciduric species. s. mutans produces lactic acid (36) which lowers the ph of the surrounding microenvironment, limits the growth of non-aciduric species (which are commonly commensal species) and produces conditions that favor the growth of other aciduric pathogens (32). in addition, the results of the snyder’s test showed that s. mitis does not have a high cariogenic potential. although s. mitis is known to metabolize carbohydrates to lactic acid like the pathogenic s. mutans (30), s. mitis is generally considered to act as a commensal species when its growth is kept in check figure 7: mean ph values in each media type over time: a) s. mitis, b) s. mutans. error bars represent standard deviation of the mean. 58 i fine focus figure 8: mean ph values of s. mutans and s. mitis over time in a) tsb, b) neuropro, and c) enflora. error bars represent standard deviation of the mean (n = 3). by competing commensals. to ensure the establishment of a beneficial microbial community early in life, it is important for infant formulae to have a nutritional composition that both inhibits the growth of pathogens like the cariogenic s. mutans and enhances the growth of commensals such as s. mitis. cell density reveals lactose-based formula favors the growth of s. mitis over s. mutans s. mutans had ca. 141x more cfus in tsb than s. mitis at the end of the 8 hour incubation, therefore tsb favored the growth of the pathogenic s. mutans over s. mitis. when cultured in infant formula, there was a decrease in cfu counts of s. mutans over time, suggesting that the infant formulae did not provide optimal growing conditions for s. mutans compared to s. mitis and that the infant formulae may have an inhibitory effect on the growth of this opportunistic pathogen. this may be in part due to the different types and concentrations of carbohydrates found in the infant formulae and tsb. tsb contains 0.025 g/ml of dextrose while neuropro contains 0.015 g/ml of lactose and enflora contains 0.014 g/ml of dextrose. previous research examined the effects of infant formula composition on the growth of the pathogen s. mutans and found that vol 8 i 59 figure 9: the relationship between cfus and ph for s. mutans grown in tryptic soy broth. error bars representing standard deviation of the mean when n = 3. figure 10: the relationship between cfus and ph of s. mitis grown in neuropro. error bars representing standard deviation of the mean are included when n = 3. figure 11: the relationship between cfus and ph of s. mitis grown in enflora. error bars representing standard deviation of the mean are included when n = 3. 60 i fine focus sucrose-based formulae yielded more growth of s. mutans when compared to formulae containing lactose (24). this is possibly due to sucrose being more readily fermentable, leading to a lowering of ph and sucrose being a substrate for the production of polysaccharides that allow bacterial adhesion and biofilm formation (45). because s. mutans is known for its rapid consumption of carbohydrates (24, 34), the higher concentration of dextrose in tsb may have allowed for greater proliferation of s. mutans compared to the infant formulae due to sucrose being more readily fermentable than lactose and rapidly lowering the ph of the formula, creating a low ph environment in which s. mutans thrives. the small difference between the cfu count of s. mutans in neuropro versus enflora at the end of the incubation (cfus for neuropro was 4x that of enflora compared to cfus for tsb being 208x and 841x higher than neuropro and enflora, respectively) is likely not due to the different carbohydrates found in each formula, since the dextrose in enflora is preferred to the lactose in neuropro (40). the magnitude of decrease in cfus of s. mutans was greater in enflora (ca. 6.8x decrease) than in neuropro (ca. 0.18x decrease) over the course of the incubation; therefore, enflora had a greater inhibitory effect on the growth of s. mutans than neuropro. since the concentrations of carbohydrates are similar between neuropro and enflora and previous research shows that s. mutans does not prefer lactose over dextrose, it may be the presence of the probiotic l. rhamnosus that caused this differential magnitude of decrease in cfus between the two infant formulae. one hypothesis for why the probiotic formula controlled the growth of both species is that the probiotic l. rhamnosus prevented uncontrolled growth by competing with the other species for resources. previous research has shown that supplementation with l. rhamnosus resulted in a decrease in dental caries and amount of s. mutans in children (ages 1-6 years), indicating that the probiotic may have the ability to outcompete the pathogen and significantly inhibit its growth and acid production (41). this same degree of interspecies competition has not been shown between l. rhamnosus and s. mitis, perhaps because commensal species both prefer similar environmental conditions (i.e. neutral ph as opposed to the low ph that s. mutans produces and thrives in) and do not threaten each other by altering the environment to a state that would inhibit the other species’ growth. this is good news in terms of how the infant formula impacts the community structure of the oral microbiome, because it indicates that the probiotic infant formula can potentially control both the overall growth of microbes (i.e. it controlled the growth of both the commensal and pathogen) and differentially (selectively) control the growth of the commensal and pathogen, with the commensal being favored and the pathogen being more strongly inhibited. the decreased amount of streptococcus growth may also have been influenced by the antimicrobial substances produced by l. rhamnosus. existing literature shows that l. rhamnosus has multiple avenues of controlling microbial growth in co-culture, including the production of various antimicrobial substances. l. rhamnosus produces microcine, a small antimicrobial peptide along with another 7 different antibacterial peptides (35). l. rhamnosus also produces two kinds of lectin proteins (antimicrobial molecules that target pathogenic microbes) which have been shown to successfully inhibit the growth and biofilm formation of salmonella species and e. coli (44). these lectin proteins have strong carbohydrate-recognition capabilities, which allows them to distinguish pathogenic microorganisms from nonpathogens based on the types and configurations of polysaccharides on the cell surfaces (44). the specificity of these probiotic lectin proteins for pathogenic microbes may explain why l. rhamnosus differentially affected the growth of s. mutans and s. mitis. these pathogen specific antimicrobial proteins may have targeted the pathogenic s. mutans, thus exerting a greater inhibitory effect on the pathogen compared to the commensal s. mitis. competitive exclusion and the production of antimicrobial compounds present potential explanations for how the probiotic suppressed growth of the two streptococcus species in this study. in addition, l. rhamnosus’ competition with s. mitis may also enhance host health by preventing the overgrowth of s. mitis. while s. mitis is generally considered a commensal in the oral microbiome, some studies have shown that an overabundance may cause the species to act as an vol 8 i 61 opportunistic pathogen (39). as previously mentioned, s. mitis can produce lactic acid like s. mutans, which in excess may lead to the formation of dental caries, giving it the potential to act as an opportunistic pathogen (1). in a study comparing the species composition of oral microbiomes of individuals with and without dental fluorosis, a dental disease associated with dental caries, individuals with dental fluorosis had a higher abundance of s. mitis than those without dental fluorosis (58). therefore, it is understood that while s. mitis typically acts as a commensal when kept in check by the cohabitants of the oral microbial community, the overgrowth of s. mitis may lead to decreased health of the oral microbiome. by competing with s. mitis and preventing the domination of the oral microbiome, l. rhamnosus may bolster host health by maintaining balance within the oral microbiome. in fact, studies on the effects of probiotic consumption on the composition of the oral microbiome show promise for boosting host health. in a study comparing the oral microbiome composition of adults administered dietary lactobacillus and streptococcus probiotics and a control group, it was found that there was a short-term increase of overall diversity of the oral microbiome (13). however, the species diversity reverted to baseline (pretreatment) levels after discontinuing probiotic intake. s. mutans thrives in sucrose-dense tsb, s. mitis thrives in lactose-based formulae the significant decline in ph of s. mutans over time in tsb compared to in the infant formula media suggests that tsb provided better growing conditions for s. mutans, allowing the organism to metabolize sugars into lactic acid at a higher rate. when s. mutans was cultured in both infant formula media, the ph actually increased slightly over time, which indicates s. mutans’ lack of growth in the media. this lack of growth observed in the infant formulae suggests that these two infant formulae limit the growth of s. mutans, perhaps in part due to lower carbohydrate concentration in the infant formulae compared to tsb. one explanation for the increasing ph of the infant formulae is s. mutans’ ability to produce alkali under stress (47). s. mutans has been shown to produce alkali by converting arginine into ammonia, co2, putrescine, and atp (34). the production of atp through this agmatine deiminase system is a protective response against starvation, among other stressors (7, 34). this stress-induced atp production provides energy for the starving cell. it is plausible that starvation caused by low carbohydrate levels in the infant formulae induced alkali production as a protective measure in s. mutans. this alkali production may have in turn caused the small increase in ph over the course of the incubation. when comparing between the two infant formula media, s. mutans had a lower ph in the non-probiotic formula, indicating that s. mutans fared better in the nonprobiotic formula than in the probiotic formula. the lower ph of the non-probiotic formula suggests that s. mutans’ growth was not as strictly inhibited as in the probiotic formula, indicating that the non-probiotic formula is less effective at preventing pathogenic growth. s. mitis’ relatively high ph in comparison to s. mutans when grown in tsb is another indication of the commensal’s low cariogenic potential, despite its shared ability to produce lactic acid as a metabolite like s. mutans. the small magnitude of the drop in ph over the course of the incubation in tsb (ca. 2.7%) compared to the ca. 15% ph drop of s. mutans shows that tsb is better suited for the growth of s. mutans than s. mitis. the fact that s. mitis had a greater ph drop over the course of the incubation when grown in nonprobiotic formula compared to the probiotic formula may indicate that s. mitis also competed with l. rhamnosus for resources. the presence of the probiotic l. rhamnosus controlled the growth of both the pathogen s. mutans and the commensal s. mitis, although to differing degrees. the probiotic inhibited the growth of the pathogen more strictly and allowed for more growth of the commensal. the manner in which the probiotic differentially affected the growth of the pathogen and commensal demonstrates that l. rhamnosus may support the establishment of a healthy oral microbial community by controlling the overall level of microbial growth while simultaneously favoring the growth of commensals over pathogens. 62 i fine focus relationship between cfus and ph over the course of the incubation, an increase in cfus is generally accompanied by a decrease in ph. this simultaneous increase in cell density and acid production relates to the overall growth of each species. for instance, when s. mutans experienced an increase in cfus between 4-6 hr and 6-8 hr over the course of the incubation in tsb, this was accompanied by a sharp decline in ph at both intervals. this shows the relationship between the increase in cell number and the increase in acid production, a byproduct of the organism’s metabolism. therefore, an increase in cfus and decrease in ph can be interpreted as an overall increase in growth rate and cellular metabolism. this pattern that connects cfus to ph is mirrored in the growth of s. mitis in both neuropro and enflora. similarly, the decrease in cfus of s. mutans grown in both of the infant formula media was accompanied by a slight increase in ph. the slow increase in ph shows that s. mutans was struggling to survive in these media, as there was no lactic acid production occurring to decrease the ph, and it is possible that the aforementioned agmatine deiminase system induced the production of alkali and atp to cope with stress. this indicates that these media did not provide optimal growing conditions for this species to enable it to be a cariogenic threat. the suboptimal conditions of the infant formulae did not allow s. mutans to reach its full cariogenic potential, indicating that the infant formulae may help reduce the risk of developing dental caries over time. this pattern was more prominent in the probiotic formula, which provided the least optimal conditions for s. mutans out of all three media types, indicating that the probiotic formula had the best potential for both inhibiting the growth of the pathogen and therefore preventing it from producing enough lactic acid to induce tooth decay (enamel demineralization begins around a ph of 5.5) (37). conclusions and directions for future research the community structure of the oral microbiome is constructed over time, beginning during gestation and continuing into adulthood (1, 59). the colonization of the oral cavity occurs sequentially: newly introduced species are dependent on the species already present in the oral cavity (52). since the established microbial community determines the species that are subsequently acquired, the state of the oral microbiome earlier in life shapes the trajectory of the microbiome later in life (59), especially since commensals and pathogens tend to exclude each other via competition (32). this study showed that a probiotic-supplemented infant formula (nutramigen enflora) was more successful at inhibiting the growth of the pathogen (s. mutans) than the non-probiotic formula (enfamil neuropro). the growth of s. mitis was greater in the non-probiotic formula, this along with the additional health benefits to the gut (i.e. preventing infection and diseases and aiding in maintaining a healthy weight) conferred by the probiotic may make nutramigen enflora the preferred infant formula for overall health. this information may aid parents in choosing an infant formula for their child if breastfeeding is not a viable option or choice. since the nutrition during infancy is a key factor in determining the structure of the oral microbiome for the rest of the child’s life (59), it is of parents’ great concern that their child is consuming the best possible nutrition source for supporting the healthy development of the oral microbiome. although this study provides preliminary data to help parents in choosing an infant formula for their child if breastfeeding is not a viable option, it is limited in its ability to predict the behavior of oral microbiota in vivo. the health of the oral microbiome is affected by a large variety of influences, including internal factors such as host genetics (23) and external factors such as diet (29). the confluence of these factors works together to mold each individual’s unique oral microbiome community (27). between 700-1,200 different species of bacteria cohabitate within the oral cavity of humans, alongside numerous fungi, archaea, and viruses that all interact with one another to shape the oral microbial community (15, 57). the commensal and pathogenic species selected for this study are merely representative of the hundreds of different species that colonize the oral cavity. future research on other prominent species, such as the commensals lactobacillus plantarum and streptococcus sanguinis and other pathogens including fusobacterium nucleatum and porphyromonas gingivalis (27) is needed to expand the understanding of the full range of interspecific interactions that shape the oral microbiome. vol 8 i 63 in addition, there are many different kinds of infant formulae on the market that may differentially affect the growth of oral microorganisms. while this study sought to understand if and how one probiotic infant formula would differentially affect the growth of streptococcus species compared to one non-probiotic infant formula of similar nutritional composition, there are many more brands and formulations of infant formula that could be studied. the two infant formulae used in this study were cow’s milk based, but many other infant formulae use alternatives such as soy or goat milk and have varying nutritional composition that may affect the growth of oral microorganisms. a variety of carbohydrates (i.e. dextrose, sucrose, lactose), lipids (i.e. milk fats, palm, coconut, & sunflower oils), and proteins (i.e. casein, whey, soy protein isolate) are present in different infant formulae (18, 43, 49). additionally, comparing how breastmilk affects the growth of oral microorganisms versus infant formulae is of interest, as breastmilk is recognized as being superior in supporting a healthy microbiome over infant formulae (40). therefore, comparing how different kinds of infant formulae compare to breastmilk in terms of supporting microbiome health may help parents choose an infant formula that is most comparable to breastmilk in that aspect. finally, while this study examined the growth of oral microorganisms over a period of 8 hours, the oral microbiome is acquired sequentially over the years of childhood (52). therefore, additional longitudinal studies are needed to capture how the community changes over time. one example of an important time point in the establishment of the oral microbiome is the eruption of the first tooth at ca. 6 months of age (59). the dental surface provides a new substrate for the colonization of the oral microflora and also happens to be s. mutans’ preferred colonization niche within the mouth. therefore, the time of primary dental eruption may be a critical inflection point in the trajectory of the development of the oral microbiome (12). therefore, a deeper understanding of factors that influence the oral microflora during the predentate and primary dental eruption stages of the infant oral microbiome are of great importance. acknowledgements i would like to thank kloe borja for her assistance in collecting measurements throughout the experiments and dr. son nguyen for his guidance throughout the development of methodology for the 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of the adolescent patients with moderate or severe dental fluorosis. sci rep. 11:2897. https://www.ncbi.nlm.nih.gov/pmc/articles/pmc7859183/ 59. xiao, j., fiscella, k.a., & gill, s.r. 2020. oral microbiome: possible harbinger for children’s health. internat j sci. 12:12. https://www.nature.com/articles/s41368-020-0082-x 60. ying, s., zeng, d., chi, l., tan, y., galzote, c., cardona, c., lax, s., gilbert, j. & quan, z. 2015. the influence of age and gender on skin-associated microbial communities in urban and rural human populations. plos one. 10:e0141842. https://pubmed.ncbi.nlm.nih.gov/26510185/ 61. zhang, g., chen, r. & rudney, j.d. 2008. streptococcus cristatus attenuates fusobacterium nucleatum-induced interleukin-8 expression in oral epithelial cells. j periodontal res. 43:408416. https://pubmed.ncbi.nlm.nih. gov/18942189/ vol 8 i 69 carbohydrate fat protein iron enfamil neuropro nutramigen enflora lactose: 11.3 g palm olein, coconut, soy, nonfat milk, 1.8 mg and high oleic sunflower oils: 5.3 g whey protein: 2 g corn syrup solids: palm olein, coconut, soy, casein 1.8 mg 10.3 g and high oleic sunflower oils: 5.3 g hydrolysate: 2.8 g table 2: mean cfus of s. mutans and s. mitis over time. data points without standard deviation had insufficient replicates to calculate standard deviation. media hour 0 hour 2 hour 4 hour 6 hour 8 4.31x106± 6.58x106 2.91x106 3.50x106 1.69x107 4.23x107 5.07x106 ± 5.37x106± 4.87 x106± 1.52 x107 3.00x105± 1.80x106 4.03x106 2.51x106 6.05x104 tsb neuropro enflora 2.40x105 4.02x105± 1.16x106± 9.15x104 2.03x105± 4.37x105 1.59x106 1.02x105 2.59x107± 1.71x107± 4.25x107± 7.89x107± 1.04x108 ± 2.80x107 1.82x106 6.72x107 2.60x107 1.57x107 3.90x105 1.82x105± 9.30x104± 6.80x104 5.03x104± 1.80x105 1.31x104 4.81x104 2.40x107 ± 2.77x107± 4.42x107± 3.35x107± 4.99x107± 5.70x107 2.91x107 2.14x107 3.35x107 3.50x107 tables table 1: composition of infant formulae. the nutritional composition (per 100 kcal) of the non-probiotic (enfamil neuropro) and probiotic (nutramigen enflora) infant formulae. 70 i fine focus table 3: mean ph values of s. mutans and s. mitis over time. reported error is standard deviation of the mean (n = 3). media hour 0 hour 1 hour 2 hour 3 hour 4 hour 5 hour 6 hour 7 hour 8 tsb neuropro enflora 7.28 ± 7.18 ± 7.18 ± 7.15 ± 7.11 ± 7.03 ± 6.88 ± 6.70 ± 6.32 ± 0.03 0.03 0.03 0.01 0.02 0.02 0.02 0.04 0.03 7.15 ± 7.10 ± 7.10 ± 7.07 ± 7.08 ± 7.08 ± 7.05 ± 7.01 ± 6.96 ± 0.01 0.01 0.03 0.01 0.02 0.02 0.01 0.01 0.02 6.6 ± 6.61 ± 6.60 ± 6.60 ± 6.61 ± 6.59 ± 6.62 ± 6.62 ± 6.62 ± 0.04 0.01 0.01 0.02 0.01 0.01 0.03 0.03 0.03 6.69 ± 6.65 ± 6.63 ± 6.64 ± 6.64 ± 6.62 ± 6.63 ± 6.59 ± 6.56 ± 0.01 0.02 0.03 0.03 0.02 0.02 0.02 0.03 0.02 6.69 ± 6.69 ± 6.68 ± 6.68 ± 6.70 ± 6.69 ± 6.72 ± 6.73 ± 6.72 ± 0.01 0.01 0.01 0.01 0.01 0.01 0.00 0.01 0.01 6.74 ± 6.73 ± 6.73 ± 6.74 ± 6.72 ± 6.72 ± 6.72 ± 6.68 ± 6.66 ± 0.01 0.00 0.01 0.02 0.01 0.00 0.01 0.02 0.02 table 4: p-values from the independent t-tests comparing the ph of media between cultures inoculated with s. mutans and s. mitis at different intervals throughout the incubation. non-normally distributed data were not included (denoted by nn). ph was the same for each replicate in enflora at hour 6 (denoted by var= 0). media hour 0 hour 2 hour 4 hour 6 hour 8 tsb neuropro enflora nn 0.026 nn nn <0.001 nn nn 0.029 0.315 0.033 nn nn nn var=0 0.003 vol 8 i 71 supplemental tables table s1: p-values from mann-whitney test for non-normally distributed data comparing the ph of media between cultures inoculated with s. mutans and s. mitis at different intervals throughout the incubation. normally distributed data were not included (denoted by nd). media hour 0 hour 2 hour 4 hour 6 hour 8 tsb neuropro enflora 0.077 nd 0.164 0.077 nd 0.077 0.184 nd nd nd 0.072 0.077 0.077 nd nd table s2: post-hoc p-values of an anova comparing the ph of tsb between time points throughout the incubation. reported post-hoc p-values from an anova comparing the ph of s. mutans and s. mitis in tsb over time indicate if the difference in ph between time points is significant for a given species. hour 0 1 2 3 4 5 6 7 8 0 1 2 3 4 5 6 7 8 0.015 0.026 <0.001 <0.001 <0.001 <0.001 <0.001 <0.001 0.004 1 0.45 0.895 0.771 0.015 <0.001 <0.001 0.004 1 0.31 0.771 0.612 0.008 <0.001 <0.001 <0.001 0.925 0.925 0.995 1 0.612 0.005 <0.001 <0.001 0.049 0.049 0.423 1 0.202 <0.001 <0.001 <0.001 <0.001 <0.001 <0.001 0.049 0.31 0.002 <0.001 <0.001 <0.001 <0.001 <0.001 <0.001 <0.001 0.202 <0.001 <0.001 <0.001 <0.001 <0.001 <0.001 <0.001 <0.001 0.005 <0.001 <0.001 <0.001 <0.001 <0.001 <0.001 <0.001 <0.001 72 i fine focus table s3: post-hoc p-values of an anova comparing the ph of neuropro between time points throughout the incubation. reported post-hoc p-values from an anova comparing the ph of s. mutans and s. mitis in neuropro over time indicate if the difference in ph between time points is significant for a given species. hour 0 1 2 3 4 5 6 7 8 0 1 2 3 4 5 6 7 8 0.285 0.029 0.098 0.204 0.008 0.029 <0.001 <0.001 0.996 0.931 0.999 1 0.628 0.931 0.29 0.001 1 0.985 0.999 0.975 0.999 1 0.285 0.019 1 1 1 1 0.931 0.999 0.098 0.005 1 1 1 1 0.75 0.975 0.044 0.002 1 0.957 1 1 0.996 0.999 0.628 0.066 1 1 0.996 1 1 0.985 0.285 0.019 0.957 1 0.904 0.985 0.996 0.825 1 0.855 0.957 1 0.904 0.985 0.996 0.825 1 1 s4: post-hoc p-values of an anova comparing the ph of enfamil between time points throughout the incubation. reported post-hoc p-values from an anova comparing the ph of s. mutans and s. mitis in enflora over time indicate if the difference in ph between time points is significant for a given species. hour 0 1 2 3 4 5 6 7 8 0 1 2 3 4 5 6 7 8 0.791 0.559 0.995 0.337 0.18 0.088 <0.001 <0.001 1 1 0.995 0.995 0.946 0.791 <0.001 <0.001 0.977 0.821 0.946 1 0.995 0.946 0.002 <0.001 1 0.977 1 0.791 0.559 0.337 <0.001 <0.001 0.527 0.821 0.11 0.263 1 0.995 0.003 <0.001 0.977 1 0.527 0.821 0.977 1 0.008 <0.001 0.002 0.005 <0.001 <0.001 0.11 0.015 0.018 <0.001 <0.001 <0.001 <0.001 <0.001 0.015 0.002 0.977 0.337 <0.001 0.002 <0.001 <0.001 0.041 0.005 1 1 leah m. sanchez 0009-0008-3367-2475 abilene christian university abilene, tx usa jennifer a. hennigan 0009-0009-2839-7998 abilene christian university abilene, tx usa abstract tetracycline resistance genes have been reported to be abundant in bacteria in environments impacted by agriculture. the fort phantom hill reservoir watershed in jones and taylor counties in texas includes feedlots, rangeland, and row crop agriculture with manure-treated soils. we hypothesized that tetracycline-resistant coliforms could be present throughout the watershed. to investigate our hypothesis, samples were taken from the sediment of seven sites within the watershed between june 2022-august 2023. tetracycline-resistant coliforms and non-coliforms were isolated from six out of seven sites. the highest relative abundance of tetracycline-resistant isolates was found at a site at cedar creek. additionally, the highest relative abundance of tetracycline resistance among lactose fermenters was at a site at elm creek. from a representative sample of tetracycline-resistant isolates across all sites, the tetb genotype was the most common. additionally, four multi-drug resistant strains of escherichia were identified: ccn-113, ccn-251, ccn-109, and elm-161. a resistance plasmid was extracted from ccn-251 carrying dhfr1 and sulii resistance genes. monitoring of the spread of antimicrobial resistance genes through surface water in the fort phantom hill watershed provides data to support the need for antibiotic stewardship in this region. keywords: antimicrobial resistance (amr); tetracycline; watershed; surface water corresponding author email: jah20c@acu.edu manuscript received 11 september, 2024; accepted 13 april, 2025. distribution of tetracycline-resistant bacteria within the fort phantom hill reservoir watershed © 2025 sanchez, hennigan. fine focus, 11(1) 24-40. doi: 10.33043/2qda84q3 shared with cc-by-nc-nd 4.0 license. https://orcid.org/0009-0008-3367-2475 https://orcid.org/0009-0009-2839-7998 mailto:jah20c%40acu.edu?subject= https://creativecommons.org/licenses/by-nc-nd/4.0/ sanchez & hennigan | distribution of tetracycline-resistant bacteria within the fort phantom hill reservoir watershed 25 introduction the world health organization has declared the spread of antibiotic resistance a leading crisis in medicine (1). agriculture and human-based activities in urban areas are primary factors in the rise of antimicrobial resistance genes (args) in the environment. args from wastewater treatment plants, agriculture runoff, and animal husbandry can contaminate surface waters, then spread through horizontal gene transfer in the natural environment (2). application of animal manures to land significantly increases the level of and diversity of args in the soil. additionally, these args can persist in manure-treated soils for up to 120 days (3). in livestock waste, the most frequently detected classes of args include those that confer resistance to tetracyclines (tet) and sulfonamides (4). of the medically important drug classes approved for use in food-producing animals in the united states, tetracyclines comprised the highest percentage of total kilograms of antimicrobials sold in 2022 (65%; ~4 million kg) (5). a study in portugal found that tet resistance genes were present in all multidrug resistant (mdr) enterobacteriaceae isolated from poultry, swine, and cattle manures (6). likewise, a study in korea found that all commensal e. coli strains isolated from cattle farms were resistant to tetracyclines (7). the fort phantom hill reservoir watershed is a mixed-use watershed containing agricultural land and urban areas in jones and taylor counties in west texas in the united states. the area is largely dominated by agribusiness, including feedlots, rangeland, and row crop agriculture with manure-treated soils. additionally, the land is used by oil industries for exploration, refining, and drilling operations. ten creeks, three reservoirs, and the fort phantom hill reservoir make up the watershed. all of these lies within the brazos river drainage system. because of topology and urban development, rain in the watershed often produces excessive, rapid runoff and flash floods. surface water flows from south to north along creeks, ending in the fort phantom hill reservoir. therefore, nonpoint source pollution poses a concern for water quality. row crop agriculture and the expansion of the city of abilene increases the possibility of anthropogenic compounds entering streams and reservoirs through runoff (8). the goal of this study was to investigate the abundance of tet-resistant bacteria at seven sites throughout fort phantom hill watershed (figure 1). this study is unique in that it provides evidence of tet-resistance from multiple sites within a mixed-use watershed. materials and methods sample collection two replicates of 50 grams of sediment and water were obtained from kirby lake (32.373,-99.728), fort phantom hill reservoir (32.614, -99.676), buck creek (32.541, -99.709), elm creek (32.463, -99.777), cedar creek (32.471, -99.721 and 32.453, -99.721), and lytle creek (32.441, -99.715) using a previously described method (10). all sites were on public property. samples were obtained at least five days after any rainfall event to reduce potential storm effects on the sites. each site was sampled on at least two different days. physical characteristics for sites were obtained using a vernier labquest2 probe (beavertown, or), including ph, temperature, conductivity, turbidity, and dissolved oxygen. the metadata for each site was stored using the application epicollect5 v 7.0.3 (center for genomic pathogen surveillance, university of oxford) enrichment and selection of tet-resistant coliforms enrichment of the samples was completed in buffered peptone water as previously described (10). serial dilutions were spread onto macconkey agar (sigma-aldrich, usa) and macconkey agar supplemented with 16 µg/ml tetracycline (ibi scientific, road dubuque, ia) in accordance with the 2022 clinical laboratory standards institute concentration for tetracycline resistance for enterobacterales (11). plates were incubated at 37°c for 18-24 hours before colonies were counted. isolates were streaked to isolation on fine focus | volume 1126 macconkey agar with 16 µg/ml tetracycline, grown in tryptic soy broth (neogen, lansing, mi) supplemented with tetracycline, and stored in 50% glycerol at -80°c. metabolic profiling and antibiotic screening tet-resistant bacteria were inoculated in sim deeps (himedia laboratories, kennett square, pa) and citrate slants (carolina biological supply, burlington, nc), grown at 37°c for 18-24 hours before analyzing. isolates were patched on tryptic soy agar (neogen) supplemented with either 4 µg/ ml cefotaxime (mp biomedicals, solon, oh) or µg/ml 32 nalidixic acid (amresco, solon, oh) and grown at 37°c for 18-24 hours. detection of tet resistance genes genomic dna was isolated using a wizard genomic dna purification kit (promega, madison, wi). multiplex pcr of teta, tetb, tetc, tetd, tetm and teto genes was conducted using primers as previously described (12) with the following modifications. briefly, reactions were completed for teta, tetm, and teto or tetb, tetd, and tetc genes using platinum™ pcr supermix (thermo fisher scientific, usa) or accuprime supermix (thermo fisher scientific) for 35 cycles of the following program: 94° for 5 min, 94° for 1 min, 55° for 1 min, 68° for 1:30 min, 68°c for 10 min. rections were visualized on 1-1.5% agarose gels using a 100 bp standard ladder (neb). seven sites were selected from six different lakes and creeks within the fort phantom hill watershed: fort phantom hill reservoir (1), buck creek (2), cedar creek (3, north; 4, central) lytle creek (5), kirby lake (6) and elm creek (7). a creek map was generated using usgs steamer (9). solid blue indicated streams and dashed lines indicate intermittent streams. red solid lines represent roadways. black triangles show usgs stream gages. figure 1. map of sampling sites in the fort phantom hill watershed sanchez & hennigan | distribution of tetracycline-resistant bacteria within the fort phantom hill reservoir watershed 27 16s pcr and sanger sequencing 16s rdna was amplified using 27f and 1492r primers as previously described (13). amplicons were purified and concentrated using a monarch® pcr & dna cleanup kit (neb, ipswich, ma) and submitted to mc biolabs (san francisco, ca) for sanger sequencing. sequences were analyzed and edited in sequencher 5.4.6 (gene codes corporation, ann arbor, mi) and queried in the ncbi 16s ribosomal rna sequence database using megablast. genus level identities were assigned to isolates with consistent percent identities of no less than 95%. 96 well plate antibiotic susceptibility testing environmental tet-resistant isolates and escherichia coli atcc 25922 were inoculated in mueller-hinton broth (sigma-aldrich) and grown overnight at 37°c. cultures were standardized to 0.12-0.14 od600 using a nanodrop one (thermo scientific), equivalent to 0.5 mcfarland standard. in triplicate wells of a 96-well plate (celltreat, usa), 100 ul of standardized cultures were added to 200 ul of mueller-hinton broth supplemented with antibiotics to produce the following final concentrations at 300 ul: 4 µg/ml cefotaxime, 16 µg/ml tetracycline, 32 µg/ml nalidixic acid, 64 µg/ml kanamycin (alfa aesar, ward hill, ma), 32 figure 2. composition of the tet-resistant population per site the proportion of tet-resistant lactose-fermenting (black) and non-lactose fermenting colonies (gray) was calculated as a percentage of total tet-resistant cfus. the bars represent the average percentage of four samples for each site. fine focus | volume 1128 µg/ml ampicillin (sigma aldrich), and 16 µg/ml trimethoprim (sigma aldrich). concentrations are consistent with the 2022 clinical laboratory standards institute concentration for resistances for enterobacterales (clinical and laboratory standards institute (11). plates were statically incubated at 37 °c for 24 ±2 hours of growth and read at od630 using a smartreader™ 96 (accuris, edison, nj). od630 readings ≥ 0.25 were classified as resistant. plasmid isolation and sequencing the plasmids from ccn-251 and ccn-113 were isolated using a monarch™ plasmid miniprep kit (neb) and visualized on a 1.5% gel using a 10 kb supercoiled dna ladder (neb). whole plasmid sequencing was performed by plasmidsaurus (eugene, or) using oxford nanopore technology with custom analysis and annotation. results to determine the relative abundance of tet resistance in the fort phantom hill reservoir watershed, sites were selected to represent the ten creeks and three reservoirs that comprise the watershed (figure 1). not all creeks could be sampled due to lack of accessibility and drought conditions. sites at indian creek to the northwest and catclaw creek were dry, except immediately following rainfall. because bacterial and arg loads have been shown to increase with rainfall events, these sites were excluded from this study (14, 15). of the seven sites investigated, tet-resistant isolates were found at six sites; however, lactose-fermenting tet-resistant isolates were only isolated at five sites (figure 2). no tet-resistant isolates were obtained from the southernmost site, kirby lake, even though the number of colony forming units (cfus) on macconkey agar plates for the same samples did not differ from other sites. only non-lactose fermenting isolates were cultured from the fort phantom hill reservoir. tet-resistant lactose-fermenting bacteria composed the majority of the tet-resistant population at two sites, cedar creek-north (98.4%) and elm creek tet-resistance in gram-negatives tet-resistance in lactose-fermenters kirby lake 0% 0% lytle creek 0.00019% 0.0005% cedar creek-central 0.14676% 0.01829% cedar creek-north 0.00347% 0.06531% elm creek 0.04718% 3.38440% buck creek 0.00275% 0.00879% fort phantomhill reservoir 0.00012% 0% table 1. mean relative abundance of tet-resistance at each site the mean relative abundance of tet-resistant lactose-fermenting and non-lactose fermenting colonies was calculated as a percentage of resistant cfus to total cfus on macconkey agar lacking tetracycline for four samples for each site. sanchez & hennigan | distribution of tetracycline-resistant bacteria within the fort phantom hill reservoir watershed 29 (56.1%) (figure 2). the site with the highest overall tet-resistance was cedar creek-central (0.146%), followed by elm creek (0.047%) (table 1). however, the site with the highest tet resistance in lactose-fermenters was elm creek (3.384%), followed by cedar creek-north (0.065%). since the total number of tet-resistant potential coliform colonies for all sites was over 3000, every isolate could not be tested further. instead, a random sample of up to ten well isolated colonies from each replicate was selected and metabolic profiles obtained for 148 tet-resistant isolates using the following criteria: nalidixic acid resistance, cefotaxime resistance, h2s production, motility, indole production, lactose fermentation, and citrate utilization. using these criteria, 23 different metabolic types were found throughout the watershed and 16s sequencing was performed to determine the genera of 37 representative strains for each metabolic type at each site (table 2). the most prominent genus identified throughout the watershed was escherichia; however, tet-resistant strains of pseudomonas, klebsiella, providencia, serratia, enterobacter and raoultella were also identified. to determine the tet genotypes for the representative strains of each metabolic type, multiplex pcr was used to detect the presence of the teta, tetb, tetc, tetd, tetm, and teto genes. of the 37 representative tet-resistant strains genotyped, isolates with the genotypes teta, tetb, tetd, teta tetb, and teta tetb tetd were identified (figure 3). figure 3. tet genotypes represented in the fort phantom hill watershed multiplex pcrs for tet genes tetb, tetd, and tetc (top panel) and teto, tetm, and teta (bottom) panels are shown on a 1% agarose gel. the band sizes for each gene product are indicated. a 100 bp ladder (neb) is included in the first lane for comparison. fine focus | volume 1130 si te genus #o f i so la te s r ep s tr ai n # te tr ac yc lin e re si sta nc e na lid ix ic a ci d re si sta nc e ce fo ta xi m e re si st an ce h 2s pr od uc ti on m ot ili ty in do le p ro du ct io n la ct os e fe rm en ta ti on ci tr at e ut ili za ti on el m c re ek (e lm ) klebsiella 3 201 klebsiella 10 218 escherichia 26 227 escherichia 1 237 escherichia 1 170 enterobacter 1 152 providencia 2 164 escherichia 1 161 pseudomonas 1 215 ce da r c re ek -c en tr al (c cc ) escherichia 2 112 klebsiella 3 268 providencia 1 272 providencia 4 st-f4 providencia 4 st-f10 serratia 17 275 klebsiella 3 267 providencia 1 271 table 2. metabolic characteristics of representative tet-resistant strains from fort phantom hill watershed sites. positive characteristics are indicated by black squares, negative results are indicated by white squares, and ambiguous results are indicated by gray squares. genus of representative strain for each metabolic type is listed. fp, fort phantom hill reservoir table 2 continues “serratia” on page 3131. sanchez & hennigan | distribution of tetracycline-resistant bacteria within the fort phantom hill reservoir watershed 31 si te genus #o f i so la te s r ep s tr ai n # te tr ac yc lin e re si sta nc e na lid ix ic a ci d re si sta nc e ce fo ta xi m e re si st an ce h 2s pr od uc ti on m ot ili ty in do le p ro du ct io n la ct os e fe rm en ta ti on ci tr at e ut ili za ti on fp serratia 2 224 ce da r c re ek -n or th (c cn ) escherichia 11 113 escherichia 14 251 escherichia 1 140 pseudomonas 1 142 escherichia 1 109 bu ck c re ek (b c) escherichia 12 146 escherichia 1 181 raoultella 3 171 raoultella 2 184 raoultella 1 178 raoultella 1 179 raoultella 1 182 pseudomonas 1 149 pseudomonas 3 263 ly tle c re ek (l c) escherichia 1 110 escherichia 1 189 escherichia 9 145 serratia 2 156 pseudomonas 1 225 table 2 (continued from page 30) fine focus | volume 1132 genotypes including tetc, tetm, or teto genes were not detected in any isolate. none of the six genes were detected in one isolate, elm-164, indicating that it harbors a different tet gene. of the represented group, the tetb genotype was the most abundant at 45.95% (17/37) and was present at all sites (table 3). the teta tetb genotype was the second most abundant at 29.73% (11/37). two isolates harboring tetd only were also detected at elm creek; tetd was found in combination with teta and tetb at elm creek and buck creek. altogether, 35.14% (13/37) of the representative isolates carried more than one tet resistance gene. the genotype found in the most different genera was tetb, being detected in escherichia, enterobacter, klebsiella, providencia, serratia, and raoultella. in contrast, the tetd genotype was only found in klebsiella. when the 148 tet-resistant isolates were screened for multi-drug resistance, seven isolates grew on macconkey agar supplemented with cefotaxime and nalidixic acid: one escherichia isolate (ccn-109) and six pseudomonas. additionally, one escherichia isolate (elm-161) showed resistance to cefotaxime only. the resistance profiles of these two isolates were further investigated using antibiotic sensitivity microdilution assays. isolate ccn-109 shows additional phenotypic resistance to ampicillin, kanamycin, and trimethoprim (figure 4). elm-161 was further determined to be resistant to ampicillin only. since ccn-109 appeared to have a unique resistance profile, other escherichia isolates from the cedar creek-north site were further investigated to determine if others showed mdr phenotypes. ccn-251 showed additional resistances to ampicillin and trimethoprim, and ccn-113 showed an additional resistance to ampicillin only (figure 5). other escherichia isolates were not phenotypically resistant to the other antibiotics tested. because args can be transmitted horizontally, we investigated whether ccn-109, ccn-113, and ccn-251 strains harbored small plasmids that are responsible for these phenotypes. using a plasmid isolation kit, we found that ccn-251 carried a 6.7 kb plasmid and ccn-113 carried a 4.6 kb plasmid (figure 6a and b, respectively), but no plasmid was detected in ccn-109 using these methods. the plasmid isolated from ccn-251 contains sulii and dfr1 genes, which encode sulfonamide-resistant dihydropteroate synthase and teta tetb tetd teta tetb teta tetb tetd undetermined elm (n=9) 0.00% 22.22% 22.22% 33.33% 11.11% 11.11% cedar creekcentral (n=8) 0.00% 100.00% 0.00% 0.00% 0.00% 0.00% fort phantom hill reservoir (n=1) 0.00% 100.00% 0.00% 0.00% 0.00% 0.00% buck (n = 9) 33.33% 11.11% 0.00% 44.44% 11.11% 0.00% cedar creek-north (n=5) 20.00% 80.00% 0.00% 0.00% 0.00% 0.00% lytle (n=5) 0.00% 20.00% 0.00% 80.00% 0.00% 0.00% total (n=37) 10.81% 45.95% 5.41% 29.73% 5.41% 2.70% table 3. tet genotypes identified at each site the percentage of tet genotypes for representatives of each metabolic group at each site are shown (n=37). sanchez & hennigan | distribution of tetracycline-resistant bacteria within the fort phantom hill reservoir watershed 33 figure 4. antibiotic susceptibility of ccn-109 and elm-161 od630 readings are shown for e. coli atcc 25922, ccn-109, and elm-161 grown in mueller-hinton with and without antibiotics at concentrations in accordance with clsi breakpoints for enterobacterales (11). standard deviations for three replicates are indicated. fine focus | volume 1134 figure 5. antibiotic susceptibility of escherichia isolates from cedar creek-north od630 readings are shown for e. coli atcc 25922 and escherichia isolates from cedar creeknorth. all were grown in mueller-hinton broth with or without antibiotics at concentrations in accordance with clsi breakpoints for enterobacterales (11). standard deviations for three replicates are indicated. sanchez & hennigan | distribution of tetracycline-resistant bacteria within the fort phantom hill reservoir watershed 35 trimethoprim-resistant dihydrofolate reductase enzymes, respectively. the aphe gene on the plasmid is incomplete, but encodes an aminoglycoside phosphotransferase which would inactivate aminoglycoside antibiotics like kanamycin. the plasmid from ccn-113 harbors the fepe gene, but no args. conclusions our investigation of the fort phantom hill reservoir watershed found a low level of tet resistance throughout, consistent with our hypothesis. the highest relative percentage of tet-resistant lactose-fermenters was 3.38% at a central location where elm creek flows through a neighborhood. a correlation was not observed between greater tet-resistance and the direction of waterflow in the watershed (figure 2). reported frequencies of tet resistance in environmental surface water samples varies; for example, in e. coli from the water and sediments of two rivers in austria, the frequency was between 1% and 11% of isolates (17). therefore, tet-resistance in the fort phantom hill watershed was similar to or less than that found in other locations. while no sites were on active farmland, all sites were no more than 8 km downstream of land zoned as agriculture-open space by the city of abilene. future studies comparing tet-resistance in sediments from ponds on nearby ranchland would provide a valuable comparison for our data set. most of the representative tet-resistant isolates that were genotyped in this study carried teta and/ or tetb genes (table 3), which is consistent with a previous report that found tetb was the most frequent resistance gene in non-clinical e. coli isolates from animal and human sources (63%), and teta was the second most frequent (35%) (18). other studies also found that teta and tetb were the most common tet resistance genes in commensal e. coli isolates from cattle (7) and e. coli from meat and meat products (19). the genotypes teta, tetb, tetc, teto, tetw, and tetm have all been detected in hospital wastewater collected from wastewater treatment plants, and teta, tetq, and tetw have been detected in municipal wastewater (2). other studies show the average abundance of tetm to be higher in manure and wastewater samples than teta or tetb (20). perez-valera and colleagues found that the treatment of soils with manure increased the abundance of tetm, and, moreover, tetm most likely originated from the manure (21). in a study that compared the presence of genes in the outlets of manure applied catchments, tetm was significantly higher than non-manure catchments (22). the tetm gene was not detected in any isolate in our study. of the isolates that were genotyped in this study, 35.14% (n=13) carried more than one tet gene (table 3). in contrast, another study found that only 8.7% of tet-resistant e. coli cattle commensals carried more than one tet gene. the presence of more than one gene for tet resistance was thought to indicate selective pressure due to the high level of tetracycline in an environment (7). further investigation is needed to determine concentration of tetracycline in the fort phantom hill surface water and if the isolates with more than one tet gene are more resistant to tetracycline; these isolates were not challenged with tetracycline concentrations greater than 16 µg/ ml. additionally, since the methods in this study only tested viable isolates that were phenotypically tet-resistant, it is possible that additional non-functional tet genes in the populations were missed. in addition to tet resistance, mdr resistant e. coli were also recovered, including two isolates that carried small non-conjugative plasmids (figure 6). the dhfr gene encoded in the plasmid recovered from ccn-251 provides an explanation for its phenotypic trimethoprim resistance (figures 5 & 6). likewise, the presence of sulii would be expected to confer resistance to other sulfonamides but must be tested experimentally. plasmids encoding both sul and dhfr genes have been readily isolated from sulfamethoxazole and trimethoprim-resistant e. coli in stream water. moreover, these e. coli isolates were found to harbor multiple sul and dhfr genes, which is likely due to the influence of sub-inhibitory concentrations of trimethoprim and sulfamethoxazole fine focus | volume 1136 figure 6. plasmid maps of plasmids isolated from escherichia isolates plasmid maps generated from plasmids isolated from escherichia isolates 113 (a) and 251 (b). gc content is mapped in black, genes in dark gray, gene fragments in white, and origins of replication in gray. plasmid maps were generated using plasmapper 3.0 (16). sanchez & hennigan | distribution of tetracycline-resistant bacteria within the fort phantom hill reservoir watershed 37 found in the water (23). the association of sul and dhfr genes on mobile elements, such as the ccn-251 plasmid isolated in this study, is highly relevant to the spread of resistance in aquatic environments like the fort phantom hill watershed and has a potential impact on human health and agriculture. in contrast, sequencing of the plasmid recovered from ccn-113 did not reveal any args, only one full-length coding sequence for fepe. in pathogenic e. coli o157:h7, the fepe protein (also called wzzfepe) is responsible for very long o-antigen chain lengths in lipopolysaccharide (>80 repeat units) (24). in both salmonella typhimurium and shigella flexneri, wzzfepe and homologue cldphs-2, respectively, were found to be essential for serum resistance (25, 26). importantly, fepe was found to be positively selected in uropathogenic e. coli (upec) clinical isolates (27). more investigation of ccn-113 is necessary to determine the contribution of the plasmid-borne fepe to its potential virulence and to conduct core genome multilocus sequence typing analysis. mic values for all isolates with the antimicrobial agents tested must be determined to follow-up on mdr isolates, particularly ccn-109 and elm-161. of notable interest is the mechanism of cefotaxime resistance in these isolates since resistance is often due to the production of extended-spectrum beta-lactamases (esbls). certain esbls, such as blactx-m, blatem and blashv are associated with clinical infections caused by enterobacterales, with blactx-m being the most prevalent type. the highly successful mobilization of esbl genes on conjugative plasmids has led to the rapid spread of esbl-producing enterobacterales globally over the last ten years (28). esbl-producing enterobacterales are not limited to the clinic, but have been detected in livestock, wildlife, companion animals, wastewater, environmental waters, and healthy carriers. considering the data on the presence of esbls in aquatic environments are lacking in north america, more research should be conducted to examine the prevalence of esbl-producing enterobacterales in surface waters (29). we recommend additional studies be completed on the prevalence of esbls within the lake fort phantom hill reservoir watershed, particularly in areas impacted by agriculture, to further elucidate their impact on the region. acknowledgements we are thankful to j.r. huddleston for the use of antibiotic stocks and d.r. denton for his assistance in metabolic profiling. this research was supported through the mcnair scholars program (gan p217a220092) and a faculty fellowship with supporting structures: innovative partnerships to enhance bench science at cccu member institutions program, run by scholarship and christianity in oxford, the uk subsidiary of the council for christian colleges and universities, with funding by the john templeton foundation and the mj murdock charitable trust. fine focus | volume 1138 references 1. world 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(2023. freshwater environment as a reservoir of extended-spectrum β-lactamase-producing enterobacteriaceae. journal of applied microbiology, 134(3). https://doi.org/10.1093/jambio/lxad034 http://doi.org/10.1046/j.1365-2958.2003.03383.x http://doi.org/10.1046/j.1365-2958.2003.03383.x https://doi.org/10.1046/j.1365-2958.1997.3731744.x https://doi.org/10.1073/pnas.0600938103 https://doi.org/10.1016/j.ijmm.2013.02.008 https://doi.org/10.1093/jambio/lxad034 distribution of tetracycline-resistant bacteria within the fort phantom hill reservoir watershed abstract introduction materials and methods results conclusions acknowledgements andrew j. gaetano elizabeth s. danka st. norbert college, division of natural sciences, de pere, wi keywords: borrelia burgdorferi, ixodes scapularis, lyme disease, spirochete, vector-borne infection © 2024 gaetano, danka. fine focus, 10(1), 9-37. doi: 10.33043/ff.10.1.9-37. shared with cc-by-nc-nd 4.0 license. manuscript received: 28 feb 2023; accepted 13 july 2023 abstract since its recent discovery in the late 1970s, lyme disease (ld) has been a growing public health concern, especially in the united states where it accounts for the majority of vector-borne infections each year. the causative agent, borrelia burgdorferi, is transmitted to humans through the bite of an infected ixodes tick. this pathogen uses many unique mechanisms to both shield itself from the host immune response and cause disease. clinically, ld presents in successive phases, with each increasing in severity as the bacterial cells migrate to new tissues and organ systems. on the epidemiological and ecological fronts, limitations in reporting, ecological changes, and a lack of public support hinder accurate surveillance and enhance the spread of the disease. the goal of this literature review is to increase public knowledge of b. burgdorferi, its vector, and the disease it causes, along with suggesting preventative measures to protect individuals who reside in high-risk areas. a collective and coordinated public health effort represents our greatest chance of restraining the ld-causing pathogen. borrelia burgdorferi: the deer tick’s dark secret https://creativecommons.org/licenses/by-nc-nd/4.0/ fine focus | volume 1010 discovery of the causative agent of lyme disease in 1976, the connecticut state department of health reported an outbreak of an unusual form of arthritis near lyme, connecticut (37). the affected individuals experienced recurrent bouts of pain and swelling in large synovial joints (such as the knee) without prior injury. other clinical presentations included flu-like symptoms and unusual cutaneous lesions called erythema migrans (em), which had first been described by the german physician alfred buchwald in 1883 (75). although a causative agent had not yet been discovered, this unique combination of signs and symptoms was termed lyme disease (ld) (37). most patients with the disease lived in heavily wooded areas away from the centers of towns, and the onset of their symptoms was often in the summer and early fall. the spatial and temporal distribution of cases hinted that the disease was likely transmitted through an insect vector, but the state department of health did not have any additional information. it would take six more years to identify the ld-causing pathogen. in 1982, wilhelm “willy” burgdorfer isolated a spirochete from ixodes scapularis, the blacklegged deer tick (7). this microorganism was soon shown to be responsible for the unique disease in lyme, connecticut. when ticks harboring this pathogen fed on new zealand white rabbits, long-lasting em-like lesions developed. indirect immunofluorescence also confirmed that the rabbits produced antibodies specific to these spirochetes. a causal relationship between the newly discovered spirochete and ld in humans was established when the serum of clinically diagnosed patients revealed antibodies specific to the pathogen, which was subsequently named borrelia burgdorferi. despite its recent characterization, the earliest confirmed case of ld occurred in the 5,300-year-old similaun iceman (“ötzi”) found preserved frozen in the italian alps (31). arthritis was observed upon clinical examination, and dna sequencing of samples from the iceman confirmed the presence of b. burgdorferi. while a great deal of work has focused on characterizing the pathogen, many challenges exist on the clinical and epidemiological fronts. diagnosis and treatment of ld is complicated by b. burgdorferi’s wide array of virulence strategies that allow it to infect multiple organ systems, lie dormant for long periods of time, and resist and suppress the host immune response (20, 47, 48). additionally, the clinical manifestations can vary widely, which further heightens the challenge for clinicians to make a timely and accurate diagnosis. while a vaccine represents the most effective preventative measure, there are not any currently available on the market. due to limitations in disease surveillance, the reported number of ld cases in the united states is thought to be significantly lower than the centers for disease control and prevention’s (cdc) annual estimate (34). despite this, ld represents more than 80% of vectorborne illnesses making it the most common vector-borne disease in the country (3). making matters worse, the home range of b. burgdorferi’s vector and its reservoirs are expanding as the ecological landscape continues to change gaetano & danka | borrelia burgdorferi: the deer tick’s dark secret 11 (73). approximately 90 million americans live in states deemed “high risk” by the cdc, and even this high value is likely an underestimate of the total number of individuals at risk (13). disease prevention depends on increasing public knowledge and awareness of b. burgdorferi and its disease-causing ability, ixodes genus ticks and their life cycle, ld and its clinical manifestations, and of available precautionary measures for individuals in high-risk areas. introduction to b. burgdorferi b. burgdorferi is a gram-negative bacterium with an inner and outer membrane. unlike most gram-negative organisms, b. burgdorferi lacks lipopolysaccharides (lps) in the outer membrane and instead displays other immunogenic glycolipids (70). the spirochete bores through host tissues using internal periplasmic flagella that confer swimming motility and flat-wave morphology (32). while this flat-wave structure dominates, other pleomorphic forms exist under certain environmental conditions (see “pleomorphic forms” and figure 6) (44). the b. burgdorferi genome consists of a single 910,725 base pair linear chromosome with 853 open reading frames whose products are involved in the basic processes of dna replication, transcription, translation, solute transport, and energy metabolism (23). due to a lack of biosynthetic genes, the spirochete is an obligate parasite and depends on an arthropod or mammalian host for survival. aside from hemolysins and drug efflux pumps, b. burgdorferi lacks common virulence factors and instead relies on dynamic gene regulation to evade and suppress the host immune response (3). as an obligate parasite, b. burgdorferi can be difficult to maintain in common laboratory cultures that do not closely mimic the host environment (1). to overcome this challenge, optimized barbour-stoenner-kelly (bsk) media that contains 6% rabbit serum and a collagen matrix is used to support the growth of b. burgdorferi (62). in vitro cultivation consists of a two-step process whereby a rapidly growing starter culture is used to initiate the growth of a long-term culture with a high bacterial yield. while in vivo studies most accurately represent the conditions that b. burgdorferi naturally encounters, the ability to work with isolated, parasitic bacteria in the laboratory increases the feasibility of research. lyme disease the unique virulence and immune evasion strategies of b. burgdorferi manifest clinically in humans as a complex, multi-stage disease. in this section of the review, the life cycle of the arthropod vector, transmission to the mammalian host, and clinical manifestations of ld will be described. 1. vector life cycle vectors are organisms, often insects, that harbor and transmit pathogens to other hosts. within the ixodes genus, black-legged deer ticks (ixodes scapularis) are the main vectors that transmit b. burgdorferi to reservoirs such as mice and hosts such as humans. these reservoirs vary in their degree of competency to transmit b. burgdorferi back into an uninfected arthropod vector. ixodes ticks have a complex, two-year, four-stage life cycle, which consists of egg, larva, fine focus | volume 1012 nymph, and adult forms (figure 1) (19). in the spring, adult female ticks at the end of their life cycle lay eggs that hatch into six-legged larvae within about 60 days. the females lay the eggs on grasses where the larvae will be exposed to mammals like deer and mice after hatching. to progress to the next stage of the life cycle, the larvae require a first blood meal which often comes from the reservoir-competent, white-footed mouse, but may also come from other small animals such as chipmunks, shrews, squirrels, and birds. after larvae feed in the late summer to early fall, they molt into eight-legged nymphs that remain inactive during the winter months. in the spring, the nymphs take a second blood meal which allows them to develop into adults in the summer. as adults, a third feeding, often from the reservoir-incompetent white-tailed deer (odocoileus virginianus), is required to reproduce. after feeding, adult male and female ticks copulate on the deer, and females lay their eggs the following spring to complete the life cycle. 2. route of transmission for humans to develop ld, a tick harboring b. burgdorferi must bite the human and take a blood meal (7). the tick’s first feeding as a larva is unable to cause infection because the bacteria cannot be passed down in eggs and the arthropod can only acquire the spirochete through feeding (19). therefore, larvae must take a blood meal from an infected, reservoir-competent organism to obtain the pathogen before transmission to humans is possible. if this first blood meal contains b. burgdorferi, the pathogen will colonize the tick midgut and the bacterial cells lose their motility. a secondary feeding during the nymph stage is required figure 1 the ixodes genus tick life cycle. note. a red blood cell indicates that a blood meal is required to progress to the following stage. the blue, yellow, red, and orange bars represent winter, spring, summer, and fall, respectively. timeline is based on reference 19. gaetano & danka | borrelia burgdorferi: the deer tick’s dark secret 13 for b. burgdorferi to replicate, restore motility, and migrate to the tick’s salivary glands where it becomes primed for transmission. thus, nymphs represent the earliest stage of the tick life cycle where b. burgdorferi transmission resulting in ld in humans is possible. to locate a host, ticks use a maneuver called questing where they climb to the top of grasses and other small plants, extend their front legs, and latch on to a passing animal (figure 2b) (18). after finding a host, the tick migrates to a suitable location to take a blood meal. for humans, one study determined the distribution of tick attachment to be 9% head-neck, 5% arm, 24% stomach/groin, 7% back, 18% chest/shoulder, 25% leg/foot, and 12% hip (figure 2a) (25). after a tick begins feeding, transmission of b. burgdorferi is not immediate. there is a positive correlation between the duration of vector attachment and the probability of b. burgdorferi colonization and disease (57). in a murine study exploring this relationship, infection was established in 7% of mice after 36 hours, 25% after 42 hours, and 75% after 48 hours. this figure 2 tick questing behavior leads to attachment to hosts. note. distribution of tick attachment sites on humans (a) and representation of the tick questing behavior that is used to seize a suitable host for a blood meal (b) (18, 25). fine focus | volume 1014 time-dependent transmission demonstrates the importance of rapid tick removal after attachment as a preventative measure against ld. while there is no evidence that human-to-human transmission of b. burgdorferi is possible, there are many published cases of gestational ld with negative outcomes such as miscarriage, death following birth, and congenital abnormalities (72). however, a systematic review of these published cases concluded that most reports contained blinding issues, had missing or limited information on the mother’s clinical symptoms, or used diagnostic methods that are no longer considered reliable. therefore, additional research using reliable methods is necessary to determine the effects of gestational ld and the consequences it may have on women in their childbearing years. 3. disease progression ld progresses in three distinct phases termed early localized, early disseminated, and late disseminated infection (48). the basic signs, symptoms, and commonly affected tissues corresponding to each phase are presented in figure 3, and each phase is described in depth below. the first clinical manifestation of early localized b. burgdorferi infection is usually a slowly expanding cutaneous rash called erythema migrans (em) (48). em presents 7-14 days after exposure at the site of tick attachment in 70-90% of cases. the rash begins as a small red papule that takes on a bullseye appearance as it expands to an average diameter of 15 centimeters. em is usually asymptomatic, but other flu-like symptoms such as headache, fatigue, malaise, and fever may occur. early localized ld usually lasts for a few days to a month. early disseminated infection usually occurs three weeks to several months after the onset of primary em, and it typically lasts 3-10 weeks (48). during this phase, b. burgdorferi figure 3 ld signs, symptoms, and commonly affected tissues. note. lyme disease progresses through three distinct stages: early localized (a), early disseminated (b), and late disseminated infection (c) (48). each stage is characterized by different signs and symptoms that reflect the spread of the pathogen from the initial site of infection. gaetano & danka | borrelia burgdorferi: the deer tick’s dark secret 15 disseminates to the central nervous system (cns), the cardiovascular system, and other cutaneous regions which leads to secondary em away from the initial site. cns involvement, termed lyme neuroborreliosis, occurs in about 15% of untreated cases (22). colonization of the nervous system can occur through penetration of the blood-brain barrier, migration from the peripheral nervous system, or movement through the cerebrospinal fluid. common signs of neurologic involvement include bilateral cranial nerve palsies of the face, meningitis, and encephalitis. cognitive impairment, psychiatric disturbances, and even seizures have been observed in patients with early disseminated ld (41). cardiac involvement, termed lyme carditis, typically occurs a few weeks to several months after em onset in about 4-10% of untreated cases (21). signs of lyme carditis include prolonged pr interval, atrioventricular (av) block, myocarditis, intraventricular conduction disturbances, bundle branch block, and congestive heart failure (50). patients presenting with lyme carditis may require electrocardiogram (ecg) monitoring and temporary or permanent pacemakers for av block (74). general flu-like symptoms also accompany the early disseminated phase of infection (48). late disseminated infection, also called chronic ld, occurs months to years after the onset of primary em and can last many years if untreated (48). late-stage disease is marked by chronic, intermittent arthritis which occurs in about 80% of untreated individuals, along with continued neurologic, cardiac, and cutaneous manifestations (76, 30). lyme-associated arthritis results from acute swelling and erythema in the joints, most commonly the knee (5). excessive inflammation, infection-induced autoimmunity, and failure to down-regulate the inflammatory response are factors that contribute to chronic ld. virulence of b. burgdorferi in hosts bacterial pathogens utilize several methods to spread through and damage host tissues. b. burgdorferi is no exception, and in this section of the review a few of the many disease-causing characteristics of this spirochete will be discussed. 1. plasmid-derived virulence in addition to the single, linear chromosome, the b. burgdorferi genome contains at least 17 linear and circular plasmids (lp and cp, respectively) (23). while some of these extrachromosomal dna molecules have demonstrated critical roles in disease progression, others remain uncharacterized. a cell’s plasmid profile is its unique collection of plasmids. long-term in vitro cultivation of b. burgdorferi results in concurrent changes to both plasmid profile and murine infectivity (64). over time, the total number of plasmids within each cell decreases, which coincides with a drop in virulence. the degree to which the virulence of b. burgdorferi depends on its plasmid profile was expanded upon in a study that examined the infective phenotypes of a collection of clonal mutants, each with a different combination of plasmids (60). high-infectivity was observed in the presence of both linear plasmid 25 (lp25) and 28-1 (lp28-1), intermediate-infectivity was observed in the presence of lp25 and the absence of lp28-1, and fine focus | volume 1016 low-infectivity was observed in the absence of lp25 independent of lp28-1 (table 1). while some b. burgdorferi plasmids have been linked to highly infective phenotypes, others take on different roles (60). for example, cp26 is required to cause disease, but its presence does not affect the degree of infectivity. other plasmids such as cp9 and lp21 are not associated with infectivity at all. further characterization of b. burgdorferi plasmids linked to infectivity will lead to a greater understanding of the organism’s requirements for infection and disease. 2. temperature-dependent gene regulation b. burgdorferi relies on both an arthropod vector and a mammalian host for survival. upon transmission from vector to host, the spirochete experiences a temperature change from 23˚c to 37˚c. this change activates genetic regulatory mechanisms that allow b. burgdorferi to adapt to its surrounding environment and successfully establish a mammalian infection (65). outer surface proteins (osp) are immunogenic lipoproteins found on the cell surface of b. burgdorferi. ospc, a known antiphagocytic factor, is required to establish a mammalian infection (8). in unfed ticks at 23˚c, b. burgdorferi predominantly expresses ospa (65). after the tick takes a 37˚c mammalian blood meal, the spirochete’s osp expression profile changes to ospc. this was confirmed by examining b. burgdorferi-infected murine serum which contained antibodies specific to ospc. one explanation for this temperature-dependent gene regulation relies on the topology of the ospc-containing cp26 plasmid. at 23˚c, the plasmid is in a supercoiled state which blocks the transcriptional machinery from accessing the ospc locus. in vitro work demonstrated that upon exposure to warm (37˚c) mammalian serum the supercoiling of cp26 is reversed permitting expression of ospc. another explanation for the temperature-dependent regulation of ospc involves small regulatory rnas (srna) (40). rpos is an alternative sigma factor involved in the initiatable 1 b. burgdorferi infectivity as it relates to linear and circular plasmid profile. infectivity-associated not infectivity-associated always present infective phenotype*, # lp25 lp28-1 cp9 lp21 cp26 high + + +/+/+ intermediate + +/+/+ low +/+/+/+ note. * + plasmid must be present for the given infective phenotype, plasmid must be absent for the given phenotype, +/plasmid does not affect infective phenotype #data from reference 60 gaetano & danka | borrelia burgdorferi: the deer tick’s dark secret 17 tion of ospc transcription. expression of rpos is regulated by dsraab, a temperature-sensitive srna. at 23˚c, the rpos transcript contains a secondary stem-loop structure that occludes the ribosome binding site (rbs) preventing translation of the sigma factor. also at 23˚c, dsraab exhibits secondary structure which prevents interactions between the srna and the rpos transcript that are necessary for high levels of gene expression (figure 4a). at 37˚c, dsraab loses its secondary structure which allows it to post-transcriptionally regulate the rpos transcript. this results in loss of the rpos stem-loop structure, rbs availability, and active translation of rpos sigma factors (figure 4b). the high level of rpos expression at 37˚c greatly enhances the expression of ospc, which helps facilitate transmission of b. burgdorferi from the arthropod vector to the mammalian host. since the discovery of this molecular thermometer, over 1000 other b. burgdorferi-srnas have been identified, many of which demonstrate temperature-sensitivity (59). these molecules likely contribute to genetic regulatory mechanisms that promote b. burgdorferi transmission by allowing the pathogen to adapt to changing environmental conditions. 3. motility b. burgdorferi possesses periplasmic bundles figure 4 temperature-dependent expression of ospc. note. a temperature shift accompanies the transmission of b. burgdorferi from the arthropod vector to the mammalian host (40). at 23˚c in the unfed tick, secondary structure of the srna dsraab prevents translation of the rpos transcript, resulting in low levels of ospc expression (a). at 37˚c in the mammalian host, dsraab secondary structure is reversed, allowing for the production of rpos and ospc (b). fine focus | volume 1018 of flagella that originate from basal bodies at its terminal ends (32). these structures confer flat-wave morphology and corkscrew swimming motility, which allow the spirochete to move both forward and backward as it bores through host tissues. this unique ability is integral to pathogenicity because it allows b. burgdorferi to disseminate throughout the human body, which results in the wide range of clinical manifestations associated with ld. the flab and flig genes are required for flagellar functioning (68). the flab gene encodes the major flagellar filament protein flab. flab mutants are nonmotile, have a straight, bacillus morphology rather than the classic flat-wave morphology, and exhibit decreased viability in both the mammalian host and arthropod vector. the flig gene encodes the c-ring at the base of the flagellar basal body, which is important for rotational torque generation. inactivation of flig results in reduced motility and infectivity despite proper assembly of the flagellar filament (38, 39). these results indicate that periplasmic flagella of b. burgdorferi play an important role in transmission and infectivity. 4. chemotaxis some microorganisms use chemotaxis to migrate toward a chemoattractant or away from a chemorepellent in the environment. b. burgdorferi utilizes chemotaxis to colonize arthropod vectors and to infect mammalian hosts. figure 5 salp12 salivary protein serves as a chemoattractant for b. burgdorferi. note. attachment of the i. scapularis tick to a host allows for the release of salp12 salivary protein into the host (46). b. burgdorferi swims up the salp12 concentration gradient to encounter the tick. this process promotes colonization of the tick midgut, which will allow b. burgdorferi to be transmitted to the next mammal that the tick feeds on. gaetano & danka | borrelia burgdorferi: the deer tick’s dark secret 19 the salp12 salivary protein of the ixodes scapularis tick serves as a chemoattractant for the bacterial cells that helps them colonize the arthropod vector (46). when uninfected ticks take a blood meal from an infected reservoir such as the white-footed mouse (peromyscus leucopus), salp12 diffuses into the host, attracting resident b. burgdorferi cells (figure 5). this allows the bacteria to enter and colonize the arthropod midgut. knockdown of salp12 results in a significant reduction of b. burgdorferi colonization of the tick, which demonstrates the importance of chemotaxis for transmission of the pathogen to its vector. to colonize the mammalian host, a chemotactic response that involves the b. burgdorferi chea2 gene is required (69). this gene encodes the histidine kinase of a two-component regulatory system that controls the directional rotation of b. burgdorferi flagella. mutations in chea2 result in unidirectional movement and failure to be attracted into the mammalian host upon tick attachment. interestingly, while chea2 mutants are unable to establish an infection in mammals, they retain the ability to colonize ticks. together, these studies show the key role that chemotaxis plays during transmission of b. burgdorferi to the human host. innate immune evasion the innate immune system is a nonspecific, noninducible line of defense that protects fungi, animals, and plants against a broad range of pathogens. b. burgdorferi uses many methods to evade this first line of defense. 1. complement inactivation the complement cascade is a tightly regulated pathway of sequentially activated proteins used to identify and eliminate pathogens through opsonization, phagocytosis, and formation of the membrane attack complex (mac). b. burgdorferi expresses several osps that disrupt this pathway including the surface lipoprotein bbk32, which binds and inactivates the c1 protease complex (2). this is the initiating component of the complement cascade, and its inactivation prevents all downstream steps. bbk32 mutants exhibit decreased virulence, which demonstrates the importance of complement inactivation for successful infection. other osps involved in complement disruption such as ospa, ospc, and cspa function by converting the blood protein plasminogen to plasmin, which is a known inhibitor of the cascade (24, 27, 55). 2. antimicrobial peptide resistance antimicrobial proteins and peptides (amps) are produced by the host immune system to defend against pathogenic bacteria. lactoferrin is an amp that inhibits microbial growth by scavenging free iron, which is a cofactor required by most bacteria (9). b. burgdorferi avoids the effects of lactoferrin by using a manganese cofactor for biological redox reactions instead of iron (2). cathelicidin is another amp produced by many mammalian cells. while this molecule usually functions by interacting with cell surface components to disrupt microbial membrane integrity, it exhibits limited binding to the b. burgdorferi outer membrane (63). additionally, the b. burgdorferi bba57 surface protein has demonstrated the ability to downregulate the expression of some amps such as bactericidal/ permeability-increasing protein (bpi), further promoting its virulence in hosts (6). fine focus | volume 1020 3. phagocyte interference the host immune response relies on phagocytic macrophages and dendritic cells to engulf and destroy foreign matter. while phagocytes effectively clear b. burgdorferi when exposed to purified bacterial cells in vitro, the pathogen can successfully evade these effects in vivo (16). this is likely due to the upregulation of the anti-inflammatory cytokine il-10 in phagocytes upon b. burgdorferi engulfment. normally, il-10 functions to dampen the host immune response after pathogen clearance to prevent endogenous tissue damage. b. burgdorferi-induced premature overproduction of il-10 inhibits the production of proinflammatory immune factors that are critical to the host’s defense. macrophages deficient in the ability to produce il-10 generate greater levels of proinflammatory cytokines during b. burgdorferi infection, which promote phagocytic events that aid in removal of the pathogen (16). these findings demonstrate the important role that reprogramming the host immune response plays during b. burgdorferi infection. 4. pleomorphic forms b. burgdorferi shows pleomorphism, which is the ability to alter cellular morphology. this is often used by organisms to survive in extreme environments. in addition to the dominant flat-wave morphology, b. burgdorferi has been observed in other forms including blebs, round bodies (rb), and biofilm-like (bfl) aggregates (figure 6) (44). at 37˚c, nearly all b. burgdorferi cells are found in their dominant flat-wave or spirochetal form. environmental stress signals such as extreme ph, high temperatures, and high levels of reactive oxygen species (ros) result in a conversion of flat-wave cells to other forms such as rbs, which have reduced metabolic requirements (47). subsequent removal of b. burgdorferi from these unfavorable conditions causes a reversion back to the dominant figure 6 b. burgdorferi cells can be found in four distinct morphologies. note. schematic representation of b. burgdorferi pleomorphic forms based on images obtained from differential interference contrast (dic) microscopy (44). flat-wave spirochetes (a), blebs (b), round bodies (rb; c), and biofilm-like aggregates (bfl; d) are not drawn to scale. gaetano & danka | borrelia burgdorferi: the deer tick’s dark secret 21 flat-wave morphology. additionally, low levels of bfl aggregates are thought to exist in all environmental conditions where, like biofilms, they promote attachment to host tissues and resist phagocytosis (44). these findings support the idea that b. burgdorferi enhances its survival by altering its morphology as environmental conditions change. the human body contains many microenvironments where b. burgdorferi may exist in different pleomorphic forms. to more successfully diagnose and treat ld, it will be important to continue exploring the physiological niches where each morphology dominates. this will guide the pharmacological development of novel treatments that more precisely and accurately target each distinct morphology of b. burgdorferi. adaptive immune evasion unlike the nonspecific innate immune system, the adaptive immune system defends against specific pathogens using antibodies and immune cells produced in response to a past exposure. b. burgdorferi disrupts the normal functioning of the adaptive immune system in many ways, three of which will be described in this review. 1. germinal center disruption during an infection, antigen-presenting cells (apc) display segments of immunogenic proteins from a phagocytosed pathogen on the major histocompatibility complex class ii (mhc ii) molecules on their cell surface. next, these apcs present the antigens to band t-lymphocytes in secondary lymphoid tissues such as the spleen and lymph nodes. in these tissues, germinal centers form, and it is within these structures that antibody-producing plasma cells and b-lymphocytes develop to confer long-term immunity. during a b. burgdorferi infection, the host immune system generates structurally defective, short-lived germinal centers that are unable to generate high quantities of antibody-producing immune cells (20). this leaves the host immunosuppressed and allows b. burgdorferi to cause further infection. 2. antibody class switching several classes of immunoglobulins (igs) exist, each with a unique function in the host immune response. during an infection, the host’s ability to shift production from one ig class to another is useful in targeting pathogenic microorganisms located in multiple tissue types. upon b. burgdorferi infection, pentameric igm molecules are produced in high quantities, while monomeric igg production is suppressed (28). igg is the major circulating ig found in the blood, and downregulating its production results in a less effective immune response. this is yet another way that b. burgdorferi manipulates the host immune response to further propagate an infection. 3. antigenic variation b. burgdorferi modifies its immunogenic cell surface proteins in a process called antigenic variation, which promotes evasion from the host’s adaptive immune response (15, 79). for example, the immunoreactive vlse surface lipoprotein encoded on lp28-1 undergoes frequent modification (figure 7). upstream of the vlse locus are 15 silent vls cassettes that randomly recombine into the expressed region fine focus | volume 1022 of the gene during b. burgdorferi infection. this results in a mosaic vlse with an estimated 1040 possible variants. continuous modification of the structure of this surface lipoprotein prevents previously generated immune factors from functioning properly. incidence and reporting of lyme disease understanding the epidemiology of vectorborne diseases such as ld is critical for disease prevention. the incidence rate and geographic distribution of infections provides health officials with pertinent information that can be used to organize public health efforts in high-risk areas. this section of the review will focus on ld surveillance, limitations in reporting, and ecological challenges that exacerbate the spread of disease. 1. cdc case definition the cdc has published clinical guidelines that define a ld diagnosis (12). the 2022 case definition requires specific clinical and laboratory criteria to be met. clinically, a patient must present at least one early or late-stage manifestation including em, arthritis in one or more joints, nervous system abnormalities (lymphocytic meningitis, facial palsy, or unexplainable encephalomyelitis), or cardiovascular involvement (atrioventricular conduction defects). the laboratory criteria include at least one of the following: isolation of b. burgdorferi in culture, detection of b. burgdorferi by polymerase chain reaction (pcr), detection of b. burgdorferi antigens by immunohistochemical assay, or a positive two-tier serology test. 2. surveillance data since its discovery, ld has been recognized throughout the world, particularly in europe and asia, and outside of the northeast or new england region of the united states (67). although surveillance in other countries is difficult, there are an estimated 85,000 cases annually in europe, with the majority occurring in germany, austria, slovenia, and sweden. figure 7 vls cassette recombination leads to highly varied vlse proteins. note. antigenic variation of the b. burgdorferi immunogenic vlse surface lipoprotein encoded on lp28-1 is accomplished through recombination events between the upstream, silent vls cassettes and the downstream expressed region (15). this allows the pathogen to continuously modify the structure of the expressed antigen, which provides a mechanism of immune evasion. https://ndc.services.cdc.gov/case-definitions/lyme-disease-2022/ https://ndc.services.cdc.gov/case-definitions/lyme-disease-2022/ https://ndc.services.cdc.gov/case-definitions/lyme-disease-2022/ gaetano & danka | borrelia burgdorferi: the deer tick’s dark secret 23 in the us, ld is the most common vectorborne disease affecting an estimated 476,000 people annually from 2010-2018 (33, 34, 52). most cases present in just a few states, with the highest incidences being reported in the northeast, mid-atlantic, and upper midwest regions (figure 8) (67). according to the cdc, most cases occur in the summer months from june to august, which coincides with nymphal scavenging (13). in the northeast, there have been drastic spikes in disease occurrence, particularly in maine where the incidence quadrupled between the years 2005 and 2015 (67). northern expansion of ld is occurring, and this trend is expected to persist as the suitable habitat for ticks continues to expand (54). in the midwest, cases are on the rise with the majority concentrated in wisconsin, minnefigure 8 3-year (2018-2020) average ld incidence by state. note. the cdc considers a state “high risk” if it has greater than 10 confirmed cases of ld per 100,000 persons for three reporting years. in this review, states with 1-10 cases per 100,000 persons are considered to have moderate risk. this figure is based on data reported in reference 13. fine focus | volume 1024 sota, and northern illinois (67). according to the cdc, wisconsin had the fourth highest incidence of ld in 2019 behind pennsylvania, new york, and new jersey (13). past models predicted further spread of ixodes ticks throughout the midwest into northern michigan, the ohio river valley, and northwest minnesota, which has indeed been observed in recent years (26). in the southeast, ld incidence is relatively low despite vectors being well established in areas such as coastal florida, south carolina, north carolina, and georgia (67). in this region, ixodes affinis and i. minor ticks harbor b. burgdorferi, but these species lack the questing ability of i. scapularis and rarely feed on humans (4). reported north to south gene flow of i. scapularis raises the possibility of altered southeastern tick behavior to that of the questing northern ticks, which could lead to a greater incidence of ld in this region in the future (78). 3. limitations in reporting ld has been a nationally reportable disease since 1991 (10). this means that physicians are required to report cases to state and local health departments who relay this information to the cdc. due to the disease’s recent characterization, the cdc points out limitations in surveillance that prevent the accurate estimation of ld incidence. these include both under-reporting in high incidence areas and over-reporting in low incidence areas due to clinical misclassification, inconsistencies in the funding and practices of health departments from one state to the next, and the collection of ld data based on area of residence rather than the location of exposure. the latter leads to the misinterpretation of surveillance data for tourists who account for a significant proportion of cases. furthermore, the ld case definition has undergone five modifications since deemed nationally reportable in 1991, which makes it more difficult for clinicians to stay current with reporting guidelines. due to these limitations, the cdc estimates that the actual number of annual ld cases is about 10 times greater than what is reported. the recent covid-19 pandemic has also greatly affected the reporting of many diseases including ld (36). while surveys indicated that americans spent more time outdoors in 2020 than in 2019 putting them at greater risk for ld, the cdc reported about half the number of confirmed cases (13). one group of researchers successfully predicted this discrepancy before the cdc published their 2020 incidence data (43). their study looked at the online traffic of the cdc’s tick removal website as an indirect quantifier of tick encounters (14). in 2020, the most recent year with published ld data, there were 25% more online visits than in 2019 suggesting that more individuals found themselves at risk for developing ld. conversely, emergency department visits for tick bites and the frequency of ld diagnostic testing were significantly reduced in 2020. this was likely due to health officials and clinicians being preoccupied during the initial spread of sars-cov-2 in the spring and early summer of 2020, which coincided with the peak seasons for tick bites. on top of this, many patients delayed or avoided seeking out healthcare for more minor affiliations, due to fear of contracting covid-19 or contributing to overburdened healthcare systems (36). this gaetano & danka | borrelia burgdorferi: the deer tick’s dark secret 25 sudden change in ld reporting was not consistent with long-term, epidemiological trends, which suggests that the covid-19 pandemic significantly affected reporting. an organized public health effort is needed to address this discrepancy in ld reporting and restore a high standard of surveillance. 4. climate and ecological challenges after the colonial period in north america, deforestation, agricultural expansion, and urbanization drastically altered the landscape, which primed the area for ld spread (73). one way this has occurred is through shifts in predator community dynamics (35). coyotes have replaced populations of both large and small predators such as wolves, bears, and foxes. the reduction in the number of foxes, which are more efficient predators of small mammals than coyotes, has decreased the amount of predation faced by b. burgdorferi’s main reservoir, the white-footed mouse. this has allowed the white-footed mouse to expand its home range, which has broadened the potential area of b. burgdorferi transmission to humans. additionally, the reduction of large predator populations along with vast agricultural expansion has allowed deer populations to flourish, increasing the reproductive range of ld vectors. the gradual increase in the temperature of the earth’s atmosphere also plays a key role in host/reservoir expansion. models used to study these effects suggest dramatic expansions of ixodes tick populations, which will broaden the range of b. burgdorferi (56). over the next four decades, the geographic distribution of the white-footed mouse is predicted to expand northward by about 300 kilometers (61). if this manifests, it will have significant effects on the distribution of ld. disease treatment and prevention complete eradication of b. burgdorferi is implausible given its many hosts, reservoirs, and vectors. rather, proper treatment of affected individuals and public health efforts to increase awareness of preventative measures in high-risk areas represent our best strategy to minimize the incidence of ld. 1. treatment early localized infection is often treated with oral antibiotics such as doxycycline, amoxicillin, cefuroxime, or azithromycin (76). pregnant women and children under the age of eight should avoid the use of doxycycline due to its adverse effects on bone development (29). early disseminated infection is treated based on the affected tissues. patients with lyme carditis or severe lyme neuroborreliosis often receive intravenously administered ceftriaxone or cefotaxime, followed by one of the oral antibiotic regimens used to treat early localized infections (77). when only mild nervous system involvement such as isolated facial nerve palsy presents, oral antibiotic treatment usually suffices (76). treatment for late disseminated, chronic ld also depends on the presented signs and symptoms. if arthritis occurs without neurologic involvement, oral antibiotics are usually administered. if cardiac or neurologic involvement persists from the early disseminated phase of infection, intravenous antibiotic treatment may be necessary (76). fine focus | volume 1026 prophylactic antibiotics are often prescribed in cases of suspected b. burgdorferi exposure after a tick bite. in a randomized clinical trial with patients that had removed an ixodes genus tick within 72 hours, a single dose of doxycycline was 87% effective at preventing em (49). this demonstrates the importance of immediate medical attention after a possible b. burgdorferi exposure to prevent long-term ld. 2. vector-focused approach while the elimination of ixodes genus ticks is highly unlikely due to their vast, expanding range, local measures can be taken to greatly reduce the chances of human-tick interactions. one such method involves the use of carbaryl, an insecticide that is also lethal to arachnids, which has proven extremely effective at eliminating tick populations (66). unfortunately, this is a broad-spectrum insecticide that also kills moths, beetles, cockroaches, ants, and mosquitoes, which could have negative ecological effects (51). this downside of the use of carbaryl should be weighed against the positive impact of treating outdoor public gathering spaces and private landscapes to limit b. burgdorferi exposures (figure 9a). permethrin is another insecticide that can be used against ticks, which poses little to no ecological threat since it is applied to clothing rather than broadly to the environment. permethrin-based treatment of clothes and shoes has proven effective at preventing tick bites, killing ticks upon attachment, and preventing the transmission of pathogens such as b. figure 9 ld preventative measures. note. the common methods for preventing ld include targeting reservoirs and vectors as well as best practices for humans who may have encountered ticks. these measures include the treatment of landscapes with insecticides (a), frequent self-examination and prompt removal of attached ticks (b), immediate medical intervention after exposure (c), and vaccine development (d) (17, 49, 57, 66). gaetano & danka | borrelia burgdorferi: the deer tick’s dark secret 27 burgdorferi (45). in a controlled study of the effectiveness of permethrin-treated outdoor gear, individuals wearing treated clothing received 3.36 times less tick bites than those wearing untreated clothing. additionally, of the ticks attached to subjects, 97.6% were alive upon removal from individuals wearing untreated clothing, while only 22.6% of ticks were alive upon removal from those wearing permethrin-treated clothing. outdoor gear can be purchased pre-treated with permethrin, or the insecticide can be applied at home carefully following the cdc’s recommendations which include wearing protective gloves, reading the directions before application, allowing clothing to dry before use, and avoiding direct exposure to skin (11). other behavioral preventative measures should be taken to minimize the chances of acquiring ld. frequent self-examination for ticks during and after outdoor activities in high-risk areas is critical (figure 9b). as previously mentioned, there is a positive, nonlinear relationship between the duration of tick attachment and ld outcomes (57). therefore, prompt removal of ticks and immediate prophylactic antibiotic treatment significantly decreases the chances of b. burgdorferi transmission (figure 9c). additionally, tucking pant legs into socks and covering bare skin in the outdoors are good strategies to prevent tick bites. insect repellents that contain n, n-diethyl-meta-toluamide, commonly known as deet, may also be helpful, but the effectiveness and duration of efficacy are inconsistent compared to other insecticides like permethrin (45). 3. reservoir-focused approach small rodent reservoirs such as the white-footed mouse play a critical role in transmission of b. burgdorferi to its arthropod vector. while culling reservoir populations has been proposed, this would likely have negative ecological consequences. instead, researchers have turned to the treatment of reservoirs to reduce populations of ticks and therefore the ld-causing spirochete (42). this has been attempted through the dispersal of permethrin-treated cotton in tick-infected areas, which is used by mice as nesting material. while this strategy significantly reduced b. burgdorferi transmission, additional research and development is needed to optimize the efficacy of this reservoir-focused treatment. 4. vaccination efforts while vectoror reservoir-focused approaches may be beneficial, immunization represents the most effective method of disease prevention (figure 9d). in 1998, the united states food and drug administration (fda) approved a recombinant b. burgdorferi ospa vaccine called lymerix (58). this vaccine functions by inducing the production of antibodies that target and eliminate b. burgdorferi as it enters the body during a tick bite. less than four years after becoming available to the public, lymerix was removed from the market due to low sales, anti-vaccine backlash, safety concerns, and class-action lawsuits. although the fda investigated the safety concerns and concluded there was a lack of evidence for the claims, the vaccine has not been available to the public since early 2002 (53). fine focus | volume 1028 currently, companies such as valneva are developing new ld vaccines designed to specifically protect individuals against north american and european strains of b. burgdorferi (17). these vla15 protein subunit vaccines use the immunoreactive c-terminus of the ospa protein to induce protective immunity. they have proven effective in murine models and are currently undergoing human clinical trials. wide-spread public acceptance of these vaccines in development represents our greatest potential defense against ld. additionally, researchers have begun to explore wildlife vaccination as an approach to ld prevention (71). ospa-based oral bait vaccines for reservoir populations of white-footed mice have demonstrated the ability to reduce the risk of ld in preliminary studies. these vaccines elicit an immune response that protects these animals from infection and reduces transmission of the pathogen to its arthropod vector. decreasing the number of b. burgdorferi-harboring vectors in the environment will reduce the chances of human infection. once the efficacy of wildlife vaccination is optimized, this may represent a successful long-term strategy to disease prevention. conclusion b. burgdorferi, the causative agent of ld, is a highly complex parasite that uses an arthropod vector for transmission to mammalian hosts. the high rate of infection achieved by this spirochete is mainly derived from its unique ability to both evade and disrupt various aspects of the host immune response as it disseminates throughout the body. clinically, ld has a variable presentation which makes it difficult glossary of abbreviations amp: antimicrobial protein/peptide bfl: biofilm-like (aggregates) bsk: barbour-stoenner-kelly media cdc: centers for disease control and prevention cp: circular plasmid em: erythema migrans fda: united states food and drug administration ig: immunoglobulin lp: linear plasmid ld: lyme disease lymerix: ospa lyme disease vaccine osp: outer surface protein rb: round bodies srna: small regulatory rna for physicians to diagnose and treat their patients. in the united states, ld is a major public health concern, especially in the upper midwest and northeast regions. surveillance limitations, a lack of public awareness, and failed vaccination efforts in the past represent some of the major challenges to disease prevention. enhancing the general public’s understanding of the risks associated with outdoor activities and providing individuals in high-risk areas with everyday preventative measures can decrease the incidence of ld in the short-term. in the long-term, widespread acceptance of novel vaccines is likely our most promising solution. while ld presents several clinical and epidemiological challenges, a collective and coordinated public health effort represents our greatest chance of controlling the deer tick’s dark secret. gaetano & danka | 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