• 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, influent- EMB, 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. Erythromycin- infused 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 re- spreading 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 Luria- Delbrück 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 pre- disposed 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 penicillin- and ciprofloxacin- infused 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 trimethoprim- and ciprofloxacin- infused 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 ciprofloxacin- infused 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. 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