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, rod- shaped 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, oxidase- positive 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 oxidase- positive was kept for further testing, since Aeromonas species have cytochrome c oxidase in their electron transport chains. Then, they were Gram stained. Gram- negative, 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 Gls- 4, 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. 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