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 Esche- richia 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-produc- ing 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 multi- drug resistant (MDR) Enterobacteriaceae isolated from poultry, swine, and cattle manures (6). Likewise, a study in Korea found that all commen- sal 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, turbid- ity, 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 coli- forms 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 Corpo- ration, 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-re- sistant 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 resis- tance, 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-re- sistant strains of Pseudomonas, Klebsiella, Provi- dencia, 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 s- ta nc e na lid ix ic a ci d re si s- ta 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 s- ta nc e na lid ix ic a ci d re si s- ta 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 repre- sented 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 harbor- ing 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 Escherich- ia isolate (CCN-109) and six Pseudomonas. Additionally, one Escherichia isolate (ELM-161) showed resistance to cefotaxime only. The resis- tance profiles of these two isolates were further investigated using antibiotic sensitivity microdi- lution 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 sulfon- amide-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 Creek- Central (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 Enterobactera- les (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 Creek- North. All were grown in Mueller-Hinton broth with or without antibiotics at concentrations in accordance with CLSI break- points 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 aminogly- coside 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 Reser- voir 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 agricul- ture-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 commen- sal 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 commen- sals 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 tetracy- cline; 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 phenotypi- cally 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 explana- tion 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 concen- trations 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 Water- shed 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 pathogen- ic 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 contri- bution 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 cefotax- ime 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 assis- tance in metabolic profiling. This research was supported through the McNair Scholars Program (GAN P217A220092) and a faculty fellowship with Supporting Structures: Innovative Partner- ships 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 Health Organization. (2015). Global action plan on antimicrobial resistance. https://www. who.int/publications/i/item/9789241509763 2. Czatzkowska, M., Wolak, I., Harnisz, M., & Korzeniewska, E. (2022). Impact of anthropogenic activities on the dissemination of ARGs in the environment—a review. International Journal of Environmental Research and Public Health, 19(19), 12853. https://doi.org/10.3390/ ijerph191912853 3. Han, X.-M., Hu, H.-W., Chen, Q.-L., Yang, L.-Y., Li, H.-L., Zhu, Y.-G., Li, X.-Z., & Ma, Y.-B. (2018). Antibiotic resistance genes and associated bacterial communities in agricultural soils amended with different sources of animal manures. Soil Biology and Biochemistry, 126, 91-102. https://doi.org/10.1016/j.soilbio.2018.08.018 4. He, Y., Mathieu, J., Stadler, L., Senehi, N., Sun, R., & Alvarez, P. J.J. (2020). Antibiotic resistance genes from livestock waste: occurrence, dissemination, and treatment. npj Clean Water, 3(4). https://doi.org/10.1038/s41545-020-0051-0 5. U.S. Food and Drug Administration. (2023). 2022 Summary report on antimicrobials sold or distributed for use in food-producing animals. U.S. Food and Drug Administration, Center for Veterinary Medicine. https://www.fda.gov/animal-veterinary/antimicrobial-resis- tance/2022-summary-report-antimicrobials-sold-or-distributed-use-food-producing-animals 6. Amador, P., Fernandes, R., Prudêncio, C., & Duarte, I. (2019). Prevalence of antibiotic resistance genes in multidrug-resistant Enterobacteriaceae on Portuguese livestock manure. Antibiotics (Basel, Switzerland), 8(1), 23. https://doi.org/10.3390/antibiotics8010023 7. Belaynehe, K. M., Shin, S. W., & Yoo, H. S. (2018). Interrelationship between tetracycline resistance determinants, phylogenetic group affiliation and carriage of class 1 integrons in commensal Escherichia coli isolates from cattle farms. BMC veterinary research, 14(1), 340. https://doi.org/10.1186/s12917-018-1661-3 8. Brazos River Authority. (2003). Fort Phantom Hill Reservoir Watershed Brush Control Assess- ment and Feasibility Study. https://www.tsswcb.texas.gov/sites/default/files/files/programs/ agency-reports/Fort%20Phantom%20Hill%20Reservoir.pdf 9. USGS Texas Water Science Center. (2024, Feb 27). Streamer. https://webapps.usgs.gov/streamer/ 10. Greenman, N. A., Jurgensen, S. K., Holmes 2nd, C. P., Kapsak, C. J., Davis, R. E., Maza, W. M., Edemba, D., Esser, B. A., Hise, S. M., Keen, T. N., Larson, H. G., Lockwood, D. J., Wang, B., Harsh, J. A., & Herrick, J. B. (2021). Genomics of environmental Salmonella: Engaging students in the microbiology and bioinformatics of foodborne pathogens. Frontiers in Micro- biology, 12, 592422. https://doi.org/10.3389/fmicb.2021.592422 11. Clinical and Laboratory Standards Institute (CLSI). (2022). M100 Performance Standards for Antimicrobial Susceptibility Testing (32nd ed.). Clinical and Laboratory Standards Institute. 12. Ng, L. K., Martin, I., Alfa, M., & Mulvey, M. (2001). Multiplex PCR for the detection of tetracy- cline resistant genes. Molecular and Cellular Probes, 15(4), 209-215. https://doi.org/10.1006/ mcpr.2001.0363 https://www.who.int/publications/i/item/9789241509763 https://www.who.int/publications/i/item/9789241509763 https://doi.org/10.3390/ijerph191912853 https://doi.org/10.3390/ijerph191912853 https://doi.org/10.1016/j.soilbio.2018.08.018 https://doi.org/10.1038/s41545-020-0051-0 https://www.fda.gov/animal-veterinary/antimicrobial-resistance/2022-summary-report-antimicrobials-sold-or-distributed-use-food-producing-animals https://www.fda.gov/animal-veterinary/antimicrobial-resistance/2022-summary-report-antimicrobials-sold-or-distributed-use-food-producing-animals https://doi.org/10.3390/antibiotics8010023 https://doi.org/10.1186/s12917-018-1661-3 https://www.tsswcb.texas.gov/sites/default/files/files/programs/agency-reports/Fort%20Phantom%20Hill%20Reservoir.pdf https://www.tsswcb.texas.gov/sites/default/files/files/programs/agency-reports/Fort%20Phantom%20Hill%20Reservoir.pdf https://webapps.usgs.gov/streamer/ https://doi.org/10.3389/fmicb.2021.592422 https://doi.org/10.1006/mcpr.2001.0363 https://doi.org/10.1006/mcpr.2001.0363 Sanchez & Hennigan | Distribution of Tetracycline-Resistant Bacteria Within the Fort Phantom Hill Reservoir Watershed 39 13. Heuer, H., Krsek, M., Baker, P., Smalla, K., & Wellington, E. M. (1997). Analysis of actinomycete communities by specific amplification of genes encoding 16S rRNA and gel-electrophoretic separation in denaturing gradients. Applied and Environmental Microbiology, 63(8), 3233–3241. https://doi.org/10.1128/aem.63.8.3233-3241.1997 14. Garner, E., Benitez, R., von Wagoner, E., Sawyer, R., Schaberg, E., Hession, W. C., Krometis, L.-A. H., Badgley, B. D., & Pruden, A. (2017). Stormwater loadings of antibiotic resistance genes in an urban stream. Water Research, 123, 144-152. http://dx.doi.org/10.1016/j. watres.2017.06.046 15. Lee, S., Suits, M., Wituszynski, D., Winston, R., Martin, J., & Lee, J. (2020). Residential urban stormwater runoff: A comprehensive profile of microbiome and antibiotic resistance. Science of the Total Environment, 723, 138033. https://doi.org/10.1016/j.scitotenv.2020.138033 16. Wishart, D. S., Ren, L., Leong-Sit, J., Saha, S., Grant, J. R., Stothard, P., Singh, U., Kropielnicki, A., Oler, E., Peters, H., Gautam, V. (2023), PlasMapper 3.0 - A web server for generating, editing, annotating and visualizing publication quality plasmid maps. Nucleic Acids Research, https://doi.org/10.1093/NAR/GKAD276 17. Skof, A., Koller, M., Baumert, R., Hautz, J., Treiber, F., Kittinger, C., & Zarfel, G. (2024). Compar- ison of the antibiotic resistance of Escherichia coli populations from water and biofilm in river environments. Pathogens (Basel, Switzerland), 13(2), 171. https://doi.org/10.3390/patho- gens13020171 18. Bryan, A., Shapir, N., & Sadowsky, M. J. (2004). Frequency and distribution of tetracycline resistance genes in genetically diverse, nonselected, and nonclinical Escherichia coli strains isolated from diverse human and animal sources. Applied and Environmental Microbiology, 70(4), 2503-7. https://doi.org/10.1128/AEM.70.4.2503-2507.2004 19. Koo, H.-J., & Woo, G.-J. (2011). Distribution and transferability of tetracycline resistance deter- minants in Escherichia coli isolated from meat and meat products. International Journal of Food Microbiology, 145(203), 407-13. https://doi.org/10.1016/j.ijfoodmicro.2011.01.003 20. Cheng, W., Chen, H., Su, C., & Yan, S. (2013). Abundance and persistence of antibiotic resistance genes in livestock farms: A comprehensive investigation in eastern China. Environmental International, 61, 1-7. https://doi.org/10.1016/j.envint.2013.08.023 21. Pérez-Valera, E., Kyselková, M., Ahmed, E., Sladecek, F. X. J., Goberna, M., & Elhottová, D. (2019). Native soil microorganisms hinder the soil enrichment with antibiotic resistance genes following manure applications. Scientific Reports, 9(1), 6760. https://doi.org/10.1038/ s41598-019-42734-5 22. Neher, T. P., Ma, L., Moorman, T. B., Howe, A. C., & Soupir, M. L. (2020). Catchment-scale export of antibiotic resistance genes and bacteria from an agricultural watershed in central Iowa. PLoS One, 15(1). https://doi.org/10.1371/journal.pone.0227136 23. Suhartono, S., Savin, M., & Gbur, E. E. (2016). Genetic redundancy and persistence of plasmid-mediated trimethoprim/sulfamethoxazole resistant effluent and stream water Esche- richia coli. Water Research, 15(103), 197-204. http://doi.org/10.1016/j.watres.2016.07.035 24. Tocilj, A., Munger, C., Proteau, A., Morona, R., Purins, L., Ajamian, E., Wagner, J., Papadopoulos, M., Van Den Bosch, L., Rubinstein, J. L., Féthière, J., Matte, A., & Cygler, M. (2008). Bacterial polysaccharide co-polymerases share a common framework for control of polymer length. Nature Structural and Molecular Biology, 15(2), 130-8. http://doi.org/10.1038/nsmb.1374 https://doi.org/10.1128/aem.63.8.3233-3241.1997 http://dx.doi.org/10.1016/j.watres.2017.06.046 http://dx.doi.org/10.1016/j.watres.2017.06.046 https://doi.org/10.1016/j.scitotenv.2020.138033 https://doi.org/10.1093/NAR/GKAD276 https://doi.org/10.3390/pathogens13020171 https://doi.org/10.3390/pathogens13020171 https://doi.org/10.1128/AEM.70.4.2503-2507.2004 https://doi.org/10.1016/j.ijfoodmicro.2011.01.003 https://doi.org/10.1016/j.envint.2013.08.023 https://doi.org/10.1038/s41598-019-42734-5 https://doi.org/10.1038/s41598-019-42734-5 https://doi.org/10.1371/journal.pone.0227136 http://doi.org/10.1016/j.watres.2016.07.035 http://doi.org/10.1038/nsmb.1374 Fine Focus | Volume 1140 25. Murray, G. L., Attridge, S. R., & Morona, R. (2003). Regulation of Salmonella typhimurium lipopolysaccharide O antigen chain length is required for virulence; identification of FepE as a second Wzz. Molecular Microbiology, 47(5), 1395-406. http://doi.org/10.1046/j.1365- 2958.2003.03383.x 26. Hong, M., & Payne, S. M. (2003). Effect of mutations in Shigella flexneri chromosomal and plasmid-encoded lipopolysaccharide genes on invasion and serum resistance. Molecular Microbiology, 24(4), 779-791. https://doi.org/10.1046/j.1365-2958.1997.3731744.x 27. Chen, S. L., Hung, C.-S., Xu, J., Reigstad, C. S., Magrini, V., Sabo, A., Blasiar, D., Bieri, T., Meyer, R. R., Ozersky, P., Armstrong, J. R., Fulton, R. S., Latreille, J. P., Spieth, J., Hooton, T. M., Mardis, E. R., Hultgren, S. J., & Gordon, J. I. (2006). Identification of genes subject to positive selection in uropathogenic strains of Escherichia coli: a comparative genomics approach. Proceedings of the National Academy of Sciences of the United States of America, 103(15), 5977-5982. https://doi.org/10.1073/pnas.0600938103 28. D’Andrea, M. M., Arena, F., Pallecchi, L., & Rossonlin, G. M. (2013). CTX-M-type β-lactamases: a successful story of antibiotic resistance. International Journal of Medical Microbiology, 303(6-7), 305–317. https://doi.org/10.1016/j.ijmm.2013.02.008 29. Cho, S., Jackson, C. R., & Frye, J. G. (2023. Freshwater environment as a reservoir of extend- ed-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