Susceptibility Profile and Multiple Antibiotics Resistance of Escherichia coli, Klebsiella spp., and Enterococci from Small-Scale Cattle Farms in Tennessee

<b>Background/Objectives</b>: Antimicrobial resistance in food-animal environments threatens sustainable production and public health, yet small farms remain poorly characterized as potential reservoirs of antimicrobial resistant bacteria. To address this, we investigated the prevalence and antimicrobial resistance profiles of <i>Escherichia coli</i>, <i>Klebsiella</i> spp., and <i>Enterococcus</i> spp. from small-scale cattle farms in Tennessee, USA. <b>Methods</b>: Over one year, 153 environmental samples (soil, manure, water) were collected from 17 farms. Target bacteria were isolated and confirmed using selective agar, biochemical tests, and PCR, and tested against 12 antibiotics using the Kirby-Bauer disk diffusion test. Multiple Antibiotic Resistance Index (MARI) and multidrug resistance (MDR) profiles were summarized. A complementary farmer survey of 26 farmers captured veterinary access, antibiotic use, manure handling, record keeping, and awareness of antimicrobial resistance. <b>Results</b>: Prevalence was highest for <i>Enterococcus</i> spp. (41.8%), followed by <i>E. coli</i> (23.5%) and <i>Klebsiella</i> spp. (12.4%). Seasonal variation was significant for <i>E. coli</i> and <i>Enterococcus</i> (<i>p</i> < 0.05). Winter manure yielded highest detection of <i>E. coli</i> (55.6%) and <i>Enterococcus</i> (53.8%), whereas <i>Klebsiella</i> peaked in Fall soil (19.1%). Resistance patterns varied across species, with <i>Enterococcus</i> showing consistent resistance to all three. <i>E. coli</i> frequently resisted erythromycin, ampicillin, and azithromycin; and <i>Klebsiella</i> commonly resisted erythromycin, ampicillin, and cefotaxime, though some of these reflect intrinsic resistance rather than acquired clinical resistance. MARI values were 0.92 in manure and soil, identifying them as high-risk reservoirs. We identified 29 distinct MDR pattern. Bipartite network visualization highlighted "resistance hubs" around erythromycin, ampicillin, and vancomycin, particularly in <i>Enterococcus</i>. In our study, 76.9% of farmers consulted veterinarians before antibiotic use, 57.7% kept written antibiotic records, and 65.4% were aware of AMR as a public health issue. Small-scale cattle farms are potential reservoirs of multidrug resistant commensal bacteria. <b>Conclusions</b>: These findings provide an evidence-based foundation to guide targeted antimicrobial stewardship and promote sustainable management practices in small-scale food animal farms.