Surface waters are important sources of water for drinking, industrial, agricultural, and recreational uses; hence, contamination of water by fecal, pathogenic, or antimicrobial resistant (AR) bacteria is a major environmental and public health concern. However, very little data is available on prevalence of these bacteria in surface water throughout a watershed. This study aimed to characterize Escherichia coli present in the Upper Oconee Watershed, a mixed-use watershed in Athens, GA, USA for potential pathogenicity and AR. E. coli were enumerated by colony counts, cultured by enrichment and direct plating, and characterized by phylo-groups, diarrheagenic pathotypes, and antimicrobial susceptibility. From the analysis, 99.3% (455/458) of the total samples were positive for E. coli resulting in 496 isolates. E. coli counts were as high as 1.2×104 CFU/100 ml, which is above the United States Environmental Protection Agency (U.S. EPA) threshold for recreational water (235 CFU/100 ml based on a one-time measurement). Phylo-groups B2 (31.7%; 157/496) and B1 (30.8%; 153/496) were the most prevalent among the isolates. Enteropathogenic E. coli (EPEC) (19/496) and Shiga toxin-producing E. coli (STEC) (1/496) were the only diarrheagenic pathotypes detected. AR was observed in 6.9% (34/496) of the isolates, 15 of which were multidrug resistant (MDR; resistance to two or more classes of antimicrobials). Tetracycline resistance was most often detected (76.5%; 26/34), followed by ampicillin (32.4%; 11/34), streptomycin (23.5%; 8/34), sulfisoxazole (23.5%; 8/34), and nalidixic acid (14.7%; 5/34). Results from this study showed that E. coli is prevalent in high levels in the Upper Oconee Watershed, suggesting possible widespread fecal contamination. The presence of pathogenic, AR E. coli in the watershed indicates that environmental water can serve as a reservoir of resistant bacteria that may be transferred to humans through drinking and recreational activities.

Poultry is a major Salmonella reservoir, but conventional culture-based methods typically identify the most abundant serovars while those less abundant remain undetected. Choice of enrichment procedure also introduces bias, and for broiler carcasses, a 1-min rinse before preenrichment is insufficient to release all Salmonella present. The inability to assess serovar diversity means that serovars more often associated with human illness may be masked by more abundant Salmonella. CRISPR-SeroSeq (serotyping by sequencing clustered regularly interspaced short palindromic repeats), an amplicon-based, next-generation sequencing tool, allows detection of multiple serovars and maps the relative serovar frequencies in a sample. To address the preceding limitations, CRISPR-SeroSeq was used on broiler carcasses collected prechilled at a commercial plant. Standard carcass rinse aliquot preenrichments and whole carcass preenrichments that were enriched in Rappaport-Vassiliadis (RV) and tetrathionate (TT) broths were compared. On average, five serovars were observed per carcass, including nine on one carcass. CRISPR-SeroSeq detected serovars comprising as little as 0.005% of the population. CRISPR-SeroSeq data matched (28 of 32) standard culture analysis for abundant serovars. Salmonella serovars Kentucky, Typhimurium, and Schwarzengrund were found on each carcass. Overall, serovar diversity was higher in whole carcass preenrichments that were enriched in RV (P < 0.05). Serovar Schwarzengrund was present at higher frequencies in whole carcass preenrichments compared with rinse aliquot preenrichments (t test, P < 0.05), suggesting it adheres more strongly to the carcass. Salmonella serovar Enteritidis was enriched eightfold more in TT than in RV, and serovars Schwarzengrund and Reading were preferentially enriched in RV. Comparison of preenriched and enriched samples suggests that selective enrichment in RV or TT was inhibitory to some serovars. This article addresses limitations of Salmonella surveillance protocols and provides information related to Salmonella population dynamics.

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The reported increase in antibiotic-resistant bacteria in humans has resulted in a major shift away from antibiotic use in food animal production. This shift has been driven by the assumption that removing antibiotics will select for antibiotic susceptible bacterial taxa, which in turn will allow the currently available antibiotic arsenal to be more effective.

Aim It is well‐known that enterococci are abundant in the environment; however, the role of surface water as a reservoir of antimicrobial‐resistant enterococci remains largely undefined. In this study, surface water samples were collected over a 2‐year period from the Upper Oconee watershed, Athens, GA to examine enterococci and their antimicrobial resistance. Methods and Results Approximately 97% (445/458) of the samples were positive for enterococci and a total of 637 enterococci were isolated. The predominant species were Enterococcus casseliflavus (33·6%) followed by Enterococcus faecalis (26·5%) and Enterococcus hirae (13·2%). Regardless of species, the highest levels of resistance were to lincomycin (88·5%) and tetracycline (13%); isolates also exhibited resistance to newer antimicrobials, daptomycin (8·9%) and tigecycline (6·4%). Multidrug resistance (resistance ≥3 antimicrobial classes) was observed to as many as five classes of antimicrobials. Resistant enterococci appeared to be randomly dispersed over the seasons rather than clustered by species or antimicrobial resistance. Conclusions This study demonstrated that surface waters contain a large population of diverse species of antimicrobial‐resistant enterococci, including resistance to new antimicrobials. Significance and Impact of the Study These results may indicate the potential of human intestinal illness and/or colonization of the human gut with resistant enterococci as enterococci correlate with increased disease risk to humans during recreational exposure to water.

Salmonella enterica serovar Heidelberg (SH) is one of the prolific serovars causing poultry-associated food-borne illness in the world. Their ability to cause invasive infections and their promiscuity to plasmids that confer multidrug resistance to antibiotics of human health importance makes them a public health threat. Although, horizontal gene transfer (HGT) is recognized as the major mechanism used by Salmonella for acquiring antimicrobial resistance (AR) and virulence genes, the biology behind acquisition of new genes in SH is still unknown. In this study, we show that one day old broiler chicks challenged orally or via the cloaca with an antibiotic susceptible SH strain and raised without antibiotics carried susceptible and multidrug resistance SH strains 14 days after challenge. SH infection perturbed the bacterial community of broiler chicks and orally challenged chicks acquired AR at a higher rate than chicks challenged through the cloaca. Furthermore, SH strains lost and gained new genes, while some inverted their chromosome after colonizing the gut of broiler chicks. The acquisition of IncI1 plasmid multilocus sequence type 26 (pST26) from commensal Escherichia coli population present in the gut of broiler chicks conferred multidrug resistance phenotype to SH recipients and carriage of pST26 increased the fitness of SH under acidic selection pressure. Our results suggest that HGT shapes the evolution of AR in SH and that antibiotic use reduction alone is insufficient to limit AR plasmid transfer from commensal bacteria to Salmonella.

Salmonella has been traditionally considered a foodborne pathogen, as it is one of the most common etiologies of foodborne illnesses worldwide; however, recent Salmonella outbreaks attributed to fresh produce and water suggest a potential environmental source of Salmonella that causes some human illnesses. Here, we investigated the prevalence, diversity, and antimicrobial resistance of Salmonella isolated from a mixed-use watershed in Georgia, USA, in order to enhance the overall understanding of waterborne Salmonella .

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