Antimicrobial susceptibility of Salmonella and Escherichia coli from equids sampled in the NAHMS 2015-16 equine study and association of management factors with resistance.

Several studies have investigated antimicrobial resistance (AMR) in Salmonella spp. and Escherichia coli isolated from hospitalized horses, but studies conducted on community-based populations of equids are limited. The factors associated with AMR in these bacteria in the general horse population are not well understood. The primary objective of our study was to estimate the prevalence of Salmonella and describe antimicrobial susceptibility of Salmonella and E. coli from equids across the United States. The second objective was to identify associations between health management and biosecurity practices and AMR. Fecal samples submitted from 1357 equids on 199 operations were tested for Salmonella, identifying 27 positive samples with 29 isolates belonging to 18 serotypes. Fecal sample and operation-level prevalence of Salmonella was 2.0% (27/1357) and 7.0% (14/199), respectively. Most (25/29) isolates were pan-susceptible while four isolates exhibited resistance, three of which were multidrug resistant. Of the 721 samples cultured for E. coli, 85% (613/721) were positive. Eighty-six percent of the E. coli isolates recovered were pan-susceptible (529/612). Ten isolates were intermediate to one antimicrobial drug and susceptible to all others. Seventy-three E. coli isolates (11.9%, SE=1.3) were resistant to one or more antimicrobials, corresponding to a 33.0% (64/194) operation-level prevalence. Resistance to sulfonamide drugs was most common with 63 isolates (10.3%) resistant to sulfisoxazole, 57 of which (9.3%) were resistant to trimethoprim-sulfamethoxazole. MDR in E. coli was rare (1.8%, SE=0.5). Univariate and multivariable regression were used to evaluate associations between health management and biosecurity questionnaire items and AMR in E. coli. The outcome modeled was resistance to any of the 14 tested antimicrobials. Depending on the operation type, operations with greater than 20 resident equids were significantly associated with resistance. In addition, performance operations were significantly associated with resistance when compared to farm/ranch operations. Operations with feed containers that prevent fecal contamination and those that had treated any equids for illness or injury were associated with a lower AMR. The study results suggest that equids in the general population appear to pose low risk of shedding antimicrobial resistant strains of Salmonella and E. coli, and therefore low transmission potential to other equids, animals, humans, or the environment. However, it is prudent to practice good hand hygiene to prevent spread of Salmonella as well as AMR, and to protect both animal and human health. Despite study limitations, potential management factors that may influence prevalence and prevent spread of AMR shed by equids were identified.

Management and environmental factors influence the prevalence and abundance of food-borne pathogens and commensal bacteria in peanut hull-based broiler litter.

In this study, we conducted a longitudinal sampling of peanut hull-based litter from a farm under a "no antibiotics ever" program. Our objective was to determine broiler management practices and environmental factors that are associated with the occurrence of food-borne pathogens (Salmonella and Campylobacter) and the abundance of commensal bacteria (Escherichia coli, Enterococcus spp., and Staphylococcus spp.). Litter (n = 288) was collected from 4 broiler houses over three consecutive flocks, starting with a complete house cleanout and fresh peanut hull. Litter was sampled at the beginning of each grow-out cycle and at the end of the cycle. Logistic and linear regression models were used to model the relationships between pathogen prevalence, commensal abundance and management practices, and environmental factors. The number of flocks raised on litter, grow-out period, broiler house, litter pH, litter moisture, and house temperature were associated with the prevalence of pathogens and the abundance of commensal bacteria in litter. The final logistic model for pathogens showed that a higher probability of detecting Salmonella in litter was associated with the number of flocks raised on litter and the grow-out period. A higher probability of detecting Campylobacter in litter was associated with the number of flocks raised on litter, broiler house and the sections of the house, and the pH of litter. Our results suggest that management practices and environmental factors affect Salmonella and Campylobacter differently and suggest that each pathogen will require its own tailored intervention to stop their persistence in broiler litter.

Distribution and Transfer of Plasmid Replicon Families among Multidrug-Resistant Enterococcus faecalis and Enterococcus faecium from Poultry.

The presence and transfer of plasmids from commensal bacteria to more pathogenic bacteria may contribute to the dissemination of antimicrobial resistance. However, the prevalence of plasmids from commensal bacteria, such as the enterococci, in food animals remains largely unknown. In this study, the diversity and prevalence of plasmid families from multidrug-resistant (MDR; resistance to three or more antimicrobials) enterococci from poultry carcasses were determined. Plasmid-positive MDR enterococci were also tested for the ability to transfer plasmids to other enterococci using conjugation. MDR Enterococcus faecalis (n = 98) and Enterococcus faecium (n = 696) that were isolated from poultry carcass rinsates between 2004 and 2011 were tested for the presence of 21 plasmid replicon (rep) families using multiplex PCR. Approximately 48% of E. faecalis (47/98) and 16% of E. faecium (110/696) were positive for at least one rep-family. Fourteen rep-families were detected overall, and ten rep-families were shared between E. faecalis and E. faecium. The rep7 and rep17 families were unique to E. faecalis, while the rep5 and rep8 families were unique to E. faecium. The rep9 family was predominant in both E. faecalis and E. faecium for all the years tested. The greatest number of rep-families detected was in 2005 (n = 10), and the least was in 2009 (n = 1). Eight rep-families were transferred from E. faecalis donors to the E. faecalis JH2-2 recipient using conjugation. Results from this study showed that E. faecalis and E. faecium from poultry carcasses contain numerous and diverse rep-families that are capable of conjugal transfer.

Analysis of Salmonella enterica Isolated from a Mixed-Use Watershed in Georgia, USA: Antimicrobial Resistance, Serotype Diversity, and Genetic Relatedness to Human Isolates.

As the cases of Salmonella enterica infections associated with contaminated water are increasing, this study was conducted to address the role of surface water as a reservoir of S. enterica serotypes. We sampled rivers and streams (n = 688) over a 3-year period (2015 to 2017) in a mixed-use watershed in Georgia, USA, and 70.2% of the total stream samples tested positive for Salmonella. A total of 1,190 isolates were recovered and characterized by serotyping, antimicrobial susceptibility testing, and pulsed-field gel electrophoresis (PFGE). A wide range of serotypes was identified, including those commonly associated with humans and animals, with S. enterica serotype Muenchen being predominant (22.7%) and each serotype exhibiting a high degree of strain diversity by PFGE. About half (46.1%) of the isolates had PFGE patterns indistinguishable from those of human clinical isolates in the CDC PulseNet database. A total of 52 isolates (4.4%) were resistant to antimicrobials, out of which 43 isolates were multidrug resistant (MDR; resistance to two or more classes of antimicrobials). These 52 resistant Salmonella isolates were screened for the presence of antimicrobial resistance genes, plasmid replicons, and class 1 integrons, out of which four representative MDR isolates were selected for whole-genome sequencing analysis. The results showed that 28 MDR isolates resistant to 10 antimicrobials had blacmy-2 on an A/C plasmid. Persistent contamination of surface water with a high diversity of Salmonella strains, some of which are drug resistant and genetically indistinguishable from human isolates, supports a role of environmental surface water as a reservoir for and transmission route of this pathogen. IMPORTANCE 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. The persistence and widespread distribution of Salmonella in surface water confirm environmental sources of the pathogen. A high proportion of waterborne Salmonella with clinically significant serotypes and genetic similarity to strains of human origin supports the role of environmental water as a significant reservoir of Salmonella and indicates a potential waterborne transmission of Salmonella to humans. The presence of antimicrobial-resistant and MDR Salmonella demonstrates additional risks associated with exposure to contaminated environmental water.

Horizontal Gene Transfer Is the Main Driver of Antimicrobial Resistance in Broiler Chicks Infected with Salmonella enterica Serovar Heidelberg.

The overuse and misuse of antibiotics in clinical settings and in food production have been linked to the increased prevalence and spread of antimicrobial resistance (AR). Consequently, public health and consumer concerns have resulted in a remarkable reduction in antibiotics used for food animal production. However, there are no data on the effectiveness of antibiotic removal in reducing AR shared through horizontal gene transfer (HGT). In this study, we used neonatal broiler chicks and Salmonella enterica serovar Heidelberg, a model food pathogen, to test if chicks raised antibiotic free harbor transferable AR. We challenged chicks with an antibiotic-susceptible S. Heidelberg strain using various routes of inoculation and determined if S. Heidelberg isolates recovered carried plasmids conferring AR. We used antimicrobial susceptibility testing and whole-genome sequencing (WGS) to show that chicks grown without antibiotics harbored an antimicrobial resistant S. Heidelberg population at 14 days after challenge and chicks challenged orally acquired AR at a higher rate than chicks inoculated via the cloaca. Using 16S rRNA gene sequencing, we found that S. Heidelberg infection perturbed the microbiota of broiler chicks, and we used metagenomics and WGS to confirm that a commensal Escherichia coli population was the main reservoir of an IncI1 plasmid acquired by S. Heidelberg. The carriage of this IncI1 plasmid posed no fitness cost to S. Heidelberg but increased its fitness when exposed to acidic pH in vitro. These results suggest that HGT of plasmids carrying AR shaped the evolution of S. Heidelberg and that antibiotic use reduction alone is insufficient to limit antibiotic resistance transfer from commensal bacteria to Salmonella enterica. IMPORTANCE 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. This change in practice has highlighted new questions that need to be answered to assess the effectiveness of antibiotic removal in reducing the spread of antibiotic resistance bacteria. This research demonstrates that antibiotic-susceptible Salmonella enterica serovar Heidelberg strains can acquire multidrug resistance from commensal bacteria present in the gut of neonatal broiler chicks, even in the absence of antibiotic selection. We demonstrate that exposure to acidic pH drove the horizontal transfer of antimicrobial resistance plasmids and suggest that simply removing antibiotics from food animal production might not be sufficient to limit the spread of antimicrobial resistance.

Population Analyses Reveal Preenrichment Method and Selective Enrichment Media Affect Salmonella Serovars Detected on Broiler Carcasses.

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.

Plasmid Replicons and ß-Lactamase-Encoding Genes of Multidrug-Resistant Escherichia coli Isolated from Humans and Food Animals in Lagos, Southwest Nigeria.

As resistance to the ß-lactam class of antibiotics has become a worldwide problem, multidrug-resistant (MDR) human (n = 243) and food animal (n = 211) isolates from Lagos, Nigeria were further tested to characterize ß-lactamase-encoding genes and plasmid replicons. Four ß-lactamase-encoding genes (blaCMY, blaCTX-M, blaOXA, and blaTEM) were detected using PCR-based replicon typing, 13 and 17 different replicons were identified using a subset of MDR E. coli from humans (n = 48) and animals (n = 96), respectively. Replicon types FIB and X2 were detected in equal numbers (2/48; 4.2% each) from human isolates, while type Y (16/96; 16.7%) was the most common type from animals. Only two replicon types, FIB and Y, were detected in both groups; all other types were confined to one group or the other, but not both. Using conjugation, replicon type Y, present in three donors, transferred in all three instances, whereas FIA transferred in 75% (3/4) of the matings. This study showed that ß-lactamase genes were prevalent in MDR E. coli from both humans and animals in Nigeria and also contained diverse plasmid replicons. As the replicon-associated genes were mobile, they are likely to continue disseminating among E. coli and facilitating transfer of associated ß-lactamase genes in this region.

Prevalence and characterization of Escherichia coli isolated from the Upper Oconee Watershed in Northeast Georgia.

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.

Draft Genome Sequences of Eight Streptogramin-Resistant Enterococcus Species Isolated from Animal and Environmental Sources in the United States.

Here, we present the draft genome sequences of eight streptogramin-resistant Enterococcus species isolated from animals and an environmental source in the United States from 2001 to 2004. Antimicrobial resistance genes were identified conferring resistance to the macrolide-lincosamide-streptogramins, aminoglycosides, tetracyclines, beta-lactams, and glycopeptides.