Genomic Characterization of Antimicrobial Resistant Salmonella Serotypes across Human, Animal and Environmental Sources Reveals Genome Relatedness and Interserovar Exchange of Resistance Determinant

Salmonella is a significant foodborne pathogen. Spread of antimicrobial resistant (AMR) foodborne pathogens due to the antimicrobial misuse in livestock production results in the development of AMR bacterial strains. Land application of animal manure, a practical use in recent sustainable agricultural management, potentially leads to the dissemination of AMR pathogens in the environment and poses a serious public health threat.;To assess the impact of manure application in commercial swine farms on the transmission of AMR Salmonella in the environment, we collected environmental samples from farms in North Carolina (n=6) and Iowa (n=7). The manure and soil samples (before/after manure application) were collected on day 0. Subsequent soil samples were recollected on days 7, 14, 21 from the same plots. A total of 1,300 soil samples and 130 manure samples were included. The overall Salmonella prevalence was 13.22%, represented by 10.69% and 38.46% prevalence in soil and manure, respectively. The prevalence in NC (25.45%) was significantly higher than in IA (2.73%) (P<0.001). Salmonella serotypes detected in NC were not detected in IA. Antimicrobial susceptibility was tested by broth microdilution. A high frequency of isolates (58.73%) were multidrug resistant and the most frequent resistance was detected against streptomycin (88.36%), sulfisoxazole (67.2%), and tetracycline (57.67%). Genotypic characterization by pulsed-field gel electrophoresis (PFGE) revealed clonally related Salmonella in manure and soil at multiple time points.;The fourteen isolates of multiple Salmonella serotypes including Johannesburg (n=2), Ohio (n=2), Rissen (n=1), Typhimurium var5- (n =5), Worthington (n=3), and 4,12:i:- (n=1), representing different farms in NC, were selected for characterization of the plasmids carrying AMR determinants. The 14 confirmed transconjugants were tested for antimicrobial susceptibility. The plasmids were isolated by modified alkaline lysis, and PCRs were performed to identify the AMR determinants and the plasmid replicon types. The plasmids were sequenced for further analysis. A class 1 integron with an ANT(2")-Ia-aadA2 cassette was detected in the 50-kb IncN plasmids identified in S. Worthington isolates. We identified 100-kb and 90-kb IncI1 plasmids in S. Johannesburg and S. Rissen isolates carrying the blaCMY-2 and tet(A) genes, respectively. An identical 95-kb IncF plasmid was widely disseminated among the different serotypes and across different farms.;Whole genome sequencing (WGS) was applied to study the association between human Salmonella clinical isolates and the environmental/animal reservoirs. Salmonella isolates recovered from different sources including human (n=44), swine (n=32), poultry ( n=22), and environment (n=102) were sequenced. The assembled genomes were used for in silico analysis of virulence genes in the VFDB database, and phylogenetically clustered using core genome single nucleotide polymorphism (SNP) and feature frequency profiling. Furthermore, AMR was analyzed by genotypic prediction using five curated AMR databases, and compared to phenotypic AMR using broth microdilution. Both core genome SNP-based and FFP-based phylogenetic trees showed consistent clustering of isolates into the respective serotypes, and suggested clustering of isolates based on the source of isolation. The overall correlation of phenotypic and genotypic AMR was 87.61% and 97.13% for sensitivity and specificity, respectively. AMR and virulence genes clustered with the Salmonella serotypes, while there were also associations between the presence of virulence genes present in both animal/environmental isolates and human clinical samples.;In conclusion, the study highlights the potential role of swine manure application in the dissemination and persistence of AMR Salmonella in the environment and provided the important evidence of horizontal dissemination of resistance determinants through plasmids of multiple Salmonella serotypes across commercial swine farms after land application. Finally, our study supports that the isolates from human had a close relationship with animal and environment isolates which are considered to be the sources for dissemination of AMR and virulence genes between Salmonella serotypes.