- AMR and virulence genes clustered with the Salmonella serovars, while there were also associations between the presence of virulence genes in both animal/environmental isolates and human clinical samples.. - Full list of author information is available at the end of the article. - 2018 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0. - Human foodborne salmonellosis causes an estimated 100,000 domestic cases and 40 deaths annually in the United States [1].. - In the European Union, Salmonella-infected gastroenteritis was the sec- ond most frequently reported foodborne illness with 91,408 clinical cases reported by thirty EU/EEA coun- tries, and a confirmed case rate of 25.4 cases per 100,000 population in 2014 compared to 21.4 cases per 100,000 population in 2013, which represented a 19% in- crease in the notification rate [3].. - Inappropriate use of antimicrobials in livestock produc- tion and the association to resistant Salmonella infection in humans are a growing concern to public health agen- cies, and have led to the rise of new multidrug resistant (MDR) bacteria and transferable genetic loci, such as colistin resistance mediated by the MCR-1 gene [5, 6].. - Our previous studies reported the persistence and dissemination of mul- tiple resistant Salmonella serovars along with their deter- minants in the environment of commercial swine operation due to the manure application on land [12, 13].. - which is in the top 4 of the most frequently reported Salmonella serovars, continue to rise while the incidence of the other serovars is decreasing [16]. - The increase in the incidence of this serovar in human cases is paralleled by a similar increase in swine and environmental detection of this serovar variant . - our understanding of the temporal and spatial con- nection of resistant Salmonella transmission within humans, animals, and the environment sources.. - The use of whole genome sequencing (WGS) has had a major impact on the study of the molecular epidemiology of AMR bacterial pathogens as- sociated and transmitted between human, animal and environmental sources. - A WGS study in Denmark re- ported that SNP, pan-genome, k-mer and nucleotide dif- ference trees were superior to the classical typing method and evaluated the association of the isolates to specific outbreaks of S. - The objectives of this study were to use WGS to analyze multiple Salmonella serovars isolated from human, food-animals and environments in the two states of the US and to clarify the epidemiological transmission of AMR Salmonella within these studied populations. - In addition, the capability of WGS to predict antimicrobial resistance and virulence genes in antimicrobial resistant Salmonella retrieved from different sources was evaluated.. - Comparison of FFP with SNP-based phylogeny of Salmonella isolates. - The 200 Salmonella enterica genomes were assessed for their phylogenetic relationships using core genome SNPs with the ParSNP program [27] and feature frequency profiling with the FFPry program [28]. - according to serotype with both analysis methods, and the topology of the resulting phylogenetic trees was very similar (Fig. - The differences in order of the clusters between the FFPry and parSNP trees may be explained by the parSNP tree being based on the core genome only, thus excluding phages, plas- mids and regions of horizontal gene transfer. - In addition, many major serovar clusters were comprised of the genomes from different sources of origin including human, animal, and the environment. - Therefore, these differ- ences have a relatively small effect on the general struc- ture of the trees and the clustering observed. - These clusters were comprised of the genomes. - The isolates with serotype Derby showed little variation in the core genome, nor was any specific clustering linked with human, swine, and environmental sources (Fig. - The swine fecal genomes were grouped together, while the soil and lagoon genomes even collected from the different Table 1 Number of Salmonella isolates (n = 200) from human, animal, and environment by serotype sequenced for comparison Salmonella. - Detection of AMR genes, plasmid replicons, and virulence genes using WGS. - The WGS data was used to detect the presence and absence of AMR genes, plasmid replicon, and virulence genes in the 200 Salmonella genomes (Figs. - In addition, the 200 Salmonella genomes were also screened for virulence genes. - One hundred and seventy-five virulence genes were detected in this study using WGS (Additional file 2: Table S2). - 1 Comparison of Salmonella enterica phylogenetic trees based on core genome single nucleotide polymorphisms using ParSNP [27] and the alignment-free whole genome comparison with feature frequency profiling of purine-pyrimidine words (FFPry) with a word length (L) of 18 [28], using the 200 Salmonella genomes included here, visualised using the serovars (colored bar) and a tanglegram in the middle to indicate position of individual genomes in both phylogenetic trees. - Association of AMR genes, plasmid replicons, and virulence genes with different Salmonella serotypes using WGS. - Serotypes were found to vary with regard to the pres- ence/absence of AMR coding gene, plasmid replicon, and virulence gene using WGS approach based on the odds ratio to evaluate their associations (Table 3). - As highlighted previously, several major virulence genes were detected in all Salmonella isolates in our study (Additional file 2: Table S2). - The objective of this study was to characterize Salmon- ella serovar, AMR determinants and virulence genes using whole genome sequencing. - The 200 Salmonella enterica genomes were isolated from different sources of origin including human, swine, poultry, and environ- ment, and were analyzed using the core genome SNP-based analysis and the alignment-free analysis method FFP. - The major difference of the SNP- and FFP-based analyses was in the order of the serovar clusters within the tree, however, the overall approach selected had relatively little effect on the top- ology of the phylogenetic trees (Fig. - ParSNP was run for all the 200 Salmonella genomes and separately for each of the serovars. - Although the separate comparisons indeed in- creased the core genome component as would be pre- dicted, this was not a major increase, and we do not expect that the removal of the outliers will have a signifi- cant effect on the trees. - Typhimurium isolates (HS71549, HS51537, and HS51628) were closely related to the environmental, swine, and chicken isolates, respectively. - 4 showed that the Salmonella isolates from chicken. - However, the human Salmonella isolates included in our analysis were only from the North Carolina State Public Health La- boratory which might not be represent all the human clinical cases. - 6 Distribution of phenotypic antimicrobial resistance in the genomes of the 200 S. - The isolates were clustered based on core genome SNPs using ParSNP, and the antimicrobial resistances are shown by black lines in the respective bars below.. - In addition, this method is still limited to the intragenus analysis of closely related species and strains [27, 41].. - Salmonella isolates (n = 200). - indicates that the mentioned gene was detected only in a specific serotype and none of the other serotypes. - These studies have revealed that the FFP method can contribute to the phylogenetic clusters based on geographic relation and outbreak detection, and could provide a complementary analysis approach. - Our study is the first to utilize the alignment-free FFP analysis in Sal- monella and compare it to the core genome SNP-based analysis. - In accordance to the high percentages of phenotypic tetracycline and sulfisoxazole resistance were reported in our result. - Overall, the resulting resist- ance genotypes correlated with 87.61% sensitivity and 97.13% specificity to the resistance phenotype (Table 2).. - The streptomycin discrepancies have been commonly detected in other studies too because streptomycin is not used to treat enteric infections, and as such, results in the absence of precise clinical breakpoint for streptomycin susceptibility in Salmonella and E. - In addition, the mechanism of streptomycin resistance is frequently due to lacking of the gene expression as well as mutations in the 16S rRNA gene leading to difficulty of phenotypic predic- tion [50, 52]. - The Salmonella serovars significantly correlated with the presence/absence of AMR genes, plasmid replicons, and virulence genes. - Plasmids were observed specific to Salmonella serovar that was very similar to the AMR genes (Table 3). - Our data showed that Salmonella isolates re- covered from animal or/and environmental sources contained the same virulence genes as carried by hu- man clinical isolates. - Derby (Table 3) which may relate to the non-source-dependent clustering found in this homogenous serovar as mentioned previously (Fig. - One of the typhoid-associated virulence factors, the cytolethal distending toxin cdtB, was detected in all isolates of Schwarzengrund, Johannesburg, and Muen- ster (Additional file 2: Table S2), which was similar to a previous study that detected this gene in S. - However, there were a few reports of the prevalence of this virulence gene in several NTS, in- cluding Javiana [57], Montevideo, Schwarzengrund, and Bredeney [61]. - This data suggested that the cdtB toxin may contribute to the pathogenicity in human and animal.. - WGS is a helpful tool to assess the phylogenetic relations among multiple serotypes, AMR and virule- nuce gene evaluation and assist in the molecular epi- demiological studies of foodborne pathogens. - Salmonella isolates selection. - The human Salmonella isolates were from stool samples from clinical cases received from the North Carolina State Pub- lic Health Laboratory (n = 44). - The Salmonella iso- lates with MICs in the intermediate level were categorized into susceptible to avoid overestimation of resistance.. - Genome library preparation and sequence assembly The Salmonella isolates (n = 200) were cultured over- night at 37 °C on Luria-Bertani (LB) agar. - The animal and environmental Salmonella isolates were initially sent to the National Veterinary Ser- vices Laboratories (NVSL) at Ames, Iowa for sero- typing using the Kauffman-White scheme, while the human serotyping was conducted at the North Caro- lina State Public Health Laboratory. - The results from SISTR interpretation were compared to the trad- itional Kauffman-White serotyping. - The Illumina raw reads were mapped against chromosomal and plasmid virulence genes found in the Virulence Factor Database for Sal- monella (VFDB) which currently contains 2017 genes database associated with virulence in Salmonella [http://www.mgc.ac.cn/VFs/status.htm] [74]. - Paired-end reads for the 200 Salmonella isolates in this study have been deposited in the National Center for Biotechnology Information (NCBI)’s under the Bioproject accession number PRJNA293224. - Distribution of phenotypic antimicrobial resistance of Salmonella isolates based on FFPry. - AMR, plasmid, and virulence genes. - AMR: Antimicrobial resistance. - WGS: Whole genome sequencing. - We gratefully appreciate the swine and poultry producers in the state of NC and TN for allowing access to their farms for collecting the animal, manure and environmental samples. - We thank Joy Horovitz for generating the WGS profile of the Salmonella isolates.. - The funding agencies did not contribute to the study design, data collection, analysis and interpretation of the data in writing the manuscript. - ST: He was the PI of the grant that funded the study and he was involved in data analysis, manuscript editing and submission.. - Antibiotic resistance threats in the United States. - Detection of the plasmid- mediated mcr-1 gene conferring colistin resistance in human and food isolates of Salmonella enterica and Escherichia coli in England and Wales. - Dissemination of the mcr- 1 colistin resistance gene. - 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