ABSTRACT
The aim of this study was to characterize the plasmids carrying antimicrobial resistance (AMR) determinants in multiple Salmonella serotypes recovered from the commercial swine farm environment after manure application on land. Manure and soil samples were collected on day 0 before and after manure application on six farms in North Carolina, and sequential soil samples were recollected on days 7, 14, and 21 from the same plots. All environmental samples were processed for Salmonella, and their plasmid contents were further characterized. A total of 14 isolates including Salmonella enterica serotypes 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, were selected for plasmid analysis. Antimicrobial susceptibility testing was done by broth microdilution against a panel of 14 antimicrobials on the 14 confirmed transconjugants after conjugation assays. The plasmids were isolated by modified alkaline lysis, and PCRs were performed on purified plasmid DNA to identify the AMR determinants and the plasmid replicon types. The plasmids were sequenced for further analysis and to compare profiles and create phylogenetic trees. 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. Our study provides evidence on the importance of horizontal dissemination of resistance determinants through plasmids of multiple Salmonella serotypes distributed across commercial swine farms after manure application.
IMPORTANCE The horizontal gene transfer of antimicrobial resistance (AMR) determinants located on plasmids is considered to be the main reason for the rapid proliferation and spread of drug resistance. The deposition of manure generated in swine production systems into the environment is identified as a potential source of AMR dissemination. In this study, AMR gene-carrying plasmids were detected in multiple Salmonella serotypes across different commercial swine farms in North Carolina. The plasmid profiles were characterized based on Salmonella serotype donors and incompatibility (Inc) groups. We found that different Inc plasmids showed evidence of AMR gene transfer in multiple Salmonella serotypes. We detected an identical 95-kb plasmid that was widely distributed across swine farms in North Carolina. These conjugable resistance plasmids were able to persist on land after swine manure application. Our study provides strong evidence of AMR determinant dissemination present in plasmids of multiple Salmonella serotypes in the environment after manure application.
INTRODUCTION
The emergence of antimicrobial resistance (AMR) in bacterial pathogens has threatened the sustainability of an effective global public health response to infectious diseases (1, 2). There are major gaps in our understanding of AMR transmission within agricultural sites and the potential impacts on humans, animals, and the environment due to a lack of studies conducted on actual commercial food animal farms (3–5). A number of studies have documented the abundance of AMR pathogens associated with livestock production due to the intensive use of antimicrobials in animal husbandry practices for therapeutic and nontherapeutic purposes (6–9). However, there is limited knowledge about the effect of manure application on the spread of AMR pathogens and AMR genes by means of horizontal gene transfer (HGT), such as by plasmids, transposons, and integrons, in the environment (4, 10, 11). Exposure of bacterial pathogens to antimicrobials in the environment increases the evolution of resistance and has an influence on the abundance, distribution, and transfer of AMR genes into different bacterial species (9, 12). We recently reported the dissemination of AMR Salmonella isolates in manure from commercial swine farms that were able to persist on land for at least 21 days after manure application, and it was clearly observed that Salmonella bacteria were rarely present in the soil before the land application (13). Given the potential risk of disseminating AMR Salmonella bacteria into the environment during manure application, we further characterized the plasmids that were detected in the multiple Salmonella serotypes isolated in our previous study.
The dissemination of undesirable AMR genes in Gram-negative pathogenic bacteria has been mainly regarded as the acquisition of multiple plasmid-located AMR genes by HGT (14, 15). Conjugation is considered the main mode of HGT of AMR genes among the Enterobacteriaceae family and helps to increase bacterial genetic diversity (16, 17). Plasmids conferring resistance have been identified as hindering antimicrobial therapy, including the use of extended-spectrum cephalosporins and fluoroquinolones, which are regarded as drugs of choice for bacterial infection in human clinical cases (14, 18, 19). Studies from several parts of the world have demonstrated the distribution of plasmids harboring extended-spectrum β-lactamase (ESBL) genes (blaCTX, blaSHV, blaCMY, and blaTEM) or ampC and plasmid-mediated quinolone resistance (PMQR) genes (qnrA, qnrB, and qnrS) in Escherichia coli and Salmonella among animal, human, and environmental sources (16, 20–22). The presence of plasmid-mediated transfer of a recently identified mobile colistin resistance gene (mcr-1) is another example of the threat posed to public health (23, 24). The comparative analysis of mcr-1-containing plasmids maintained in the Enterobacteriaceae family revealed that they are disseminated in a broad host range, including human, animal, and food sources, and are now being reported from different countries worldwide (25–27). Plasmids that confer resistance in carbapenem-resistant Enterobacteriaceae (CRE) pose an urgent threat to public health with their global expansion (28, 29). Mollenkopf et al. (30) reported that the CRE carrying blaIMP-27 plasmids were recovered from the environment of a swine production area in the United States. The farm environment is considered a potential reservoir of AMR Salmonella strains that probably exchange AMR determinants with humans and animals by plasmid horizontal transfer (13, 22, 31, 32).
The objective of this study was to determine and characterize the resistance plasmid profiles isolated from multiple AMR Salmonella serotypes recovered in manure and environmental samples after land application of manure on commercial swine farms in North Carolina. To address this, we performed antimicrobial susceptibility testing (AST), plasmid replicon typing, conjugation assays, and plasmid sequencing to fully understand the role of these plasmids in transferring AMR determinants in the environment.
RESULTS
Salmonella serotypes and plasmid characterization.A total of 14 different Salmonella serotypes isolated from commercial swine farms in North Carolina were selected to determine whether the AMR genes were located on transmissible plasmids. We also wanted to find out whether dissemination of AMR Salmonella bacteria through manure application assists in the transmission of genes via plasmids to other susceptible bacterial populations. Salmonella isolates collected from the swine farm environment after manure application were selected from each farm based on type of sample (lagoon and soil), sampling day, serotype, and resistance phenotype (Table 1). All 14 Salmonella donors harbored at least one large plasmid larger than 40 kb in size, and their plasmid profiles were dependent on farm origin and donor Salmonella serotype. PCR-based replicon typing (PBRT) revealed four plasmid replicons (FI, FII, I1, and N) among the 14 isolates carrying plasmids (Table 1). IncN plasmids (n = 3) of 50 kb in size were found in Salmonella enterica serotype Worthington isolates from both lagoon and soil samples in North Carolina farm 1 (NCF1). In NCF3, 100-kb IncI1 (n = 2) plasmids were isolated from S. enterica serotype Johannesburg while S. enterica serotype Typhimurium var5− was the predominant serotype at this farm and carried IncFII plasmids (n = 4) of 95 kb in size. Furthermore, IncFII plasmids were also found in S. Typhimurium var5− from NCF5 and 4,12:i:− from NCF6. A single S. enterica serotype Rissen isolate from a lagoon sample in NCF6 carried an IncI1 plasmid of 90 kb in size. The heterogeneous IncF group was the predominant replicon type detected in this study. Within the IncF group, we detected the subgroups FIA, FIB, FIC, FIIA, and Frep, with IncFIC and Frep being the most prevalent subgroups. The IncFI plasmid group found in 10 Salmonella isolates (Tab