In this work, we demonstrate that antimicrobial drug resistance in S.
Kentucky CVM29188 is mediated by two of three large plasmids. Resistance phenotypes were transferable to susceptible S.
Newport, and E. coli
strains, demonstrating that both pCVM29188_146 and pCVM29188_101, alone and combined, have the potential for conjugative transfer across both serovar and species boundaries. Transfer assays showed that tetracycline resistance is conferred by pCVM29188_146 and encoded as part of a larger, Tn10
-containing resistance fragment with similarity to drug resistance plasmids from Yersinia ruckeri
) and Erwinia amylovora
). A Tn5393
transposon within the same resistance fragment carries the strAB
gene cluster for streptomycin resistance. A transposon-like resistance element with wide distribution among salmonellae and other Enterobacteriaceae
) is responsible for ceftiofur resistance mediated by pCVM29188_101. The high degree of sequence conservation of these resistance elements from unrelated plasmids from different bacterial hosts supports the concept that a shared horizontal resistance gene pool is available to diverse bacterial communities from environmental, agricultural, and clinical settings (11
). The annotation of pCVM29188_46 suggests that the smallest plasmid is not involved in the antimicrobial resistance phenotype of S
. Kentucky CVM29188.
Comparative sequence analysis suggests that both pCVM29188_146 and pCVM29188_101 evolved from common plasmid backbones by acquisition and integration of horizontally transferred resistance gene cassettes into larger mosaic plasmid resistance islands. Notably, the backbone of pCVM29188_146 bears strong resemblance to a group of APEC virulence plasmids, previously found only in E. coli
and thought responsible for causing colibacillosis in chickens (17
). Acquisition of one of these two plasmids, pAPEC-O2-ColV, by commensal E. coli
strains has been shown to result in increased abilities to kill avian embryos, grow in human urine, and colonize the murine kidney (36
). Shared sequence regions encode putative virulence factors involved in avian pathogenesis (5
), some of which may also play a role in uropathogenic E. coli
infections in humans (30
). APEC-like resistance plasmids similar to pCVM29188_146 were also found in a group of S.
Kentucky isolates that, according to PFGE-based phylogenetic predictions, constitute a highly related group of S.
Kentucky strains that is predominant in chicken. Given the shared intestinal habitat, it is likely that S.
Kentucky acquired APEC-like plasmids from commensal and/or pathogenic E. coli
strains in the chicken intestine. To our knowledge, transfer of virulence genes between E. coli
on mobile genetic elements has previously been shown only once, in the case of the heat-stable toxin gene astA
, which is likely to have been acquired by a gifsy-2-related prophage in Salmonella enterica
serovar Abortusovis from a pathogenic E. coli
In 94% of all tested S.
Kentucky chicken strains, the presence of APEC-like plasmids was associated with two different antimicrobial resistance phenotypes, tetracycline and streptomycin. Both resistance phenotypes could be encoded by APEC-like resistance plasmids similar in composition to pCVM29188_146. In this context, it is noteworthy that within the last decade the prevalence of S.
Kentucky in chickens, compared to that of other Salmonella
serovars, has constantly been increasing (40
) and that tetracycline and streptomycin resistances represent the two most common resistance phenotypes in S.
Kentucky from chickens (10
). Together, these results indicate the possibility of a newly emerging S.
Kentucky lineage with APEC-like resistance plasmids similar to pCVM29188_146 in chickens.
The physical linkage of genetic determinants for both antimicrobial resistance and APEC virulence on the same plasmid, as seen for pCVM29188_146, allows for at least two different evolutionary models to explain the wide distribution of similar plasmids in S.
Kentucky chicken populations: the spread of APEC-like plasmids in S.
Kentucky from chickens could have been facilitated through the selection for either antimicrobial resistance or phenotypes associated with putative APEC virulence factors. In both cases, a positive selection in the Darwinian sense would describe the results of a discrimination within the S.
Kentucky populations against strains lacking APEC-like plasmids. In the first scenario, antimicrobial selection could have promoted the integration of resistance gene cassettes into APEC virulence plasmids from pathogenic E. coli
strains. In a second step, mediated through antimicrobial selection, APEC-derived resistance plasmids could have been mobilized within enterobacterial communities, leading to their presence in S.
Kentucky strains. Tetracycline is used in chicken production to treat secondary E. coli
infections in poultry (25
). According to this scenario, similar APEC-like resistance plasmids should also be present in other enterobacteria from the same environment and also from other settings under similar antimicrobial selection. In this study, however, the majority of APEC-like plasmids were identified in S.
Kentucky isolates from chicken but rarely in other Salmonella
serovars from chicken or in S
. Kentucky isolates from other sources.
In the second scenario, the prevalence of APEC-like plasmids in S.
Kentucky strains from chicken could result from selective benefits that these plasmids confer to their host in the colonization of the chicken gut. Infection with S.
Kentucky is usually not associated with disease patterns in chicken (John Maurer, personal communication). However, plasmid-encoded APEC virulence factors have been associated with resistance to serum (iss
) and oxidative stress (sitABCD
) as well as with the biosynthesis of the siderophores salmochelin (14
) and aerobactin (9
) and other transport mechanisms, such as those encoded by sitABCD
) and etsABC
). These APEC virulence factors could provide S.
Kentucky strains with selective advantages in coping with stress factors associated with the intestinal environment or in competing with other enterobacteria for limited resources.
The results presented in this study show that antimicrobial resistance determinants and APEC virulence factors implied in avian and possibly human pathogenesis can be encoded by the same plasmid. Under antimicrobial selection, the propagation of these virulence factors within bacterial communities could potentially lead to the emergence of new virulent strains from the commensal microflora. We therefore suggest that future sequencing studies focus on plasmid population dynamics within host-associated microbiota in order to examine the prevalence and spread of combined virulence and resistance plasmids in bacteria from agricultural, environmental, and clinical settings. Horizontal transfer of plasmids not directly selected for by a given antimicrobial agent, as seen for the ceftiofur resistance plasmid pCVM29188_101 in conjugation assays selecting for the tetracycline resistance plasmid pCVM29188_146, is another area worthy of exploration and may be important to consider in addition to cotransfer of resistance genes linked on the plasmid under direct selection.