A Second Large Plasmid Encodes Conjugative Transfer and Antimicrobial Resistance in O119:H2 and Some Typical O111 Enteropathogenic Escherichia coli Strains

Abstract

A novel and functional conjugative transfer system identified in O119:H2 enteropathogenic Escherichia coli (EPEC) strain MB80 by subtractive hybridization is encoded on a large multidrug resistance plasmid, distinct from the well-described EPEC adherence factor (EAF) plasmid. Variants of the MB80 conjugative resistance plasmid were identified in other EPEC strains, including the prototypical O111:NM strain B171, from which the EAF plasmid has been sequenced. This separate large plasmid and the selective advantage that it confers in the antibiotic era have been overlooked because it comigrates with the virulence plasmid on conventional gels.Enteropathogenic Escherichia coli (EPEC) continues to be a major cause of diarrhea and death, predominantly in developing countries (8,22). During infection, EPEC strains attach intimately to the intestinal epithelium and efface the absorptive microvilli, initiating a complex signaling cascade that ultimately leads to diarrhea by mechanisms that are only partially understood (8). These pathogenic effects manifest as an "attaching and effacing" histopathological hallmark, which is conferred by a large chromosomal island called the locus of enterocyte effacement (LEE). The LEE encodes a virulence gene regulator and a type III secretion system and effectors, as well as the intimin adhesin and its translocated receptor. The LEE is present in all EPEC strains, as well as in enterohemorrhaghic E. coli and some animal pathogens (reviewed in references 8 and 22).EPEC strains are classified as being either typical or atypical based on their ability to form densely packed three-dimensional clusters, or microcolonies, a phenotype known as localized adherence (LA). LA is conferred by a large EPEC adherence factor (EAF) plasmid, associated with virulence in epidemiological and volunteer challenge studies, which is present in all typical EPEC strains and absent or altered in atypical EPEC strains (3,16,25,35). The specific virulence factor responsible for LA is an EAF plasmid-encoded bundleforming pilus (Bfp), which also contributes to antigenicity, autoaggregation, and biofilm formation (3,11,15,20). On a separate region of the EAF plasmid are the perABC genes, encoding the plasmid-encoded regulator (a transcriptional activator for the bfp operon and the LEE, as well as its own promoter) and other virulence loci (9, 19). The EAF plasmid and the LEE are believed to have been acquired by different EPEC lineages in separate horizontal events (30).EAF plasmids from two well-studied EPEC strains have been sequenced. The major difference between the EAF plasmids from isolates E2368/69 (of the EPEC1 lineage [30]) and B171 (representing the EPEC2 lineage [30]), is the presence of conjugative transfer (tra) genes on pMAR7, the E2348/69 EAF plasmid (7). The B171 EAF plasmid, pB171, does not contain tra genes (36), even though at least one earlier paper reported conjugative transfer of pB171 (31). Other than with respect to tra genes, the two sequenced EAF plasmids are highly conserved, and low-resol...

Characterization of a Large Antibiotic Resistance Plasmid Found in Enteropathogenic Escherichia coli Strain B171 and Its Relatedness to Plasmids of Diverse E. coli and Shigella Strains

“…The resistance plasmid, pB171_90, also carries two genes that were present on the EPEC resistance plasmid and not on the EPEC virulence plasmid and were previously suggested to have a role in pathogenesis (36). One potential pathogenicity gene is csi, a putative calcium sequestration inhibitor (36), whose function in virulence is poorly understood.…”

“…The traI and traC genes of the conjugative transfer system were absent from the virulence plasmid of EPEC reference strain E2348/69; however, they were detected in other EPEC strains, including the EPEC reference strain B171 (30,36,37). While portions of the MB80 plasmid were sequenced (36) and the draft genome sequence of EPEC strain B171 has been generated (30), the complete sequence of the resistance plasmid from these EPEC strains has not been previously described. Thus, to increase our understanding of EPEC antibiotic resistance plasmids, we sequenced the large, ϳ90-kb antibiotic and heavy metal resistance plasmid, pB171_90, from the EPEC reference strain B171, which was previously determined to confer resistance to tetracycline, streptomycin, chloramphenicol, and sulfonamides (30,36,37).…”

“…A large multidrug resistance plasmid from EPEC strain MB80 was previously characterized using subtractive hybridization (36). The traI and traC genes of the conjugative transfer system were absent from the virulence plasmid of EPEC reference strain E2348/69; however, they were detected in other EPEC strains, including the EPEC reference strain B171 (30,36,37).…”

“…A large multidrug resistance plasmid from EPEC strain MB80 was previously characterized using subtractive hybridization (36). The traI and traC genes of the conjugative transfer system were absent from the virulence plasmid of EPEC reference strain E2348/69; however, they were detected in other EPEC strains, including the EPEC reference strain B171 (30,36,37). While portions of the MB80 plasmid were sequenced (36) and the draft genome sequence of EPEC strain B171 has been generated (30), the complete sequence of the resistance plasmid from these EPEC strains has not been previously described.…”

“…While portions of the MB80 plasmid were sequenced (36) and the draft genome sequence of EPEC strain B171 has been generated (30), the complete sequence of the resistance plasmid from these EPEC strains has not been previously described. Thus, to increase our understanding of EPEC antibiotic resistance plasmids, we sequenced the large, ϳ90-kb antibiotic and heavy metal resistance plasmid, pB171_90, from the EPEC reference strain B171, which was previously determined to confer resistance to tetracycline, streptomycin, chloramphenicol, and sulfonamides (30,36,37). We describe the distribution of the pB171_90 genes among a publicly available collection of 4,798 E. coli genome assemblies in GenBank, which includes E. coli of diverse pathotypes as well as commensal E. coli that lack canonical pathotype-specific virulence genes.…”