Molecular characterization of bla ESBL-harboring conjugative plasmids identified in multi-drug resistant Escherichia coli isolated from food-producing animals and healthy humans

Abstract

Background: Extended-spectrum β-lactamase (ESBL)-encoding genes are frequently mapped to plasmids, yet few of these structures have been characterized at the molecular level, to date.

Methods: Eighty-seven ESBL-producing Escherichia coli were isolated from fecal samples of food-producing animals and healthy humans in Switzerland from 2009 to 2011. Plasmid DNA of all isolates was purified. Broth mating assays were carried out individually for 32 isolates to determine if the ESBL marker could be transferred by conjugation. The plasmid sizes were determined by S1-nuclease pulsed-field gel electrophoresis (PFGE) and the plasmids were typed by PCR-based replicon typing. Susceptibility tests by disk diffusion followed with a re-analysis S1-nuclease PFGE and PCRs were performed to confirm plasmid transfer. Microarray was performed to detect additional antibiotic resistance markers and multi-locus sequence typing was also performed in selected donor strains. The phylotypes were identified by triplex PCR.

Results: About half (n = 46) of the 87 isolates carried small (<20-kb) plasmids. All selected 32 isolates contained large plasmids (ranging in sizes from 20- to 600-kb). Eleven plasmid replicon types were detected. Of these, IncFIA (n = 5), IncFIB (n = 9), and IncK/B (n = 4) were common. Nine isolates demonstrated the ability to transfer their cefotaxime resistance marker at high transfer rates. Plasmid profile re-analysis of these transconjugants identified 16 plasmids. IncFIB and IncI1 were the most prevalent replicon types. Phylogenetic grouping showed that five of the nine donor strains belonged to phylogroup B1. Nine different sequence types were identified in nine tested donor strains.

Conclusion: Characterization of these ESBL-encoding conjugative plasmids extends our understanding on these resistance markers in multi-drug resistant E. coli cultured from healthy human and animal sources.

Keywords: E. coli; ESBLs; S1-nuclease PFGE; conjugation; horizontal gene transfer; plasmid profiling; plasmids; replicon typing.

FIGURE 1

A heat-map summary of the sources; a schematic showing the S1 nuclease plasmid profile; ESBL present/absent profiles; the resistance profile and the corresponding Inc plasmid type(s) for all 32 ESBL-positive E. coli previously reported from healthy food-producing animals and humans in Switzerland. Black squares shown indicate a feature present in that isolate denoting its original source the ESBL marker(s) detected, its corresponding antimicrobial resistance profile and the Inc types detected. White squares denote features that are lacking in the corresponding bacterial isolate. Isolate numbers denotes by the grayed boxes on the left of the heat-map indicate those bacteria that contained self-transmissible ESBL-harboring plasmids (they are investigated further, as described in Figure 2). Antimicrobial compounds (it is known that many ESBL producers may appear susceptible or intermediate to certain oxyimino cephalosporins in vitro, if CLSI criteria are applied strictly, but do not respond to the respective therapies. Consequently, for clinical reporting these results have to be corrected to “resistant.”) used are abbreviated as follows: AM, ampicillin; AMC, amoxicillin–clavulanic acid; CAZ, ceftazidime; CF, cephalothin; CPD, cefpodoxime; CIP, ciprofloxacin; CTX, cefotaxime; CXM, cefuroxime; FEP, cefepime; GM, gentamicin; S, streptomycin; SXT, trimethoprim-sulfamethoxazole; TE, tetracycline.

FIGURE 2

A heat-map showing the comparison of ESBL-positive E. coli donors and the resultant transconjugants, characterized on the basis of their plasmid profiles; ESBL-markers identified by PCR; antimicrobial resistance profile; and plasmid replicon type(s). The MIC for cefotaxime in each pairwise combination is also shown along with the fold increase in this value. (T) in the first column typifies the transconjugant. Black and white squares denote the presence and absence, respectively of a particular feature. The symbol “–” signifies no fold-change in the MIC for the transconjugant. Antimicrobial compounds are abbreviated as follows: AM, ampicillin; AMC, amoxicillin–clavulanic acid; C, chloramphenicol; CPD, cefpodoxime; CIP, ciprofloxacin; CTX, cefotaxime; KF, cephalothin; NA, nalidixic acid; S, streptomycin; SXT, trimethoprim-sulfamethoxazole; TE, tetracycline; W, trimethoprim.

Figure A1

A heat-map summary of the original 87 ESBL-positive E. coli isolates previously reported from healthy food-producing animals and humans in Switzerland. Black squares shown indicate a feature present in that isolate denoting its original source and the ESBL marker(s) detected; and white squares denote features that are lacking in the corresponding bacterial isolate. The approximate sizes of small plasmids are also showing in separate columns. The symbol “–” signifies no small (<30-kb) plasmid in the corresponding strain. Isolate numbers denoted by the grayed boxes on the left of the heat-map indicate those bacteria that were selected for conjugation experiments and S1-nuclease-based PFGE studies (as described in Figure 1).