Title: Characterization of multidrug-resistant Escherichia coli by antimicrobial resistance profiles, plasmid replicon typing, and pulsed-field gel electrophoresis Authors
Submitted to: Microbial Drug Resistance
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: January 25, 2011
Publication Date: June 1, 2011
Citation: Lindsey, R.L., Frye, J.G., Thitaram, S.N., Meinersmann, R.J., Cray, P.J., Englen, M.D. 2011. Characterization of multidrug-resistant Escherichia coli by antimicrobial resistance profiles, plasmid replicon typing, and pulsed-field gel electrophoresis. Microbial Drug Resistance. 17(2):157-163. Interpretive Summary: Bacterial plasmids are circular double-stranded DNA molecules that can replicate by themselves in a host cell. Characterization of plasmids is important from a clinical perspective because genes encoding important traits such as antimicrobial resistance are frequently present in plasmids. Incompatibility (Inc) replicon types are identified based on the inability of two plasmids to exist in the same cell. The molecular DNA technique known as multiplex polymerase chain reaction (PCR) can be used to classify 18 of the 27 known plasmid Inc types in genus Enterobacteriaceae. The objective of this study was to analyze plasmid replicon types, antimicrobial phenotypes, and genetic relationships of 35 multidrug-resistant (MDR) Escherichia coli. A total of 35 E. coli isolates were selected based on antimicrobial profile of clinically important drugs from 7 different food and companion animals. The DNA molecule separation technique known as pulsed-field gel electrophoresis (PFGE) was used to determine possible genetic relationships among E. coli isolates. Multiplex PCR was used to examine the Inc replicon types. Linkage analysis was conducted with isolate antimicrobial resistances, replicon types, and animal sources. A high degree of genotypic diversity was observed, with 34 distinct PFGE clusters found among the 35 isolates examined. A total of 15 different plasmid replicon types were detected and all isolates carried multiple replicon types. Overall Inc B/O plasmids were the most common type identified followed by Inc FIB. Significant linkage of Inc B/O was seen with sulfamethoxazole and ceftriaxone while Inc FIB was significantly linked with amoxicillin-clavulanic acid, ampicillin, chloramphenicol, and tetracycline. As expected, significant linkage of E. coli isolates was seen with amoxicillin-clavulanic acid, ampicillin, cefoxitin, tetracycline, and ceftiofur since they were used in isolate selection. The animal source of the isolate did not appear to have a correlation to cluster analysis based on PFGE. Inc B/O and FIB were the predominant replicon types among MDR E. coli isolates in this study. Additional research is warranted to determine the diversity and linkages associates with plasmids carrying antimicrobial resistance genes in E. coli.
Technical Abstract: Aim: Plasmid characterization has particular clinical importance because genes encoding significant traits including antimicrobial resistance are frequently carried on plasmids. The objective of this study was to examine the distribution of multidrug resistance (MDR) in Escherichia coli in relation to plasmid replicon types, and isolate sources and genotypes. Methods: A total of 35 E. coli isolates from 7 different animal sources were selected based on resistance to 15 clinically important drugs: amikacin, amoxicillin/clavulanic acid, ampicillin, cefoxitin, ceftiofur, ceftriaxone, chloramphenicol, ciprofloxacin, gentamicin, kanamycin, nalidixic acid, streptomycin, sulfamethoxazole , tetracycline and trimethoprim/sulfamethoxazole. Pulsed-field gel electrophoresis (PFGE) was used to determine genetic relationships among the E. coli isolates. A PCR-based replicon typing assay consisting of three multiplex PCR reactions was used to detect 18 of the 27 known plasmid types in Enterobacteriaceae based on their incompatibility (Inc) replicon types. Linkage analysis was performed for antimicrobial resistance profiles, replicon types and animal host source. Results: A high degree of genotypic diversity was observed: 34 different PFGE types were identified among the 35 isolates examined. Fifteen different plasmid replicon types were detected and all isolates carried multiple replicon types. Inc B/O was the most common replicon type followed by Inc FIB and Inc Frep. Significant linkage of Inc B/O with sulfamethoxazole was found while Inc FIB was linked with chloramphenicol. Significant linkage was also seen with amoxicillin-clavulanic acid, ampicillin, cefoxitin, tetracycline, and ceftiofur since resistance to these drugs were used as criteria for isolate selection. The isolate source was not correlated with PFGE genotype. Conclusions: Cluster analysis based on PFGE demonstrated a highly variable population genotypically. Commensal E. coli are reservoirs of a diverse array of plasmids carrying antimicrobial resistance. The full extent of resistance plasmid diversity in E. coli and the associated linkages with antimicrobial resistance genes requires additional study.