Mechanisms of cephalosporin resistance in indicator Escherichia coli isolated from food animals

Abstract

Resistance to β-lactams is considered one of the major global problems and recently it became the most frequently studied topic in the area of antimicrobial resistance. The study was focused on phenotypic and genetic characterisation of commensal Escherichia coli (E. coli), including those producing cephalosporinases, isolated from gut flora of healthy slaughter animals. E. coli were cultured simultaneously on MacConkey agar (MCA) and cefotaxime supplemented MCA. The isolates were confirmed with ONPG and indol tube tests as well as PCR targeting uspA gene. Microbroth dilution method was applied for determination of Minimal Inhibitory Concentrations and interpreted according to EUCAST epidemiological cut-off values. Cephalosporin resistance phenotypes were defined by E-tests (BioMerieux) and relevant gene amplicons from selected strains were sequenced. A total of 298 E. coli isolates with cephalosporin resistance (ESC) found in 99 ones, were obtained from 318 cloacal or rectal swabs deriving from broilers, layers, turkeys, pigs and cattle. Both extended spectrum β-lactamase (ESBL) and ampC-cephalosporinase resistance phenotypes were noted in all tested animal species but cattle. At least one of the analysed genes was identified in 90 out of 99 cephalosporin-resistant isolates: bla_TEM (n=44), bla_CMY (n=38), bla_CTX-M (n=33) and bla_SHV (n=12). None of the phenotypes was identified in nine isolates. Sequencing of PCR products showed occurrence of ESBL-genes: bla_CTX-M-1/-61, bla_SHV-12, bla_{TEM-1,-52/-92,-135} and ampC-gene bla_CMY-2. They were located on numerous and diverse plasmids and resistance transferability was proved by electroporation of bla_SHV-12 and bla_CTX-M-1/-61 located on X1 plasmids. Detection of cephalosporin resistant E. coli confirms the existence of resistance genes reservoir in farm animals and their possible spread (i.e. via IncX1 plasmids) to other bacteria including human and animal pathogens. The identified genetic background indicates on ecological aspects of selection and dissemination of cephalosporin resistance in E. coli isolated from food-producing animals rather than its potential role for public health threats.

Keywords: Antibiotic resistance; CTX-M-1; ESBL; Escherichia coli; IncX1 plasmids; SHV-12; pAmpC.