Molecular epidemiology of Escherichia coli producing CTX-M and plasmid AmpC-type β-lactamases from dairy farms identifies a dominant plasmid encoding CTX-M-32 but no evidence for transmission to humans in the same geographical region

Abstract

ObjectivesThe objective of this study was to identify the mechanisms of cefotaxime resistance (CTX-R) in 1226 Escherichia coli from 4581 environmental samples collected on 53 dairy farms over a 2-year period in South West England and to characterise a blaCTX-M-32-producing plasmid, pMOO-32, found to be widely distributed.MethodsCTX-R isolates were identified using MIC breakpoint agar plates. β-lactamase genes of interest (GOIs) were detected by PCR. WGS was performed and analysed using the Center for Genomic Epidemiology platform. A plasmid-specific multiplex PCR was designed to indicate the presence of plasmid pMOO-32.ResultsAmongst 1226 CTX-R isolates, PCR identified blaCTX-M group 1 (549 isolates), blaCTX-M group 9 (100 isolates), blaCMY (12 isolates), blaDHA (1 isolate) and no GOI (566 isolates). WGS analysis of 184 representative isolates identified blaCTX-M (131 isolates; encoding CTX-M-1, -14, -15, -32 and the novel variant, CTX-M-214), blaCMY-2 (6 isolates), blaDHA-1 (one isolate) and presumed AmpC-hyperproduction in 46 isolates that were PCR negative for GOIs. A highly conserved plasmid was identified in 73 isolates, representing 27 E. coli STs. This ∼220 kb IncHI2 plasmid carrying blaCTX-M-32 was designated pMOO-32, was found to be stable in cattle and human transconjugant E. coli even in the absence of selective pressure, and was found by multiplex PCR to be present on 26/53 study farms.Conclusionsβ-lactamases capable of conferring resistance to third generation cephalosporins were evident on 47/53 farms within this study. This was largely because of the widespread dissemination of an IncHI2 plasmid carrying blaCTX-M-32.

Characterization of AmpC-hyperproducing Escherichia coli from humans and dairy farms collected in parallel in the same geographical region

“…Confirmation of AmpC hyper-production and identification of porin loss and marR mutations in E. coli from dairy farms Our first aim was to investigate putative AmpC hyper-producing E. coli isolates from dairy farms identified in our recent surveillance study. 10,11 We decided to focus on a 50 x 50 km subregion of the study area, in which 25 farms were found to be positive for putative AmpC hyperproducers: defined as cefotaxime-resistant isolates that were PCR-negative for known mobile cephalosporinase genes. First, antibiograms were determined for one putative AmpC hyperproducing isolate from each of 4 randomly selected farms.…”

“…Twenty-five test E. coli isolates came from dairy farms located within a 50 x 50 km region of the South West of England, part of the wider area of our earlier study. 10,11 Isolates came, variously, from faecally contaminated sites around calves, heifers, cows and the near-farm environment. Samples were collected between January 2017 and December 2018.…”

“…Positivity for AmpC hyper-producing E. coli in a sample was defined by the growth of E. coli on tryptone bile X-glucuronide agar containing 2 mg/L cefotaxime which were PCR-negative for known horizontally-acquired cefotaxime resistance genes: blaCTX-M, blaSHV, blaCMY and blaDHA. 10,11 The risk factor analysis methodology used has been described previously, including the use of a novel method using a logistic link function to account for measurement error. 10 Ethics All farmers gave fully informed consent to participate in the study.…”

“…10 We then reported that 566/1226 of these CTX-R E. coli isolates (from 186 samples from 38 farms) were PCR-negative for mobile cephalosporinases and so were presumed to be chromosomal AmpC hyper-producers. 11 If this presumption was correct, AmpC hyper-production was the mechanism of resistance in 46.2% of CTX-R E. coli from dairy cattle in this region of the UK. This figure is comparable with the 42.9% presumed AmpC hyper-producers seen in CTX-R E. coli from dairy cattle in a recent nationwide Dutch study 12 and contrasts with the 3.8% of AmpC hyper-producers seen in CTX-R isolates in our recent study of human urinary E. coli.…”

“…This figure is comparable with the 42.9% presumed AmpC hyper-producers seen in CTX-R E. coli from dairy cattle in a recent nationwide Dutch study 12 and contrasts with the 3.8% of AmpC hyper-producers seen in CTX-R isolates in our recent study of human urinary E. coli. 3 One aim of the work reported here was to characterise putative AmpC hyper-producing E. coli from our recent survey of dairy farms 10,11 and to identify risk factors for the presence of AmpC hyper-producers on these farms. Another aim was to investigate potential zoonotic transmission of AmpC hyper-producers by using WGS-based phylogenetic analysis to compare isolates from farms with human urinary E. coli collected in parallel from the same 50…”