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Objectives The 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 bla CTX-M-32 -producing plasmid, pMOO-32, found to be widely distributed. Methods CTX-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. Results Amongst 1226 CTX-R isolates, PCR identified bla CTX-M group 1 (549 isolates), bla CTX-M group 9 (100 isolates), bla CMY (12 isolates), bla DHA (1 isolate) and no GOI (566 isolates). WGS analysis of 184 representative isolates identified bla CTX-M (131 isolates; encoding CTX-M-1, -14, -15, -32 and the novel variant, CTX-M-214), bla CMY-2 (6 isolates), bla DHA-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 bla CTX-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. Conclusions β-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 bla CTX-M-32 .

Little is known about the drivers of critically important antibacterial resistance in species with zoonotic potential present on farms (e.g. CTX-M β-lactamase-positive Escherichia coli ). There is also debate about the influence of farms on the circulation of resistance in local human populations. This was a two-year surveillance study on 53 dairy farms. E. coli positive for bla CTX-M were detected in 224/4145 (5.4%) of all samples from faecally-contaminated sites. E. coli positive for bla CTX-M were more prevalent (98/631; 15.5%) in calf samples and less prevalent (12/630; 1.9%) in samples collected from pastureland, including publicly accessible sites. Multilevel, multivariable logistic regression showed antibiotic dry cow therapeutic choice to be associated with risk of bla CTX-M positivity, including use of cefquinome or framycetin; 74% of bla CTX-M -positive E. coli were framycetin-resistant. Low temperature was associated with low risk of bla CTX-M positivity. This was additional to the effect of temperature on total E. coli density, a finding with profound implications for surveillance. There was no evidence that study farms had a significant impact on circulating bla CTX-M plasmids in the local human population: across 296 fully sequenced E. coli isolates, two cattle isolates shared bla CTX-M plasmids with eight urinary isolates collected in parallel.

Background Bicyclic boronates are a new and potentially important class of β-lactamase inhibitor, with the ability to inhibit β-lactamases from all molecular classes, including mobile metallo-β-lactamases. Objective Our objective was to identify mutants resistant to the actions of the bicyclic boronate inhibitor 2 , when being used in combination with aztreonam. Methods Overnight cultures were plated on to agar containing increasing concentrations of aztreonam with a fixed 10 mg/L concentration of the inhibitor. Resistant derivatives and parent strains were analysed by whole genome sequencing and LC-MS/MS proteomics to identify mechanism of resistance. Results When using a mixed overnight culture containing one Escherichia coli (TEM-1, CTX-M-15, CMY-4 producer) and one Klebsiella pneumoniae (SHV-12, CTX-M-15, NDM-1 producer) mobilisation of an IncX3 plasmid carrying bla SHV-12 from the K. pneumoniae into the E. coli generated an aztreonam/boronate resistant derivative. Conclusions High-level production of three bicyclic boronate susceptible enzymes (CMY-4, CTX-M-15, SHV-12) capable of hydrolysing aztreonam plus TEM-1, which binds the inhibitor, overcomes the fixed inhibitor dose used. This was only identified when using a mixed culture for selection. It would seem prudent that to allow for coalescence of the myriad β-lactamase genes commonly found in bacterial populations colonising humans, this mixed culture approach should be the norm when testing the potential for generating β-lactamase inhibitor resistance in pre-clinical analysis.