Sublethal concentrations of antibiotics enhance transmission of antibiotic resistance genes in environmental Escherichia coli

Charlotte J Gray-Hammerton^1,2*Steven P Hooton²Kirsty Sands^1,3Timothy R Walsh^1,3Claudia Orbegozo Rubio¹Edward A R Portal^1,3Chris Hudson⁴Dov Joseph Stekel^2,5Christine Elizabeth Ruth Dodd²Jon L Hobman²

• ¹Ineos Oxford Institute for Antimicrobial Research, University of Oxford, Oxford, United Kingdom
• ²University of Nottingham School of Biosciences, Sutton Bonington, United Kingdom
• ³Department of Medical Microbiology, Division of Infection and Immunity, Cardiff University, Cardiff, United Kingdom
• ⁴School of Veterinary Science and Medicine, University of Nottingham, Sutton Bonington Campus, Sutton Bonington, United Kingdom
• ⁵Department of Mathematics and Applied Mathematics, University of Johannesburg, Johannesburg, South Africa

Third generation cephalosporin resistant Enterobacterales are ranked second on the World Health Organization (WHO)'s Bacterial Priority Pathogens List. Amongst them, extended spectrum β-lactamase producing Escherichia coli (ESBL-Ec) are used by the WHO as sentinel organisms to monitor the spread of antibiotic resistance worldwide and are often associated with mobilizable multi-drug resistance (MDR). However, we know less about how ESBL-producing genes spread in environmental E. coli. This study investigates how the blaCTX-M-15 gene from ESBL-Ec isolated on a UK dairy farm could transfer between strains. For this study 39 E. coli were isolated from a single dairy farm over four months, using cefotaxime supplemented selective media. All had similar antibiotic susceptibility test phenotypes, and PCR, whole genome sequencing (WGS), and resistance gene transmission experiments demonstrated they were all closely related. In silico multi-locus sequence typing and single nucleotide polymorphism analysis showed that all 39 strains were Sequence Type 2325, but plasmid carriage differed. Thirty five of the 39 ESBL-Ec strains were multidrug resistant, displaying blaCTX-M type cephalosporin resistance and resistance to fluoroquinolones and tetracyclines. WGS confirmed all 39 isolates carried the ISEcp1 mobile genetic element carrying the blaCTX-M-15 ESBL producing gene, and the qnrS1 partial quinolone resistance gene in the chromosome. Thirty-five strains also carried tetAR within this ISEcp1 element. We found that sub-lethal levels of ampicillin, cloxacillin and ceftazidime could enhance the transfer of ISEcp1 blaCTX-M-15 from the chromosome of these dairy farm strains into endogenous self-transmissible plasmids, which can themselves then transfer into and confer phenotypic antibiotic resistance in a recipient E. coli K-12 strain. In conclusion, we saw not only clonal dissemination of these environmentally occurring ESBL producing strains within the farm environment but also showed experimentally that these strains had the ability to mobilize their ESBL producing genes, and that these and other resistance genes can be acquired or lost on transfer. This shows the importance of longitudinal monitoring of antibiotic resistance, especially in places with high prevalence or selective pressure for resistant bacteria.