Phenotypic and genotypic analysis of blaCTX-M encoding plasmids isolated from bovine E. coli samples in the United Kingdom

The purpose of this study was to characterize blaCTX-M plasmids originating from bovine Escherichia coli and investigate their contribution to bacterial host fitness. In this study 52 bovine Escherichia coli strains collected between March and October 2007 encoding blaCTX-M, an extended spectrum β-lactamase (ESBL) gene conferring resistance to 3rd generation cephalosporins, were characterized. The majority of strains belonged to E. coli commensal phylogroups A and B1 expressing a multi-drug resistance (MDR) phenotype and harboured multiple plasmids of which 90% were transferred by conjugation. Transconjugants or transformants were made successfully from all 52 strains when selecting for resistance to cefotaxime. All plasmids were shown by PCR and sequence analysis to harbour blaCTX-M and nearly 80 % encoded multiple resistances. Plasmid sequence analysis of four plasmids encoding blaCTX-M-14b (IncI1-X1), -15 (IncFII-FIA-FIB) and -32 (IncX1 and IncB), identified genes necessary for stable plasmid maintenance and spread. Five representative plasmids encoding blaCTX-M-1, -15, -14b and -32 were assayed for their fitness impact upon the host. Efficiencies of β-lactam hydrolysis using whole cell extracts were determined in the same E. coli BL21 host strain with the most efficient encoded by blaCTX-M-14b and blaTEM-1 ESBL genes and least efficient encoded blaCTX-M-15 only. A 160 kb plasmid encoding 13 resistance genes was grown in the presence of 380 different metabolites and differences in metabolite utilisation between this and the plasmid-free BL21 strain determined. The plasmid-harbouring strain utilized less phosphor-sulphur compounds, suggesting the metabolic cost incurred by acquiring the plasmid may have implications of cellular utilization of alternative phosphate sources. There were no differences in growth was observed in nutrient rich media. The contribution of active efflux to resistance was investigated using L-phenylalanyl-L- 4 arginyl-b-naphthylamide (PAβN) in combination with ampicillin, cefotaxime or ceftazidime. Minimum inhibitory concentration (MIC) values were found to decrease ≥ 2 fold in the presence of the efflux pump inhibitor (EPI), in some cases becoming completely susceptible to ampicillin. This indicates that the possible use of EPIs in combination with previously failed antimicrobial drugs to potentially restore efficacy of treatment