Abstract
Enzymes belonging to the CTX-M family are now the most prevalent extended spectrum β-lactamases, conferring resistance to multiple β-lactams. In 2009 an outbreak of CTX-M harbouring Enterobacteriaceae occurred at UK pig farm in which the pigs had received multiple rounds of antibiotic treatment. The aim of this study was to characterise the outbreak CTX-M strains and to identify the fitness contribution of the outbreak CTX-M plasmids beyond antimicrobial resistance. CTX-M producing Escherichia coli, Klebsiella pneumoniae and Salmonella enterica of serovars 4,5,12,i:- and Bovismorbificans were isolated indicating an instance of non-clonal spread of resistant organisms. Plasmid characterisation revealed that a CTX-M-14 plasmid of 50kb and unknown incompatibility group was harboured by two E. coli strains. A second plasmid type was a CTX-M-1, sul2 and floR Incl1 plasmid of 104kb found in all three species of bacteria. Sequencing of the CTX-M-1 plasmid revealed that it was a novel plasmid that had arisen by the acquisition of two resistance islands by a ColIb-p9-like plasmid. A curing method specific for Incl1 plasmids was developed and successfully used to cure lab strains harbouring the outbreak plasmids, outbreak K. pneumoniae strain B3791 and wild type E. coli strains harbouring unrelated Incl1 plasmids. Additionally, a plasmid free Salmonella 4,5,12,i:- strain was obtained from the outbreak farm and used, together with B3791, to investigate the phenotypic contribution of the outbreak plasmids. Competitive fitness studies showed that carriage of the outbreak plasmid imposed a burden on K. pneumoniae strain B3791 whilst being neutral to the growth of Salmonella 4,5,12,i:- S348/11. No effect was seen in the virulence or biofilm formation in either strain. In conclusion, the outbreak of CTX-M within this farm was caused by the incursion and spread of a successful, novel CTX-M-1-Incl1 plasmid, pIFM3804. Relating to the prevalence of this plasmid, carriage of multiple resistance genes together with the use of multiple antibiotics could partly explain its widespread presence within this farm.