Comparative genomics of blaCTX-M plasmids from veterinary and human ' Escherichia coli ' and methods for their identification and differentiation

The blaCTX-M gene confers resistance to penicillins and cephalosporins and is now the most widely disseminated plasmid mediated Extended Spectrum beta-lactamase (ESBL). Plasmids harbouring blaCTX-M have been recovered from both human and animals isolates, with increasing evidence for the transmission between hosts which is a major public health concern. The aim of this study was to investigate the relationship of blaCTX-M plasmids from UK human and veterinary E. coli isolates with plasmids previously sequenced around the world, and develop molecular markers to identify and differentiate plasmids. Molecular markers were first established as a suitable method for identifying plasmids by studying the prevalence of IncK pCT-like plasmids, which were found to be associated with 30% of CTX-M-14 producers in the UK, with plasmids mobilising the gene between unrelated isolates from cattle, turkeys and humans. Seven blaCTX-M plasmids, belonging to four incompatibility groups, from E. coli were isolated, fully sequenced and annotated. The only human sequenced plasmid was pH19 the first IncZ blaCTX-M-14 to be sequenced. The plasmids sequenced from animals included pSAM7 from cattle, the first IncX4 plasmid to be sequenced with blaCTX-M-14b in a novel transposition unit. Plasmid pCH01 was isolated from a chicken isolate and harboured the blaCTX-M-3 and is the first IncA/C group CTX-M to be sequenced. The four IncI1╬│ blaCTX-M-1 plasmids from chicken (pCH02 and pCH03), cattle (pCT01) and turkey (pT01), are the first ST3 and IncI1╬│ blaCTX-M-1 plasmids to be sequenced. Comparative analysis found UK plasmids shared approximately 70-99% sequence coverage with previously published sequences from different hosts and bacterial species around the world. This demonstrated that plasmids in the UK were closely related to plasmids found elsewhere, and no genetic characteristics were identified why these plasmid could not exist in either human or animals isolates, with the main differences observed in the inserted resistance regions. In all plasmids the blaCTX-M was associated with ISEcp1, and included a novel blaCTX-M-14b and blaCTX-M-3 transposition unit in pSAM7 and pCH01 respectively. In 6/7 plasmids the ISEcp1-blaCTX-M was not associated with any other resistance regions, inserting as separate events. Molecular markers were designed from the comparative analysis between plasmids that were capable of both identifying and differentiating plasmids belonging to the same incompatibility group. Five groups were identified for IncX4, eight for IncZ, B and K, 12 for IncI1╬│ and 14 for IncA/C. Markers were used in screening of field isolates to identify similar plasmids, with novel combinations being observed, not previously identified in silico. These markers represent a new non-sequencing based tool to identify and characterise plasmids further, benefitting the study of plasmids and their epidemiology.