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A generic toolbox for the molecular epidemiology of CTX-M bearing verotoxigenic E. coli and vaccine trial - OD2028

Extended spectrum beta lactamase (ESBL) enzymes are capable of breaking down certain antibiotics called cephalosporins which are commonly used in hospitals as front line treatments (Bonnet, 2004). Until recently these were mainly found in bacteria of the Klebsiella or Enterobacter species from hospital specialist care units, and had mutant beta lactasmases of the TEM or SHV varieties (Livermore et al., 2007). Subsequently, CTX-M group ESBLs have become prominent and are common in Escherichia coli, and with many infections in 'complicated community' patients (Canton and Coque, 2006; Livermore and Hawkey, 2005). The first isolation of a CTX-M ESBL in E. coli from food producing animals occurred in the autumn of 2004 at a Welsh dairy farm (Liebana et al., 2006) and there have now been more than 50 further farm cases. Farm animals are now recognised as important carriers of ESBL E. coli and Salmonella (Caratolli, 2008). Recently a verotoxin (VTEC) producing O26 E. coli isolate bearing the CTX-M type 14 ESBL enzyme was isolated from a calf on a dairy farm in North Yorkshire. The O26 type is quite common in food producing animals and often associated with virulence genes including vtx (verocytotoxin) and eae (intimin) (Pearce et al., 2006). This serotype is acknowledged as an emerging pathogen associated with disease in both ruminants and humans (Aktan et al., 2007). Any exposure to third generation cephalosporins would provide a means for selection and proliferation of these potential pathogens.
CTX-M genes are carried on circular pieces of DNA called plasmids. The plasmids themselves are now recognised as the epidemic agents of this CTX-M not the host bacteria. CTX-M genes have been found in a wide range of enteric bacteria (Woodford, 2005). Some plasmids are highly promiscuous (Liebana et al., 2006) while others are host restricted. The molecular epidemiology of these resistance plasmids is now a major issue for understanding where the CTX-M genes come from, their spread (Carattoli et al., 2006; Canton and Coque, 2006) and therefore the discovery and introduction of measures to prevent their dispersal.
A year long study of the CTX-M14 VTEC O26 E. coli at the farm in North Yorkshire and associated heifer rearing unit will be used to assess transmission of the CTX-M 14 plasmid to other gut bacteria and zoonotic pathogens (e.g. Salmonella if present) and the effect of animal husbandry. The survival of the CTX-M14 VTEC O26 E. coli and the plasmid in different farm environments, like manure stacks, will be determined. A model for plasmid spread to other bacteria will be developed. This model would be used in subsequent cases to predict the risk of transmission to other disease causing bacteria and assess the likely impact of any control measures such as vaccination or animal husbandry. Such assessment would enable the application of suitable control measures to at least limit the spread of CTX-M E. coli and reduce their introduction into the food chain.
A vaccine prepared with CTX-M14 E. coli was applied for the control of diarrhoea at the first outbreak farm in Wales (Liebana et al., 2006). Monitoring of this farm by the VLA suggested a drop in the prevalence of CTX-M14 E. coli from 57 to 3% after vaccination and the farmer reported reduced scouring in calves and of metritus in calving cows. However this vaccination was not part of a scientific study and this drop in bacteria could have been the result of several unknown factors. These issues have been discussed with Defra who consider that it is essential to pursue the potential for the control of CTX-M ESBL E. coli by vaccination. In the present proposal the effect of vaccination on the prevalence of E. coli bearing CTX-M14 in calves will be evaluated within a controlled trial at the VLA. A vaccine trial on a working farm is considered too high a risk as changes in husbandry or antibiotic therapy may jeopardise interpretation of any observed effects.
The development and application of appropriate DNA based methods for distinguishing between plasmids is key to the evaluation of their potential risk. Such risks may include transfer to different hosts, such as VTEC E. coli or Salmonellae, or the carriage of other virulence or antibiotic resistance genes. Appreciation of these factors would enable control measures on farms to be focused towards such higher risk plasmids (e.g. highly transferable with antibiotic resistance and virulence genes). The molecular make up of a range of 20 plasmids will be compared following DNA sequencing and representative genetic markers selected for translation to a DNA detection platform (array) for plasmid characterisation in existing monitoring activities. The molecular attributes of CTX-M plasmids will be collated in a public database which will provide a resource for international surveillance and tracing of epidemic strains. This would greatly help determining the source of ESBLs in the food chain and better understanding of the link between animal and human cases. This project will provide Defra with the following:-
1) Survival and persistence of CTX-M14 VTEC O26 E. coli on the farm and of this plasmid in different bacteria. A mathematical model to assess plasmid transmission dynamics and the effect that any interventions, such as vaccination, may have on prevalence in cattle.
2) A trial for the effect of an vaccination on the prevalence of CTX-M14 bearing E. coli in cattle.
3) A generic tool box for the molecular characterisation of plasmids will be developed based on the Identibac DNA array platform. This will provide simultaneous typing of plasmids, virulence and antibiotic resistance genes in ongoing VLA monitoring activities (e.g. Defra project FZ2200).
1) Epidemiological investigation of CTX-M 14 gene in E. coli, gut commensals and survivability in different farm environments.

2) Detailed molecular characterisation of plasmids for marker selection.

3) Production of a European database for CTX-M plasmid molecular data collation.

4) Development and evaluation of an autogenous vaccine for E. coli bearing the CTX-M14 plasmid.

5) Development of a generic molecular tool box for molecular typing of plasmids.
Project Documents
• EVID4 - Final project report : OD2028 Final Report   (2581k)
Time-Scale and Cost
From: 2009

To: 2012

Cost: £905,939
Contractor / Funded Organisations
Veterinary Laboratories Agency
Animal Health              
Antimicrobial Resistance              
Method Development              
Plants and Animals              
Veterinary Medicines              
Fields of Study
Animal Health