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Loss of Antibiotic Resistance: Analysis of Phenotype and Related gene expression - OD2004

Description
Policy relevance- Antibiotic resistance in zoonotic pathogens isolated from man has been an ncreasing world-wide issue, such that recent World Health Organisation meetings (October 1997 & June 1998) discussed the role that the use of antimicrobials in animals may contribute to the antibiotic resistance of these pathogens entering the food chain. With food safety a priority issue for the U.K. government, a recent report by the House of Lords on antibiotic resistance has stimulated discussion and the forthcoming report by a working party of the ACMSF investigating antibiotic resistance in food-borne pathogens is awaited. Of particular current concern is the increasing numbers of multiply antibiotic-resistant (MAR) zoonoses, such as S. typhimurium DT104 abd Campylobacter, from animals with the potential risk to public health that such bacteria pose. Recently, EU legislation has banned use of certain antibiotics from use as growth promotion on the assumption that removal of the selective pressure will result in loss of MAR bacteria. Monitoring the outcome of removal requires confirmation that phenotypic loss of resistance is associated with loss of genes rather than loss of expression of resistance genes.

Objective- Until recently, MAR bacteria were presumed to contain transferable genetic elements that contained multiple promiscuous genetic elements (i.e. plasmids, transposons, tnegrons). Recent advances have shown that MAR can be due to mutations within native chromosomal non-mobile genes. Indeed, fluoroquinolones select MAR mutants. Thus, this proposal seeks to inform the Ministry of the phenotypic and genotypic outcome of removal of antibiotic selective pressure upon MAR genes in zoonotic pathogens by analysis of expression of these genes from resistant and sensitive isolates.

Use of results- The ministry will be informed of the risks associated with MAR associated with chromosomal genes and there provide data additional to those gained for similar studies with mobile genetic elements and thereby inform policies relating to removal of antibiotics as growth promoters.
Objective
01 To describe bacterial isolates from field studies and collaborative in vivo studies in terms of antimicrobial sensitivity and to determine the number of isolates which possess the mAR phenotype.

02 To determine whether upregulated expression of mar,sox, acr or rob of the panel isolates of (a) salmonella, (b) E. coli and (c) Campylobacter collected in 01 above is responsible for the MAR phenotype.

03 To determine the expression of mar, sox, acr or rob genes of defined (i.e. NCTC typed strains) (a_ Salmonella, (b) E.coli and (c) Campylobacter after exposure and subsequent removal from to antibiotics used for growth promotion and therapeutic use.

04 To quantify by QRT-PCR, the levels of expression of mar, sox, acr and rob leading to multiple antibiotic resistance in isoltes of (a) Salmonella, (b) E. coli and (c) Campylobacter.

05 To determine the precise nature of the mutations in mar, sox acr and rob leading to multiple antibiotic resistance in isolates of (a) Salmonella, (b) E. coli and (c) Campylobacter.

06 To determine the precise nature of the mutations in mar, sox, acr and rob leading to higher-level multiple antibiotic resistance in isolates of (a) Salmonella, (b) E. coli and (c) Campylobacter and to determine the MICs in comparison with standard disc diffusionb results and the rate of foward mutation to second-stage, high level resistance with and without selective pressure.

07 To provide the Ministry with a definitive account of the risk associated with the MAR-like phenotype in isolates from all sources deemed sensitive by traditional approaches to determining relative sensitivities.


higher-level multiple antibiotic resistance in isolates of
Project Documents
• Final Report : Loss of antibiotic resistance: Analysis of phenotype and related gene expression   (104k)
Time-Scale and Cost
From: 2000

To: 2003

Cost: £240,869
Contractor / Funded Organisations
Veterinary Laboratories Agency
Keywords
Animal Diseases              
Animal Health              
Antimicrobial Resistance              
Bacteria              
Biotechnology              
Campylobacter              
Plants and Animals              
Salmonella              
Fields of Study
Animal Health