Characterisation of triclosan resistance in Salmonella enterica serovar Typhimurium.

Disinfectants are routinely used to sterilize surfaces and materials which may be at risk of bacterial contamination and provide a route by which humans can become infected. Recently a large number of domestic products have been marketed as antimicrobial and many contain triclosan which is an antimicrobial compound with a wide range of activity against many bacteria. Triclosan is commonly found in handwashes, washing up liquids, toothpaste and cosmetics amongst others. It is also impregnated into chopping boards and other surfaces as it can be incorporated into plastics. In the USA domestic triclosan use has become so common it can be detected in streams and rivers in the environment. Although triclosan kills bacteria effectively it is possible to select bacterial strains which are resistant to triclosan. Many of the mechanisms of antibiotic resistance in bacteria can also give resistance to other compounds including disinfectants. Efflux pumps are proteins that pump toxic molecules including antibiotics and disinfectants out of the cell making the cell resistant. Salmonella is a major cause of human disease and were responsible for over 12,500 cases of enteritis in the U.K in 2004. Most salmonella infections in humans are associated with consumption of poultry products (i.e. chicken and eggs). Our preliminary work has demonstrated that triclosan resistance can be easily generated in salmonella after exposure to triclosan but that the type of resistance differs. Three different levels of resistance to triclosan have been produced from various strains of salmonella in the laboratory. This is a new observation. This project proposes to determine the genes and proteins involved in triclosan resistance in salmonella and to evaluate whether unrestricted use of triclosan is likely to result in selection of antibiotic resistance as a byproduct. I propose to study examples of each of the three different types of triclosan resistance and determine what genes are involved in each phenotype. I also propose to determine whether the triclosan resistant strains are more or less able to become antibiotic resistant than their parents and determine what the prevalence of triclosan resistance is amongst a large collection of various salmonella isolated from animals and humans.

Recently the potential for biocide exposure to promote cross resistance to antibiotics has been identified. Triclosan is a broad spectrum antimicrobial incorporated in a wide array of domestic products which acts as an inhibitor of fatty acid biosynthesis by binding to FabI an enoyl-acyl carrier protein. S. Typhimurium is a major cause of human disease and was responsible for over 12,500 cases of enteritis in the U.K in 2004. I have selected triclosan resistant salmonella from a panel of strains including wild-type strains, DT104 isolates, cyclohexane tolerant strains, gyrA mutants and MAR strains. Analysis of these mutants revealed three distinct phenotypes: low level (MIC of triclosan <4mg/L), intermediate level (MIC of triclosan 8-32mg/L) and high-level mutants (MIC of triclosan >32mg/L). Sequencing of fabI revealed a substitution of glycine with valine at position 93 of FabI in various mutants. However this substitution did not correlate with susceptibility to triclosan. I propose to investigate the underlying genetic mechanism(s) of each distinct triclosan resistance phenotype by studying exemplars of each phenotype. The role of FabI in resistance will be defined by complementation of mutants with a FabI G93V substitution and by artificially introducing this substitution into a wild-type strain. Mutants will be studied by microarray analysis and any differences in gene expression between the three phenotypes identified. The contribution of active efflux to each resistance phenotype will be defined and the ability of each class of mutants to develop antibiotic resistance in the presence and absence of efflux pump inhibitors determined. Genomic libraries derived from each class of resistant mutant will be generated and expressed in E. coli, triclosan resistant colonies will be identified and the effector gene(s) characterized. The prevalence of triclosan resistance in a large panel of animal and human isolates of salmonella enterica will be determined.