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Title: A study of the mechanism of action of triclosan and PCMX
Author: Otter, Rachel.
Awarding Body: University of Brighton
Current Institution: University of Brighton
Date of Award: 2006
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Triclosan and PCMX are two broad-spectrum antimicrobial agents of particular interest to Reckitt Benckiser. Triclosan is one of the most widely used biocides today and is incorporated into increasing numbers of household products such as toothpaste, mouth washes and underann deodorants. It is generally accepted that biocides have multiple target sites within the bacterial cell; however, recent reports have identified mutations in the E. coli fabI gene as being linked to decreased susceptibility to triclosan. The fabI gene encodes for enoyl reductase, an important enzyme in fatty acid biosynthesis. Conclusions were drawn that triclosan had a single, primary target in bacteria and the previously reported effects of triclosan on membrane structure were secondary effects arising from specific inhibition of the fatty acid biosynthetic pathway. In this study the mechanisms of action of both triclosan and PCMX were investigated against the hygiene indicators E. coli NCTC 8196 and S. aureus NCTC 10788. Initially, interactions of both biocides with the bacterial cell were investigated using adsorption isotherms. Triclosan was found to possess an unusual Z-shaped isotherm, indicative of a concentration dependant breakdown of cellular structure. Disruption of E. coli cells by sonication also increased the cells sorptive capacity for triclosan, indicating that exposure to concentrations of triclosan above 6ppm induced a structurally damaging event to the cell. PCMX was shown to have a complex S-shaped isotherm, with 2 saturation plateuxs. The termination of the primary uptake phase corresponded to the threshold concentration required for bactericidal activity and leakage of cellular constituents, the secondary uptake was thought to represent penetration to new sorptive sites within the cell itself. The bacteriostatic and bactericidal properties of both biocides were probed by calculation of MICs and MBCs and by production of time survivor curves. Both compounds were found to have bacteriostatic or bactericidal activity against E. coli NCTC 8196 and S. aureus NCTC 10788 depending on concentration. Concentration exponents were detennined and were found to be in the region of S for triclosan and 6 for PCMX, indicating that their activity is greatly reduced on dilution. Non-growing cells were used to investigate membrane damage caused by the two biocides. The leakage of intracellular potassium and changes in the permeability of the membrane to specific ions and protons were used as subtle indicators of membrane damage. PCMX was shown to cause substantial membrane damage, indicated by rapid, gross potassium leakage and by the rapid loss of absorbance of E. coli spheroplasts stabilised in salt solutions, when exposed to bactericidal concentrations of the agent. The effect of triclosan on the membrane appeared to be more subtle, with cells exhibiting a more concentration dependant loss of potassium and spheroplast stability over time. Bactericidal concentrations of triclosan were shown to induce the translocation of protons and the uncoupling of oxidative phosphorylation. Both compounds were able to manifest significant damage to the bacterial cell under conditions which limit the significance of the ENR system to bacterial survival. Whilst ENR may be a target for the growth inhibitory properties of triclosan, this study indicates that the disinfectant potential of the agent derives from additional target damage. Combinations of triclosan and PCMX were also investigated for possible signs of synergy. Whilst adsorption profiles for either compound in the presence of the other clearly indicated changes in the sorptive capacity of the cell, the enhanced bacteriostatic and bactericidal effects of combinations of the biocides were thought to have arisen from the non-linear relationship between antibacterial activity and concentration, rather than 'true' synergy itself.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available