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Development and horizontal gene transfer of triclosan resistance in Staphylococcus aureus.

Seaman, Paul Francis 2007. Development and horizontal gene transfer of triclosan resistance in Staphylococcus aureus. PhD Thesis, Cardiff University.

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<italic>Staphylococcus aureus</italic> is a major cause of hospital-acquired infections that are becoming increasingly difficult to treat because of the organism's ability to acquire resistance to current antimicrobial agents. Particular attention has been focussed on the evolution of methicillin-resistant <italic> S. aureus</italic> (MRSA) - strains of <italic>S. aureus</italic> that are, in some cases, resistant to almost all known antibiotic classes. One method used to control the spread of MRSA has been the use of topical washes that include triclosan, a potent antimicrobial with particular activity against Gram-positive organisms. Triclosan has traditionally been classed as a biocide and is also used in a broad spectrum of consumer healthcare products, including toothpastes and deodorants. It has also been used to prevent bacterial growth through incorporation into plastics used during food preparation or sutures used to close wounds following surgery. However, in 1991 resistance to triclosan was reported and was described to transfer in association with mupirocin resistance. This was followed by reports that resistance was present in 7.5% of <italic> S. aureus</italic> isolates and that MRSA was less susceptible to triclosan than methicillin- sensitive <italic>S. aureus</italic> (MSSA). It later emerged that, contrary to previous thinking, triclosan targets a specific bacterial protein, Fabl. We aimed to characterize the development of reduced susceptibility to triclosan in MSSA and MRSA and to identify whether triclosan does have a single, specific target. We also set out to elucidate the potential for triclosan resistance to be disseminated by horizontal gene transfer (HGT). By using extensive microbiological and genetic techniques we found that <italic> S. aureus</italic> can evolve reduced susceptibility to triclosan through spontaneous mutation. MICs of 1-4 mg/L were achieved by a C284T mutation offabl, compared to wild-type MICs of -0.03 mg/L. However, reduced susceptibility was also observed in non-fabl mutants, implying that other mechanisms of resistance are available (and that triclosan has targets other than Fabl). We have shown that triclosan induces the leakage of potassium ions from cells, an indication that triclosan targets the cytoplasmic membrane. However, whilst reduced susceptibility to triclosan did confer reduced susceptibility to the lethal effects of 7.5 mg/L triclosan, this effect was ameliorated by higher concentrations of triclosan. Indeed, in-use concentrations of the commercial preparation of triclosan, Irgacide LP 10, are equally active against reduced susceptibility <italic> S. aureus</italic> and wild-type. Therefore, the evolution of reduced-susceptibility to triclosan is of ambiguous clinical significance. We found that spontaneous mutation to reduced susceptibility was not associated with a significant fitness cost, augmenting its potential for emergence in nature. Evolution of reduced susceptibility did not confer co-resistance to other antimicrobials and MRSA and MSSA strains were equally susceptible. An assessment of commensal <italic> S. aureus</italic> carried amongst the student population of Cardiff revealed that reduced susceptibility to triclosan is rare in this population. However, coagulase-negative staphylococci (CoNS) showed consistently higher MICs for triclosan and may represent an amenable reservoir of resistance. There was no indication that mupirocin and triclosan resistance have co-transferred in the past. Indeed, there appeared to be no relationship between resistance to either of these compounds in <italic>S. aureus</italic>. Reduced susceptibility to triclosan could not be disseminated amongst 5". aureus, or related Gram-positive bacteria by transduction, conjugation or transformation. Importantly, during the course of this work we discovered that triclosan could select for <italic> S. aureus</italic> small-colony variants (SCVs) that were coincidently resistant to gentamicin and penicillin. These were slow growing and illustrated the typical SCV phenotype. SCVs were more readily transformable than wild-type cells and may represent an enduring reservoir of resistance determinants. In conclusion, we found that <italic>S. aureus</italic> could develop reduced susceptibility to triclosan by spontaneous mutation or the evolution of SCVs. However, the level of resistance is of ambiguous significance. We propose that triclosan does target Fabl, but also has other cellular targets, particularly at higher concentrations. Whilst the evolution of reduced susceptibility and the occurrence of SCVs should be monitored these should not preclude the use of triclosan as part of infection control procedures. However, to reduce the opportunities for resistance, infection control procedures should not rely upon a single antimicrobial to provide the panacea for nosocomial infections.

Item Type: Thesis (PhD)
Status: Unpublished
Schools: Biosciences
Subjects: Q Science > QH Natural history > QH301 Biology
Q Science > QH Natural history > QH426 Genetics
ISBN: 9781303209314
Date of First Compliant Deposit: 30 March 2016
Last Modified: 12 Feb 2016 23:12

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