N i Development and horizontal gene transfer of triclosan resistance in Staphylococcus Thesis presented for the Degree of Philosophiae Doctor by Paul F. Seaman, BSc (Hons) Cardiff School of Biosciences Cardiff University March 2007 UMI Number: U584980 All rights reserved INFORMATION TO ALL USERS The quality of this reproduction is dependent upon the quality of the copy submitted. In the unlikely event that the author did not send a complete manuscript and there are missing pages, these will be noted. Also, if material had to be removed, a note will indicate the deletion. Dissertation Publishing UMI U584980 Published by ProQuest LLC 2013. Copyright in the Dissertation held by the Author. Microform Edition © ProQuest LLC. All rights reserved. This work is protected against unauthorized copying under Title 17, United States Code. ProQuest LLC 789 East Eisenhower Parkway P.O. Box 1346 Ann Arbor, Ml 48106-1346 If we knew what it was we were doing, it would not be called research, would it? Albert Einstein ii Acknowledgements I have received great support from many quarters throughout this project. Firstly I would like to acknowledge Prof A. Denver Russell, who very sadly died in September 2004 - one year into this project. Prof. Russell’s enthusiasm, knowledge, friendliness and ideas have been a constant inspiration and I count myself very lucky to have worked with him. I would like to say a huge thank you to my supervisor, Dr Martin J Day, for his constant advice, enthusiasm and infinite ideas. It has been great to have a supervisor who has encouraged me to follow my own ideas and provided support at every opportunity. I would also like to thank Dr Dietmar Ochs of Ciba Speciality Chemicals for his advice and regular consultations. Of course, I must thank my fellow 0.30 people. In particular I would like to acknowledge Dr Kevin Ashelford, who, in addition to providing many cups of tea and an excellent selection of biscuits, gave advice on several of the bioinformatics aspects of this work. My thanks also go to Dr Gordon Webster and Dr Deborah Simpson who kindly offered me advice upon many occasions. I must also thank Dr Ant Hann for his help with the electron microscopy and entertaining chats and Mr Alan Pauli, PHLS, UHW, Cardiff, for supplying many of the clinical strains. An extra special thank you must to go to Mrs Joan Hubbard, who constantly looked after me, kept the lab running smoothly and always saved me the green sweets! I am hugely grateful to my parents for supporting and encouraging me through both my first degree and PhD. It hasn’t been the easiest few years for any of us, but your love and strength has been inspirational. Finally... Siwan, my girlfriend, proof reader, financier and friend. Diolch yn fawr. Dwi’n amau fydden i erioed wedi dechrau y PhD heb dy gymorth di, heb son am ei orffen. Diolch yn fawr am dy gariad a gymorth.. .ac am adael i mi chwarae rygbi. Scientific Publications and Presentations Parts of this work have been published in scientific journals and presented orally as detailed below: Seaman, P, Day, MJ, Russell, AD & Ochs, D. (2004) Susceptibility of capsular Staphylococcus aureus strains to some antibiotics, triclosan and cationic biocides. J Antimicrob Chemother 54, 696-698. Seaman, PF. (2005) Gene transfer of biocide resistance in methicillin-resistant Staphylococcus aureus. All-Wales Microbiology Meeting, Gregynog Hall, Newtown, UK. Seaman, PF, Ochs, D. & Day, MJ (2007) Small colony variants; a novel mechanism for triclosan resistance in methicillin-resistant Staphylococcus aureus. J Antimicrob Chemother. 59,43-50. Seaman, PF, Ochs, D. & Day, MJ (2007) FabI mutations, drug resistance and fitness costs of reduced triclosan susceptibility in Staphylococcus aureus. Submitted to J Antimicrob Chemother. Seaman, PF, Ochs, D. & Day, MJ (2007) Gene transfer of biocide resistance in staphylococci. Submitted to J Antimicrob Chemother Seaman, PF, Ochs, D. & Day, MJ (2007) Carriage and antimicrobial susceptibility of Staphylococci in an undergraduate teaching class Submitted to FEMS Microbiol Lett Seaman, PF, Ochs, D. & Day, MJ (2007) Comment on: Triclosan resistance in methicillin-resistant Staphylococcus aureus expressed as small colony variants: a novel mode of evasion of susceptibility to antiseptics. J Antimicrob Chemother. In Print. iv I have also published and presented additional work: Seaman, PF, Miller, RV & Day, MJ. (2005) Isolation and characterization of three novel bacteriophages and the host from the Great Salt Plains, Oklahoma. American Society of Microbiology 105th General Meeting, Atlanta GA, USA. Murray, TZ, Seaman, PF, Day, MJ & Miller RV. (2006) Halomonas spp. bacteriophages from the Great Salt Plains Microbial Observatory. American Society of Microbiology 106th General Meeting, Orlando, FL. USA. Seaman, PF & Day MJ. (2007) Isolation and characterization of a bacteriophage with an unusually large genome from the Great Salt Plains National Wildlife Refuge, Oklahoma, USA. FEMS Microbiol Ecol. 60, 1-13. v Summary Staphylococcus aureus 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 S. aureus (MRSA) - strains of S. aureus 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 S. aureus isolates and that MRSA was less susceptible to triclosan than methicillin- sensitive S. aureus (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 S. aureus can evolve reduced susceptibility to triclosan through spontaneous mutation. MICs of 1-4 mg/L were achieved by a C284T mutation of fabl, 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 LP10, are equally active against reduced susceptibility S. aureus 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 S. aureus 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 S. aureus. Reduced susceptibility to triclosan could not be disseminated amongst S. 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 S. aureus 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 S. aureus 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,