Mechanisms of tolerance to Melaleuca alternifolia (tea tree) oil in Pseudomonas aeruginosa

Chelsea Papadopoulos

Research output: ThesisDoctoral Thesis

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Abstract

[Truncated abstract] Pseudomonas aeruginosa, an important opportunistic pathogen, is resistant to a wide array of functionally and structurally diverse antimicrobial agents including antibiotics, disinfectants and biocides. P. aeruginosa is more resistant than other Gram negative bacteria to tea tree oil (TTO), the essential oil steam distilled from the leaves of Melaleuca alternifolia and comprised of over 100 terpene hydrocarbon components and their oxygenated derivatives. TTO is an established topical antimicrobial agent, with antibacterial, antiviral and antifungal properties. Intrinsic antimicrobial resistance mechanisms in P. aeruginosa include the low permeability of the outer membrane and expression of multi-drug efflux pumps. A series of multi-drug efflux mutants from the resistance-nodulation-cell division family was obtained and their susceptibility to TTO and several components examined. This demonstrated that TTO and the components terpinen-4-ol, 1,8-cineole and α-terpineol were substrates of MexAB-OprM, using both pump deletion mutants and the pump inhibitor Phe-arg β-naphthylamide dihydrochloride. In complementation studies, the addition of mexAB-oprM to deletion mutants restored susceptibility to these agents to that of the wild-type, confirming the role of MexAB-OprM in tolerance to TTO and these three components. ... An increase in susceptibility to ticarcillin and Timentin occurred in PAO1 following serial subculture in terpinen-4-ol. Susceptibility to ticarcillin has been associated with expression of the MexCD-OprJ system in P. aeruginosa. A library of transposon mutants was created to find additional mechanisms by which P. aeruginosa could tolerate TTO. The library yielded a total of 20 mutants that were more susceptible than parental strains to TTO and/or terpinen-4-ol. The insertion site of the transposon was identified in 14 mutants and, in four mutants, this was a gene related to flagellar biosynthesis. Flagella deficient mutants have previously demonstrated enhanced susceptibility to the membrane-disrupting surfactant sodium dodecyl sulfate and this echoes the increased susceptibility to TTO and terpinen-4-ol observed. Three non-sibling surA mutants were also identified. SurA is involved in the correct folding of outer membrane proteins, including porins, in Gram negative bacteria: surA mutants of Escherichia coli have phenotypes that are characteristic of a defective cell envelope, including an increased susceptibility to hydrophobic agents. The increase in susceptibility to hydrophobic TTO and terpinen-4-ol in the surA mutants is consistent with this and represents the first report linking SurA function to antimicrobial resistance in P. aeruginosa. In conclusion, several Mex efflux systems of P. aeruginosa including MexAB-OprM, MexCD-OprJ and MexEF-OprN, as well as the LPS core, outer membrane integrity and a functioning flagella biosynthetic pathway contribute to the tolerance of this organism to TTO and/or several components.
Original languageEnglish
QualificationDoctor of Philosophy
Publication statusUnpublished - 2008

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