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JAC Advance Access originally published online on May 30, 2006
Journal of Antimicrobial Chemotherapy 2006 58(2):449-451; doi:10.1093/jac/dkl200
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© The Author 2006. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved. For Permissions, please e-mail: journals.permissions@oxfordjournals.org

Susceptibility of pseudomonads to Melaleuca alternifolia (tea tree) oil and components

Chelsea J. Papadopoulos1,*, Christine F. Carson1, Katherine A. Hammer1 and Thomas V. Riley1,2

1 Microbiology and Immunology Discipline, School of Biomedical, Biomolecular and Chemical Sciences, The University of Western Australia 35 Stirling Highway, Crawley, Western Australia 6009, Australia 2 Division of Microbiology and Infectious Diseases, PathWest Laboratory Medicine WA Nedlands, Western Australia 6009, Australia

*Corresponding author. Tel: +61-8-9346-4730; Fax: +61-8-9346-2912; E-mail: chelsea{at}cyllene.uwa.edu.au

Received 1 November 2005; returned 18 February 2006; revised 16 April 2006; accepted 26 April 2006


   Abstract
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Objectives: Thirty isolates of Pseudomonas aeruginosa, 15 isolates of Pseudomonas putida and 11 isolates of Pseudomonas fluorescens were tested for susceptibility to tea tree oil (TTO), the essential oil of Melaleuca alternifolia, and the components terpinen-4-ol, {alpha}-terpineol, cineole, {gamma}-terpinene and {rho}-cymene.

Methods: MICs were determined by broth microdilution in Mueller–Hinton medium supplemented with 0.002% (v/v) Tween 80.

Results: The MIC90 of TTO for all isolates tested was 4% (v/v) or less. Susceptibility to components tested varied between species.

Conclusions: Pseudomonas spp. are susceptible to TTO and some of its components although they are less susceptible than many other bacteria tested previously.

Keywords: terpenes , terpinen-4-ol , Pseudomonas aeruginosa , essential oils , antimicrobials


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Tea tree oil (TTO), derived from the Australian native plant Melaleuca alternifolia (Myrtaceae), is becoming established as an alternative topical antimicrobial agent, with antibacterial, antiviral and antifungal properties ascertained both in vitro and in a growing number of clinical trials.1 Whilst organisms such as Staphylococcus aureus (including methicillin-resistant S. aureus) and Escherichia coli are relatively susceptible to the antimicrobial action of TTO and its components,1–3 with MICs of 0.5% and 0.25% respectively, one organism that appears to be more resistant is Pseudomonas aeruginosa. Broth microdilution MICs for P. aeruginosa range from 1% to 8%; however, only small numbers of strains have been tested with most sample sizes less than 5.4 An opportunistic pathogen that demonstrates a high and increasing level of multi-drug resistance,5,6 P. aeruginosa remains a major causative agent of nosocomial infections. Given the current deficiency of published data on TTO susceptibility in the genus Pseudomonas, and the resistance to conventional antibiotics in P. aeruginosa, the objectives of this study were to elucidate a clear susceptibility profile for P. aeruginosa as well as Pseudomonas putida and Pseudomonas fluorescens.


   Materials and methods
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Thirty P. aeruginosa isolates were obtained from the Sterile Site Culture Collection (SSCC) of the Microbial Contamination Unit of PathWest Laboratory Medicine WA, Nedlands. Two P. fluorescens isolates and one P. putida isolate were also obtained from the SSCC. The reference strains P. fluorescens NCTC 4755 and P. putida NCTC 10936 were obtained from the culture collection of the Microbiology and Immunology Discipline of The University of Western Australia. The remaining eight P. fluorescens isolates and 13 P. putida isolates were soil-derived isolates identified in our laboratory. Six soil samples originating from a tea tree plantation and two soil samples from local garden beds were used to isolate species of Pseudomonas. One gram of soil was mixed with 5 mL of saline, and following vigorous vortexing, samples were left at room temperature for 1 h. After further vortexing, samples were serially diluted and spread plated onto pre-dried Pseudomonas agar base plates supplemented with cetrimide, fucidin and cephalosporin (Oxoid, Basingstoke, UK). Isolates were identified to genus level via a series of biochemical tests and were identified to species level using API20NE strips (bioMérieux, Marcy l'Étoile, France).

TTO (batch W/E504) was kindly provided by Australian Plantations Pty Ltd, Wyrallah, New South Wales, Australia. The levels of the components determined by gas chromatography mass spectrometry analysis and the range specified by the international standard7 (shown in brackets) were as follows: 40.3% (>30%) terpinen-4-ol, 19.7% (10–28%) {gamma}-terpinene, 8.6% (5–13%) {alpha}-terpinene, 3.2% (trace–15%) 1,8-cineole, 3.2% (1.5–5%) terpinolene, 3.1% (1.5–8%) {alpha}-terpineol, 2.4% (1–6%) {alpha}-pinene, 2.4% (0.2–12%) {rho}-cymene, 1.6% (trace–7%) aromadendrene, 1.2% (trace–8%) {delta}-cadinene, 1.0% (0.5–4%) limonene, 0.5% (trace–3%) globulol, 0.4% (trace–1.5%) viridiflorol and 0.1% (trace–3.5%) sabinene. Terpinen-4-ol (purity 100%) was provided by SNP Natural Products (Sydney, NSW, Australia). Cineole (purity 99%) and {alpha}-terpineol (purity 95%) were purchased from Sigma Chemical Company (St Louis, MO, USA). {gamma}-Terpinene (purity 97%) and {rho}-cymene (purity 99%) were purchased from Aldrich Chemical Company (Milwaukee, WI, USA). Terpinen-4-ol and {alpha}-terpineol were selected on the basis of their known antimicrobial activity2 and the fact that they comprise ~40% of TTO. Cineole was selected since it has some antimicrobial activity and its concentration in TTO has been manipulated partly in response to the misconception that it is a skin irritant.1,2 {gamma}-Terpinene and {rho}-cymene were selected since they constitute up to 30% of TTO.

The susceptibility of Pseudomonas isolates to TTO, terpinen-4-ol, {gamma}-terpinene, cineole, {alpha}-terpineol and {rho}-cymene was determined using a broth microdilution method based on CLSI guidelines.8 Mueller–Hinton broth (MHB; Oxoid) was supplemented with 0.002% (v/v) Tween 80 (Sigma) (MHB-T) to enhance dispersion of the TTO or components. A stock of TTO or component at twice the desired final concentration was prepared in MHB-T and mixed vigorously by vortex. This was used to prepare serial doubling dilutions in a 96-well tray over the range 0.03–8% (v/v). Overnight broth cultures of organisms in MHB were adjusted in MHB-T so that the final concentration in each well following inoculation of the microtitre tray was ~5.0 x 105 cfu/mL. Inocula concentrations were confirmed by viable counts; after serial 10-fold dilution of the inoculum in sterile distilled water, four 10 µL samples from the appropriate dilution were inoculated onto well-dried blood agar plates. After incubation at 37°C for 18–24 h the number of cfu was counted and the concentration of the inocula calculated. After 18 h of incubation of trays at 37°C, the MIC was the lowest concentration of TTO that resulted in a clear well. Ten microlitres from each well was spot-inoculated onto pre-dried nutrient agar (NA) and incubated overnight at 35°C to determine the MBC. The MBC was defined as the concentration of TTO or component that killed 99.9% of the inoculum. Experiments were performed at least three times and the modal value selected.

The antibiotic susceptibility profiles of the isolates were determined using an agar dilution method with approved breakpoints for determining susceptibility and resistance.8 The antibiotics tested were cefepime, ceftazidime, ciprofloxacin, gentamicin, meropenem, ticarcillin/clavulanate and tobramycin.


   Results and discussion
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Table 1 shows susceptibility results for the Pseudomonas isolates to TTO and two of the five oil components tested. There was no difference in the susceptibility of the soil and clinical isolates. Both P. aeruginosa and P. fluorescens had MIC90 and MBC90 values of 4% for TTO. Similar values were obtained for {alpha}-terpineol. The MIC90 and MBC90 for terpinen-4-ol against P. aeruginosa were both >8% while for P. fluorescens the values were 2% and >8%, respectively. P. putida isolates had lower MBCs for TTO (1%) and {alpha}-terpineol (2%), while the MBC90 for terpinen-4-ol was 4%. MIC and MBC values for {gamma}-terpinene, cineole and {rho}-cymene were greater than 8% for all pseudomonads tested. As in previous studies,2 terpinen-4-ol and {alpha}-terpineol were the most active while cineole, {gamma}-terpinene and {rho}-cymene were less so.


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  		Table 1. Broth microdilution MICs and MBCs (%) of TTO and components against pseudomonad species

 
The antibiotic susceptibility profiles of the three species are shown in Table 2. All 56 isolates were susceptible to cefepime and all the P. fluorescens and P. putida isolates were susceptible to ciprofloxacin, gentamicin, meropenem and tobramycin.


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  		Table 2. Antibiotic susceptibility profiles of the Pseudomonas spp. determined by an agar dilution method (% susceptible)

 
P. aeruginosa and other Pseudomonas spp. are notorious for their involvement in nosocomial infections and their incidence of resistance to antibiotics. Adjunct or alternative treatments for Pseudomonas skin and wound infections that fall outside the realm of conventional antibiotics are needed. TTO is emerging as an alternative antimicrobial agent that is safe for topical applications.9 A large number of products containing TTO as the active antimicrobial agent are available but there are limited in vitro data on the susceptibility of pseudomonads to TTO and no clinical data on the efficacy of these products in cutaneous infections involving Pseudomonas spp. Many TTO products intended for wound management or hand-washing contain 5–10% (w/v) TTO and, given that the MBC90 of TTO for P. aeruginosa was 4%, it is possible that the use of such topical agents in both the treatment of wounds and other skin washing situations may be of benefit in preventing and reducing infection and transmission. Whether TTO products can effectively treat cutaneous infections in which Pseudomonas spp. are involved remains to be determined in clinical studies. The possibility that Pseudomonas spp. may develop resistance to TTO also remains and requires clarification.


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None to declare.


   Acknowledgements
 
The assistance of the Division of Microbiology & Infectious Diseases, PathWest Laboratory Medicine WA is greatly appreciated. This work was supported by a PhD scholarship (UWA-82A) from the Rural Industries Research and Development Corporation (to C. J. P.).


   References
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1 Carson CF, Hammer KA, Riley TV. (2006) Melaleuca alternifolia (tea tree) oil: a review of antimicrobial and other medicinal properties. Clin Microbiol Rev 19:50–62.[Abstract/Free Full Text]

2 Carson CF and Riley TV. (1995) Antimicrobial activity of the major components of the essential oil of Melaleuca alternifolia. J Appl Bacteriol 78:264–9.[Medline]

3 Hammer KA, Carson CF, Riley TV. (1999) Antimicrobial activity of essential oils and other plant extracts. J Appl Microbiol 86:985–90.[CrossRef][Medline]

4 Banes-Marshall L, Cawley P, Phillips CA. (2001) In vitro activity of Melaleuca alternifolia (tea tree) oil against bacterial and Candida spp. isolates from clinical specimens. Br J Biomed Sci 58:139–45.[Web of Science][Medline]

5 Jung R, Fish DN, Obritsch MD, et al. (2004) Surveillance of multi-drug resistant Pseudomonas aeruginosa in an urban tertiary-care teaching hospital. J Hosp Infect 57:105–11.[CrossRef][Web of Science][Medline]

6 Deshpande LM, Fritsche TR, Jones RN. (2004) Molecular epidemiology of selected multi-drug resistant bacteria: a global report from the SENTRY Antimicrobial Surveillance Program. Diagn Microbiol Infect Dis 49:231–6.[CrossRef][Web of Science][Medline]

7 International Organization for Standardization. (2004) ISO 4730:2004, Oil of Melaleuca, terpinen-4-ol type (tea tree oil). Geneva, Switzerland: ISO.

8 Clinical Laboratory Standards Institute. (2006) Methods for Dilution Antimicrobial Susceptibility Tests for Bacteria That Grow Aerobically: Approved Standard M7-A7 (CLSI, Villanova, PA, USA).

9 Hammer KA, Carson CF, Riley TV, et al. (2006) A review of the toxicity of Melaleuca alternifolia (tea tree) oil. Food Chem Toxicol 44:616–25.[CrossRef][Web of Science][Medline]


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