Selection of resistance to the essential oil of Melaleuca alternifolia in Staphylococcus aureus

Robert R. S. Nelson

Department of Clinical Microbiology, Western Infirmary, Dumbarton Road, Glasgow G11 6NT, UK

Sir,

Interest has arisen in the use of the essential oil of Melaleuca alternifolia (tea-tree) as a topical agent, active against methicillin-resistant Staphylococcus aureus (MRSA).1 It possesses proven in vitro activity against S. aureus1,2 and it has been suggested that it may have a role to play in the eradication of MRSA carriage.13 This report describes the first detection of resistant sub-populations and in vitro selection of resistance to tea-tree oil (TTO) in S. aureus.

Essential oil of M. alternifolia complying with ISO 4730 (Health Imports, Bradford, UK) was used. To detect naturally occurring resistance, 100 isolates of MRSA from individual hospital in-patients were incubated overnight in brain–heart infusion broth. The culture was diluted 1 in 100 and 1 µL (approximately 104 cfu) was inoculated on to Mueller–Hinton agar (Oxoid, Basingstoke, UK) supplemented with 2.5% TTO and 0.5% Tween 20 (Sigma Chemicals, Poole, UK). Twenty isolates were inoculated per plate using a multi-point inoculator (Denley, Sussex, UK). Plates were incubated at 37°C for 48 h in air and examined for visible signs of growth.

Three clinical MRSA isolates (denoted as A, B and C) together with S. aureus NCTC 6571 and ATCC 25923 were used to detect resistant variants derived from a single clone. MICs of TTO were determined for each isolate. Mueller–Hinton agar supplemented with 0.5% Tween 20 (v/v) and 0.5, 1.0 or 2.0% TTO (v/v), was flooded with 1 mL of an overnight broth culture (approximately 109 cfu) of the test isolate. Plates were incubated at 37°C in air for 4 days and inspected for growth. Plate counts were determined by a colony counter (Gallenkamp, London, UK) and checked for the presence of free coagulase. A single colony for each isolate, from the plate with the highest TTO concentration demonstrating visible growth, was retained for MIC determination immediately and after 90 days' storage on Columbia agar slopes to determine stability.

To induce resistance, Mueller–Hinton agar supplemented with 0.5% Tween 20 (v/v) and TTO in the following concentrations (% v/v): 0.2, 0.4, 0.8, 1.0, 1.4, 2.0, 2.5, 3.0 and 4.0 was prepared. The above strains were subcultured on to agar with the lowest TTO concentration and incubated in air at 37°C for 4 days. Any growth present was subcultured to the next incremental plate and reincubated. This cycle was repeated until visible growth no longer occurred. Isolates from the highest incremental plate were checked for free coagulase production and retained for MIC determination immediately and after 90 days' storage as before. A microbroth method1 was used to determine the MIC, which was defined as the lowest concentration resulting in a significant decrease in the inoculum (>90%).

None of the 100 MRSA strains produced visible growth on agar containing 2.5% TTO. The remaining results are shown in the Table.Go


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Table. Frequency of occurrence of resistant sub-populations and MIC of TTO pre- and post-induction of resistance in five strains of S. aureus
 
Interest in TTO as a possible agent to eradicate MRSA carriage has been increasing and mention of it is given in the current UK guidelines for MRSA control.3 However, no studies have been performed to determine whether resistance is likely to arise, or is already present. Screening of 100 MRSA isolates revealed no resistance to 2.5% TTO (v/v), a concentration 10-fold that previously reported as the MIC for MRSA1,2 and a value which represents that found in lower-strength TTO preparations. This finding may be expected in a hospital in which TTO is not used on the general wards. Relatively resistant sub-populations do occur however, with all five test strains producing isolates with MICs from 0.5 to 16% (v/v) at a frequency of 1.2 x 10-8 to 1 x 10-5, this resistance proving to be relatively stable at 3 months. The presence of a sub-population of resistant mutants in isolates unexposed to TTO suggests that exposure will lead to selection of resistance. Stepwise induction of resistance was possible on solid media with a minimum four-fold increase in MIC and one strain attaining a stable MIC of 16% (v/v), greater than the 5 to 10% (v/v) concentration advocated in topical formulations. This stepwise induction of low-level resistance to TTO is analogous to the position of mupirocin at the time of its introduction in 1985,4 with induction of a stable MIC to mupirocin of 40 mg/L.4 Such low-level resistance has not subsequently proved to be clinically relevant,5 although it should be noted that commercial preparations of mupirocin contain 2000 mg/L, 50 times the highest MIC induced, whereas commercial 10% TTO preparations are unlikely to inhibit the growth of the most resistant strain induced in this study. Additionally, TTO is widely used in nonpharmaceutical preparations, frequently at concentrations identical to those used in this study to induce resistance. Tea-tree oil may well prove valuable as the basis for a topical agent to eradicate MRSA colonization but resistance will almost inevitably arise.

Notes

J Antimicrob Chemother 2000;45: 549–550

Tel: +44-141-211-2246; Fax: +44-141-211-2138; E-mail: rnelson{at}wghut-nhs.org.uk

References

1 . Carson, C. F., Cookson, B. D., Farrelly, H. D. & Riley, T. V. (1995). Susceptibility of methicillin-resistant Staphylococcus aureus to the essential oil of Melaleuca alternifolia. Journal of Antimicrobial Chemotherapy 35, 421–4.[Abstract]

2 . Nelson, R. R. (1997). In-vitro activities of five plant essential oils against methicillin-resistant Staphylococcus aureus and vancomycin-resistant Enterococcus faecium. Journal of Antimicrobial Chemotherapy 40, 305–6.[Free Full Text]

3 . Combined Working Party of the Hospital Infection Society and the British Society for Antimicrobial Chemotherapy. (1990). Revised guidelines for the control of epidemic methicillin-resistant Staphylococcus aureus. Journal of Hospital Infection 16, 351–77.[ISI][Medline]

4 . Casewell, M. W. & Hill, R. L. (1985). In-vitro activity of mupirocin (‘pseudomonic acid’) against clinical isolates of Staphylococcus aureus. Journal of Antimicrobial Chemotherapy 15, 523–31.[Abstract]

5 . Cookson, B. D. (1998). The emergence of mupirocin resistance: a challenge to infection control and antibiotic prescribing practice. Journal of Antimicrobial Chemotherapy 41, 11–8.[Abstract]