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 brainheart infusion broth. The culture was diluted 1 in 100 and 1 µL (approximately 104 cfu) was inoculated on to MuellerHinton 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. MuellerHinton 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, MuellerHinton 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.
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Notes
J Antimicrob Chemother 2000;45: 549550
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, 4214.[Abstract]
2
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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, 3056.
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, 35177.[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, 52331.[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, 118.[Abstract]