a Microbiology Department, St Thomas' Hospital, London SE1 7EH, UK; b The Oil Fields, Chatswood, PO Box 767, NSW 2067, Australia
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Abstract |
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Introduction |
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A recent major review of antibiotic resistance emphasized the importance of hospital infection control, and the control of these organisms,4 and many authorities have reiterated the key role of hand-washing with appropriate disinfectants in this process.5,6
Mupirocin nasal ointment (for staphylococci) and hand disinfectants are widely used to control the carriage and spread of these organisms, but resistance is increasing and eradication with current agents is not always successful.712 Alternative strategies are required and more effective agents are needed.
Tea tree oil is obtained by steam distillation of the leaves of Melaleuca alternifolia, a tree native to Australia, and is reported to have antibacterial, antifungal, antiviral, anti-inflammatory and analgesic properties.13 Currently, tea tree oil is used in cosmetics and healthcare products and has recently re-emerged as an effective antiseptic.14 A limited number of published controlled clinical trials support this latter use.1517
The concentrations of tea tree oil found in commercially available products range from 2 to 5%.18 Terpinen-4-ol is the main antimicrobial component but other components, such as -terpineol, also have antimicrobial activities similar to those of terpinen-4-ol.19,20 Previous studies of the in vitro activity of tea tree oil have employed broth dilution and disc diffusion techniques.14,18,21 We have performed timekill studies with a standard tea tree oil and an oil extracted from the superior tree (Clone 88) with increased concentrations of terpinen-4-ol,22 to determine the killing effect of the oils against clinically significant bacterial isolates.
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Materials and methods |
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We examined clinical isolates from Guy's and St Thomas' Hospitals, chosen for their varying degrees of sensitivity and resistance to a range of antibiotics. Isolates of MRSA, GRE and multidrug-resistant Klebsiella species, isolated from outbreaks of hospital infection, were included. The isolates included were S. aureus (four isolates: two methicillin sensitive, two methicillin resistant/vancomycin tolerant), Enterococcus faecium (four: one vancomycin sensitive, three vancomycin resistant), Enterococcus faecalis (three: two vancomycin sensitive, one vancomycin resistant), Pseudomonas aeruginosa (four: two gentamicin sensitive, two gentamicin resistant), Stenotrophomonas maltophilia (two) and Klebsiella pneumoniae (three: one gentamicin sensitive, two gentamicin resistant). Organisms were stored at 70°C in glycerol broth. Fresh subcultures were used for each experiment.
Tea tree oils
We tested two types of tea tree oil supplied by The Oil Fields (Sydney, NSW, Australia). Standard oil had a 34.8% terpinen-4-ol and 5.5% cineole content, whereas the Clone 88 oil had a 43.1% terpinen-4-ol and a 1% cineole content.
Preparation of inocula
Inocula for the timekill determinations were prepared following NCCLS guidelines.23 Organisms were grown overnight at 37°C in BrainHeart Infusion broth (BHI; Oxoid, Basingstoke, UK). The overnight broth was adjusted to a 0.5 McFarland standard in IsoSensitest broth (ISB; Oxoid, Basingstoke, UK) and a further dilution was made by inoculating 200 µL in glass flasks containing 20 mL of ISB. The final bacterial concentration in the flasks was (15) x 105 cfu/mL. The flasks for all isolates were shaken at 150 rpm (Certomat, B. Braun Biotech, Aylesbury, UK) for 90 min at 37°C (120 min for the pseudomonads) to ensure that the organisms were out of their lag phases and into their logarithmic phases.
Timekill studies
The timekill studies were performed according to NCCLS guidelines.23 Each isolate was inoculated into three flasks, one as a growth control and one for each oil type. After the 90 min (or 120 min) initial incubation period, an aliquot of the broth was taken from each flask to determine the initial inoculum. The tea tree oil and Tween 80 (SigmaAldrich Ltd, Poole, UK) were pre-mixed and added to each designated flask; Tween 80 only was added to the control flask. The final concentrations of the oil and Tween 80 were 5% and 0.5%, respectively. All flasks were shaken at 150 rpm at 37°C, and samples subcultured at 0, 0.5, 1, 1.5, 2, 4 and 6 h for all isolates except the enterococci. The enterococci were subcultured at 0, 10, 20, 30, 60, 90 and 120 min. Colony counts were performed by making appropriate dilutions in physiological saline, plating 100 µL of each dilution on to pre-warmed blood agar (Columbia base agar, Oxoid) supplemented with 7% sterile defibrinated horse blood (TCS Botolph, Clayton, UK) and incubated for 48 h at 37°C. Viable counts were calculated to give cfu/mL, and kill curves were plotted with time against the logarithm of the viable count. Each experiment was performed twice on separate occasions.
Determining timekill endpoints
A bactericidal effect is defined as a 3 log decrease in the cfu/mL or a 99.9% kill over a specified time.23 The definition of kill for this study has been described by May et al.,24 together with modifications based on a suggestion by Handwerger & Tomasz that a kill can be determined at 6 h.25 A constant logarithmic rate of kill has been assumed during a timekill. A 90% kill at 6 h is equivalent to a 99.9% kill at 24 h. In this study the kill measurement was determined by the actual reduction in viable counts at 6 h for each isolate.
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Results |
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The two tea tree oils were more active against methicillin-sensitive S. aureus (MSSA) than against MRSA. Both isolates of MSSA were killed within 30 min by both oils. The standard oil killed the MRSA by 6 h (Figure 1). The cloned oil killed one of the two isolates of MRSA by 1.5 h but the other isolate was not killed until 3.5 h.
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Discussion |
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Many claims have been made about the wonders of tea tree oil but few clinical studies have been published. However, in vitro susceptibility studies of its activity against a variety of organisms suggest that tea tree oil may have a place in modern medicine.
Tea tree oil is a complex mixture of terpenes and related alcohols with over 100 components.26 However, up to 90% of the whole oil content is made up by the following components: terpinen-4-ol, 1,8-cineole, -terpineol, terpinolene and
- and
-terpinene. International standards of tea tree oil have been most recently defined by ISO 4730.27 The main antimicrobial component is terpinen-4-ol. Recent studies indicate that the component 1,8-cineole, previously held responsible for undesirable side effects (mucus and skin irritation), is now thought not to be the agent responsible for hypersensitivity reactions.28,29 Cytotoxicity through the use of high concentrations of tea tree oil has been reported; however, limited data are available, suggesting that more in vivo work is required to confirm this.30,31 In this study an oil concentration of 5% in broth was chosen to reflect the normal concentration found in some commercially available products.18
The two oils provided were chemically different. The standard oil provided a concentration of terpinen-4-ol and 1,8-cineole found in commercial preparations of tea tree oil. The oil of Clone 88 was selected for its higher content of the active ingredient terpinen-4-ol and its lower content of 1,8-cineole.14 Oils with increased concentrations of terpinen-4-ol have displayed enhanced antimicrobial activity20 and our results support this.
Most reported studies on tea tree oil are based on the measurement of MICs and MBCs. One limitation of these procedures is the inability of the method to determine how quickly an agent acts on the organisms. In our studies, we assessed the viability of the organisms against tea tree oil with timekill determinations. This method determines the viability of the organisms after contact with the oil for a specified time period.
The results of the timekill studies show the oil of Clone 88 to be more or equally effective compared with the standard oil. Some organisms such as Klebsiella spp. and S. maltophilia were rapidly killed by both oils but organisms that were killed more slowly showed a greater susceptibility to the cloned oil than to the standard oil.
There were differences in the activity of the oils against the different genera of bacteria and even within some genera, notably the enterococci. The standard oil produced a significantly more rapid decrease in viable count during the first 10 min for E. faecium than for E. faecalis but the cloned oil produced a rapid kill within 10 min for both species. MRSA isolates were killed more slowly than MSSA, and the standard oil was less effective than the cloned oil. Although we have not tested S. epidermidis, previous MIC studies found S. aureus to be more susceptible to tea tree oil than was S. epidermidis.21
The antibiotic susceptibility pattern for the organisms did not appear to predict the activity of the oil against most of the organisms. For enterococci and klebsiellae there was no difference in kill rate if the organism was multi-resistant. However, the oils were less active against antibioticresistant isolates of staphylococci and P. aeruginosa and, against these isolates, the cloned oil was more active than the standard oil.
Previous studies have reported MICs and MBCs of tea tree oil against the same species of organisms that we have tested by the timekill approach.13,21 These report tea tree oil MICs of 0.120.5% for S. aureus (including MRSA) and K. pneumoniae, 0.51% for vancomycin-resistant enterococci and 25% for P. aeruginosa, and MBCs of 0.120.5% for K. pneumoniae, 0.252% for S. aureus and 25% for P. aeruginosa. These differences in MIC and MBC results for tea tree oil against the species are reflected in our timekill studies.
There were problems with timekill studies of P. aeruginosa. Despite the extra time provided for the initial inoculum to achieve exponential phase, some isolates of P. aeruginosa could not be used in our tests because they had not reached their logarithmic phase of growth in that time. Results of isolates exhibiting regrowth were repeated to ensure reproducibility. It has been demonstrated previously that P. aeruginosa required a higher concentration of oil to produce an effective kill.21,32
There are also inherent problems to performing MIC, MBC and timekill determinations with tea tree oil. The oil is insoluble in water and must be solublized by the use of detergents or emulsifiers.33 In this study, Tween 80 was used, and it is important to mix the Tween 80 with the oil before the addition of the oil into the shaking flask. Unfortunately, the oilTween 80 mixture forms a turbid suspension. This prevents visual determination of growth in the flasks and hinders the reading of endpoints in broth MICs.34
The mode of action of tea tree oil is unclear. A recent study has shown that tea tree oil stimulates autolysis in exponential and stationary phase cells of Escherichia coli.35 This study also showed that exponentially growing cells were more susceptible to autolysis by tea tree oil than are stationary cells.
Multi-resistant organisms are difficult to eradicate from skin, and staphylococci, enterococci and klebsiellae are transmitted by direct contact.36 Adherence to infection control protocol (e.g. hand-washing) is critical to reduce transmission but effective hand disinfectants are also required. One study has shown that tea tree oil is more active against the organisms associated with transient carriage than against commensal skin flora and thus may be useful in eliminating the transient flora while suppressing but maintaining commensal flora.21 The rapid kill rate of Clone 88 compared with standard tea tree oil shown for most of the organisms tested in the present study is encouraging and suggests that further clinical studies should be carried out. Tea tree oil in a topical formulation might eliminate organisms from carriage sites such as the hairline, axilla, nares, groin and perineum, and incorporation of tea tree oil in hand-washing formulations may reduce the transmission of many multi-resistant organisms associated with nosocomial infections.
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Acknowledgments |
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Notes |
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References |
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Received 23 July 1999; returned 15 November 1999; accepted 20 December 1999