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Broth microdilution assays were used to determine minimum inhibitory concentrations (MICs) and fractional inhibitory concentration indices (FICIs) of tea tree oil (TTO), tobramycin, colistin and aztreonam (ATM) against clinical cystic fibrosis-associated Pseudomonas aeruginosa (CFPA) isolates (n = 20). The minimum biofilm eradication concentration (MBEC) and fractional biofilm eradication concentration index (FBECI) were also determined using a similar microbroth dilution checkerboard assay, with biofilms formed using the MBEC device®. TTO was effective at lower concentrations against multidrug-resistant (MDR) CFPA isolates (n = 3) in a biofilm compared to in a planktonic state (MBEC 18·7-fold lower than MIC). CFPA within biofilm was less susceptible to ATM, colistin and tobramycin compared to planktonic cells (MBEC 6·3-fold, 9·3-fold, and 2·1-fold higher than MIC respectively). All combinations of essential oil and antibiotic showed indifferent relationships (FICI 0·52–1·72) when tested against planktonic MDR CFPA isolates (n = 5). Against CFPA isolates (n = 3) in biofilm, combinations of TTO/aztreonam and TTO/colistin showed indifferent relationships (mean FBECI 0·85 and 0·60 respectively), whereas TTO/tobramycin showed a synergistic relationship (mean FBECI 0·42). The antibiofilm properties of TTO and the synergistic relationship seen between TTO and tobramycin against CFPA in vitro make inhaled TTO a promising candidate as a potential therapeutic agent.

Background: The efficacy, tolerability and acceptability of a tea tree oil gel (200 mg/g) and face wash (7 mg/g) were evaluated for the treatment of mild to moderate facial acne. Methods: In this open-label, uncontrolled phase II pilot study, participants applied tea tree oil products to the face twice daily for 12 weeks and were assessed after 4, 8 and 12 weeks. Efficacy was determined from total numbers of facial acne lesions and the investigator global assessment (IGA) score. Tolerability was evaluated by the frequency of adverse events and the mean tolerability score determined at each visit. Product acceptability was assessed via a questionnaire at the end of the study period. Results: Altogether 18 participants were enrolled, of whom 14 completed the study. Mean total lesion counts were 23.7 at baseline, 17.2 at 4, 15.1 at 8 and 10.7 at 12 weeks. Total lesion counts differed significantly over time by repeated measures anova (P < 0.0001). The mean IGA score was 2.4 at baseline, 2.2 at 4, 2.0 at 8 and 1.9 at 12 weeks, which also differed significantly over time (P = 0.0094). No serious adverse events occurred and minor local tolerability events were limited to peeling, dryness and scaling, all of which resolved without intervention. Conclusion: This study shows that the use of the tea tree oil products significantly improved mild to moderate acne and that the products were well tolerated.

Essential oils from the Western Australian (WA) Eucalyptus mallee species Eucalyptus loxophleba, Eucalyptus polybractea, and Eucalyptus kochii subsp. plenissima and subsp. borealis were hydrodistilled from the leaves and then analysed by gas chromatography&ndash, mass spectrometry in addition to a commercial Eucalyptus globulus oil and 1,8-cineole. The main component of all oils was 1,8-cineole at 97.32% for E. kochii subsp. borealis, 96.55% for E. kochii subsp. plenissima, 82.95% for E. polybractea, 78.78% for E. loxophleba 2, 77.02% for E. globulus, and 66.93% for E. loxophleba 1. The Eucalyptus oils exhibited variable antimicrobial activity determined by broth microdilution, with E. globulus and E. polybractea oils showing the highest activities. The majority of microorganisms were inhibited or killed at concentrations ranging from 0.25% to 8.0% (v/v). Enterococcus faecalis and Candida albicans were the least susceptible organisms, whilst Acinetobacter baumannii was the most sensitive. In conclusion, all oils from WA Eucalyptus species showed microorganism inhibitory activity, although this varied according to both the Eucalyptus species and the microorganism tested. These data demonstrate that WA Eucalyptus oils show activity against a range of medically important pathogens and therefore have potential as antimicrobial agents.

This study investigated the hypothesis that the salt adaptation response of Enterococcus faecalis alters susceptibility to tea tree oil (TTO). Six E. faecalis isolates were adapted to 6.5 % NaCl, and then exposed to TTO in phosphate-buffered saline (PBS). One isolate was also exposed to TTO in Brain Heart Infusion Broth (BHIB). The viability of salt-adapted and non-adapted control cells was determined at 0, 45 and 90 min and compared. MICs for several antibiotics and TTO were also determined by E test and broth microdilution, respectively. Results showed that susceptibility to TTO in PBS was significantly reduced after salt adaptation for five isolates (83 %) (P < 0.05). Mean differences between salt-adapted and non-adapted cell counts were 2.51 log at 45 min and 2.13 log at 90 min. However, when E. faecalis ATCC 19433 was exposed to TTO in BHIB, no significant differences were seen. In conclusion, salt adaptation resulted in reduced susceptibility to TTO in PBS for the majority of isolates, indicating that cross protection had occurred. This effect was absent in BHIB, suggesting that the uptake of compatible solutes from the growth medium protected non-adapted cells from TTO. Whether this has implications for the clinical effectiveness of TTO remains to be determined.

Over-the-counter acne treatments containing tea tree oil from the plant Melaleuca alternifolia are widely available, and evidence indicates that they are a common choice amongst those self-treating their acne. The aims of this review were to collate and evaluate the clinical evidence on the use of tea tree oil products for treating acne, to review safety and tolerability and to discuss the underlying modes of therapeutic action.

The aim of this study was to seek additional data on the antimicrobial susceptibility of Staphylococcus spp. after habituation to low levels of the topical antimicrobial agent tea tree (Melaleuca alternifolia) oil. Meticillin-susceptible Staphylococcus aureus (MSSA), meticillin-resistant S. aureus (MRSA) and coagulase-negative staphylococci (CoNS) were habituated to 0.075% tea tree oil for 3 days. Subsequently, the susceptibility of five isolates each of MSSA, MRSA and CoNS to fusidic acid, mupirocin, chloramphenicol, linezolid and vancomycin was determined by Etest, and susceptibility to tea tree oil, terpinen-4-ol, carvacrol and triclosan was determined by agar dilution. Following habituation to 0.075% tea tree oil, antimicrobial MICs differed between control and habituated isolates on 33 occasions (out of a possible 150), with MICs being higher in habituated isolates on 22 occasions. Using clinical breakpoint criteria, one MSSA isolate changed susceptibility category from vancomycin-susceptible (MIC = 2 mu g/mL) to intermediate susceptibility (MIC = 3 mu g/mL) after habituation in one of two replicates. For the non-antibiotic antimicrobial agents, MICs of habituated and control isolates differed on 12 occasions (out of a possible 120); 10 occasions in MRSA and 2 occasions in MSSA. MICs were higher for habituated isolates on five occasions. However, all the differences were one serial dilution only and were not regarded as significant. Habituation to sublethal concentrations of tea tree oil led to minor changes in MICs of antimicrobial agents, only one of which may have been clinically relevant. There is no evidence to suggest that tea tree oil induces resistance to antimicrobial agents. (C) 2013 Elsevier B.V. and the International Society of Chemotherapy. All rights reserved.

This study investigated the effects of the volatile terpene-rich oil from Melaleuca alternifolia (tea tree oil) on the formation of biofilms and the adhesion of C. albicans cells to both biotic and abiotic surfaces. Biofilm formation on polystyrene was significantly inhibited for 70% of the isolates at the lowest test concentration of 0.016% of tea tree oil (TTO) when quantified by XTT and 40% of isolates when measured by crystal violet staining. Adhesion to polystyrene, quantified by crystal violet staining, was significantly reduced for 3 isolates at 0.031%, 6 isolates at 0.062% and 0.125% and for all 7 isolates at 0.25% TTO. Reductions in adhesion were not due to loss of viability (at concentrations of ≤ 0.125%) or interactions between the TTO and polystyrene. Similarly, adhesion to buccal epithelial and HeLa cells was also significantly reduced in the presence of 0.016-0.062% TTO. Treatment with 0.125% TTO, but not 0.062%, decreased the cell surface hydrophobicity of C. albicans, indicating one potential mechanism by which adhesion may be reduced. These data demonstrate that sub-inhibitory TTO reduces the adhesion of C. albicans to both human cells and polystyrene, inhibits biofilm formation and decreases cell surface hydrophobicity.

This study was conducted to determine the frequencies at which single-step mutants resistant to tea tree oil and rifampicin occurred amongst the Gram-positive organisms Staphylococcus aureus, Staphylococcus epidermidis and Enterococcus faecalis. For tea tree oil, resistance frequencies were very low at

Due to the lack of antiseptics with sporicidal properties, the activity of tea tree oil (TTO)-containing products was investigated according to the EN 14347 European suspension method. The activity of different concentrations of TTO in 0.001% Tween 80, as well as an alcoholic hygienic skin wash (AHSW) and an alcoholic handrub (AHR), was compared to the sporicidal disinfectants sodium hypochlorite (SH) and hydrogen peroxide (HP), and the antiseptic povidone iodine (PVI). These formulations were assessed after 30, 60 and 120 minutes against two strains of spore-forming bacteria: Bacillus cereus and Bacillus subtilis . The TTO formulated products and PVI did not achieve >4 log 10 reduction in spore counts, thus did not meet the European standard requirements. Nevertheless, 5%, 10% and 15% TTO in 0.001% Tween 80 significantly reduced the concentration of bacterial spores when compared with the water control, and were generally more effective than PVI. At a concentration of 15%, TTO was more active than 0.05% SH against spores of both strains after 30 minutes' contact time, while 5% TTO in Tween 80 was generally more active than the AHSW that also contained 5% TTO. These findings emphasise the fact that it is necessary to assess final formulations rather than the active component by itself as excipients may interfere with the activity. The alcohol-containing AHSW and AHR did not demonstrate any sporicidal properties, confirming previous studies showing that alcohol is not sporicidal and suggesting no synergy between TTO and alcohol against spores. Hence these should not be used for hand hygiene or surface decontamination if spores are involved. Further studies should be carried out to investigate the potential sporicidal activity of TTO directly on human skin in vivo with volunteers andlor by using an ' ex vivo ' test that uses freshly excised human skin and which would allow testing of any biocide as well as any spore-forming bacterial strain, including pathogens.

The activity of tea tree oil (TTO) and TTO-containing products was investigated according to the EN 1276 and EN 12054 European suspension methods. The activity of different concentrations of TTO, a hygienic skin wash (HSW), an alcoholic hygienic skin wash (AHSW) and an alcoholic hand rub (AHR) was investigated. These formulations were assessed in perfect conditions with the EN 12054 test, and in perfect conditions as well as in the presence of interfering substances with the EN 1276 test, against Staphylococcus aureus, Acinetobacter baumannii, Escherichia coli and Pseudomonas aeruginosa. With the latter test, the activity of the same formulations without TTO was also assessed as a control. With the EN 1276 test, the AHR achieved a >10(5)-fold reduction against all four test organisms within a 1-min contact time. The AHSW achieved a >or=10(5)-fold reduction against A. baumannii after a 1-min contact time and against S. aureus, E. coli and P. aeruginosa after a 5-min contact time. The efficacy of TTO appeared to be dependent on the formulation and the concentration tested, the concentration of interfering substances and, lastly, the organism tested. Nevertheless, 5% TTO achieved a >10(4)-fold reduction in P. aeruginosa cell numbers after a 5-min contact time in perfect conditions. TTO (5%) in 0.001% Tween 80 was significantly more active against E. coli and P. aeruginosa than against S. aureus and A. baumannii. With the EN 12054 test, after a 1-min contact time, 5% TTO in 0.001% Tween 80 and the AHSW achieved a >10(4)-fold reduction in E. coli and A. baumannii cell numbers, respectively, and the AHR achieved a >4 log10 reduction against all organisms tested. The formulations used in this study are now being tested using a novel ex vivo method as well as the in vivo European standard handwashing method EN 1499.

The in vitro activity of Melaleuca alternifolia (tea tree) oil against 161 isolates of oral bacteria from 15 genera was determined. Minimum inhibitory concentrations (MIC) and minimum bactericidal concentrations (MBC) ranged from 0.003 to 2.0% (v/v). MIC90 values were 1.0% (v/v) for Actinomyces spp., Lactobacillus spp., Streptococcus mitis and Streptococcus sanguis, and 0.1% (v/v) for Prevotella spp. Isolates of Porphyromonas, Prevotella and Veillonella had the lowest MICs and MBCs, and isolates of Streptococcus, Fusobacterium and Lactobacillus had the highest. Time kill studies with Streptococcus mutans and Lactobacillus rhamnosus showed that treatment with ≥0.5% tea tree oil caused decreases in viability of >3 log colony forming units/ml after only 30 s, and viable organisms were not detected after 5 min. These studies indicate that a range of oral bacteria are susceptible to tea tree oil, suggesting that tea tree oil may be of use in oral healthcare products and in the maintenance of oral hygiene.

The in vitro activity of Melaleuca alternifolia (tea tree) oil against dermatophytes (n = 106) and filamentous fungi (n = 78) was determined. Tea tree oil MICs for all fungi ranged from 0.004% to 0.25% and minimum fungicidal concentrations (MFCs) ranged from

Globally, scabies affects more than 130 million people at any time. In the developed world, outbreaks in health institutions and vulnerable communities result in a significant economic burden. A review of the literature demonstrates the emergence of resistance toward classical scabicidal treatments and the lack of effectiveness of currently available scabicides in reducing the inflammatory skin reactions and pyodermal progression that occurs in predisposed patient cohorts. Tea tree oil (TTO) has demonstrated promising acaricidal effects against scabies mites in vitro and has also been successfully used as an adjuvant topical medication for the treatment of crusted scabies, including cases that did not respond to standard treatments. Emerging acaricide resistance threatens the future usefulness of currently used gold standard treatments (oral ivermectin and topical permethrin) for scabies. The imminent development of new chemical entities is doubtful. The cumulative acaricidal, antibacterial, antipruritic, anti-inflammatory, and wound healing effects of TTO may have the potential to successfully reduce the burden of scabies infection and the associated bacterial complications. This review summarizes current knowledge on the use of TTO for the treatment of scabies. On the strength of existing data for TTO, larger scale, randomized controlled clinical trials are warranted. Refereed/Peer-reviewed

The effect of tea tree oil (TTO) on the formation of germ tubes by Candida albicans was examined. Two isolates were tested for germ tube formation (GTF) in the presence of TTO concentrations (% v/v) ranging from 0.25% (1/2 minimum inhibitory concentration [MIC]) to 0.004% (1/128 MIC). GTF at 4 h in the presence of 0.004 and 0.008% (both isolates) and 0.016% (one isolate) TTO did not differ significantly (P > 0.05) from controls. At all other concentrations at 4 h, GTF differed significantly from controls (P < 0.01). A further eight isolates were tested for GTF in the presence of 0.031% TTO, and at 4h the mean GTF for all 10 isolates ranged 10.0-68.5%. Two isolates were examined for their ability to form germ tubes after 1 h of pre-exposure to several concentrations of TTO, prior to induction of germ tubes in horse serum. Cells pre-exposed to 0.125 and 0.25% TTO formed significantly fewer germ tubes than control cells at 1 h (P < 0.05), but only those cells pre-exposed to 0.25% differed significantly from control cells at later time points (P < 0.01). GTF by C. albicans is affected by the presence of, or pre-exposure to, sub-inhibitory concentrations of TTO. This may have therapeutic implications.

The effect of some potentially interfering substances and conditions on the antimicrobial activity of Melaleuca alternifolia (tea tree) oil was investigated. Agar and broth dilution methods were used to determine minimum inhibitory and cidal concentrations of tea tree oil in the presence and absence of each potentially interfering substance. Activity was determined against Gram-positive and -negative bacteria, and Candida albicans. Minimum inhibitory or cidal concentrations differed from controls by two or more dilutions, for one or more organisms, where Tween-20, Tween-80, skim-milk powder and bovine serum albumin were assessed. These differences were not seen when assays were performed in anaerobic conditions, or in the presence of calcium and magnesium ions. The effect of organic matter on the antimicrobial activity of tea tree oil was also investigated by an organic soil neutralization test. Organisms were exposed to lethal concentrations of tea tree oil ranging from 1-10% (v/v), in the presence of 1-30% (w/v) dry bakers' yeast. After 10 min contact time, viability was determined. Ator = 1%, organic matter compromised the activity of each concentration of tea tree oil against Staphylococcus aureus and C. albicans. At 10% or more, organic matter compromised the activity of each tea tree oil concentration against Pseudomonas aeruginosa. Organic matter affected 1 and 2% tea tree oil, but not 4 and 8%, against Escherichia coli. In conclusion, organic matter and surfactants compromise the antimicrobial activity of tea tree oil, although these effects vary between organisms.

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.MICs were determined by broth microdilution in Mueller-Hinton medium supplemented with 0.002% (v/v) Tween 80.The MIC90 of TTO for all isolates tested was 4% (v/v) or less. Susceptibility to components tested varied between species.Pseudomonas spp. are susceptible to TTO and some of its components although they are less susceptible than many other bacteria tested previously.

Complementary and alternative medicines occupy a privileged marketing position in many countries, including Australia, being able to carry low-level health claims while being relatively unfettered by regulatory requirements. Consequently, many enjoy widespread use for therapeutic purposes in the absence of definitive efficacy or safety data. Occasionally, the therapeutic properties of complementary and alternative medicines are scrutinised more closely and more thoroughly than their conventional counterparts, leading to the confirmation or discrediting of their properties.

This study examined the effect of subinhibitory Melaleuca alternifolia (tea tree) essential oil on the development of antibiotic resistance in Staphylococcus aureus and Escherichia coli . Frequencies of single-step antibiotic-resistant mutants were determined by inoculating bacteria cultured with or without subinhibitory tea tree oil onto agar containing 2 to 8 times the MIC of each antibiotic and with or without tea tree oil. Whereas most differences in resistance frequencies were relatively minor, the combination of kanamycin and tea tree oil yielded approximately 10-fold fewer resistant E. coli mutants than kanamycin alone. The development of multistep antibiotic resistance in the presence of tea tree oil or terpinen-4-ol was examined by culturing S. aureus and E. coli isolates daily with antibiotic alone, antibiotic with tea tree oil, and antibiotic with terpinen-4-ol for 6 days. Median MICs for each antibiotic alone increased 4- to 16-fold by day 6. Subinhibitory tea tree oil or terpinen-4-ol did not greatly alter results, with day 6 median MICs being either the same as or one concentration different from those for antibiotic alone. For tea tree oil and terpinen-4-ol alone, day 6 median MICs had increased 4-fold for S. aureus ( n = 18) and 2-fold for E. coli ( n = 18) from baseline values. Lastly, few significant changes in antimicrobial susceptibility were seen for S. aureus and S. epidermidis isolates that had been serially subcultured 14 to 22 times with subinhibitory terpinen-4-ol. Overall, these data indicate that tea tree oil and terpinen-4-ol have little impact on the development of antimicrobial resistance and susceptibility.

Objectives: The aim of this study was to investigate the antimicrobial activity of a range of commercially available tea tree oil (TTO) products and to evaluate whether formulation plays a significant part in their antiseptic activity. Methods: The antimicrobial activity of the purchased products and control TTO solutions was assessed against Escherichia coli, Staphylococcus aureus, Salmonella typhimurium, Pseudomonas aeruginosa, and Candida albicans using well diffusion, broth microdilution, and broth macrodilution assays. Results: Zone sizes obtained by the agar well diffusion assay ranged from 0 to 49.8mm, with the more viscous and lipophilic products producing the smallest zones. Micro- and macrodilution methods showed that eight products had minimum inhibitory concentrations that were lower than the nonformulated TTO control. The remaining three products showed activity equivalent to the TTO control. Conclusions: In general, the commercially available antiseptic TTO products showed antimicrobial activity that was equivalent to, or greater than the nonformulated TTO control. This suggests that the TTO within these products has retained its antimicrobial activity. Furthermore, the enhanced activity of the products may be attributed to other antimicrobial excipients within the products such as preservatives, or to synergistic antimicrobial interactions between the TTO and other product excipients. The observation that the commercially available antiseptic TTO products tested in this study retained adequate antimicrobial activity emphasizes the importance of considering how product bases and excipients may interact with the active compound during formulation to ensure efficacy of the final product. Finally, the current data suggest that these TTO products may also be active in vivo. However, this can only be determined through further studies and in clinical trials.

SUMMARYComplementary and alternative medicines such as tea tree (melaleuca) oil have become increasingly popular in recent decades. This essential oil has been used for almost 100 years in Australia but is now available worldwide both as neat oil and as an active component in an array of products. The primary uses of tea tree oil have historically capitalized on the antiseptic and anti-inflammatory actions of the oil. This review summarizes recent developments in our understanding of the antimicrobial and anti-inflammatory activities of the oil and its components, as well as clinical efficacy. Specific mechanisms of antimicrobial and anti-inflammatory action are reviewed, and the toxicity of the oil is briefly discussed.

The susceptibility of 64 Malassezia furfur isolates to Melaleuca alternifolia oil was determined. The minimum inhibitory concentration for 90% of isolates was 0·25% by agar dilution and 0·12% by broth dilution. These data indicate that tea tree oil may be useful in the treatment of skin conditions involving M. furfur.

The essential oil of Melaleuca alternifolia, also known as tea tree or melaleuca oil, is widely available and has been investigated as an alternative antimicrobial, anti-inflammatory and anti-cancer agent. While these properties are increasingly well characterised, relatively limited data are available on the safety and toxicity of the oil. Anecdotal evidence from almost 80 years of use suggests that the topical use of the oil is relatively safe, and that adverse events are minor, self-limiting and occasional. Published data indicate that TTO is toxic if ingested in higher doses and can also cause skin irritation at higher concentrations. Allergic reactions to TTO occur in predisposed individuals and may be due to the various oxidation products that are formed by exposure of the oil to light and/or air. Adverse reactions may be minimised by avoiding ingestion, applying only diluted oil topically and using oil that has been stored correctly. Data from individual components suggest that TTO has the potential to be developmentally toxic if ingested at higher doses, however, TTO and its components are not genotoxic. The limited ecotoxicity data available indicate that TTO is toxic to some insect species but more studies are required.

Objectives: The essential oil of Melaleuca alternifolia (tea tree oil) and its components have antimicrobial activity against a wide range of Gram-positive and Gram-negative bacteria, fungi and viruses. The mechanism(s) by which Pseudomonas aeruginosa NCTC 10662 maintains a decreased susceptibility to tea tree oil and components was investigated. Results: Ethylene diamine tetraacetic acid enhanced the antimicrobial activity of tea tree oil and terpinen-4-ol against stationary phase P. aeruginosa while polymyxin B nonapeptide enhanced the activity of tea tree oil and γ-terpinene. Pre-treatment with the protonophore carbonyl cyanide m-chlorophenylhydrazone increased the susceptibility of exponential phase cells to sub-inhibitory concentrations of tea tree oil, terpinen-4-ol and γ-terpinene, indicating that intrinsic tolerance to tea tree oil and components is substantially energy dependent. Conclusions: Increased tolerance to tea tree oil in P. aeruginosa is directly related to the barrier and energy functions of the outer membrane, and may involve efflux systems.

Objectives: The aim of this study was to investigate the mechanism of action of tea tree oil and its com- ponents against Candida albicans, Candida glabrata and Saccharomyces cerevisiae. Methods: Yeast cells were treated with tea tree oil or components, at one or more concentrations, for up to 6 h. During this time, alterations in permeability were assessed by measuring the leakage of 260 nm absorbing materials and by the uptake of Methylene Blue dye. Membrane fluidity was measured by 1,6-diphenyl-1,3,5- hexatriene fluorescence. The effects of tea tree oil on glucose-induced medium acidification were quantified by measuring the pH of cell suspensions in the presence of both tea tree oil and glucose. Results: The treatment of C. albicans with tea tree oil and components at concentrations of between 0.25 and 1.0% (v/v) altered both permeability and membrane fluidity. Membrane fluidity was also increased when C. albicans was cultured for 24 h with 0.016%-0.06% (v/v) tea tree oil, as compared with control cells. For all three organisms, glucose-induced acidification of the external medium was inhibited in a dose-dependent manner in the presence of 0.2%, 0.3% and 0.4% tea tree oil. Conclusions: Data from this study support the hypothesis that tea tree oil and components exert their anti- fungal actions by altering membrane properties and compromising membrane-associated functions.

The efficacy of formulations containing tea tree oil (TTO) has been assessed in vitro in previous studies. Products that passed the European suspension test guidelines were investigated further in this study, in vivo with volunteers using the European handwashing method (EN 1499) and ex vivo using freshly excised human skin samples. The activity of 5% TTO in 0.001% Tween 80, in a hygienic skin wash (HSW) and in an alcoholic hygienic skin wash (AHSW) was investigated and compared with that of a non-medicated soft soap (SS, control). These formulations were assessed against Escherichia coli K12 as recommended by the European standard. In-vivo results showed that 5% TTO in Tween 80 and the AHSW were significantly more active than the SS after 1 min of handwashing. When assessed ex vivo, these two products were also significantly more active than the reference soap after 1 min of rubbing. Both methods showed that 5% TTO in Tween 80 was generally, although not always, more active than a handwash formulation, and that the AHSW was generally more active than the HSW, although this difference was not significant. The formulations tested, as well as the SS, were more active when assessed in vivo than ex-vivo against E. coli, although only the SS and the HSW were significantly more active in vivo. There appeared to be a pattern in the comparison between ex vivo and in vivo results. The antiseptics tested were, on average, 1.28+/-0.06 times more active when assessed in-vivo than when assessed ex vivo. Nevertheless, the main outcome of the European handwashing method is for the formulation tested to be significantly more active than the SS; both 5% TTO in Tween 80 and the AHSW achieved this both in-vivo and ex-vivo. TTO in Tween 80 and in formulations met the European in-vivo method requirements.

K . A . H A M M E R , C . F . C A R S O N A N D T . V . R I L E Y . 2003. Aims: To investigate the in vitro antifungal activity of the components of Melaleuca alternifolia (tea tree) oil. Methods and Results: Activity was investigated by broth microdilution and macrodilution, and time kill methods. Components showing the most activity, with minimum inhibitory concentrations and minimum fungicidal concentrations of £0AE25%, were terpinen-4-ol, a-terpineol, linalool, a-pinene and b-pinene, followed by 1,8-cineole. The remaining components showed slightly less activity and had values ranging from 0AE5 to 2%, with the exception of b-myrcene which showed no detectable activity. Susceptibility data generated for several of the least watersoluble components were two or more dilutions lower by macrodilution, compared with microdilution. Conclusions: All tea tree oil components, except b-myrcene, had antifungal activity. The lack of activity reported for some components by microdilution may be due to these components becoming absorbed into the polystyrene of the microtitre tray. This indicates that plastics are unsuitable as assay vessels for tests with these or similar components. Significance and Impact of the Study: This study has identified that most components of tea tree oil have activity against a range of fungi. However, the measurement of antifungal activity may be significantly influenced by the test method.

The in vitro activities of ketoconazole, econazole, miconazole, and tea tree oil against 54 Malassezia isolates were determined by agar and broth dilution methods. Ketoconazole was more active than both econazole and miconazole, which showed very similar activities. M. furfur was the least susceptible species. M. sympodialis , M. slooffiae , M. globosa , and M. obtusa showed similar susceptibilities to the four agents.

The in-vitro activity of a range of essential oils, including tea tree oil, against the yeast candida was examined. Of the 24 essential oils tested by the agar dilution method against Candida albicans ATCC 10231, three did not inhibit C. albicans at the highest concentration tested, which was 2.0% (v/v) oil. Sandalwood oil had the lowest MIC, inhibiting C. albicans at 0.06%. Melaleuca alternifolia (tea tree) oil was investigated for activity against 81 C. albicans isolates and 33 non-albicans Candida isolates. By the broth microdilution method, the minimum concentration of oil inhibiting 90% of isolates for both C. albicans and non-albicans Candida species was 0.25% (v/v). The minimum concentration of oil killing 90% of isolates was 0.25% for C. albicans and 0.5% for non-albicans Candida species. Fifty-seven Candida isolates were tested for sensitivity to tea tree oil by the agar dilution method; the minimum concentration of oil inhibiting 90% of isolates was 0.5%. Tests on three intra-vaginal tea tree oil products showed these products to have MICs and minimum fungicidal concentrations comparable to those of non-formulated tea tree oil, indicating that the tea tree oil contained in these products has retained its anticandidal activity. These data indicate that some essential oils are active against Candida spp., suggesting that they may be useful in the topical treatment of superficial candida infections.

Objectives: The purpose of this study was to determine the susceptibility of a range of transient and commensal skin flora to the essential oil of Melaleuca alternifolia , or tea tree. Methods: A modified broth microdilution method was used. Polyoxyethylene sorbitan mono-oleate detergent was added to the test medium to enhance solubility of the tea tree oil. Results: Serratia marcescens had the lowest minimum inhibitory concentration (MIC 90 ) of 0.25%. The highest MIC 90 was 3% for Pseudomonas aeruginosa . The lowest minimum bactericidal concentration (MBC 90 ) was 0.25% for S. marcescens and Klebsiella pneumoniae , whereas the highest was 8% for Staphylococcus capitis . Conclusions: S. aureus and most of the gram-negative bacteria tested were more susceptible to tea tree oil than the coagulase-negative staphylocci and micrococci. These results suggest that tea tree oil may be useful in removing transient skin flora while suppressing but maintaining resident flora.

Tea-tree oil (TTO) is the essential oil derived from an Australian native plant that has been used for hundreds of years, mainly for its antimicrobial and anti-inflammatory properties. With the advent of ever-increasing resistance to antibiotics and biocides amongst organisms, TTO may play an important role in the health industry in the fight against resistance.

Tea tree oil, an essential oil from the Australian plant Melaleuca alternifolia, has broad antimicrobial activity and is incorporated into a diverse range of pharmaceutical and cosmetic products (2, 3). Blackwell (1) described a patient with typical signs and symptoms of bacterial vaginosis (BV) who treated herself with tea tree oil vaginal pessaries. After treatment, the patient was symptom free and the vaginal flora comprised predominantly gram-positive bacilli. It was suggested that the use of tea tree oil for the treatment of BV be further assessed. Therefore, the aim of this study was to evaluate the activities of tea tree oil against lactobacilli and a range of organisms associated with BV. Reference (n = 9) and stored clinical (n = 24) isolates were obtained from the culture collections of the Western Australian Centre for Pathology and Medical Research (PathCentre) and the Department of Microbiology at The University of Western Australia. Recent clinical isolates (n = 69) were recovered from vaginal swabs submitted to PathCentre and identified according to the Wadsworth Anaerobic Bacteriology Manual (5). Three isolates of Gardnerella vaginalis were kindly provided by Helen MacDonald of the Women’s and Children’s Hospital, Adelaide, Australia. Organisms were tested against doubling dilutions of tea tree oil (batch 93/04, Australian Plantations Pty. Ltd., Wyrallah, Australia) prepared in agar or broth medium. Bacteroides, Prevotella, fusobacteria, and anaerobic gram-positive cocci were tested in supplemented brucella laked-blood agar (5), with 0.5% (vol/vol) Tween 20 added to enhance oil solubility. G. vaginalis and Mobiluncus spp. were tested in Columbia agar base supplemented with 1% Proteose Peptone 3 and 5% chocolatized sheep blood. Lactobacilli (n = 26) were tested by the broth microdilution method using de Man-Rogosa-Sharpe (MRS) broth (Unipath Ltd., Basingstoke, United Kingdom). Selected BV isolates (n = 13) were tested by a broth macrodilution method (5) using brain heart infusion broth (BHIB) supplemented with 1 μg of vitamin K and 5 μg of hemin per ml, with a final test volume of 2 ml. A final concentration of 0.001% (vol/vol) Tween 80 was added to both MRS and supplemented BHIB, to enhance oil solubility. Preparation of inocula, inoculation of susceptibility tests, incubations, and determination of MICs and/or MBCs were carried out as described elsewhere (5). Table ​Table11 lists MIC data obtained by the agar dilution assay. In addition, for five G. vaginalis isolates tea tree oil MICs were 0.06%, and for three of the four Mobiluncus isolates the MICs were 0.03%, with 0.06% for the fourth. By broth macrodilution, MICs for all six Bacteroides isolates were 0.06%. The MBCs for five isolates were 0.06%, while the MBC for the remaining isolate was 0.12%. MICs and MBCs for two Prevotella isolates were 0.03%. The MICs for four of the five Peptostreptococcus anaerobius isolates were 0.06%, with 0.03% for the fifth. MBCs were 0.03% for one isolate, 0.06% for three isolates, and 0.12% for the remaining isolate. MICs for the lactobacilli ranged from 0.12 to 2.0% and the MIC at which 90% of isolates were inhibited (MIC90) was 2.0%. MBCs for lactobacilli ranged from 0.25 to 2.0%, and the MBC90 was 2.0%. TABLE 1 Tea tree oil MICs for organisms associated with BV These MIC data are similar to those obtained in previous studies which indicated that a variety of anaerobic and aerobic bacteria were susceptible to tea tree oil (2–4). The data also show that all lactobacilli tested were appreciably more resistant to tea tree oil than organisms known to be associated with BV, with at least a twofold difference in MIC90 results. Therefore, the clinical success reported by Blackwell (1) may be due, in part, to the susceptibility of BV-associated organisms to tea tree oil and the relative resistance of commensal lactobacilli. This difference in susceptibility may allow formulation of products that will selectively kill or inhibit certain organisms while having a minimal effect on the commensal lactobacilli. Appropriate trials are now urgently needed to determine whether these theoretical benefits will translate into clinical practice.