AAC JoumalsASMorg Effects of Melaleuca alternifolia (Tea Tree)Essential Oil and the Major Monoterpene Component Terpinen-4-ol on the Development of Single-and Multistep Antibiotic Resistance and Antimicrobial Susceptibility Katherine A.Hammer,Christine F.Carson,and Thomas V.Rileya.b Discipline of Microbiology and Immunology,School of Biomedical,Biomolecular and Chemical Sciences,The University of Western Australia,Crawley,Westem Australia, 6009,Australia,and Division of Microbiology and Infectious Diseases,PathWest Laboratory Medicine WA,Queen Elizabeth ll Medical Centre,Nedlands,Western Australia, Downloaded 6009,Australia 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 deter- from mined 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 寻 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. ://aac. 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 asm. 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) 叵 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. 心 Dlants have long been recognized as a valuable source of medic- ences the development of de novo antibiotic resistance in medi- inal agents.In particular,secondary plant metabolites such as cally important bacteria. essential oils have been used throughout history for therapeutic g purposes.The essential oil that is steam distilled from the Austra- MATERIALS AND METHODS lian native plant Melaleuca alternifolia(Myrtaceae),also known as Bacteria and antimicrobials.Reference and clinical isolates of Staphylo- melaleuca oil or tea tree oil (TTO),is used topically for its antimi- coccus aureus (n 18),Escherichia coli (n 21),and Staphylococcus epi- SHAN crobial and anti-inflammatory effects(5).The oil contains pre- dermidis (n=1),including antibiotic-resistant strains,were obtained dominantly monoterpenes and related alcohols,and its composi- from the Division of Microbiology and Infectious Diseases at PathWest 工 Laboratory Medicine WA.References strains were S.aureus NCTC 6571, tion is regulated by the international standard ISO 4730:2004(20). NCTC29213,and ATCC 25923,E.coli NCTC10418,ATCC25922,ATCC MICs of tea tree oil are typically between 0.125 and 2%(vol/vol) 43889,ATCC 43894,and ATCC 11775,and S.epidermidis ATCC 12228. (5,9),and bactericidal activity is largely attributable to nonspecific Ciprofloxacin,vancomycin,mupirocin,kanamycin,ampicillin,and ri- membrane effects(6,9).Clinical studies with tea tree oil products fampin were purchased from Sigma-Aldrich(St.Louis,MO).Benzalko- have shown efficacy for a range of superficial infections,including nium chloride (>95%pure)and triclosan (Irgasan;297%)were pur- acne,cold sores,tinea,and oral candidiasis,as well as for the de- chased from Fluka (Buchs,Switzerland).Terpinen-4-ol (97.0%)was ▣ colonization of methicillin-resistant Staphylococcus aureus car- obtained from Acros Organics(Geel,Belgium).Tea tree oil (batch A352) riage(5).Irritant reactions and contact allergy have been reported was provided by P.Guinane Pty.Ltd.,Cudgen,New South Wales,Austra- infrequently and can be minimized by avoiding the use of neat oil lia.The composition was determined by gas chromatography-mass spec- trometry,which was performed by Diagnostic and Analytical Services and storing oil correctly (5). Environmental Laboratory,Wollongbar,New South Wales,Australia, 刀 Two recent studies suggested that several bacteria that had and complied with ISO 4730(20).The major components of the oil were been exposed to tea tree oil subsequently were less susceptible to terpinen-4-ol (37.0%),y-terpinene (18.6%),a-terpinene (10.0%),and antibiotics in vitro(23,24).Although decreases in antibiotic sus- ceptibility were transient,this nonetheless raises concerns that tea tree oil hinders the effectiveness of conventional antibiotics by Received 16 September 2011 Retumned for modification 10 October 2011 either reducing susceptibility or influencing the development of Accepted 7 November 2011 resistance.This is particularly important if tea tree oil is to become Published ahead of print 14 November 2011 more widely used in hospital environments or in long-term care Address correspondence to K.Hammer,katherine.hammer@uwa.edu.au. facilities,such as for the decolonization of MRSA carriers (3,11, Copyright01,American Society for Microbiology.All Rights Reserved 30).The purpose of this study therefore was to examine whether dot101128/AAC.05741-11 tea tree oil or its major component,terpinen-4-ol(T4ol),influ- 0066-4804/12/$12.00 Antimicrobial Agents and Chemotherapy p.909-915 aac.asm.org 909Effects of Melaleuca alternifolia (Tea Tree) Essential Oil and the Major Monoterpene Component Terpinen-4-ol on the Development of Single- and Multistep Antibiotic Resistance and Antimicrobial Susceptibility Katherine A. Hammer, a Christine F. Carson, a and Thomas V. Rileya,b Discipline of Microbiology and Immunology, School of Biomedical, Biomolecular and Chemical Sciences, The University of Western Australia, Crawley, Western Australia, 6009, Australia,a and Division of Microbiology and Infectious Diseases, PathWest Laboratory Medicine WA, Queen Elizabeth II Medical Centre, Nedlands, Western Australia, 6009, Australiab 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 deter￾mined 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. Plants have long been recognized as a valuable source of medic￾inal agents. In particular, secondary plant metabolites such as essential oils have been used throughout history for therapeutic purposes. The essential oil that is steam distilled from the Austra￾lian native plant Melaleuca alternifolia (Myrtaceae), also known as melaleuca oil or tea tree oil (TTO), is used topically for its antimi￾crobial and anti-inflammatory effects (5). The oil contains pre￾dominantly monoterpenes and related alcohols, and its composi￾tion is regulated by the international standard ISO 4730:2004 (20). MICs of tea tree oil are typically between 0.125 and 2% (vol/vol) (5, 9), and bactericidal activity is largely attributable to nonspecific membrane effects (6, 9). Clinical studies with tea tree oil products have shown efficacy for a range of superficial infections, including acne, cold sores, tinea, and oral candidiasis, as well as for the de￾colonization of methicillin-resistant Staphylococcus aureus car￾riage (5). Irritant reactions and contact allergy have been reported infrequently and can be minimized by avoiding the use of neat oil and storing oil correctly (5). Two recent studies suggested that several bacteria that had been exposed to tea tree oil subsequently were less susceptible to antibiotics in vitro (23, 24). Although decreases in antibiotic sus￾ceptibility were transient, this nonetheless raises concerns that tea tree oil hinders the effectiveness of conventional antibiotics by either reducing susceptibility or influencing the development of resistance. This is particularly important if tea tree oil is to become more widely used in hospital environments or in long-term care facilities, such as for the decolonization of MRSA carriers (3, 11, 30). The purpose of this study therefore was to examine whether tea tree oil or its major component, terpinen-4-ol (T4ol), influ￾ences the development of de novo antibiotic resistance in medi￾cally important bacteria. MATERIALS AND METHODS Bacteria and antimicrobials. Reference and clinical isolates of Staphylo￾coccus aureus (n
18), Escherichia coli (n
21), and Staphylococcus epi￾dermidis (n
1), including antibiotic-resistant strains, were obtained from the Division of Microbiology and Infectious Diseases at PathWest Laboratory Medicine WA. References strains were S. aureus NCTC 6571, NCTC 29213, and ATCC 25923, E. coli NCTC 10418, ATCC 25922, ATCC 43889, ATCC 43894, and ATCC 11775, and S. epidermidis ATCC 12228. Ciprofloxacin, vancomycin, mupirocin, kanamycin, ampicillin, and ri￾fampin were purchased from Sigma-Aldrich (St. Louis, MO). Benzalko￾nium chloride (95% pure) and triclosan (Irgasan;
97%) were pur￾chased from Fluka (Buchs, Switzerland). Terpinen-4-ol (97.0%) was obtained from Acros Organics (Geel, Belgium). Tea tree oil (batch A352) was provided by P. Guinane Pty. Ltd., Cudgen, New South Wales, Austra￾lia. The composition was determined by gas chromatography-mass spec￾trometry, which was performed by Diagnostic and Analytical Services Environmental Laboratory, Wollongbar, New South Wales, Australia, and complied with ISO 4730 (20). The major components of the oil were terpinen-4-ol (37.0%), -terpinene (18.6%), -terpinene (10.0%), and Received 16 September 2011 Returned for modification 10 October 2011 Accepted 7 November 2011 Published ahead of print 14 November 2011 Address correspondence to K. Hammer, katherine.hammer@uwa.edu.au. Copyright © 2012, American Society for Microbiology. All Rights Reserved. doi:10.1128/AAC.05741-11 0066-4804/12/$12.00 Antimicrobial Agents and Chemotherapy p. 909 –915 aac.asm.org 909 on May 12, 2015 by SHANGHAI JIAOTONG UNIVERSITY http://aac.asm.org/ Downloaded from