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 909
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, 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 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. Plants have long been recognized as a valuable source of medicinal 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 Australian native plant Melaleuca alternifolia (Myrtaceae), also known as melaleuca oil or tea tree oil (TTO), is used topically for its antimicrobial and anti-inflammatory effects (5). The oil contains predominantly monoterpenes and related alcohols, and its composition 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 decolonization of methicillin-resistant Staphylococcus aureus carriage (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 susceptibility 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), influences the development of de novo antibiotic resistance in medically important bacteria. MATERIALS AND METHODS Bacteria and antimicrobials. Reference and clinical isolates of Staphylococcus aureus (n 18), Escherichia coli (n 21), and Staphylococcus epidermidis (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 rifampin were purchased from Sigma-Aldrich (St. Louis, MO). Benzalkonium chloride (95% pure) and triclosan (Irgasan; 97%) were purchased 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, Australia. The composition was determined by gas chromatography-mass spectrometry, 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
Hammer et al. 1,8-cineole(3.6%).Solutions of tea tree oil and terpinen-4-ol (measured for each combination then were determined.If the median fell be- in %vol/vol)were prepared daily and used within 2 h. tween dilution values,the higher of the two values was selected. Single-step resistance studies.MICs for each antibiotic,tea tree oil, Effect of terpinen-4-ol serial passage on antimicrobial susceptibil- and terpinen-4-ol were determined by agar dilution using the Clinical and ity.These experiments were conducted to (i)further attempt to induce Laboratory Standards Institute method(8),with the inclusion of 0.5% terpinen-4-ol resistance by broth macrodilution and (ii)determine antibiotic Tween 20 in the agar as a solubilizer for the latter two antimicrobials susceptibility after serial subculture.Overnight cultures of the three test or- Inocula were prepared by culturing E.coli and S.aureus isolates overnight ganisms,S.aureus NCTC 6571,S.aureus ATCC 25923,and S.epidermidis in trypticase soy broth(TSB)and then diluting them 1:10 into fresh TSB ATCC 12228,in TSB were diluted 1:10 into TSB containing 0.05%terpinen- with 0.001%Tween 80,both without (treatments A and B)and with 4-ol with 0.001%Tween 80.Cultures were incubated at 37C on a Stuart SFI (treatments C and D)0.03125%tea tree oil.This tea tree oil concentration flask shaker(Bibby Scientific,Staffordshire,United Kingdom)with wrist- was determined in preliminary growth curve experiments to be the high- action shaking equivalent to 500 rpm for 24 h.Cultures then were diluted 1:10 est concentration allowing approximately normal growth (data not into fresh TSB containing 0.1 and 0.2%terpinen-4-ol and were incubated as shown).Cultures were incubated at 37C with shaking until mid-late described above for I to 4 days.Bacteria from the highest concentration that logarithmic phase.Cells then were collected,washed,and resuspended was visibly turbid then were diluted into two fresh terpinen-4-ol solutions at ownloaded in 1/10 of the original volume in 0.85%saline.The cell suspension then the same concentration and a slightly higher concentration,using 0.1%in- was diluted in a series of 10-fold dilutions in 0.85%saline,and viable crements.This process was repeated until organisms failed to grow.S.aureus counts were performed on each cell suspension on Mueller-Hinton ATCC 25923 also was cultured as described above with tea tree oil,and a control culture of TSB with 0.001%Tween 80 but without tea tree oil or from agar (MHA)both without (treatments A and C)and with tea tree oil (treatments B and D). terpinen-4-ol was maintained identically throughout in parallel(passaged control).The susceptibility of serially passaged isolates was determined by 寻 Agar plates were prepared containing each antibiotic in 20 ml MHA with a final concentration of 0.5%(vol/vol)Tween 20.A second set removing an aliquot from a serial-passage culture that had been incubated for no more than 24 h (control,tea tree oil,and/or terpinen-4-ol),collecting cells was prepared in parallel containing antibiotic with tea tree oil.For S. /aac by centrifugation,washing them twice,and then resuspending them in 0.85% aureus,final antibiotic concentrations were 2X MIC for ciprofloxacin saline.The cell concentration was adjusted to approximately 108 CFU/ml, and vancomycin and 8x MIC for mupirocin and rifampin.Where and susceptibility was determined by the broth microdilution method(8). 3 relevant,0.25%(1/2X MIC)tea tree oil was included in the agar.For E. Inocula for the nonpassaged control were prepared by culturing bacteria from coli,final antibiotic concentrations were 2X MIC for kanamycin and a stock stored at-80C onto blood agar,incubating overnight,then inocu- 回 ampicillin,1X MIC for ciprofloxacin,and 8X MIC for rifampin.Agar lating into TSB and culturing organisms until mid-exponential phase.MICs contained 0.125%(1/4X MIC)tea tree oil.These tea tree oil concen- of all antimicrobial agents were determined according to CLSI criteria(8). g trations were determined in preliminary experiments (data not Statistical analyses.Frequencies of resistance data were first trans- shown).Plates containing antibiotic alone were stored for a maximum formed to their corresponding log values.However,for ease of representa- 更 of 7 days at 4C before use,whereas plates containing tea tree oil were tion,frequencies are shown as the geometric means.Transformed resistance prepared on the day of the experiment.Agar plates were inoculated by frequencies then were analyzed by a repeated-measure one-way analysis of 心 spreading 100-ul volumes from the appropriate dilution of cell sus- variance (ANOVA)with the Bonferroni post hoc test(P8 ug/ml;vancomycin,0.5 to 2 ug/ml;mupirocin,0.06 to 0.12 prepared in Mueller-Hinton broth in triplicate in a 96-well microtiter ug/ml;rifampin,0.004 to 0.008 ug/ml;tea tree oil,0.5%;and tray.The first dilution series contained antibiotic alone,the second terpinen-4-ol,0.25%.For E.coli,baseline MICs were ciprofloxa- contained the antibiotic with a final concentration of 0.062%tea tree cin,0.008 to >32 ug/ml;kanamycin,2 to >32 ug/ml;ampicillin, O oil,and the third contained the antibiotic with a final concentration of I to >32 ug/ml;rifampin,4 to >64 ug/ml;tea tree oil,0.25 to 0.031%terpinen-4-ol.All wells contained a final concentration of 0.5%;and terpinen-4-ol,0.12 to 0.25%.Resistance frequencies for ① 0.001%Tween 80 to enhance the solubility of tea tree oil/terpinen-4- vancomycin and ciprofloxacin did not differ significantly in the ol.A minimum of 10 isolates of each species was examined per antibi- presence and absence of tea tree oil for S.aureus (Table 1).For otic.Additional microtiter trays containing doubling dilutions of tea rifampin,significant differences were found between treatments B tree oil or terpinen-4-ol alone also were prepared to determine and C and for mupirocin between treatments A and B,B and C, 刃 whether susceptibility to either substance changed over the course of and Cand D.However,differences were minor,i.e.,less than 1 log the assay.Each triplicate dilution series was inoculated with in magnitude.For E.coli,frequencies of resistance to rifampin did exponential-phase cultures adjusted to result in final inoculum con- not differ significantly in the presence of tea tree oil.Kanamycin centrations of~5 X 105 CFU/ml.All trays were incubated for 24 h at resistance frequencies differed significantly for all treatments with 37C with shaking at 120 rpm and examined visually.The MIC was recorded as the lowest concentration resulting in a significant decrease the exception of treatments A and C.Approximately 1 log fewer in growth.To perform the serial subculture,an aliquot of culture from kanamycin-resistant mutants were detected when tea tree oil was the concentration immediately below the MIC (i.e.,1/2X MIC)was present in the agar than when it was absent. removed,diluted 1:5,and used to inoculate a fresh tray containing the For multistep assays,MICs for S.aureus increased by more identical combination of antibiotic with or without TTO or terpinen- than double(4-fold)from the baseline for ciprofloxacin,mupiro- 4-ol prepared as described above.This procedure was repeated for a cin,and vancomycin alone after 2 to 4 days and on day 6 for TTO total of 6 days.The medians and geometric means of MICs obtained and terpinen-4-ol (alone)(Table 2).On day 6,median MICs for 910 aac.asm.org Antimicrobial Agents and Chemotherapy
1,8-cineole (3.6%). Solutions of tea tree oil and terpinen-4-ol (measured in %, vol/vol) were prepared daily and used within 2 h. Single-step resistance studies. MICs for each antibiotic, tea tree oil, and terpinen-4-ol were determined by agar dilution using the Clinical and Laboratory Standards Institute method (8), with the inclusion of 0.5% Tween 20 in the agar as a solubilizer for the latter two antimicrobials. Inocula were prepared by culturing E. coli and S. aureus isolates overnight in trypticase soy broth (TSB) and then diluting them 1:10 into fresh TSB with 0.001% Tween 80, both without (treatments A and B) and with (treatments C and D) 0.03125% tea tree oil. This tea tree oil concentration was determined in preliminary growth curve experiments to be the highest concentration allowing approximately normal growth (data not shown). Cultures were incubated at 37°C with shaking until mid-late logarithmic phase. Cells then were collected, washed, and resuspended in 1/10 of the original volume in 0.85% saline. The cell suspension then was diluted in a series of 10-fold dilutions in 0.85% saline, and viable counts were performed on each cell suspension on Mueller-Hinton agar (MHA) both without (treatments A and C) and with tea tree oil (treatments B and D). Agar plates were prepared containing each antibiotic in 20 ml MHA with a final concentration of 0.5% (vol/vol) Tween 20. A second set was prepared in parallel containing antibiotic with tea tree oil. For S. aureus, final antibiotic concentrations were 2 MIC for ciprofloxacin and vancomycin and 8 MIC for mupirocin and rifampin. Where relevant, 0.25% (1/2 MIC) tea tree oil was included in the agar. For E. coli, final antibiotic concentrations were 2 MIC for kanamycin and ampicillin, 1 MIC for ciprofloxacin, and 8 MIC for rifampin. Agar contained 0.125% (1/4 MIC) tea tree oil. These tea tree oil concentrations were determined in preliminary experiments (data not shown). Plates containing antibiotic alone were stored for a maximum of 7 days at 4°C before use, whereas plates containing tea tree oil were prepared on the day of the experiment. Agar plates were inoculated by spreading 100-l volumes from the appropriate dilution of cell suspension onto each agar plate. Plates then were incubated at 30 to 35°C for 24 to 72 h, and colonies (single-step mutants) were counted. Frequencies of resistance were calculated by dividing the number of mutants (in CFU/ml) by the number of CFU in the inoculum. The assay was repeated at least three times on separate occasions for each isolate and each antibiotic. Geometric means of resistance frequencies then were determined for each isolate. Multistep resistance studies. Multistep resistance was selected for by using the CLSI broth microdilution method (8) with minor modi- fications. Briefly, a series of doubling dilutions of each antibiotic was prepared in Mueller-Hinton broth in triplicate in a 96-well microtiter tray. The first dilution series contained antibiotic alone, the second contained the antibiotic with a final concentration of 0.062% tea tree oil, and the third contained the antibiotic with a final concentration of 0.031% terpinen-4-ol. All wells contained a final concentration of 0.001% Tween 80 to enhance the solubility of tea tree oil/terpinen-4- ol. A minimum of 10 isolates of each species was examined per antibiotic. Additional microtiter trays containing doubling dilutions of tea tree oil or terpinen-4-ol alone also were prepared to determine whether susceptibility to either substance changed over the course of the assay. Each triplicate dilution series was inoculated with exponential-phase cultures adjusted to result in final inoculum concentrations of 5 105 CFU/ml. All trays were incubated for 24 h at 37°C with shaking at 120 rpm and examined visually. The MIC was recorded as the lowest concentration resulting in a significant decrease in growth. To perform the serial subculture, an aliquot of culture from the concentration immediately below the MIC (i.e., 1/2 MIC) was removed, diluted 1:5, and used to inoculate a fresh tray containing the identical combination of antibiotic with or without TTO or terpinen- 4-ol prepared as described above. This procedure was repeated for a total of 6 days. The medians and geometric means of MICs obtained for each combination then were determined. If the median fell between dilution values, the higher of the two values was selected. Effect of terpinen-4-ol serial passage on antimicrobial susceptibility. These experiments were conducted to (i) further attempt to induce terpinen-4-ol resistance by broth macrodilution and (ii) determine antibiotic susceptibility after serial subculture. Overnight cultures of the three test organisms, S. aureus NCTC 6571, S. aureus ATCC 25923, and S. epidermidis ATCC 12228, in TSB were diluted 1:10 into TSB containing 0.05% terpinen- 4-ol with 0.001% Tween 80. Cultures were incubated at 37°C on a Stuart SF1 flask shaker (Bibby Scientific, Staffordshire, United Kingdom) with wristaction shaking equivalent to 500 rpmfor 24 h.Cultures thenwere diluted 1:10 into fresh TSB containing 0.1 and 0.2% terpinen-4-ol and were incubated as described above for 1 to 4 days. Bacteria from the highest concentration that was visibly turbid then were diluted into two fresh terpinen-4-ol solutions at the same concentration and a slightly higher concentration, using 0.1% increments. This process was repeated until organisms failed to grow. S. aureus ATCC 25923 also was cultured as described above with tea tree oil, and a control culture of TSB with 0.001% Tween 80 but without tea tree oil or terpinen-4-ol was maintained identically throughout in parallel (passaged control). The susceptibility of serially passaged isolates was determined by removing an aliquotfrom a serial-passage culture that had been incubatedfor no more than 24 h (control, tea tree oil, and/or terpinen-4-ol), collecting cells by centrifugation,washing them twice, and then resuspending them in 0.85% saline. The cell concentration was adjusted to approximately 108 CFU/ml, and susceptibility was determined by the broth microdilution method (8). Inoculafor the nonpassaged controlwere prepared by culturing bacteriafrom a stock stored at 80°C onto blood agar, incubating overnight, then inoculating into TSB and culturing organisms until mid-exponential phase. MICs of all antimicrobial agents were determined according to CLSI criteria (8). Statistical analyses. Frequencies of resistance data were first transformed to their corresponding log10 values. However, for ease of representation, frequencies are shown as the geometric means. Transformed resistance frequencies then were analyzed by a repeated-measure one-way analysis of variance (ANOVA) with the Bonferroni post hoc test (P 0.05). MICs from the multistep experiments were log transformed (base 2) to approximate normal distributions. Log2 values then were analyzed by repeated-measure one-way ANOVA with the Bonferroni post hoc test (P 0.05). All statistical analyses were performed using GraphPad Prism (version 3.03) software, and differences were considered significant when P 0.05. RESULTS Baseline MICs for S. aureuswere the following: ciprofloxacin, 0.06 to 8 g/ml; vancomycin, 0.5 to 2 g/ml; mupirocin, 0.06 to 0.12 g/ml; rifampin, 0.004 to 0.008 g/ml; tea tree oil, 0.5%; and terpinen-4-ol, 0.25%. For E. coli, baseline MICs were ciprofloxacin, 0.008 to 32 g/ml; kanamycin, 2 to 32 g/ml; ampicillin, 1 to 32 g/ml; rifampin, 4 to 64 g/ml; tea tree oil, 0.25 to 0.5%; and terpinen-4-ol, 0.12 to 0.25%. Resistance frequencies for vancomycin and ciprofloxacin did not differ significantly in the presence and absence of tea tree oil for S. aureus (Table 1). For rifampin, significant differences were found between treatments B and C and for mupirocin between treatments A and B, B and C, and C and D. However, differences were minor, i.e., less than 1 log in magnitude. For E. coli, frequencies of resistance to rifampin did not differ significantly in the presence of tea tree oil. Kanamycin resistance frequencies differed significantly for all treatments with the exception of treatments A and C. Approximately 1 log fewer kanamycin-resistant mutants were detected when tea tree oil was present in the agar than when it was absent. For multistep assays, MICs for S. aureus increased by more than double (4-fold) from the baseline for ciprofloxacin, mupirocin, and vancomycin alone after 2 to 4 days and on day 6 for TTO and terpinen-4-ol (alone) (Table 2). On day 6, median MICs for Hammer et al. 910 aac.asm.org Antimicrobial Agents and Chemotherapy on May 12, 2015 by SHANGHAI JIAOTONG UNIVERSITY http://aac.asm.org/ Downloaded from
Tea Tree Oil and Antibiotic Resistance TABLE 1 Frequencies of single-step antibiotic-resistant mutants occurring in the presence and absence of tea tree oil Frequency of mutants in: Control culture with: TTO culture with: Organism and Fold increase Antibiotic alone Antibiotic TTO Antibiotic alone Antibiotic TTO no.of isolates Antibiotic in MIC (treatment A) (treatment B) (treatment C) (treatment D) P valueb S.aureus 10 RIF 8 8.9×10-8 5.9×10-8 1.1×10-7 7.2×10-8 0.0065 10 MUP 8 7.3×10-8 2.3×10-8 9.9×10-8 3.5X10-8 0.0002d 7 VAN 2 2.4×10-7 1.2×10-7 3.4×10-7 5.9×10-7 0.1268 4 CIP 2 8.1×10-8 5.9×10-8 8.6×10-8 3.7×10-8 0.6725 E.coli 10 RIF 8 3.7×10-8 2.9×10-8 4.3×10-8 3.8×10-8 0.2083 ownloaded 9 KAN 3.3×10-6 2.0×10-7 2.6×10-6 31×10-7 <0.0001e Values are the geometric means from 4 to 10 isolates.MUP,mupirocin;RIF,rifampin;VAN,vancomycin;CIP,ciprofloxacin;KAN,kanamycin;TTO,tea tree oil. P values were obtained by repeated-measure one-way ANOVA. from Significant differences exist between treatments B and C(P<0.01)(Bonferroni post test). Significant differences exist between treatments A and B(P<0.01).B and C(P<0.001),and Cand D(P<0.05). Significant differences exist between treatments A and B(P<0.001),A and D(P<0.01),B and C(P<0.001),and C and D(P<0.01). ://aac. antibiotic alone increased 4-fold for ciprofloxacin and vancomy- or differing by one dilution only from antibiotic alone on all days. cin and 8-fold for mupirocin compared to MICs at day 1.The The only exception was mupirocin on day 5,where the median presence ofTTO or terpinen-4-ol with antibiotic did not appear to MIC in the presence of terpinen-4-ol was 4-fold that of mupirocin 3 greatly influence MICs,with median MICs being either identical alone.However,at day 6 this difference was only 2-fold.The sta- TABLE 2 S.aureus MICs of antibiotics(ug/ml)alone,antibiotics with or without tea tree oil (0.062%)or terpinen-4-ol (0.031%),and tea tree oil or terpinen-4-ol without antibiotic,determined by serial subculture Agent(no.of isolates) MIC on day: and parameter Treatment 5 6 CIP (10) Median Alone 0.5 1 2 2 2 With TTO 0.25 1 1 2 2 2 With T4ol 0.25 0.5 1 2 2 GM Alone 0.8 0.8 1.1 1.5 1.6 2.3 With TT 0.25 1.0 1.1 1.6 2.5 3.5 SHAN With T4ol 02 06 1.0 1.5 1.7 2.3 MUP (11) Median Alone 0.12 0.25 0.5 1 1 With TTO 0.12 0.12 0.5 0.5 0.5 With T4ol 0.12 0.12 0.5 1 4 2 GM Alone 0.1 0.3 0.5 0.9 1.2 1.5 With TTO 0.1 0.2 0.5 0.6 0.5 1.4 O With T4ol 0.1 0.2 0.5 0.9 1.9 3.1 5 VAN (12) Median Alone 4 4 4 With TTO 1 ¥ 8 4 With T4ol UNIVERSI GM Alone 1.0 2.7 4.5 7.6 4.0 With TTO 1.1 50 5.0 6.3 3.8 3 With T4ol 0.9 4.0 4.0 5 3.6 50 Tea tree oil(18) Median Alone 0.5 0.5 1 2 GM Alone 0.5 0.7 1.0 0.9 1.0 1.7 Terpinen-4-ol(18) Median Alone 0.25 0.25 0.25 0.5 0.5 GM Alone 0.2 0.3 0.3 0.6 0.6 0.8 GM,geometric means.Boldface type indicates that the MIC is more than double the baseline(day 1)value.Single underlining indicates that values differed significantly from antibiotic alone on that day.Double underlining indicates significant differences between tea tree oil and terpinen-4-ol treatments on that day. February 2012 Volume 56 Number 2 aac.asm.org 911
antibiotic alone increased 4-fold for ciprofloxacin and vancomycin and 8-fold for mupirocin compared to MICs at day 1. The presence of TTO or terpinen-4-ol with antibiotic did not appear to greatly influence MICs, with median MICs being either identical or differing by one dilution only from antibiotic alone on all days. The only exception was mupirocin on day 5, where the median MIC in the presence of terpinen-4-ol was 4-fold that of mupirocin alone. However, at day 6 this difference was only 2-fold. The staTABLE 1 Frequencies of single-step antibiotic-resistant mutants occurring in the presence and absence of tea tree oila Organism and no. of isolates Antibiotic Fold increase in MIC Frequency of mutants in: P valueb Control culture with: TTO culture with: Antibiotic alone (treatment A) Antibiotic TTO (treatment B) Antibiotic alone (treatment C) Antibiotic TTO (treatment D) S. aureus 10 RIF 8 8.9 108 5.9 108 1.1 107 7.2 108 0.0065c 10 MUP 8 7.3 108 2.3 108 9.9 108 3.5 108 0.0002d 7 VAN 2 2.4 107 1.2 107 3.4 107 5.9 107 0.1268 4 CIP 2 8.1 108 5.9 108 8.6 108 3.7 108 0.6725 E. coli 10 RIF 8 3.7 108 2.9 108 4.3 108 3.8 108 0.2083 9 KAN 2 3.3 106 2.0 107 2.6 106 3.1 107 0.0001e a Values are the geometric means from 4 to 10 isolates. MUP, mupirocin; RIF, rifampin; VAN, vancomycin; CIP, ciprofloxacin; KAN, kanamycin; TTO, tea tree oil. b P values were obtained by repeated-measure one-way ANOVA. c Significant differences exist between treatments B and C (P 0.01) (Bonferroni post test). d Significant differences exist between treatments A and B (P 0.01), B and C (P 0.001), and C and D (P 0.05). e Significant differences exist between treatments A and B (P 0.001), A and D (P 0.01), B and C (P 0.001), and C and D (P 0.01). TABLE 2 S. aureus MICs of antibiotics (g/ml) alone, antibiotics with or without tea tree oil (0.062%) or terpinen-4-ol (0.031%), and tea tree oil or terpinen-4-ol without antibiotic, determined by serial subculturea Agent (no. of isolates) and parameter Treatment MIC on day: 1 2 3 456 CIP (10) Median Alone 0.5 1 1 222 With TTO 0.25 1 1 222 With T4ol 0.25 0.5 1 222 GM Alone 0.8 0.8 1.1 1.5 1.6 2.3 With TTO 0.25 1.0 1.1 1.6 2.5 3.5 With T4ol 0.2 0.6 1.0 1.5 1.7 2.3 MUP (11) Median Alone 0.12 0.25 0.5 1 1 1 With TTO 0.12 0.12 0.5 0.5 0.5 1 With T4ol 0.12 0.12 0.5 1 4 2 GM Alone 0.1 0.3 0.5 0.9 1.2 1.5 With TTO 0.1 0.2 0.5 0.6 0.5 1.4 With T4ol 0.1 0.2 0.5 0.9 1.9 3.1 VAN (12) Median Alone 1 2 4 844 With TTO 1 4 4 844 With T4ol 1 4 4 844 GM Alone 1.0 2.7 4.5 7.6 4.0 3.8 With TTO 1.1 5.0 5.0 6.3 3.8 4.3 With T4ol 0.9 4.0 4.0 9.5 3.6 5.0 Tea tree oil (18) Median Alone 0.5 0.5 1 1 1 2 GM Alone 0.5 0.7 1.0 0.9 1.0 1.7 Terpinen-4-ol (18) Median Alone 0.25 0.25 0.25 0.5 0.5 1 GM Alone 0.2 0.3 0.3 0.6 0.6 0.8 a GM, geometric means. Boldface type indicates that the MIC is more than double the baseline (day 1) value. Single underlining indicates that values differed significantly from antibiotic alone on that day. Double underlining indicates significant differences between tea tree oil and terpinen-4-ol treatments on that day. Tea Tree Oil and Antibiotic Resistance February 2012 Volume 56 Number 2 aac.asm.org 911 on May 12, 2015 by SHANGHAI JIAOTONG UNIVERSITY http://aac.asm.org/ Downloaded from
Hammer et al. TABLE 3 Summary of E.coli MICs of antibiotics (ug/ml)alone,antibiotics with tea tree oil (0.062%)or terpinen-4-ol (0.031%),and tea tree oil or terpinen-4-ol without antibiotics,determined by serial subculture MIC on day: Agent(no.of isolates) and parameter Treatment 1 2 3 5 6 CIP (12) Median Alone 0.008 0.016 0.016 0.03 0.03 0.12 With TTO 0.008 0.016 0.03 0.06 0.06 0.12 With T4ol 0.008 0.016 0.06 0.06 0.12 0.12 GM Alone 0.010 0.025 0.034 0.070 0.070 0.140 With TTO 0.002 0.014 0.029 0.045 0.064 0.122 With T4ol 0.009 0.018 0.048 0.078 0.100 0.147 KAN (11) ownloaded Median Alone 32 4 64 128 With TTO ¥ 16 32 3 32 4 +With T4ol Y 16 3 32 64 GM Alone 6.7 45.3 20.2 60.4 50.8 107.6 from With TTO 麷 12.7 33.9 38.1 42.7 47.9 With T4ol 151 28.5 33.9 32.0 53.8 AMP (10) http://aac. Median Alone 8 16 With TTO 6 16 6 With T4ol 2 16 GM Alone 2.1 4.9 8.0 10.6 11.3 16.0 asm.or With TTO 2.1 4.6 8.6 9.8 12.1 12.1 口 With T4ol 2.1 4.9 11.3 11.3 17.1 243 Tea tree oil (18) Median Alone 0.5 1 1 1 GM Alone 0.65 0.73 0.96 1.00 1.12 0.96 Terpinen-4-ol (18) Median Alone 0.12 0.25 0.25 0.25 0.25 0.25 吕 GM Alone 0.13 0.17 0.22 0.29 0.25 0.26 GM,geometric means.Boldface type indicates that the MIC is more than double the baseline (day 1)value.Single underlining indicates that values differed significantly from antibiotic alone on that day.Double underlining indicates significant differences between tea tree oil and terpinen-4ol treatments on that day. SHA Z tistical analysis of MICs obtained on each day under the three grow consistently in concentrations greater than 0.1%terpinen- different conditions (antibiotic alone,with tea tree oil,or with 4-ol after 18 to 20 passages,demonstrating that resistance to terpinen-4-ol)demonstrated significant differences for cipro- terpinen-4ol could not be induced in vitro (Table 4).Similarly,S. floxacin on days 1(P<0.0001)and 2 (P=0.02),for vancomycin epidermidis ATCC 12228 would not grow at concentrations above on days 2(P<0.0001),4(P=0.0017),and 6(P<0.0001),and 0.2%terpinen-4-ol,and S.aureus ATCC 25923 would not grow for mupirocin on day 2(P 0.0082). above 0.1%tea tree oil.Serial passage with terpinen-4-ol resulted For E.coli,increases in the MIC of more than two doubling in few changes in antimicrobial susceptibility(Table 1).Changes dilutions occurred for all three antibiotics alone on days 2 to 3 in MICs of two or more dilutions were evident for ciprofloxacin, (Table 3).Increases in median MICs from days 1 to 6 for antibiotic gentamicin,tetracycline,and benzalkonium chloride only.How- alone were 16-fold for ciprofloxacin and kanamycin and 8-fold for ever,with the exception of benzalkonium chloride and S.aureus ampicillin.Similarly to S.aureus,the presence of TTO or ATCC 25923,differences were not observed consistently for every terpinen-4-ol with antibiotic did not appear to greatly influence passage number.The susceptibility of multiply passaged S.aureus 刀 MICs,with median MICs obtained under the three conditions NCTC6571 to tetracycline reverted to 0.25 ug/ml after the organ- being either the same or differing by one dilution only on each ism was stored at-80C and then recultured.MICs for S.aureus day.The exception was ciprofloxacin with terpinen-4-ol,where ATCC 25923 passaged in 0.1%TTO did not differ by more than 1 the median MIC was 4-fold higher than that of ciprofloxacin alone dilution from that of the control.Passaging in TSB alone did not on days 3 and 5.The analysis of MICs showed significant differ- produce significant changes in MICs,as susceptibility data for the ences between the three conditions for ciprofloxacin on day 1(P< passaged and nonpassaged controls did not vary by more than 1 0.0001),for kanamycin on days1(P<0.0001),2(P<0.0001), dilution for all three strains (data not shown). and 6(P=0.0288),and for ampicillin on day 6(P=0.0383).For tea tree oil and terpinen-4-ol alone,the median MIC increased DISCUSSION 2-fold during the 6 days. There are many examples in the literature of the presence of a Lastly,using a macrodilution method,S.aureus strains did not second antimicrobial agent or nonantibiotic drug preventing or 912 aac.asm.org Antimicrobial Agents and Chemotherapy
tistical analysis of MICs obtained on each day under the three different conditions (antibiotic alone, with tea tree oil, or with terpinen-4-ol) demonstrated significant differences for cipro- floxacin on days 1 (P 0.0001) and 2 (P 0.02), for vancomycin on days 2 (P 0.0001), 4 (P 0.0017), and 6 (P 0.0001), and for mupirocin on day 2 (P 0.0082). For E. coli, increases in the MIC of more than two doubling dilutions occurred for all three antibiotics alone on days 2 to 3 (Table 3). Increases in median MICs from days 1 to 6 for antibiotic alone were 16-fold for ciprofloxacin and kanamycin and 8-fold for ampicillin. Similarly to S. aureus, the presence of TTO or terpinen-4-ol with antibiotic did not appear to greatly influence MICs, with median MICs obtained under the three conditions being either the same or differing by one dilution only on each day. The exception was ciprofloxacin with terpinen-4-ol, where the median MIC was 4-fold higher than that of ciprofloxacin alone on days 3 and 5. The analysis of MICs showed significant differences between the three conditions for ciprofloxacin on day 1 (P 0.0001), for kanamycin on days 1 (P 0.0001), 2 (P 0.0001), and 6 (P 0.0288), and for ampicillin on day 6 (P 0.0383). For tea tree oil and terpinen-4-ol alone, the median MIC increased 2-fold during the 6 days. Lastly, using a macrodilution method, S. aureus strains did not grow consistently in concentrations greater than 0.1% terpinen- 4-ol after 18 to 20 passages, demonstrating that resistance to terpinen-4ol could not be induced in vitro (Table 4). Similarly, S. epidermidis ATCC 12228 would not grow at concentrations above 0.2% terpinen-4-ol, and S. aureus ATCC 25923 would not grow above 0.1% tea tree oil. Serial passage with terpinen-4-ol resulted in few changes in antimicrobial susceptibility (Table 1). Changes in MICs of two or more dilutions were evident for ciprofloxacin, gentamicin, tetracycline, and benzalkonium chloride only. However, with the exception of benzalkonium chloride and S. aureus ATCC 25923, differences were not observed consistently for every passage number. The susceptibility of multiply passaged S. aureus NCTC 6571 to tetracycline reverted to 0.25 g/ml after the organism was stored at 80°C and then recultured. MICs for S. aureus ATCC 25923 passaged in 0.1% TTO did not differ by more than 1 dilution from that of the control. Passaging in TSB alone did not produce significant changes in MICs, as susceptibility data for the passaged and nonpassaged controls did not vary by more than 1 dilution for all three strains (data not shown). DISCUSSION There are many examples in the literature of the presence of a second antimicrobial agent or nonantibiotic drug preventing or TABLE 3 Summary of E. coli MICs of antibiotics (g/ml) alone, antibiotics with tea tree oil (0.062%) or terpinen-4-ol (0.031%), and tea tree oil or terpinen-4-ol without antibiotics, determined by serial subculturea Agent (no. of isolates) and parameter Treatment MIC on day: 123456 CIP (12) Median Alone 0.008 0.016 0.016 0.03 0.03 0.12 With TTO 0.008 0.016 0.03 0.06 0.06 0.12 With T4ol 0.008 0.016 0.06 0.06 0.12 0.12 GM Alone 0.010 0.025 0.034 0.070 0.070 0.140 With TTO 0.007 0.014 0.029 0.045 0.064 0.122 With T4ol 0.009 0.018 0.048 0.078 0.100 0.147 KAN (11) Median Alone 8 32 16 64 64 128 With TTO 4 16 32 32 32 64 With T4ol 4 16 32 32 32 64 GM Alone 6.7 45.3 20.2 60.4 50.8 107.6 With TTO 4.8 12.7 33.9 38.1 42.7 47.9 With T4ol 2.5 15.1 28.5 33.9 32.0 53.8 AMP (10) Median Alone 2 4 8816 16 With TTO 2 4 8816 16 With T4ol 2 4 8816 32 GM Alone 2.1 4.9 8.0 10.6 11.3 16.0 With TTO 2.1 4.6 8.6 9.8 12.1 12.1 With T4ol 2.1 4.9 11.3 11.3 17.1 24.3 Tea tree oil (18) Median Alone 0.5 11111 GM Alone 0.65 0.73 0.96 1.00 1.12 0.96 Terpinen-4-ol (18) Median Alone 0.12 0.25 0.25 0.25 0.25 0.25 GM Alone 0.13 0.17 0.22 0.29 0.25 0.26 a GM, geometric means. Boldface type indicates that the MIC is more than double the baseline (day 1) value. Single underlining indicates that values differed significantly from antibiotic alone on that day. Double underlining indicates significant differences between tea tree oil and terpinen-4ol treatments on that day. Hammer et al. 912 aac.asm.org Antimicrobial Agents and Chemotherapy on May 12, 2015 by SHANGHAI JIAOTONG UNIVERSITY http://aac.asm.org/ Downloaded from
Tea Tree Oil and Antibiotic Resistance TABLE 4 MICs of antibiotics (ug/ml),biocides (ug/ml),and tea tree oil and terpinen-4-ol (%vol/vol)for three Staphylococcus strains serially subcultured with terpinen-4-ol or tea tree oil S.aureus NCTC 6571 S.aureus ATCC 25923 S.epidermidis ATCC 12228 MIC MIC MIC With 0.1% With 0.1% With 0.1% With 0.2% Agent Passage no. Control T4ol Passage no. Control T4ol TTO Passage no. Control T4ol AMX 19 0.12 0.06 18 0.12 0.12 0.12 14 0.5 22 0.25 0.25 0.5 0.25 17 1 0.5 CIP 17 0.25 0.06 公 0.12 0.12 0.25 1g 0.25 0.25 19 0.12 0.06 18 0.25 0.06 0.12 17 0.5 0.25 22 0.12 0.06 20 0.25 0.06 Downloaded GEN 1> 16 0.5 0.25 0.5 14 0.12 0.5 19 0.25 0.12 0.12 0.25 0.25 22 20 0.25 0.25 from TET 17 0.12 0.06 0.12 0.12 0.25 4 0. 0.5 19 0.25 <0.03 18 0.25 0.12 0.25 17 0.5 22 0.12 0.06 20 0.25 0.12 http://aac. VAN 1 16 2 4 19 1 0.5 18 22 0.5 0.25 20 1 .asm.org/ Benzalkonium ◇ 0.5 16 2 0.5 Chloride 19 1 18 2 0.5 2 1 20 2 0.5 May Triclosan 19 0.06 0.03 18 0.06 0.03 0.03 0.03 0.06 22 0.12 0.12 20 0.25 0.12 Tea tree oil 17 0.5 0.25 16 0.12 0.25 0.25 0.5 19 1 0.5 18 0.5 0.5 0.5 17 0.5 0.5 22 0.25 0.25 20 0.5 0.5 Terpinen-4-ol 17 0.25 0.5 16 0.25 0.5 0.5 14 0.5 0.5 19 0.5 0.5 18 0.5 0.5 0.25 17 0.5 0.5 SHAN 22 0.12 0.25 20 0.12 0.25 Boldface type indicates a difference in MIC of 4-fold or more for passaged and nonpassaged strains 工 delaying the development of antibiotic resistance(22,27).One of affected by either culturing with tea tree oil or combining antibi- the best known is the treatment of tuberculosis with combinations otic with tea tree oil.The exception was kanamycin,whereby E. of rifampin,isoniazid,pyrazinamide,and ethambutol(19,29).At coliresistance frequencies were consistently approximately 1 logo the other end of the spectrum,there are concerns that the overuse lower when cultured on kanamycin agar with tea tree oil for both of antimicrobial agents such as biocides leads to increases in anti- control cultures and tea tree oil cultures.Culturing with tea tree oil ① biotic resistance (15).These concerns relate to the use of disinfec- prior to determining resistance frequencies had no significant im- tants and antiseptics in the domestic environment and the theory pact.Two possible explanations for the differences in resistance that the increased and chronic exposure of bacteria to sublethal frequencies are that the tea tree oil is preventing mutations(and n concentrations of biocide leads to tolerance,which may also con- decreasing the overall mutation rate)or decreasing the survival of fer tolerance to antibiotics.Since several biocides have multiple, a small proportion of resistant mutants (no change in mutation nonspecific mechanisms of action,similarly to tea tree oil,this rate).There is little evidence to support the first possibility,since same concern could apply to the oil.Although decreased antibi- (i)if this was the case we would expect more differences in muta- otic susceptibility following biocide exposure has been demon- tion rates in the current study,and (ii)previous studies have strated in vitro(4,16),there isstill debate as to what impact,ifany, shown that tea tree oil neither increases(12,14)nor decreases(12) this has in clinical practice(17).The current study has demon- mutations using the bacterial reverse mutation assay.This there- strated that tea tree oil has little impact on the development of fore suggests that the decreased number of mutants is specific to antibiotic resistance,and that exposure to the major component kanamycin and its mechanism(s)of action and resistance.Amin- terpinen-4-ol does not significantly alter antimicrobial suscepti- oglycosides exert antibacterial action primarily by interfering with bility. protein synthesis by binding to rRNA in the small subunit of the Frequencies ofsingle-step antibiotic resistance were largely un- pacterial ribosome.Mechanisms of kanamycin resistance include February 2012 Volume 56 Number 2 aac.asm.org 913
delaying the development of antibiotic resistance (22, 27). One of the best known is the treatment of tuberculosis with combinations of rifampin, isoniazid, pyrazinamide, and ethambutol (19, 29). At the other end of the spectrum, there are concerns that the overuse of antimicrobial agents such as biocides leads to increases in antibiotic resistance (15). These concerns relate to the use of disinfectants and antiseptics in the domestic environment and the theory that the increased and chronic exposure of bacteria to sublethal concentrations of biocide leads to tolerance, which may also confer tolerance to antibiotics. Since several biocides have multiple, nonspecific mechanisms of action, similarly to tea tree oil, this same concern could apply to the oil. Although decreased antibiotic susceptibility following biocide exposure has been demonstrated in vitro (4, 16), there is still debate as to what impact, if any, this has in clinical practice (17). The current study has demonstrated that tea tree oil has little impact on the development of antibiotic resistance, and that exposure to the major component terpinen-4-ol does not significantly alter antimicrobial susceptibility. Frequencies of single-step antibiotic resistance were largely unaffected by either culturing with tea tree oil or combining antibiotic with tea tree oil. The exception was kanamycin, whereby E. coli resistance frequencies were consistently approximately 1 log10 lower when cultured on kanamycin agar with tea tree oil for both control cultures and tea tree oil cultures. Culturing with tea tree oil prior to determining resistance frequencies had no significant impact. Two possible explanations for the differences in resistance frequencies are that the tea tree oil is preventing mutations (and decreasing the overall mutation rate) or decreasing the survival of a small proportion of resistant mutants (no change in mutation rate). There is little evidence to support the first possibility, since (i) if this was the case we would expect more differences in mutation rates in the current study, and (ii) previous studies have shown that tea tree oil neither increases (12, 14) nor decreases (12) mutations using the bacterial reverse mutation assay. This therefore suggests that the decreased number of mutants is specific to kanamycin and its mechanism(s) of action and resistance. Aminoglycosides exert antibacterial action primarily by interfering with protein synthesis by binding to rRNA in the small subunit of the bacterial ribosome. Mechanisms of kanamycin resistance include TABLE 4 MICs of antibiotics (g/ml), biocides (g/ml), and tea tree oil and terpinen-4-ol (%, vol/vol) for three Staphylococcus strains serially subcultured with terpinen-4-ol or tea tree oila Agent S. aureus NCTC 6571 S. aureus ATCC 25923 S. epidermidis ATCC 12228 Passage no. MIC Passage no. MIC Passage no. MIC Control With 0.1% T4ol Control With 0.1% T4ol With 0.1% TTO Control With 0.2% T4ol AMX 19 0.12 0.06 18 0.12 0.12 0.12 14 1 0.5 22 0.25 0.25 20 0.5 0.25 17 1 0.5 CIP 17 0.25 0.06 16 0.12 0.12 0.25 14 0.25 0.25 19 0.12 0.06 18 0.25 0.06 0.12 17 0.5 0.25 22 0.12 0.06 20 0.25 0.06 GEN 17 1 2 16 0.5 0.25 0.5 14 0.12 0.5 19 1 1 18 0.25 0.12 0.12 17 0.25 0.25 22 1 2 20 0.25 0.25 TET 17 0.12 0.06 16 0.12 0.12 0.25 14 0.5 0.5 19 0.25 0.03 18 0.25 0.12 0.25 17 1 0.5 22 0.12 0.06 20 0.25 0.12 VAN 17 1 1 16 2 2 2 14 4 4 19 1 0.5 18 1 1 2 17 4 4 22 0.5 0.25 20 1 1 Benzalkonium 17 0.5 1 16 2 0.5 2 14 2 1 Chloride 19 1 1 18 2 0.5 1 17 2 1 22 1 1 20 2 0.5 Triclosan 19 0.06 0.03 18 0.06 0.03 0.03 17 0.03 0.06 22 0.12 0.12 20 0.25 0.12 Tea tree oil 17 0.5 0.25 16 0.12 0.25 0.25 14 0.5 0.5 19 1 0.5 18 0.5 0.5 0.5 17 0.5 0.5 22 0.25 0.25 20 0.5 0.5 Terpinen-4-ol 17 0.25 0.5 16 0.25 0.5 0.5 14 0.5 0.5 19 0.5 0.5 18 0.5 0.5 0.25 17 0.5 0.5 22 0.12 0.25 20 0.12 0.25 a Boldface type indicates a difference in MIC of 4-fold or more for passaged and nonpassaged strains. Tea Tree Oil and Antibiotic Resistance February 2012 Volume 56 Number 2 aac.asm.org 913 on May 12, 2015 by SHANGHAI JIAOTONG UNIVERSITY http://aac.asm.org/ Downloaded from
Hammer et al. the reduction of intracellular antibiotic concentration(typically ol.Little evidence was found to support the concern that the in- via efflux),the alteration of the target site (normally by spontane- creased use of tea tree oil in both domestic and health care envi- ous mutation),and enzymatic inactivation(21),and bacteria may ronments will lead to increased antimicrobial resistance possess more than one mechanism.The identification of the spe- cific gene mutation(s)resulting in kanamycin resistance in mu- REFERENCES tants obtained in both the presence and absence of tea tree oil 1.Ali SM,et al.2005.Antimicrobial activities of eugenol and cinnamalde- would allow the identification of an absent mutant subset. hyde against the human gastric pathogen Helicobacter pylori.Ann.Clin. The effects of tea tree oil or terpinen-4-ol on the development Microbiol.Antimicrob.4:20. 2.Amsterdam D.2005.Susceptibility testing of antimicrobials in liquid of multistep antibiotic resistance were minimal when evaluated by media.In Lorian V(ed),Antibiotics in laboratory medicine,5th ed.Lip- the standard MIC assessment criteria,whereby differences in the pincott Williams and Wilkins,Philadelphia,PA. MIC of one doubling dilution are not considered to be significant 3.Bowler WA,Bresnahan J,Bradfish A,Fernandez C.2010.An integrated (2,7).However,using statistical analyses,significant differences approach to methicillin-resistant Staphylococcus aureus control in a rural, were evident between treatments on some days.In the majority of regional-referral healthcare setting.Infect.Control Hosp.Epidemiol.31: 269-275. ownloaded instances,MICs were significantly lower when tea tree oil or 4.Braoudaki M,Hilton AC.2004.Adaptive resistance to biocides in Sal- terpinen-4-ol was present,and significant differences occurred monella enterica and Escherichia coli O157 and cross-resistance to antimi- mostly on days 1 and 2.This indicates synergistic antimicrobial crobial agents.J.Clin.Microbiol.42:73-78. from interactions rather than a true alteration in resistance.It also re- 5. Carson CF,Hammer KA,Riley TV.2006.Melaleuca alternifolia (tea tree) mains possible that some of the changes in antibiotic susceptibility oil:a review of antimicrobial and other medicinalproperties.Clin.Micro- biol.Rev.19:50-62. were the result of phenotypic adaptation rather than true resis- 6.Carson CF,Mee BJ,Riley TV.2002.Mechanism of action of Melaleuca tance.Similarly to the single-step studies,the combination of tea alternifolia (tea tree)oil on Staphylococcus aureus determined by time-kill, tree oil and kanamycin appears to have influenced the develop- lysis,leakage,and salt tolerance assays and electron microscopy.Antimi- :/aac crob.Agents Chemother.46:1914-1920. ment of multistep resistance in E.coli;however,testing with addi- 7.Clinical Laboratory Standards Institute.2006.Performance standards tional isolates is required to confirm this.Overall,since the pres- for antimicrobial susceptibility testing,16th informational supplement. 3 ence of tea tree oil or terpinen-4-ol resulted in only minor changes CLSI document M100-S16.Clinical and Laboratory Standards Institute, in antibiotic susceptibility,and no consistent trends were appar- Wayne,PA. 恒 ent for either S.aureus or E.coli,it is reasonable to conclude from 8.Clinical Laboratory Standards Institute.2006.M7-A7:methods for di- lution antimicrobial susceptibility tests for bacteria that grow aerobically, g these data that tea tree oil and terpinen-4ol do not have a signifi- approved standard,7th ed.Clinical and Laboratory Standards Institute, cant impact on the development of multistep antibiotic resistance. Wayne,PA. The repeated exposure of S.aureus and S.epidermidis strains to 9.Cox SD,et al.2000.The mode of antimicrobial action of the essential oil terpinen-4-ol did not induce significant changes in antimicrobial of Melaleuca alternifolia (tea tree oil).J.Appl.Microbiol.88:170-175. susceptibility,which is largely in agreement with previously pub- 10.Di Pasqua R,Hoskins N,Betts G,Mauriello G.2006.Changes in membrane fatty acids composition of microbial cells induced by addiction lished data indicating minor changes in susceptibility(of 2-fold or of thymol,carvacrol,limonene,cinnamaldehyde,and eugenol in the less)after exposure to tea tree oil for similar Gram-positive organ- growing media.J.Agric.Food Chem.54:2745-2749. isms (23,24).Furthermore,where changes of 4-fold or more oc- 11.Dryden MS,Dailly S,Crouch M.2004.A randomized,controlled trial of g curred,susceptibility was largely increased rather than decreased tea tree topical preparations versus a standard topical regimen for the clearance of MRSA colonization.J.Hosp.Infect.56:283-286. These data suggest that if adaptive measures were induced by 12.Evandri MG,et al.2005.The antimutagenic activity of Lavandula angus- terpinen-4-ol or tea tree oil,they were not sufficient to alter anti- tifolia (lavender)essential oil in the bacterial reverse mutation assay.Food Z microbial susceptibility or confer cross-protection to other anti- Chem.Toxicol.43:1381-1387. 13.Ferrini AM,et al.2006.Melaleuca alternifolia essential oil possesses po- 工 microbial agents. Of the few previous studies that have attempted to induce re- tent anti-staphylococcal activity extended to strains resistant to antibiot- ics.Int.J.Immunopathol.Pharmacol.19:539-544. = sistance to essential oils or components,most have found either 14.Fletcher JP,Cassella JP,Hughes D,Cassella S.2005.An evaluation of the minor decreases in susceptibility or no change(1,13,24,25,28). mutagenic potential of commercially available tea tree oil in the United This is similar to the present study,where minor susceptibility Kingdom.Int.J.Aromather.15:81-86. changes were seen by microdilution but not by macrodilution. 15.Fraise AP.2002.Biocide abuse and antimicrobial resistance-a cause for Precisely why changes in susceptibility were observed by one concern?J.Antimicrob.Chemother.49:11-12. 16.Fuangthong M,et al.2011.Exposure of Acinetobacter baylyi ADPI to the ① method and not the other remains to be determined.Minor biocide chlorhexidine leads to acquired resistance to the biocide itselfand changes in essential oil susceptibility most likely are explained by to oxidants.J.Antimicrob.Chemother.66:319-322. phenotypic adaptation,which confers a low level of tolerance and 17.Gilbert P,McBain AJ.2003.Potential impact of increased use of biocides has been shown to occur via reversible changes in membrane lipid in consumer products on prevalence of antibiotic resistance.Clin.Micro- biol.Rev.16:189-208. composition (10,31)and efflux(26).Organisms expressing the 西 18.Gustafson JE,Cox SD,Liew YC,Wyllie SG,Warmington JR.2001.The multiple antibiotic resistance (Mar)phenotype also have moder- bacterial multiple antibiotic resistant(Mar)phenotype leads to increased ately reduced tea tree oil susceptibility(18).Given that many es- tolerance to tea tree oil.Pathology 33:211-215. sential oil components,including monoterpenes,are lipophilic 19.Guy ES,Mallampalli A.2008.Managing TB in the 21st century:existing and target the structure,function,and integrity of microbial and novel drug therapies.Ther.Adv.Respir.Dis.2:401-408. 20.International Organisation for Standardisation.2004.ISO 4730:2004, membranes,it seems unlikely that true resistance will arise. oil of Melaleuca,terpinen-4-ol type (tea tree oil).International Organisa- In conclusion,this study found that exposure to tea tree oil did tion for Standardisation,Geneva,Switzerland. not have any global effects on the development of antibiotic resis- 21.Jana S,Deb JK.2006.Molecular understanding of aminoglycoside action tance in the tested strains of S.aureus,S.epidermidis,and E.coli. and resistance.Appl.Microbiol.Biotechnol.70:140-150. 22.Kalan L,Wright GD.2011.Antibiotic adjuvants:multicomponent anti- Furthermore,no decreases in antimicrobial susceptibility were infective strategies.Expert Rev.Mol.Med.13:e5. observed after repeated exposure to the monoterpene terpinen-4- 23.McMahon M,Blair I,Moore J,McDowell D.2007.Habituation to 914 aac.asm.org Antimicrobial Agents and Chemotherapy
the reduction of intracellular antibiotic concentration (typically via efflux), the alteration of the target site (normally by spontaneous mutation), and enzymatic inactivation (21), and bacteria may possess more than one mechanism. The identification of the specific gene mutation(s) resulting in kanamycin resistance in mutants obtained in both the presence and absence of tea tree oil would allow the identification of an absent mutant subset. The effects of tea tree oil or terpinen-4-ol on the development of multistep antibiotic resistance were minimal when evaluated by the standard MIC assessment criteria, whereby differences in the MIC of one doubling dilution are not considered to be significant (2, 7). However, using statistical analyses, significant differences were evident between treatments on some days. In the majority of instances, MICs were significantly lower when tea tree oil or terpinen-4-ol was present, and significant differences occurred mostly on days 1 and 2. This indicates synergistic antimicrobial interactions rather than a true alteration in resistance. It also remains possible that some of the changes in antibiotic susceptibility were the result of phenotypic adaptation rather than true resistance. Similarly to the single-step studies, the combination of tea tree oil and kanamycin appears to have influenced the development of multistep resistance in E. coli; however, testing with additional isolates is required to confirm this. Overall, since the presence of tea tree oil or terpinen-4-ol resulted in only minor changes in antibiotic susceptibility, and no consistent trends were apparent for either S. aureus or E. coli, it is reasonable to conclude from these data that tea tree oil and terpinen-4ol do not have a signifi- cant impact on the development of multistep antibiotic resistance. The repeated exposure of S. aureus and S. epidermidisstrains to terpinen-4-ol did not induce significant changes in antimicrobial susceptibility, which is largely in agreement with previously published data indicating minor changes in susceptibility (of 2-fold or less) after exposure to tea tree oil for similar Gram-positive organisms (23, 24). Furthermore, where changes of 4-fold or more occurred, susceptibility was largely increased rather than decreased. These data suggest that if adaptive measures were induced by terpinen-4-ol or tea tree oil, they were not sufficient to alter antimicrobial susceptibility or confer cross-protection to other antimicrobial agents. Of the few previous studies that have attempted to induce resistance to essential oils or components, most have found either minor decreases in susceptibility or no change (1, 13, 24, 25, 28). This is similar to the present study, where minor susceptibility changes were seen by microdilution but not by macrodilution. Precisely why changes in susceptibility were observed by one method and not the other remains to be determined. Minor changes in essential oil susceptibility most likely are explained by phenotypic adaptation, which confers a low level of tolerance and has been shown to occur via reversible changes in membrane lipid composition (10, 31) and efflux (26). Organisms expressing the multiple antibiotic resistance (Mar) phenotype also have moderately reduced tea tree oil susceptibility (18). Given that many essential oil components, including monoterpenes, are lipophilic and target the structure, function, and integrity of microbial membranes, it seems unlikely that true resistance will arise. In conclusion, this study found that exposure to tea tree oil did not have any global effects on the development of antibiotic resistance in the tested strains of S. aureus, S. epidermidis, and E. coli. Furthermore, no decreases in antimicrobial susceptibility were observed after repeated exposure to the monoterpene terpinen-4- ol. Little evidence was found to support the concern that the increased use of tea tree oil in both domestic and health care environments will lead to increased antimicrobial resistance. REFERENCES 1. Ali SM, et al. 2005. Antimicrobial activities of eugenol and cinnamaldehyde against the human gastric pathogen Helicobacter pylori. Ann. Clin. Microbiol. Antimicrob. 4:20. 2. Amsterdam D. 2005. Susceptibility testing of antimicrobials in liquid media. In Lorian V (ed), Antibiotics in laboratory medicine, 5th ed. Lippincott Williams and Wilkins, Philadelphia, PA. 3. Bowler WA, Bresnahan J, Bradfish A, Fernandez C. 2010. An integrated approach to methicillin-resistant Staphylococcus aureus control in a rural, regional-referral healthcare setting. Infect. Control Hosp. Epidemiol. 31: 269 –275. 4. Braoudaki M, Hilton AC. 2004. Adaptive resistance to biocides in Salmonella enterica and Escherichia coli O157 and cross-resistance to antimicrobial agents. J. Clin. Microbiol. 42:73–78. 5. Carson CF, Hammer KA, Riley TV. 2006. Melaleuca alternifolia (tea tree) oil: a review of antimicrobial and other medicinal properties. Clin. Microbiol. Rev. 19:50 –62. 6. Carson CF, Mee BJ, Riley TV. 2002. Mechanism of action of Melaleuca alternifolia (tea tree) oil on Staphylococcus aureus determined by time-kill, lysis, leakage, and salt tolerance assays and electron microscopy. Antimicrob. Agents Chemother. 46:1914 –1920. 7. Clinical Laboratory Standards Institute. 2006. Performance standards for antimicrobial susceptibility testing, 16th informational supplement. CLSI document M100-S16. Clinical and Laboratory Standards Institute, Wayne, PA. 8. Clinical Laboratory Standards Institute. 2006. M7–A7: methods for dilution antimicrobial susceptibility tests for bacteria that grow aerobically, approved standard, 7th ed. Clinical and Laboratory Standards Institute, Wayne, PA. 9. Cox SD, et al. 2000. The mode of antimicrobial action of the essential oil of Melaleuca alternifolia (tea tree oil). J. Appl. Microbiol. 88:170 –175. 10. Di Pasqua R, Hoskins N, Betts G, Mauriello G. 2006. Changes in membrane fatty acids composition of microbial cells induced by addiction of thymol, carvacrol, limonene, cinnamaldehyde, and eugenol in the growing media. J. Agric. Food Chem. 54:2745–2749. 11. Dryden MS, Dailly S, Crouch M. 2004. A randomized, controlled trial of tea tree topical preparations versus a standard topical regimen for the clearance of MRSA colonization. J. Hosp. Infect. 56:283–286. 12. Evandri MG, et al. 2005. The antimutagenic activity of Lavandula angustifolia (lavender) essential oil in the bacterial reverse mutation assay. Food Chem. Toxicol. 43:1381–1387. 13. Ferrini AM, et al. 2006. Melaleuca alternifolia essential oil possesses potent anti-staphylococcal activity extended to strains resistant to antibiotics. Int. J. Immunopathol. Pharmacol. 19:539 –544. 14. Fletcher JP, Cassella JP, Hughes D, Cassella S. 2005. An evaluation of the mutagenic potential of commercially available tea tree oil in the United Kingdom. Int. J. Aromather. 15:81–86. 15. Fraise AP. 2002. Biocide abuse and antimicrobial resistance–a cause for concern? J. Antimicrob. Chemother. 49:11–12. 16. Fuangthong M, et al. 2011. Exposure of Acinetobacter baylyi ADP1 to the biocide chlorhexidine leads to acquired resistance to the biocide itself and to oxidants. J. Antimicrob. Chemother. 66:319 –322. 17. Gilbert P, McBain AJ. 2003. Potential impact of increased use of biocides in consumer products on prevalence of antibiotic resistance. Clin. Microbiol. Rev. 16:189 –208. 18. Gustafson JE, Cox SD, Liew YC, Wyllie SG, Warmington JR. 2001. The bacterial multiple antibiotic resistant (Mar) phenotype leads to increased tolerance to tea tree oil. Pathology 33:211–215. 19. Guy ES, Mallampalli A. 2008. Managing TB in the 21st century: existing and novel drug therapies. Ther. Adv. Respir. Dis. 2:401–408. 20. International Organisation for Standardisation. 2004. ISO 4730:2004, oil of Melaleuca, terpinen-4-ol type (tea tree oil). International Organisation for Standardisation, Geneva, Switzerland. 21. Jana S, Deb JK. 2006. Molecular understanding of aminoglycoside action and resistance. Appl. Microbiol. Biotechnol. 70:140 –150. 22. Kalan L, Wright GD. 2011. Antibiotic adjuvants: multicomponent antiinfective strategies. Expert Rev. Mol. Med. 13:e5. 23. McMahon M, Blair I, Moore J, McDowell D. 2007. Habituation to Hammer et al. 914 aac.asm.org Antimicrobial Agents and Chemotherapy on May 12, 2015 by SHANGHAI JIAOTONG UNIVERSITY http://aac.asm.org/ Downloaded from
Tea Tree Oil and Antibiotic Resistance sub-lethal concentrations of tea tree oil (Melaleuca alternifolia)is associ- tions,p.365-440.In Lorian (ed),V.Antibiotics in laboratory medicine. ated with reduced susceptibility to antibiotics in human pathogens.J. Lippincott Williams Wilkins,Philadelphia,PA. Antimicrob.Chemother.59:125-127. 28 Shapira R,Mimran E.2007.Isolation and characterization of Escherichia 24.McMahon MAS,et al.2008.Changes in antibiotic susceptibility in staph- coli mutants exhibiting altered response to thymol.Microb.Drug Resist. ylococci habituated to sub-lethal concentrations of tea tree oil (Melaleuca Mech.Epidemiol.Dis.13:157-165. alternifolia).Lett.Appl.Microbiol.47:263-268 Sia IG,Wieland ML 2011.Current concepts in the management of 25.Mondello F,De Bernardis F,Girolamo A,Salvatore G,Cassone A.2003.In tuberculosis.Mayo Clin.Proc.86:348-361. vitro and in vivo activity of tea tree oil against azole-susceptible and -resistant 30. Thompson G,et al.2008.A randomized controlled trial of tea tree oil human pathogenic yeasts.J.Antimicrob.Chemother.51:1223-1229 (5%)body wash versus standard body wash to prevent colonization with 26.Papadopoulos CJ,Carson CF,Chang B),Riley TV.2008.Role of the MexAB. methicillin-resistant Staphylococcus aureus (MRSA)in critically ill adults OprM effux pump of Pseudomonas in tolerance to teatree(Melaleuca search protocol.BMC Infect.Dis.8:161. altertifolia)oil and its monoterpene components terpinen-4-ol,1,8-cineole,and 31.Ultee A,Kets EPW,Alberda M,Hoekstra FA,Smid EJ.2000.Adaptation alpha-terpineol.Appl.Environ.Microbiol.74:1932-1935. of the food-borne pathogen Bacillus cereus to carvacrol.Arch.Microbiol. 27.Pillai S,Moellering R Jr.,Eliopoulos G.2005.Antimicrobial combina 174:233-238 Downloaded from http://aac.asm.org/on May 12,2015 by SHANGHAI JIAOTONG UNIVERSITY February 2012 Volume 56 Number 2 aac.asm.org 915
sub-lethal concentrations of tea tree oil (Melaleuca alternifolia) is associated with reduced susceptibility to antibiotics in human pathogens. J. Antimicrob. Chemother. 59:125–127. 24. McMahon MAS, et al. 2008. Changes in antibiotic susceptibility in staphylococci habituated to sub-lethal concentrations of tea tree oil (Melaleuca alternifolia). Lett. Appl. Microbiol. 47:263–268. 25. Mondello F, De Bernardis F, Girolamo A, Salvatore G, Cassone A. 2003. In vitro and in vivo activity of tea tree oil against azole-susceptible and -resistant human pathogenic yeasts. J. Antimicrob. Chemother. 51:1223–1229. 26. Papadopoulos CJ, Carson CF, Chang BJ, Riley TV. 2008. Role of the MexABOprM efflux pump ofPseudomonas aeruginosa in tolerance to tea tree (Melaleuca altenifolia) oil and its monoterpene components terpinen-4-ol, 1,8-cineole, and alpha-terpineol. Appl. Environ. Microbiol. 74:1932–1935. 27. Pillai S, Moellering R Jr., Eliopoulos G. 2005. Antimicrobial combinations, p. 365–440. In Lorian (ed), V. Antibiotics in laboratory medicine. Lippincott Williams & Wilkins, Philadelphia, PA. 28. Shapira R, Mimran E. 2007. Isolation and characterization of Escherichia coli mutants exhibiting altered response to thymol. Microb. Drug Resist. Mech. Epidemiol. Dis. 13:157–165. 29. Sia IG, Wieland ML. 2011. Current concepts in the management of tuberculosis. Mayo Clin. Proc. 86:348 –361. 30. Thompson G, et al. 2008. A randomized controlled trial of tea tree oil (5%) body wash versus standard body wash to prevent colonization with methicillin-resistant Staphylococcus aureus (MRSA) in critically ill adults: research protocol. BMC Infect. Dis. 8:161. 31. Ultee A, Kets EPW, Alberda M, Hoekstra FA, Smid EJ. 2000. Adaptation of the food-borne pathogen Bacillus cereus to carvacrol. Arch. Microbiol. 174:233–238. Tea Tree Oil and Antibiotic Resistance February 2012 Volume 56 Number 2 aac.asm.org 915 on May 12, 2015 by SHANGHAI JIAOTONG UNIVERSITY http://aac.asm.org/ Downloaded from