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Diğer adı tea tree oil dir,Avusturalyada yetişen bir tür ağaçtan elde edilir.Cilt rahatsızlıklarına sebep olan mantar ve bakterileri öldürücü etkileri vardır.Ödem ve enflamasyonu engeller.Cildin enflamasyonlu hastalıklarında (sedef,dermatit,kurdeşen vs) kullanılır.kaşmpuan,krem,solusyon ve pomad gibi birçok formu vardır.Çok çeşitli hastalığın tedavisinde kullanım sonuçlarıyla ilgili sayısız bilimsel çalışma yapılmıştır.

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10.1128/AAC.05741-11.

Antimicrob. Agents Chemother. 2012, 56(2):909. DOI:

Riley

Katherine A. Hammer, Christine F. Carson and Thomas V.

Susceptibility

Antibiotic Resistance and Antimicrobial

Development of Single- and Multistep

Component Terpinen-4-ol on the

Essential Oil and the Major Monoterpene

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,

NCTC29213, andATCC25923, E. coliNCTC10418,ATCC25922,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.

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 mlMHA

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 modifications.

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 500rpmfor 24 h. Cultures then were 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 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

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).

Inocula for the nonpassaged control were 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. aureus were 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

TABLE 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 _ 10_8 5.9 _ 10_8 1.1 _ 10_7 7.2 _ 10_8 0.0065c

10 MUP 8 7.3 _ 10_8 2.3 _ 10_8 9.9 _ 10_8 3.5 _ 10_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

9 KAN 2 3.3 _ 10_6 2.0 _ 10_7 2.6 _ 10_6 3.1 _ 10_7 _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 4 5 6

CIP (10)

Median Alone 0.5 1 1 2 2 2

With TTO 0.25 1 1 2 2 2

With T4ol 0.25 0.5 1 2 2 2

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 8 4 4

With TTO 1 4 4 8 4 4

With T4ol 1 4 4 8 4 4

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

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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 ciprofloxacin

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 medianMICwas 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:

1 2 3 4 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 0.007 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 8 8 16 16

With TTO 2 4 8 8 16 16

With T4ol 2 4 8 8 16 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 1 1 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

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.

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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.

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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 significant

impact on the development of multistep antibiotic resistance.

The repeated exposure of S. aureus and S. epidermidis strains 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.

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