TTO (çay ağacı yağı)
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.
Mikrop öldürücü etkisi üzerine yapılan çalışma
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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