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Journal of Applied Microbiology 2000, 88, 170–175
The mode of antimicrobial action of the essential oil of
Melaleuca alternifolia (tea tree oil)
S.D. Cox1, C.M. Mann1, J.L. Markham1, H.C. Bell2, J.E. Gustafson3, J.R. Warmington3 and
S.G. Wyllie1
1
Centre for Biostructural and Biomolecular Research, University of Western Sydney, Hawkesbury, New South
Wales, 2Australian Tea Tree Oil Research Institute, Lismore, New South Wales and 3Genetica
Biotechnologies, Bentley, Western Australia
7236/5/99: received 14 May 1999, revised 16 august 1999 and accepted 16 August 1999
S .D . C O X, C. M . M AN N , J .L . MA RK H AM , H . C. BE L L, J. E . G US T AF SO N , J .R . WA RM I NG TO N AN D S . G.
The essential oil of Melaleuca alternifolia (tea tree) exhibits broad-spectrum
antimicrobial activity. Its mode of action against the Gram-negative bacterium Escherichia
coli AG100, the Gram-positive bacterium Staphylococcus aureus NCTC 8325, and the yeast
Candida albicans has been investigated using a range of methods. We report that exposing
these organisms to minimum inhibitory and minimum bactericidal/fungicidal concentrations
of tea tree oil inhibited respiration and increased the permeability of bacterial cytoplasmic
and yeast plasma membranes as indicated by uptake of propidium iodide. In the case of E.
coli and Staph. aureus, tea tree oil also caused potassium ion leakage. Differences in the
susceptibility of the test organisms to tea tree oil were also observed and these are interpreted
in terms of variations in the rate of monoterpene penetration through cell wall and cell
membrane structures. The ability of tea tree oil to disrupt the permeability barrier of cell
membrane structures and the accompanying loss of chemiosmotic control is the most likely
source of its lethal action at minimum inhibitory levels.
W YL LI E . 2000.
INTRODUCTION
The essential oil of Melaleuca alternifolia, commonly known
as tea tree oil, has a long history of use as a topical antiseptic
(Markham 1999). In recent times it has gained a reputation
as a safe, natural and effective antiseptic. This has led to a
resurgence in popularity and currently it is incorporated as
the principal antimicrobial or as a natural preservative in
many pharmaceutical and cosmetic products intended for
external use.
The chemical composition of tea tree oil has been well
defined and consists largely of cyclic monoterpenes (Brophy
et al. 1989) of which about 50% are oxygenated and about
50% are hydrocarbons. Tea tree oil exhibits broad-spectrum
antimicrobial activity (see Markham 1999 for a review) which
can be principally attributed to terpinen-4-ol (Southwell et al.
1993; Carson and Riley 1995).
Correspondence to: Dr S.D. Cox, Building L9, Faculty of Science and
Technology, UWS Hawkesbury, Bourke Street, Richmond, 2753, New South
Wales, Australia (e-mail: [email protected]).
A wide variety of essential oils are known to possess
antimicrobial properties and in many cases this activity is due
to the presence of active monoterpene constituents (Knobloch
et al. 1988; Beylier 1979; Morris et al. 1979). Several studies
have also shown that monoterpenes exert membrane-damaging effects (reviewed by Sikkema et al. 1995). Examination
of Escherichia coli cells using electron microscopy after
exposure to tea tree oil revealed a loss of cellular electrondense material and coagulation of cytoplasmic constituents,
although it was apparent that these effects were secondary
events that occurred after cell death (Gustafson et al. 1998).
Tea tree oil also stimulates leakage of cellular potassium ions
and inhibits respiration in E. coli cell suspensions, providing
evidence of a lethal action related to cytoplasmic membrane
damage (Cox et al. 1998).
Here we report the further investigation of the antimicrobial activity of tea tree oil against three clinically significant
micro-organisms, E. coli, Staphylococcus aureus and Candida
albicans.
© 2000 The Society for Applied Microbiology
M OD E O F AC TI O N O F T E A T RE E OI L 171
MATERIALS AND METHODS
Tea tree oil
The tea tree oil used in all assays was from a sample (Batch
6081) donated by Main Camp, Ballina, NSW, Australia.
Growth of test organisms
Cells used in all assays were twice passaged, in Iso-sensitest
Broth (Oxoid, Basingstoke, UK) in the case of E. coli strain
AG100, a K-12 derivative (George and Levy 1983) in the
case of Staph. aureus NCTC 8325, and in Malt extract broth
(Oxoid) in the case of C. albicans KEM H5 at 37 °C.
Minimum inhibitory concentrations (MIC) and minimum
bactericidal/fungicidal concentrations (MBC)
MIC/MBC assays were performed as described in Gustafson
et al. (1998) with the following exceptions. In the case of C.
albicans, Malt extract broth (Oxoid) was substituted for Isosensitest broth (Oxoid). Tween-80 was omitted from the
dilution/assay mixture. Minimum bactericidal/fungicidal
concentrations were determined by sampling 100 ml from
each tube that showed no growth into a neutralising broth
which contained 30 g l−1 Tryptone soy broth (Oxoid), 30 g l−1
neutralized liver digest (Oxoid) and 10 g l−1 lecithin (Defiance
Milling Co., Acacia Ridge, QLD). Following a 10-min room
temperature incubation, 10 ml Iso-sensitest was added to each
tube (malt extract broth was used in the case of C. albicans)
and they were then incubated at 37 °C for 72 h. The minimum
bactericidal/fungicidal concentration was determined as the
lowest concentration of tea tree oil that showed no growth.
Viability assays
An overnight culture was used to inoculate an Iso-sensitest
broth (Malt extract broth was used for C. albicans). Cells
were grown at 37 °C to exponential phase (4–5 h), washed
once and resuspended in sterile 100-ml conical flasks containing 20 ml of cell suspension and the required volume of
tea tree oil. The flask contents were continually stirred on a
magnetic stirrer to ensure uniform oil dispersion throughout
the assays. Aliquots (1 ml) were removed at the required time
intervals into 9 ml of neutralising broth and allowed to stand
at room temperature for 10 min. Serial 10-fold dilutions of
the neutralising broths were then prepared in 0·1% peptone
and pour plates prepared using Tryptone soy agar (Oxoid).
Colonies were counted after a 3 d incubation at 37 °C and the
viable cell number reported as colony-forming units (CFU)
per ml.
Measurement of respiration
Microbial respiration rates were determined using an oxygen
electrode as previously described in Cox et al. (1998). For E.
coli and C. albicans, cell suspensions were preincubated for
5 min in the indicated tea tree oil concentration prior to
measuring the respiratory activity. In the case of Staph.
aureus, cells were preincubated in the presence of tea tree oil
for 10 min prior to measurement.
Efflux of potassium ions
Potassium ion concentration in cell suspensions was measured
using a potassium ion selective electrode, as previously
described in Cox et al. (1998). The concentration of total free
potassium for Staph. aureus suspensions was measured after
incubation in lysostaphin (100 mg ml−1) at 37 °C for 60 min,
followed by sonication. To measure total free potassium in
C. albicans, cells were lysed by incubating in chitinase
(1 mg ml−1) and lyticase (1 mg ml−1) at 37 °C for 60 min,
followed by sonication. Complete lysis in each case was confirmed by microscopic examination.
Propidium iodide uptake
Cells (100 ml culture) were grown overnight as described
above, washed and resuspended in 50 mmol l−1 sodium phosphate buffer, pH 7·1. Aliquots (1 ml) were added to stirred
conical flasks containing 19 ml buffer and the required
amount of tea tree oil. The inoculum density was
¼ 108 CFU ml−1. Following a 30-min incubation at room
temperature, 50ml aliquots were transferred into Eppendorfs
containing 950 ml phosphate buffer in FACS tubes (Becton
Dickinson, Immunocytometry Systems, Mountain View,
California). These tubes were stored on ice and 5 ml of staining
solution, consisting of 2·5 mg ml−1 propidium iodide (Molecular Probes, Eugene, Oregon) dissolved in milliQ water, was
added to give a final propidium iodide concentration of
10 mg ml−1. Immediately following this, the percentage of
propidium iodide stained cells was determined using a FACScan Flow cytometer (Becton Dickinson).
Assay of tea tree oil-induced carboxyfluorescein
leakage
Multilamellar lipid vesicles were prepared following the procedure of New (1990). Phospholipids (14 mg phosphatidylethanolamine, 4 mg phosphatidylglycerol and 2 mg
cardiolipin) were dissolved in chloroform in a 100-ml roundbottom flask and evaporated to dryness. Following this, the
dried phospholipid mixture was resuspended in 2 ml of
50 mmol l−1 sodium phosphate buffer, pH 7·0, containing a
self-quenched
concentration
of
carboxyfluorescein
© 2000 The Society for Applied Microbiology, Journal of Applied Microbiology 88, 170–175
172 S .D . C O X E T A L .
(100 mmol l−1), by gentle shaking with glass beads. The
resulting suspension of liposomes (multilamellar lipid vesicles) was then dialysed overnight to remove unencapsulated
carboxyfluorescein. Liposome suspension (100 ml) was added
to an Eppendorf tube, followed by phosphate buffer and the
required amount of tea tree oil, to give 1 ml final volume.
The mixture was then vortexed and incubated for the
required time interval with repeated mixing at 5 min intervals.
When the incubation period had elapsed, 50 ml of the liposome suspension was sampled into 2 ml phosphate buffer.
Fluorescence was measured in a glass cuvette using a fluorescence spectrophotometer (Hitachi F-4500, Hitachi, San
José, CA, USA; lex 470 nm; lem 520 nm). One hundred
per cent carboxyfluorescein leakage was determined by
adding triton-X100, 1·0% (v/v).
RESULTS
Minimum inhibitory concentrations (MIC) and minimum
bactericidal/fungicidal concentrations (MBC) of tea tree
oil
The MIC and MBC of tea tree oil were 0·25% and 0·5%
(v/v), respectively, for both E. coli AG100 and Staph. aureus
NCTC 8325. MIC and MBC values for C albicans KEM H5
were a factor of two lower, at 0·125% and 0·25% (v/v),
respectively.
Fig. 1 Effects of tea tree oil on viability of test organisms. (a) E. coli
Effects of tea tree oil on cell viability
The effects of tea tree oil exposure on the viability of E. coli,
Staph. aureus and C. albicans are shown in Fig. 1(a,b,c). Each
figure is representative of three separate experiments that
gave similar results. At minimum inhibitory and minimum
lethal concentrations, E. coli was most susceptible to the
effects of tea tree oil, followed by C. albicans and then Staph.
aureus.
Effects of tea tree oil on respiration
Tea tree oil inhibited respiration in cell suspensions of E.
coli, Staph. aureus and C. albicans (Fig. 2). Inhibition of E.
coli respiration commenced in 0·25% (v/v) tea tree oil and
was complete at 0·5% (v/v). Respiration in C. albicans cells
was inhibited at 0·125% (v/v), which was the lowest concentration assayed and corresponds to the MIC for this organism. Inhibition of Staph. aureus cell respiration (after 10 min
of tea tree oil exposure) commenced at a concentration of
0·5% (v/v).
AG 100: () no tea tree oil, and (Ž) 0·50% v/v tea tree oil. (b)
Staph. aureus NCTC 100: () no tea tree oil, (ž) 0·25% v/v tea
tree oil, and (Ž) 0·50% v/v tea tree oil. (c) C. albicans KEM H6:100:
() no tea tree oil, (ž) 0·125% v/v tea tree oil, () 0·25% v/v
tea tree oil; and () 0·50% v/v tea tree oil
Effects of tea tree oil on membrane integrity
Exposing cell suspensions of E. coli, Staph. aureus and C.
albicans to 0·25% (v/v) tea tree oil for 30 min increased cell
permeability to the fluorescent nucleic acid stain, propidium
iodide (Fig. 3), relative to control suspensions that did not
contain tea tree oil. The inability of propidium iodide to
penetrate cells with intact cytoplasmic or plasma membranes
(see Brul et al. 1997; Mason et al. 1997; Wenisch et al. 1997;
Lebaron et al. 1998) was confirmed by the low level of uptake
observed in cells not exposed to tea tree oil (Fig. 3).
Tea tree oil at 0·25% (v/v) induced leakage of potassium
ions from E. coli and Staph. aureus cells (Fig. 4). The data,
which is representative of triplicate experiments that gave
similar results, shows that leakage from E. coli cells commenced immediately upon addition of tea tree oil and the
© 2000 The Society for Applied Microbiology, Journal of Applied Microbiology 88, 170–175
M OD E O F AC TI O N O F T E A T RE E OI L 173
Fig. 2 Effects of tea tree oil concentration on O2 consumption rates
in cell suspensions of E. coli AG100 (), Staph. aureus NCTC 8325
(ž) and C. albicans KEM H5 (). Error bars represent the standard
deviation (n 3) of data from replicate experiments. In some cases
the error bars are small enough to be obscured by data symbols
Fig. 4 Effects of 0·25% v/v tea tree oil on potassium ion efflux in
cell suspensions of E. coli AG100 and Staph. aureus NCTC 8325.
() E. coli, 0·25% v/v tea tree oil; (ž) E. coli, no tea tree oil; ()
S. aureus, 0·25% v/v tea tree oil; (Ž), Staph. aureus, no tea tree oil
Tea tree oil (0·25% v/v) also stimulated the leakage of
encapsulated carboxyfluorescein from a suspension of multilamellar lipid vesicles (Fig. 5).
DISCUSSION
In this study, tea tree oil inhibited respiration in E. coli,
Staph. aureus and C. albicans cells at minimum inhibitory
levels. The possibility that tea tree oil directly inhibits a
specific respiratory enzyme or metabolic event cannot be
eliminated. However, our findings also reveal that minimum
inhibitory levels of tea tree oil altered cell membrane struc-
Fig. 3 Uptake of propidium iodide in cell suspensions of E. coli
AG100, Staph. aureus NCTC 8325 and C. albicans KEM H5. Cells
were exposed to 0·25% v/v tea tree oil for 30 min () and compared
with control flasks containing no added tea tree oil (Ž). Error bars
represent standard deviations calculated from separate assays
(n 3)
extent approached 100% of total cellular free potassium after
about 30 min. Efflux from Staph. aureus cells, by comparison,
commenced after about 5 min exposure to tea tree oil and
continued at a slower rate, reaching 20% after 30 min. Leakage of potassium ions from C. albicans in the presence 0·25%
(v/v) tea tree oil over a period of two h was no greater than
background levels (data not shown). However, after exposure
to 2·5% (v/v) for 60 min the amount of potassium in cell
supernatants was 23·1% of that in supernatants from total
cell lysates.
Fig. 5 Stimulation of leakage of carboxyfluorescein from
multilamellar lipid vesicles exposed to tea tree oil. () No tea tree
oil; (ž) 0·25% v/v tea tree oil. Error bars represent standard
deviations (n 2)
© 2000 The Society for Applied Microbiology, Journal of Applied Microbiology 88, 170–175
174 S .D . C O X E T A L .
ture. Increased uptake of the nucleic acid stain propidium
iodide, to which the cell membrane is normally impermeable,
was observed. Also, leakage of potassium ions commenced
immediately upon adding tea tree oil to suspensions containing E. coli and within 5 min for Staph. aureus cells.
In the case of C. albicans, we did not detect the appearance
of potassium ions in cell supernatants containing 0·25% (v/v)
tea tree oil. However, the propidium iodide staining of C.
albicans cells exposed to tea tree oil is a clear indication of
damage to the plasma membrane. It may be that potassium
ions do not appear in cell supernatants (after up to 2 h
exposure) because they remain incorporated in the thick layer
of the C. albicans cell wall. Given the increased permeability
to propidium iodide, it seems unlikely that the plasma membrane would have remained impermeable to potassium ions.
Further confirmation of the general toxicity of tea tree oil to
membrane structures is provided by its permeabilising effect
on multilamellar liposomes.
Previously, we have shown that tea tree oil inhibits respiration and causes leakage of cellular potassium in E. coli at
minimum inhibitory levels (Cox et al. 1998). These effects,
along with the findings presented here, indicate that tea tree
oil damages cell membrane structure in E. coli, Staph. aureus
and C. albicans. The cytoplasmic membranes of bacteria and
the plasma and mitochondrial membranes of yeast provide a
barrier to the passage of small ions such as H+, K+, Na+ and
Ca2+ and allow cells and organelles to control the entry and
exit of different compounds. This permeability barrier role
of cell membranes is integral to many cellular functions,
including the maintenance of the energy status of the cell,
other membrane-coupled energy-transducing processes, solute transport, regulation of metabolism and control of turgor
pressure (Booth 1985; Poolman et al. 1987; Trumpower and
Gennis 1994).
Toxic effects on membrane structure and function have
generally been used to explain the antimicrobial action of
essential oils and their monoterpenoid components (Andrews
et al. 1980; Uribe et al. 1985; Knobloch et al. 1988). Sikkema
et al. (1994) showed that, as a result of their lipophilic character, cyclic monoterpenes will preferentially partition from an
aqueous phase into membrane structures. This resulted in
membrane expansion, increased membrane fluidity and inhibition of a membrane-embedded enzyme. In yeast cells and
isolated mitochondria, a-pinene and b-pinene destroy cellular
integrity, inhibit respiration and ion transport processes and
increase membrane permeability (Andrews et al. 1980; Uribe
et al. 1985). More recently, Helander et al. (1998) have
described effects of different essential components on outer
membrane permeability in Gram-negative bacteria. The fact
that tea tree oil-induced damage to cell membrane structure
accompanied the decline in viability for all three micro-organisms included in this study confirms it as the most likely
cause of cell death.
In spite of similar MIC/MBC values, the micro-organisms
studied here showed obvious differences in their susceptibility to tea tree oil. The rate of viability decline of C.
albicans in 0·25% (v/v) tea tree oil was less than that seen for
E. coli in the same concentration and for Staph. aureus the
rate of inactivation was slower than that of either E. coli or
C. albicans. The relative inhibition of respiration and the
extent of membrane damage of these different micro-organisms follow the same pattern. Given the broad spectrum
activity of tea tree oil and its general membrane-damaging
effect, it is likely that this variability reflects the rate at which
its active components diffuse through the cell wall and into
the phospholipid regions of cell membrane structures.
In conclusion, our observations confirm that the antimicrobial activity of tea tree oil results from its ability to disrupt
the permeability barrier of microbial membrane structures.
This mode of action is the same against E. coli, Staph. aureus
and C. albicans and is similar to that of other broad-spectrum,
membrane-active disinfectants and preservatives, such as
phenol derivatives, chlorhexidine (see McDonnell and Russell 1999) and parabenzoic acid derivatives (Sox 1997).
ACKNOWLEDGEMENT
This work was wholly funded by the Australian Tea Tree Oil
Research Institute (ATTORI), Lismore, New South Wales,
Australia.
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