The effect of the essential oil
and its components from
Melaleuca alternifolia on
endospore germination in
Bacillus cereus
By: Rachel Schmid
ASM Microblibrary.org © Weber
Picture by Geneva Foundation for Medical Education and Research
Historical Use Of Tea Tree Oil
(TTO)
• Small, summer flowering
tree native to Australia
• First used by Bundjalong
Aborigines in New South
Wales for skin problems
and respiration aliments
(Carson and Riley 1993).
• 1925: distilled oil’s
antimicrobial properties
published by Penfold and
Grant
• Since then extensive
research done on oil’s
uses
Uses of the Oil
• Published evidence of antibacterial,
antifungal, antiprotozan, antiviral, and antiinflammatory properties
• Also used to treat athlete’s foot, head lice,
acne, and other skin irritations
• Oil readily available for everyday use
without a prescription
• Found in shampoos, skin treatments, etc.
The major components of TTO
Component
terpinen-4-ol
γ-terpinene
α-terpinene
1, 8-cineole
terpinolene
α-terpineol
p-cymene
α-pinene
aromadendrene
virdiflorene
δ-cadinene
limonene
β-phellandrene
globulol
myrcene
α -thujene
β-pinene
sabinene
α -phellandrene
viridiflorol
Mean a
Min a
Max a
37.93
20.20
9.56
3.87
3.45
3.01
2.80
2.46
1.68
1.68
1.49
1.01
0.94
0.86
0.86
0.83
0.66
0.45
0.44
0.33
28.6
9.5
4.6
0.5
1.6
1.5
0.4
0.8
0.1
0.3
0.1
0.4
0.4
0.1
0.1
0.1
0.1
0.0
0.1
0.1
57.9
28.3
12.8
17.7
5.4
7.6
12.4
3.6
6.6
6.1
7.5
2.7
1.9
3.0
1.8
2.1
1.6
3.2
1.9
1.4
ISO 4730
range in % b
≥30
10-28
5-13
≤15
1.5-5
1.5-8
0.5-12
1-6
Trace-7
N/A
Trace-8
0.5-4
N/A
Trace-3
N/A
N/A
N/A
Trace-3.5
N/A
Trace-1.5
Previously Found Active Components
• terpinen-4-ol thought to be most active
ingredient (Carson and Riley 1995)
• terpinen-4-ol and α-terpineol cause majority of
the antibacterial and antifungal action (Carson et
al., 2006)
• α-pinene, linalool, and limonene also shown to
have antibacterial properties (Raman et al.,
1995)
• 1,8-cineole thought to play role in allowing active
components into cell
The present study
• TTO has many antimicrobial
abilities
• Can it prevent endospore
germination?
• If so, what component of the oil
can do this?
Endospores
• Hardy,
encapsulated
pieces of DNA
• Able to survive
through harsh
conditions
• Bacillus spp. able
to form them
Picture by textbookofbacteriology.net
Bacillus spp.
• Using B. cereus as
model for B. anthracis
• 2001 bioterrorism
attacks using anthrax
spores on mailed
envelopes
• 22 mail workers
infected and 5 died
from exposure
• Most infections from
anthrax are
cutaneous
Methods
• B. cereus bacteria placed
in LB on shaker for 8
days
• Heat treatment
• Spread on LB plate
Methods
•
•
•
•
B. cereus bacteria placed in LB on shaker for 8 days
Heat treatment
Spread on LB plate
3-4 3M discs were placed on each plate
Methods
• B. cereus bacteria placed
in LB on shaker for 8
days
• Heat treatment
• Spread on LB plate
• 4 3M discs were placed
on each plate
• Added small amount of
TTO or components:
terpinen-4-ol, γ-terpinene,
α-terpinene, 1,8-cineole,
α-pinene, p -cymene,
α-terpineol, or limonene
Methods
• B. cereus bacteria placed
in LB on shaker for 8
days
• Heat treatment
• Spread on LB plate
• 4 3M discs were placed
on each plate
• Added small amount of
TTO or components:
terpinen-4-ol, γ-terpinene,
α-terpinene, 1,8-cineole,
α-pinene, p -cymene,
α-terpineol, or limonene
• Incubated for 24 hours at
32°C
• Measured zone of
inhibition
Methods
• B. cereus bacteria placed
in LB on shaker for 8
days
• Heat treatment
• Spread on LB plate
• 4 3M discs were placed
on each plate
• Added small amount of
TTO or components:
terpinen-4-ol, γ-terpinene,
α-terpinene, 1,8-cineole,
α-pinene, p -cymene,
α-terpineol, or limonene
• Incubated for 24 hours at
32°C
• Measured zone of
inhibition
• Dose effects of active
components
• Synergistic effects
between active + active
and active + inactive
• ANOVA and Tukey
Kramer Post Hoc
performed
• Oil and components
checked for purity on
GC/MS
Results
• TTO inhibited
endospore
germination
• terpinen-4-ol,
α-terpinene, and
α-terpineol
components active
• None significantly
more active than the
others or TTO
Synergisms
2.5
Radial Cleared Zone (mm)
• Two active
components:
• terpinen-4-ol and
α-terpineol
• Combination
significantly more
effective than either
component
• F = 40.17, df = 2,
p < 0.0001
2
1.5
1
0.5
0
-0.5
terpinen-4-ol α-terpineol
both
Synergisms
8
Radial Cleared Zone (mm)
7
6
5
4
3
2
1
0
-1
1,8-cineole
α-terpinene
both
• Active and inactive:
• α-terpinene and
1,8-cineole
• F = 26.24, df = 2,
p < 0.0001
Radial Cleared Zone (mm)
5.5
• α-terpinene and
p-cymene
• F = 10.50, df = 2,
p = 0.0014
4.5
3.5
2.5
1.5
0.5
-0.5
p-cymene
α-terpinene
both
Synergisms
• Active and inactive:
• α-terpineol and
1,8-cineole
• F = 56.43, df = 2,
p < 0.0001
Radial Cleared Zone (mm)
2.5
2
1.5
1
0.5
0
-0.5
1,8-cineole
α-terpineol
both
• α-terpineol and
γ-terpinene
• and F = 19.86, df = 2,
p < 0.0001
Radial Cleared Zone (mm)
2.5
2
1.5
1
0.5
0
-0.5
γ -terpinene
α-terpineol
both
GC/MS
• The ten most
abundant components
of the commercial
sample of TTO.
• The relative
percentages in the oil
as observed by
GC/MS.
• The normal range for
α-terpinene is 5-13%.
Component
% Peak Area
Retention (min)
terpinen-4-ol
34.00%
12.836
γ-terpinene
27.14%
10.257
α-terpinene
16.23%
9.292
α-pinene
5.76%
7.351
3.77%
10.917
o-cymene
3.41%
9.469
1,8-cineole
3.12%
9.636
limonene
2.38%
9.578
α-terpineol
2.22%
13.092
α-thujene
1.96%
7.184
αterpineole
ne
GC/MS
Composition of commercially purchased
components that were active or part of a
significant synergism
Component
Purity
Contaminant
1,8-cineole
100.00%
p-cymene
99.63%
0.37%
cymene
95.24%
4.24%
o-cymene
94.18%
4.41%
cyclooctan, 1-(diethylboryl)
89.96%
10.04%
γ-terpineol
76.46%
12.92%
5.99%
2.63%
o-cymene
1,8-cineole
1,3-heptadiene
γ-terpinene
terpinen-4-ol
α-terpineol
α-terpinene
Discussion
• Terpinen-4-ol is not the only active component,
α-terpineol and α-terpinene are just as active
• Terpenes are shown to cause a loss of membrane
integrity and disrupt proton motive force (Sikkema et
al. 1995; Cox et al. 1998)
terpinen-4-ol
OH
α-terpineol
OH
α-terpinene
• These components are not active on their own
but contribute to the overall activity of the oil
• In bacteria, 1,8-cineole has been shown to
disrupt the cell membrane to allow active
components in (Carson et al. 2006)
γ-terpinene
1,8-cineole
OH
p-cymene
Suggested Studies
• Revise ISO for TTO to contain more
α-terpinene
• Use of TTO in alternative treatments of
infectious disease
• More work with TTO and anthrax
endospores in containment labs
• Clinical trials for prevention/healing of
cutaneous infections in places where
refrigeration of antibiotics is impossible
Literature Cited
• Carson, C. F., K. A. Hammer, and T. V. Riley. 2006. Melaleuca (Tea
Tree) Oil: a review of antimicrobial and other medicinal
properties. Clinical Microbiology Review 19: 50-62.
• Carson, C. F., and T. V. Riley. 1993. Antimicrobial activity of essential
oil of Melaleuca alternifolia. Letters in Applied Microbiology 16:
49-55.
• Carson, C. F., and T. V. Riley. 1995. Antimicrobial activity of the major
components of the essential oil of Melaleuca alternifolia. J. of
Applied Bacteriology 78: 264-269.
• Cox, S. D., J. E. Gustafson, C. M. Mann, J. L. Markham, Y. C. Liew, R.
P. Hartland, H. C. Bell, J. R. Warmington, and S. G. Wyllie. 1998.
Tea tree oil causes K+ leakage and inhibits respiration in
Escherichia coli. Letters Applied Microbiology 26: 355-358.
• Raman, A, U. Weir, and S. F. Bloomfield. 1995. Antimicrobial effects of
tea tree oil and its major components on Staphylococcus aureus,
Staphylococcus epidermidis, and Propionibacterium acnes.
Applied Microbiology 21: 242-245.
• Sikkema, J., J. A. De Bont, and B. Poolman. 1995. Mechanisms of
membrane toxicity of hydrocarbons. Microbiological Reviews 59:
201–222.