Transcript The effect of the essential oil and its components from Melaleuca

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.