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![Page 1: The effect of the essential oil and its components from Melaleuca alternifolia on endospore germination in Bacillus cereus By: Rachel Schmid ASM Microblibrary.org.](https://reader035.fdocuments.us/reader035/viewer/2022062307/5515155b55034673228b4b9d/html5/thumbnails/1.jpg)
The effect of the essential oil and its components from
Melaleuca alternifolia on endospore
germination in Bacillus cereusBy: Rachel Schmid
ASM Microblibrary.org © Weber
![Page 2: The effect of the essential oil and its components from Melaleuca alternifolia on endospore germination in Bacillus cereus By: Rachel Schmid ASM Microblibrary.org.](https://reader035.fdocuments.us/reader035/viewer/2022062307/5515155b55034673228b4b9d/html5/thumbnails/2.jpg)
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
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Uses of the Oil
• Published evidence of antibacterial, antifungal, antiprotozan, antiviral, and anti-inflammatory 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.
![Page 4: The effect of the essential oil and its components from Melaleuca alternifolia on endospore germination in Bacillus cereus By: Rachel Schmid ASM Microblibrary.org.](https://reader035.fdocuments.us/reader035/viewer/2022062307/5515155b55034673228b4b9d/html5/thumbnails/4.jpg)
The major components of TTO Component
Mean a
Min a
Max a
ISO 4730
range in % b terpinen-4-ol 37.93 28.6 57.9 ≥30 γ-terpinene 20.20 9.5 28.3 10-28 α-terpinene 9.56 4.6 12.8 5-13 1, 8-cineole 3.87 0.5 17.7 ≤15 terpinolene 3.45 1.6 5.4 1.5-5 α-terpineol 3.01 1.5 7.6 1.5-8 p-cymene 2.80 0.4 12.4 0.5-12 α-pinene 2.46 0.8 3.6 1-6 aromadendrene 1.68 0.1 6.6 Trace-7 virdiflorene 1.68 0.3 6.1 N/A δ-cadinene 1.49 0.1 7.5 Trace-8 limonene 1.01 0.4 2.7 0.5-4 β-phellandrene 0.94 0.4 1.9 N/A globulol 0.86 0.1 3.0 Trace-3 myrcene 0.86 0.1 1.8 N/A α -thujene 0.83 0.1 2.1 N/A β-pinene 0.66 0.1 1.6 N/A sabinene 0.45 0.0 3.2 Trace-3.5 α -phellandrene 0.44 0.1 1.9 N/A viridiflorol 0.33 0.1 1.4 Trace-1.5
![Page 5: The effect of the essential oil and its components from Melaleuca alternifolia on endospore germination in Bacillus cereus By: Rachel Schmid ASM Microblibrary.org.](https://reader035.fdocuments.us/reader035/viewer/2022062307/5515155b55034673228b4b9d/html5/thumbnails/5.jpg)
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
![Page 6: The effect of the essential oil and its components from Melaleuca alternifolia on endospore germination in Bacillus cereus By: Rachel Schmid ASM Microblibrary.org.](https://reader035.fdocuments.us/reader035/viewer/2022062307/5515155b55034673228b4b9d/html5/thumbnails/6.jpg)
The present study
•TTO has many antimicrobial abilities
•Can it prevent endospore germination?
•If so, what component of the oil can do this?
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Endospores
• Hardy, encapsulated pieces of DNA
• Able to survive through harsh conditions
• Bacillus spp. able to form them
Picture by textbookofbacteriology.net
![Page 8: The effect of the essential oil and its components from Melaleuca alternifolia on endospore germination in Bacillus cereus By: Rachel Schmid ASM Microblibrary.org.](https://reader035.fdocuments.us/reader035/viewer/2022062307/5515155b55034673228b4b9d/html5/thumbnails/8.jpg)
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
![Page 9: The effect of the essential oil and its components from Melaleuca alternifolia on endospore germination in Bacillus cereus By: Rachel Schmid ASM Microblibrary.org.](https://reader035.fdocuments.us/reader035/viewer/2022062307/5515155b55034673228b4b9d/html5/thumbnails/9.jpg)
Methods• B. cereus bacteria
placed in LB on shaker for 8 days
• Heat treatment• Spread on LB plate
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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
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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
![Page 12: The effect of the essential oil and its components from Melaleuca alternifolia on endospore germination in Bacillus cereus By: Rachel Schmid ASM Microblibrary.org.](https://reader035.fdocuments.us/reader035/viewer/2022062307/5515155b55034673228b4b9d/html5/thumbnails/12.jpg)
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
![Page 13: The effect of the essential oil and its components from Melaleuca alternifolia on endospore germination in Bacillus cereus By: Rachel Schmid ASM Microblibrary.org.](https://reader035.fdocuments.us/reader035/viewer/2022062307/5515155b55034673228b4b9d/html5/thumbnails/13.jpg)
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
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Results
• TTO inhibited endospore germination
• terpinen-4-ol, α-terpinene, and α-terpineol components active
• None significantly more active than the others or TTO
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Synergisms
• Two active components:
• terpinen-4-ol and α-terpineol
• Combination significantly more effective than either component
• F = 40.17, df = 2, p < 0.0001
- 0.5
0
0.5
1
1.5
2
2.5
terpinen- 4- ol α- terpineol both
Radia
l Cle
are
d Z
one (
mm
)
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Synergisms
• Active and inactive:• α-terpinene and
1,8-cineole• F = 26.24, df = 2,
p < 0.0001
• α-terpinene and p-cymene
• F = 10.50, df = 2, p = 0.0014
- 1
0
1
2
3
4
5
6
7
8
1,8- cineole α- terpinene both
Ra
dia
l C
leare
d Z
one
(m
m)
- 0.5
0.5
1.5
2.5
3.5
4.5
5.5
p- cymene α- terpinene both
Rad
ial C
leare
d Z
one
(m
m)
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Synergisms
• Active and inactive:• α-terpineol and
1,8-cineole• F = 56.43, df = 2,
p < 0.0001
• α-terpineol and γ-terpinene
• and F = 19.86, df = 2, p < 0.0001
- 0.5
0
0.5
1
1.5
2
2.5
1,8- cineole α- terpineol both
Radia
l Cle
are
d Z
one (
mm
)
- 0.5
0
0.5
1
1.5
2
2.5
γ- terpinene α- terpineol both
Radia
l Cle
are
d Z
one (
mm
)
![Page 18: The effect of the essential oil and its components from Melaleuca alternifolia on endospore germination in Bacillus cereus By: Rachel Schmid ASM Microblibrary.org.](https://reader035.fdocuments.us/reader035/viewer/2022062307/5515155b55034673228b4b9d/html5/thumbnails/18.jpg)
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
α-terpineol
ene
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
![Page 19: The effect of the essential oil and its components from Melaleuca alternifolia on endospore germination in Bacillus cereus By: Rachel Schmid ASM Microblibrary.org.](https://reader035.fdocuments.us/reader035/viewer/2022062307/5515155b55034673228b4b9d/html5/thumbnails/19.jpg)
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
γ-terpinene 95.24%
4.24% o-cymene
terpinen-4-ol 94.18%
4.41% cyclooctan, 1-(diethylboryl)
α-terpineol 89.96%
10.04% γ-terpineol
α-terpinene 76.46%
12.92%
5.99%
2.63%
o-cymene
1,8-cineole
1,3-heptadiene
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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 α-terpineol α-terpinene
OH OH
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• 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 p-cymene
OH
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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
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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.