The effect of the essential oil and its components from

Melaleuca alternifolia on endospore

germination in Bacillus cereusBy: Rachel Schmid

ASM Microblibrary.org © Weber

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.

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

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

• Two active components:

• terpinen-4-ol and α-terpineol

• Combination significantly more effective than either component

• F = 40.17, df = 2, p < 0.0001

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terpinen- 4- ol α- terpineol both

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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

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1,8- cineole α- terpinene both

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4.5

5.5

p- cymene α- terpinene both

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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

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1,8- cineole α- terpineol both

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γ- terpinene α- terpineol both

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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

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

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

• 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

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.