the effect of the essential oil and its components from melaleuca alternifolia on endospore...
TRANSCRIPT
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
Pic
ture
by
Ge
neva
Fou
nd
atio
n f
or
Me
dica
l Edu
catio
n a
nd
Re
sear
ch
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
- 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
)
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)
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
)
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.