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The effect of the essential oil and its components from Melaleuca alternifolia on endospore germination in Bacillus cereus By: Rachel Schmid ASM Microblibrary.org.

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Presentation on theme: "The effect of the essential oil and its components from Melaleuca alternifolia on endospore germination in Bacillus cereus By: Rachel Schmid ASM Microblibrary.org."— Presentation transcript:

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 © Weber

2 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 oils antimicrobial properties published by Penfold and Grant Since then extensive research done on oils uses Picture by Geneva Foundation for Medical Education and Research

3 Uses of the Oil Published evidence of antibacterial, antifungal, antiprotozan, antiviral, and anti- inflammatory properties Also used to treat athletes foot, head lice, acne, and other skin irritations Oil readily available for everyday use without a prescription Found in shampoos, skin treatments, etc.

4 The major components of TTO

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

6 The present study TTO has many antimicrobial abilities Can it prevent endospore germination? If so, what component of the oil can do this?

7 Endospores Hardy, encapsulated pieces of DNA Able to survive through harsh conditions Bacillus spp. able to form them Picture by textbookofbacteriology.net

8 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

9 Methods B. cereus bacteria placed in LB on shaker for 8 days Heat treatment Spread on LB plate

10 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

11 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

12 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

13 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

14 Results TTO inhibited endospore germination terpinen-4-ol, α-terpinene, and α-terpineol components active None significantly more active than the others or TTO

15 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

16 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

17 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

18 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 AreaRetention (min) terpinen-4-ol34.00%12.836 γ-terpinene27.14%10.257 α-terpinene16.23%9.292 α-pinene5.76%7.351 α- terpineole ne 3.77%10.917 o-cymene3.41%9.469 1,8-cineole3.12%9.636 limonene2.38%9.578 α-terpineol2.22%13.092 α-thujene1.96%7.184

19 GC/MS Composition of commercially purchased components that were active or part of a significant synergism ComponentPurityContaminant 1,8-cineole100.00% p-cymene99.63% 0.37%cymene γ -terpinene 95.24% 4.24%o-cymene terpinen-4-ol94.18% 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

20 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-olα-terpineolα-terpinene OH

21 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

22 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

23 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.


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