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:
2Historical Use Of Tea Tree Oil (TTO) Small, summer flowering tree native to AustraliaFirst 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 GrantSince then extensive research done on oil’s usesPicture by Geneva Foundation for Medical Education and Research
3Uses of the OilPublished evidence of antibacterial, antifungal, antiprotozan, antiviral, and anti-inflammatory propertiesAlso used to treat athlete’s foot, head lice, acne, and other skin irritationsOil readily available for everyday use without a prescriptionFound in shampoos, skin treatments, etc.
5Previously 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
6The present study TTO has many antimicrobial abilities Can it prevent endospore germination?If so, what component of the oil can do this?
7Endospores Hardy, encapsulated pieces of DNA Able to survive through harsh conditionsBacillus spp. able to form themPicture by textbookofbacteriology.net
8Bacillus spp. Using B. cereus as model for B. anthracis 2001 bioterrorism attacks using anthrax spores on mailed envelopes22 mail workers infected and 5 died from exposureMost infections from anthrax are cutaneous
9Methods B. cereus bacteria placed in LB on shaker for 8 days Heat treatmentSpread on LB plate
10Methods B. cereus bacteria placed in LB on shaker for 8 days Heat treatmentSpread on LB plate3-4 3M discs were placed on each plate
11Methods B. cereus bacteria placed in LB on shaker for 8 days Heat treatmentSpread on LB plate4 3M discs were placed on each plateAdded small amount of TTO or components: terpinen-4-ol, γ-terpinene, α-terpinene, 1,8-cineole, α-pinene, p -cymene, α-terpineol, or limonene
12Methods B. cereus bacteria placed in LB on shaker for 8 days Heat treatmentSpread on LB plate4 3M discs were placed on each plateAdded small amount of TTO or components: terpinen-4-ol, γ-terpinene, α-terpinene, 1,8-cineole, α-pinene, p -cymene, α-terpineol, or limoneneIncubated for 24 hours at 32°CMeasured zone of inhibition
13Methods Measured zone of inhibition Dose effects of active components B. cereus bacteria placed in LB on shaker for 8 daysHeat treatmentSpread on LB plate4 3M discs were placed on each plateAdded small amount of TTO or components: terpinen-4-ol, γ-terpinene, α-terpinene, 1,8-cineole, α-pinene, p -cymene, α-terpineol, or limoneneIncubated for 24 hours at 32°CMeasured zone of inhibitionDose effects of active componentsSynergistic effects between active + active and active + inactiveANOVA and Tukey Kramer Post Hoc performedOil and components checked for purity on GC/MS
14Results TTO inhibited endospore germination terpinen-4-ol, α-terpinene, and α-terpineol components activeNone significantly more active than the others or TTO
15Synergisms Two active components: terpinen-4-ol and α-terpineol Combination significantly more effective than either componentF = 40.17, df = 2, p <
16Synergisms Active and inactive: α-terpinene and 1,8-cineole F = 26.24, df = 2, p <α-terpinene and p-cymeneF = 10.50, df = 2, p =
17Synergisms Active and inactive: α-terpineol and 1,8-cineole F = 56.43, df = 2, p <α-terpineol and γ-terpineneand F = 19.86, df = 2, p <
18GC/MSComponent% Peak AreaRetention (min)terpinen-4-ol34.00%12.836γ-terpinene27.14%10.257α-terpinene16.23%9.292α-pinene5.76%7.351α-terpineolene3.77%10.917o-cymene3.41%9.4691,8-cineole3.12%9.636limonene2.38%9.578α-terpineol2.22%13.092α-thujene1.96%7.184The 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%.
19GC/MSComposition of commercially purchased components that were active or part of a significant synergismComponentPurityContaminant1,8-cineole100.00%p-cymene99.63%0.37%cymeneγ-terpinene95.24%4.24%o-cymeneterpinen-4-ol94.18%4.41%cyclooctan, 1-(diethylboryl)α-terpineol89.96%10.04%γ-terpineolα-terpinene76.46%12.92%5.99%2.63%1,3-heptadiene
20Discussion terpinen-4-ol α-terpineol α-terpinene Terpinen-4-ol is not the only active component, α-terpineol and α-terpinene are just as activeTerpenes 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-terpineneOHOH
21These 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-terpinene1,8-cineolep-cymeneOH
22Suggested Studies Revise ISO for TTO to contain more α-terpinene Use of TTO in alternative treatments of infectious diseaseMore work with TTO and anthrax endospores in containment labsClinical trials for prevention/healing of cutaneous infections in places where refrigeration of antibiotics is impossible
23Literature Cited• Carson, C. F., K. A. Hammer, and T. V. Riley Melaleuca (Tea Tree) Oil: a review of antimicrobial and other medicinal properties. Clinical Microbiology Review 19:• Carson, C. F., and T. V. Riley Antimicrobial activity of essential oil of Melaleuca alternifolia. Letters in Applied Microbiology 16:• Carson, C. F., and T. V. Riley Antimicrobial activity of the major components of the essential oil of Melaleuca alternifolia. J. of Applied Bacteriology 78:• 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 Tea tree oil causes K+ leakage and inhibits respiration in Escherichia coli. Letters Applied Microbiology 26:• Raman, A, U. Weir, and S. F. Bloomfield Antimicrobial effects of tea tree oil and its major components on Staphylococcus aureus, Staphylococcus epidermidis, and Propionibacterium acnes. Applied Microbiology 21:• Sikkema, J., J. A. De Bont, and B. Poolman Mechanisms of membrane toxicity of hydrocarbons. Microbiological Reviews 59: 201–222.