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:
2 Historical 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
3 Uses 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.
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 conditionsBacillus spp. able to form themPicture by textbookofbacteriology.net
8 Bacillus 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
9 Methods B. cereus bacteria placed in LB on shaker for 8 days Heat treatmentSpread on LB plate
10 Methods B. cereus bacteria placed in LB on shaker for 8 days Heat treatmentSpread on LB plate3-4 3M discs were placed on each plate
11 Methods 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
12 Methods 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
13 Methods 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
14 Results TTO inhibited endospore germination terpinen-4-ol, α-terpinene, and α-terpineol components activeNone 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 componentF = 40.17, df = 2, p <
16 Synergisms 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 =
17 Synergisms 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 <
18 GC/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%.
19 GC/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
20 Discussion 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
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-terpinene1,8-cineolep-cymeneOH
22 Suggested 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
23 Literature 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.