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. 2002 Jun;46(6):1914-20.
doi: 10.1128/aac.46.6.1914-1920.2002.

Mechanism of Action of Melaleuca Alternifolia (Tea Tree) Oil on Staphylococcus Aureus Determined by Time-Kill, Lysis, Leakage, and Salt Tolerance Assays and Electron Microscopy

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Mechanism of Action of Melaleuca Alternifolia (Tea Tree) Oil on Staphylococcus Aureus Determined by Time-Kill, Lysis, Leakage, and Salt Tolerance Assays and Electron Microscopy

Christine F Carson et al. Antimicrob Agents Chemother. .
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Abstract

The essential oil of Melaleuca alternifolia (tea tree) has broad-spectrum antimicrobial activity. The mechanisms of action of tea tree oil and three of its components, 1,8-cineole, terpinen-4-ol, and alpha-terpineol, against Staphylococcus aureus ATCC 9144 were investigated. Treatment with these agents at their MICs and two times their MICs, particularly treatment with terpinen-4-ol and alpha-terpineol, reduced the viability of S. aureus. None of the agents caused lysis, as determined by measurement of the optical density at 620 nm, although cells became disproportionately sensitive to subsequent autolysis. Loss of 260-nm-absorbing material occurred after treatment with concentrations equivalent to the MIC, particularly after treatment with 1,8-cineole and alpha-terpineol. S. aureus organisms treated with tea tree oil or its components at the MIC or two times the MIC showed a significant loss of tolerance to NaCl. When the agents were tested at one-half the MIC, only 1,8-cineole significantly reduced the tolerance of S. aureus to NaCl. Electron microscopy of terpinen-4-ol-treated cells showed the formation of mesosomes and the loss of cytoplasmic contents. The predisposition to lysis, the loss of 260-nm-absorbing material, the loss of tolerance to NaCl, and the altered morphology seen by electron microscopy all suggest that tea tree oil and its components compromise the cytoplasmic membrane.

Figures

FIG. 1.
FIG. 1.
Time-kill curves of S. aureus ATCC 9144 in control suspensions (♦) and after treatment with TTO (A), 1,8-cineole (B), terpinen-4-ol (C), or α-terpineol (D) at one-half the MICs (▵), the MICs (•), and two times the MICs (□). The MICs of TTO, terpinen-4-ol, and α-terpineol were 0.25% (vol/vol); and that of 1,8-cineole was 0.5%. The organisms were suspended in PBS-T. Each symbol indicates the means ± SEs for at least three replicates. The lower detection threshold was 103 CFU/ml.
FIG. 2.
FIG. 2.
Appearance of 260-nm-absorbing material in the filtrates of S. aureus control suspensions (white bars) and after treatment with the MICs of TTO (0.25%; bars with diagonal stripes), 1,8-cineole (0.5%; grey bars), terpinen-4-ol (0.25%; bars with horizontal stripes), or α-terpineol (0.25%; black bars). The organisms were suspended in PBS-T. The means ± SEs for at least three replicates are illustrated.
FIG. 3.
FIG. 3.
Proportion of S. aureus cells able to form colonies on NA (white bars), NA supplemented with 50 g of NaCl per liter (black bars), and NA supplemented with 75 g of NaCl per liter (grey bars) after 30 min of treatment with TTO (A), 1,8-cineole (B), terpinen-4-ol (C), or α-terpineol (D) at one-half the MICs, the MICs, and two times the MICs. The MICs of TTO, terpinen-4-ol, and α-terpineol were 0.25% (vol/vol); and that of 1,8-cineole was 0.5%. The organisms were suspended in PBS-T. The means ± SEs for at least four replicates are illustrated.
FIG. 4.
FIG. 4.
Electron micrographs of S. aureus cells stained with uranyl acetate after no treatment (A) and after treatment with 0.3% terpinen-4-ol for 10 min (B and C). Magnifications: ×11,500 (A) and ×14,200 (B and C).
FIG. 4.
FIG. 4.
Electron micrographs of S. aureus cells stained with uranyl acetate after no treatment (A) and after treatment with 0.3% terpinen-4-ol for 10 min (B and C). Magnifications: ×11,500 (A) and ×14,200 (B and C).

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