Propionibacterium acnes is a gram-positive anaerobic microorganism recognized as the major skin bacterium causing acne.1 Clove essential oil is the extract of Syzygium aromaticum (L) Merr et Perry. Its antimicrobial property has been reported,2 but to our knowledge its antibacterial activity toward P acnes and the mechanism of action have not been reported.
Agar diffusion and broth microdilution methods were used to evaluate the activity of clove essential oil toward P acnes, and the mechanisms of action were investigated by flow cytometry (FCM), atomic force microscopy (AFM), and sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE).
Clove essential oil exerted significant antibacterial activity toward P acnes (Table). The mean (SD) bacteriostatic diameter was 24.0 (0.5) mm. Both the minimum inhibitory concentration and minimum bactericidal concentration were 0.31 mg/mL.
Propidium iodide staining and FCM showed a dose-dependent induction of cell death by essential oil due to cell wall and membrane damage (Figure 1). The relative amount of dead cells among untreated cells was 7.85%, whereas the proportion increased to 71.67% after treatment with 0.62-mg/mL essential oil. This result indicates that the mode of action of clove essential oil toward P acnes is associated with a loss of membrane integrity.
Fluorescence cell number–per-channel (A1-D1) and light scatter–intensity (A2-D2) cytometric histograms of control Propionibacterium acnes (A) and P acnes grown in the presence of different concentrations of clove essential oil and stained with propidium iodide. B-D, Treatment with 0.04-mg/mL, 0.16-mg/mL, and 0.62-mg/mL, respectively, clove essential oil. FL3 indicates fluorescence level; FSC, forward light scatter; and Q, quadrant.
By AFM, a large concentration-dependent variation in the morphologic characteristics of bacteria was observed after treatment with clove essential oil (Figure 2). At low concentrations, the essential oil first attached to the surface of P acnes, and the surface became rougher. At higher concentrations, ruptured cell walls and membranes resulted, causing severe cell damage. Furthermore, the cytoplasmic volume decreased, ultimately leading to bacterial death.
Topographic (A1-E1) and phase imaging (A2-E2) atomic force microscopy images of control Propionibacterium acnes (A) and P acnes treated with different concentrations of clove essential oil. B-E, Treatment with 4, 16, 32, and 64 times the minimum inhibitory concentration, respectively, of clove essential oil. Scale bars represent 500 nm.
By SDS-PAGE, we found that 2 specific proteins at 18 kDa and 33 kDa disappeared after treatment with 10- or 20-mg/mL essential oil for 1 hour. At concentrations below 10 mg/mL, no changes in protein expression were observed (Figure 3). After treatment for 4 hours, a global decrease in protein expression was observed.
Gel electrophoresis of Propionibacterium acnes treated with clove essential oil for 4 hours (A) and 1 hour (B). M indicates molecular marker. Lane 1 represents control P acnes (without essential oil); lanes 2 through 8 represent P acnes treated with clove essential oil in concentrations of 0.31, 0.62, 1.25, 2.50, 5.00, 10.00, and 20.00 mg/mL, respectively.
Clove essential oil exhibited significant activity against P acnes; the minimum inhibitory concentration indicates a high lipophilicity and promises potential in vivo activity. The bacteriostatic mechanism involves damage to the cell walls and membranes of bacteria. At longer incubation times, cytoplasmic proteins may diffuse from the cytoplasm. Alternatively, essential oil might inhibit protein synthesis. Under short-term incubation, the disappearance of 18- and 33-kDa proteins might point to a specific mechanism, ultimately leading to bacterial death.
The role of P acnes in acne development seems to be related to the activity of several enzymes, including lipase and protease.3Propionibacterium acnes lipase (GehA, 33 kDa) and a heat shock protein (PPA737, 18 kDa) have been recognized as the virulence factors involved in the pathogenesis of acne.4,5 Therefore, it was supposed that the downregulated 33-kDa and 18-kDa proteins in the present analysis might be GehA and PPA737. The findings would provide a scientific basis for the application of clove essential oil as a therapeutic agent for acne, and in vivo studies should be conducted to investigate further.
Correspondence: Dr Zu, Key Laboratory of Forest Plant Ecology, Ministry of Education, Northeast Forestry University, Harbin 150040, People's Republic of China (firstname.lastname@example.org).
Author Contributions:Study concept and design: Fu, Chen, Zu, and Efferth. Acquisition of data: Chen and Z. Liu. Analysis and interpretation of data: X. Liu, Y. Liu, and Yao. Drafting of the manuscript: Fu, Chen, Z. Liu, X. Liu, Y. Liu, and Efferth. Critical revision of the manuscript for important intellectual content: Zu, Yao, and Efferth. Statistical analysis: Fu and Chen. Obtained funding: Fu and Zu. Administrative, technical, and material support: Fu. Study supervision: Fu, Chen, Zu, Z. Liu, X. Liu, Y. Liu, Yao, and Efferth.
Financial Disclosure: None reported.
Funding/Support: This study was funded by grants 30770231 from the National Natural Science Foundation of China, Beijing, China; JC200704 from the Heilongjiang Province Science Foundation for Excellent Youths, Harbin, China; 108049 from the Key Project of Chinese Ministry of Education, Beijing; 2006BAD18B0401 from the National Key Technology Research and Development Program, Beijing; 2006-4-75 from the Innovative Program for Importation of International Advanced Agricultural Science and Technology, National Forestry Bureau, Beijing; and 2006RFXXS001 from the Research Foundation for Science and Technology Innovation Talents of Harbin.
Role of the Sponsors: The sponsors had no role in the design and conduct of the study; in the collection, analysis, and interpretation of data; or in the preparation, review, or approval of the manuscript.
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