Effect of Tea Tree Oil on Methicillin-resistant Staphylococcus aureus Biofilm
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摘要: 本文研究了茶树精油对耐甲氧西林金黄色葡萄球菌（Methicillin-resistant Staphylococcus aureus，MRSA）生物被膜的影响及其作用机制。首先测定了茶树精油对1株MRSA标准菌株及12株金黄色葡萄球菌分离菌株的MIC值为0.08%~0.32%。结晶紫半定量染色实验发现0.16%的茶树精油对MRSA生物被膜形成有抑制作用，结晶紫半定量染色实验和激光共聚焦显微镜发现0.32%的茶树精油可完全清除MRSA成熟的生物被膜，0.16%可明显破坏其生物被膜。抗生物被膜机制的初步探究发现茶树精油可以抑制MRSA生物被膜形成过程中多糖黏附素（polysaccharide intercellular adhesion，PIA）和胞外DNA（extracellular DNA，eDNA）的分泌以及物被膜形成相关基因agr、ica、cid、sar的表达。因此，茶树精油具有强大的抗生物被膜作用，可能机制是抑制其PIA的合成，eDNA的分泌以及生物被膜形成相关基因的表达。Abstract: The effects of tea tree oil on the biofilm of Methicillin-resistant Staphylococcus aureus (MRSA) and its mechanism of action were studied in this paper . Firstly, the MIC values of tea tree oil against MRSA and 12 clinically isolated strains were determined to be 0.08%~0.32%. It was found that 0.16% of tea tree oil had an inhibitory effect on the formation of MRSA biofilms. Tea tree oil at 0.32% could completely remove mature biofilms of MRSA while 0.16% could significantly reduce the mature biofilms by semi-quantitative crystal violet experiments and CLSM. Preliminary exploration of the biofilm mechanism found that tea tree oil could inhibit the secretion of polysaccharide intercellular adhesion (PIA) and extracellular DNA (eDNA), and the expression level of agr, ica, cid and sar during the formation of MRSA biofilm. Therefore, tea tree essential oil would have a strong anti-biofilm effect, and could inhibite the synthesis of PIA, the secretion of eDNA and the expression of genes related to biofilm formation.
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Key words:
- tea tree oil /
- biofilm /
- semi-quantitative crystal violet experiments /
- CLSM /
- PIA /
- eDNA
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图 1 茶树精油对MRSA生物被膜生长的抑制效果
Figure 1. Inhibition effect of tea essential oil on MRSA biofilm growth
图 2 茶树精油对MRSA成熟生物被膜的清除作用
Figure 2. Clearance of tea tree oil against MRSA mature biofilm
注：与对照组相比，**表示数据差异极显著，P<0.01；图5同 。
图 3 激光共聚焦显微镜细下MRSA生物被膜图像
Figure 3. CLSM image of LIVE/DEAD stained MRSA biofilms grown on cell slide
注：A、B、C、D中茶树精油浓度分别为0、0.04%、0.08%、0.16%；绿色代表活细菌，红色代表死细菌。
图 4 茶树精油对MRSA生物被膜中PIA的影响
Figure 4. Effect of tea tree oil on PIA in MRSA biofilm
注：A、B、C、D中茶树精油浓度分别为0、0.02%、0.04%、0.08% 。
图 5 茶树精油对eDNA分泌的影响
Figure 5. Influence of tea tree oil effect on the secretion of eDNA
表 1 PCR引物
Table 1. PCR primer
引物名称 序列 长度 icaA-F GGCTGGACTCATATTTGTAAGTTGG 25 icaA-R GTATTCCCTCTGTCTGGGCTTG 22 cidA-F TGTACCGCTAACTTGGGTAGAAGAC 25 cidA-R CGGAAGCAACATCCATAATACCTAC 25 agrA-F TGCGAAGACGATCCAAAACA 20 agrA-R GGGCAATTTCCATAGGCTTTTC 22 sarA-F TGGTTCTCATCTCCCTTTGCTT 22 sarA-R GCGATGCTAATCTTCCTGGTG 21 16S-R GCTCGTGTCGTGAGATGTTGG 21 16S-F TTTCGCTGCCCTTTGTATTGT 21 
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表 2 茶树精油对金黄葡萄球菌的MIC值
Table 2. MIC of tea tree oil against Staphylococcus aureus
菌株 MIC值（%） MBC值（%） ATCC43300 0.32 0.32 S-1 0.16 0.32 S-2 0.08 0.16 S-3 0.16 0.16 S-4 0.16 0.16 S-5 0.16 0.16 S-6 0.16 0.16 S-7 0.16 0.16 S-8 0.16 0.16 S-9 0.32 0.32 S-10 0.16 0.16 S-11 0.16 0.32 S-12 0.16 0.32
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表 3 茶树精油作用后MRSA部分基因表达量的变化
Table 3. Changes in the expression of some genes in MRSA treatment by tea tree essential oil
基因名称 对照组表达量 处理组表达量 表达量变化 agrA 1.000 0.320 −3.13±0.16 cidA 1.000 0.188 −5.31±0.19 sarA 1.000 0.474 −2.10±0.22 icaA 1.000 0.242 −4.13±1.11
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