大叶桉挥发油对铜绿假单胞菌的体外抑菌效果及生物膜抑制作用

Effects of bacteriostasis in vitro and biofilm inhibition of Eucalyptus robusta-derived volatile oil on Pseudomonas aeruginosa

目的探讨大叶桉挥发油对铜绿假单胞菌的体外抑菌效果及生物膜抑制作用。方法采用水共沸蒸馏法制备大叶桉挥发油,使用气相色谱-质谱分析法分析大叶桉挥发油的成分,并制备10%~50%浓度药敏纸片。采用平板涂布法测定大叶桉挥发油对铜绿假单胞菌的最小抑菌浓度(MIC)。将10%~50%浓度药敏纸片均匀散在贴于涂有0.5 MCF细菌悬液的血平板上,观察抑菌环大小。构建24份铜绿假单胞菌生物膜模型,并随机分为观察组与对照组,各12份,观察组采用含10%大叶桉挥发油的培养基进行培养,对照组采用含等量生理盐水的培养基进行培养,通过扫描电镜观察大叶桉挥发油对细菌生物膜的干预作用。采用实时荧光定量PCR检测两组铜绿假单胞菌生物膜中感应基因LasI的mRNA表达情况。结果大叶桉挥发油中存在7种主要成分,即α-蒎烯、柠檬烯、桉油精、莰烯、邻-异丙基苯、α-松油醇、1-甲基-4-(1-甲基乙烯基)环己醇乙酸酯。20%及以上浓度的大叶桉挥发油具有理想的抑菌效果,但任何浓度的大叶桉挥发油药敏纸片在血平板上均无法产生抑菌环。大叶桉挥发油对延缓铜绿假单胞菌生物膜的形成起到了一定的作用。观察组LasI mRNA表达量低于对照组(P<0.05)。结论大叶桉挥发油可直接抑制铜绿假单胞菌的生长,延缓铜绿假单胞菌生物膜的形成,下调铜绿假单胞菌生物膜LasI基因的表达从而降低其毒力,具有临床治疗铜绿假单胞菌感染的潜在价值。

Objective To investigate the effects of bacteriostasis in vitro and biofilm inhibition of Eucalyptus robusta-derived volatile oil on Pseudomonas aeruginosa.Methods Eucalyptus robusta-derived volatile oil was prepared by the azeotropic distillation method,and components of Eucalyptus robusta-derived volatile oil were analyzed by the gas chromatography-mass spectrometry method,as well as drug-sensitive paper with concentration of 10%to 50%was prepared.The minimum inhibitory concentration(MIC)of Eucalyptus robusta-derived volatile oil on Pseudomonas aeruginosa was measured by the spread plate method.The drug-sensitive paper with concentration of 10%to 50%was evenly distributed on the blood plate coated with 0.5 MCF bacterial suspension,and the size of the bacteriostatic ring was observed.A total of 24 biofilm models of Pseudomonas aeruginosa were constructed,and they were randomly assigned to observation group or control group,with 12 models in each group.The observation group was cultured by medium containing 10%Eucalyptus robusta-derived volatile oil,whereas the control group was cultured by medium containing normal saline with equivalent volume.The intervention effect of Eucalyptus robusta-derived volatile oil on bacterial biofilms was observed by scanning electron microscope.The mRNA expression of induction gene LasI in biofilm of Pseudomonas aeruginosa in both groups was detected by the real-time fluorescent quantitative PCR.Results There were seven major components in Eucalyptus robusta-derived volatile oil,namelyα-pinene,limonene,eucalyptol,camphene,o-cymene,α-terpilenol and 1-methyl-4-(1-methylvinyl)cyclohexyl acetate.Eucalyptus robusta-derived volatile oil with concentration of 20%and above exerted an ideal bacteriostatic effect;however,drug-sensitive paper with any concentration of Eucalyptus robusta-derived volatile oil did not produce a bacteriostatic ring on blood plates.Eucalyptus robusta-derived volatile oil could delay the biofilm formation of Pseudomonas aeruginosa to a certain extent.The mRNA expression of LasI in the observation group was lower than that in the control group(P<0.05).Conclusion Eucalyptus robusta-derived volatile oil can directly inhibit the growth of Pseudomonas aeruginosa,delay the biofilm formation of Pseudomonas aeruginosa,down-regulate LasI gene expression of Pseudomonas aeruginosa biofilm to reduce its virulence,exerting potential value in clinical treatment of Pseudomonas aeruginosa infection.