异绿原酸联合抗生素对铜绿假单胞菌-烟曲霉混合生物被膜的体外协同抑菌作用

In vitro synergistic antibacterial activity of isochlorogenic acid combined with antibiotics against polymicrobial biofilms formed by Pseudomonas aeruginosa and Aspergillus fumigatus

目的研究金银花活性成分异绿原酸及联合伏立康唑+头孢他啶对早期铜绿假单胞菌-烟曲霉菌混合生物被膜的体外协同抑菌作用。方法体外构建早期24 h静止铜绿假单胞菌-烟曲霉混合生物被膜模型并建立不同浓度(250、500、1000μg/ml)的异绿原酸组及联合抗生素组(1μg/ml伏立康唑+16μg/ml头孢他啶)分别作用24 h,扫描电镜(SEM)观察生物被膜形态学改变;结晶紫染色法定量生物被膜;XTT减低法测混合生物被膜内烟曲霉及铜绿假单胞菌的总代谢活性。结果电镜观察证实异绿原酸可破坏早期混合生物被膜结构使胞外基质减少;且异绿原酸联合抗生素组较单纯抗生素联合组对混合菌体的抑制作用更加明显,使载体上生物被膜的量明显降低。结晶紫染色法提示:250、500、1000μg/ml异绿原酸组总被膜定量与250、500、1000μg/ml异绿原酸联合伏立康唑+头孢他啶组、伏立康唑+头孢他啶组、空白对照组及组间两两比较,差异均有统计学意义(P<0.05)。XTT比色法提示:对于早期混合生物被膜,异绿原酸联合抗生素组与空白组、异绿原酸组相比,联合用药组的代谢活性明显减低(抑制率达50%)。结论异绿原酸在体外不能杀死铜绿假单胞菌-烟曲霉混合生物被膜内菌体,但对早期铜绿假单胞菌-烟曲霉混合生物被膜具有破坏作用,使得异绿原酸联合伏立康唑+头孢他啶对早期混合生物被膜的破坏作用明显强于伏立康唑联合头孢他啶组,且均呈浓度依赖性。

Objective To study the synergistic antibacterial effect of honeysuckle active components isochlorogenic acid combined with voriconazole+ceftazidime on the early polymicrobial biofilms of Pseudomonas aeruginosa and Aspergillus fumigatus.Methods The polymicrobial biofilm models in the early stages were established in vitro under static condition and then treated with isochlorogenic acid at different concentrations(250,500,1000μg/ml)and antibiotics(1μg/ml of voriconazole+16μg/ml of ceftazidime)alone or in combination for 24 h in vitro.Morphological changes in the polymicrobial biofilm structure were observed under scanning electron microscope(SEM).Crystal violet staining assay was used for polymicrobial biofilm quantitation.The total metabolic activity of Pseudomonas aeruginosa and Aspergillus fumigatus in formed polymicrobial biofilms were calculated with 2,3-bis-(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide(XTT)reduction assay.Results Under the SEM,it was observed that isochlorogenic acid could destroy the structure of early polymicrobial biofilms and reduce the extracellular matrix.The inhibitory effect of isochlorogenic acid combined with antibiotics against the polymicrobial biofilms was stronger than that of the antibiotics alone,and the amount of biofilms on the carrier was significantly reduced.Crystal violet staining assay showed that the total amount of biofilms in isochlorogenic acid(250,500,1000μg/ml)groups was significantly different from that in isochlorogenic acid(250,500,1000μg/ml)combined with antibiotics groups,antibiotics group and blank control group(all P<0.05).Moreover,there were significant differences between different concentration groups(P<0.05).XTT assay suggested that the metabolic activity in the isochlorogenic acid combined with antibiotics group was significantly lower than that in the blank group and isochlorogenic acid group(the inhibition rate reached 50%).Conclusions Isochlorogenic acid could not kill the Aspergillus fumigatus and Pseudomonas aeruginosa in polymicrobial biofilms in vitro,but exhibit destructive effect on the polymicrobial biofilms in the early stages.Isochlorogenic acid combined with voriconazole and ceftazidime had inhibitory effects against the early stages of polymicrobial biofilms of Aspergillus fumigatus and Pseudomonas aeruginosa in a concentration-dependent manner and was stronger than voriconazole combined with ceftazidime.