抗微生物肽CRAMP联合抗生素分散铜绿假单胞菌生物被膜的增效作用研究

Synergistic effect of CRAMP and antibiotic against Pseudomonas aeruginosa biofilm

摘要:

【目的】研究修饰后的鼠源抗微生物肽CRAMP联用抗生素对铜绿假单胞菌PAO1成熟生物被膜的分散作用，为临床联合应用抗生物被膜药物提供理论依据。【方法】采用微量肉汤稀释法测定CRAMP修饰肽和抗生素对PAO1的最低抑菌浓度(MIC)、最低杀菌浓度(MBC)和生物被膜最小根除浓度(MBEC)；采用时间杀菌曲线(time-kill curve，TKC)法测定CRAMP修饰肽及抗生素单用和联用对PAO1成熟生物被膜的杀菌活性；采用菌落计数法和激光共聚焦扫描显微镜(CLSM)评估CRAMP修饰肽联用抗生素对PAO1成熟生物被膜的分散作用。【结果】与单用抗生素组相比，除2种碳青霉烯类药物和4种β-内酰胺类药物，其他抗生素联合CRAMP修饰肽后的MBEC值均有不同程度的下降，万古霉素、罗红霉素和阿奇霉素下降倍数最明显(4倍)。TKC试验结果表明，CRAMP修饰肽分别联用万古霉素、罗红霉素和阿奇霉素均具有比单用药更快且更强的杀菌作用，尤其是与万古霉素联用仅在3 h时杀灭了全部(100%)的生物被膜细菌。随后，通过CLSM观察发现生物被膜数量、体积、面积和单位面积荧光强度均有明显的变化。【结论】CRAMP修饰肽对铜绿假单胞菌PAO1成熟生物被膜具有分散作用，与万古霉素联用具有明显协同增效作用，且作用PAO1成熟生物被膜1 h时协同效应最大。

Abstract:

[Objective] To investigate the effect of modified mouse cathelicidin related antimicrobial peptide (CRAMP) combined with antibiotics against the mature biofilm of Pseudomonas aeruginosa PAO1 and thereby lay a theoretical basis for the combined application of anti-biofilm drugs. [Methods] Broth microdilution was employed for the determination of minimum inhibitory concentration (MIC), minimum bactericidal concentration (MBC), and minimum biofilm eradication concentration (MBEC) of CRAMP and antibiotics on PAO1. The time-kill curve (TKC) method was used to determine the bactericidal activities of CRAMP alone, antibiotics alone, and the combination of CRAMP and antibiotics against the mature biofilm of PAO1. Colony counting method and confocal laser scanning microscope (CLSM) were applied to evaluate the dispersal effect of CRAMP combined with antibiotics on the biofilm. [Results] Compared with the antibiotics alone, the combination of antibiotics (except for two carbapenems and four β-lactam antibiotics) with CRAMP recorded the decrease in MBEC, particularly vancomycin, roxithromycin, and azithromycin (1/4 that of antibiotics alone). According to the TKC, the combination of vancomycin, roxithromycin, and azithromycin with CRAMP showed faster and stronger bactericidal activity than antibiotics alone, especially the combination of vancomycin which killed all (100%) biofilm bacteria in 3 h. Under CLSM, the number, volume, area, and fluorescence intensity per unit area of biofilm were significantly changed. [Conclusion] CRAMP can disperse the mature biofilm of PAO1 and has synergistic effect with vancomycin, and the synergistic effect is the most significant at 1 h.