南方医科大学学报

南方医科大学学报 ›› 2023, Vol. 43 ›› Issue (2): 257-264.doi: 10.12122/j.issn.1673-4254.2023.02.14

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左氧氟沙星联合纤维素酶在体外能有效根除卡介苗生物被膜感染

张志飞，廖洪建，杨 敏，胡 璨，杜永洪   

  1. 重庆医科大学生物医学工程学院，重庆 400016；超声医学工程国家重点实验室，重庆 400016
  • 出版日期:2023-02-20 发布日期:2023-03-16

Levofloxacin combined with cellulase can eradicate bacille Calmette-Guerin biofilm infection

ZHANG Zhifei, LIAO Hongjian, YANG Min, HU Can, DU Yonghong   

  1. School of Biomedical Engineering, Chongqing Medical University, Chongqing 400016, China; State Key Laboratory of Ultrasound Medical Engineering, Chongqing 400016, China
  • Online:2023-02-20 Published:2023-03-16

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摘要： 目的 探究左氧氟沙星（LEV）和纤维素酶（CL）对结核杆菌模式菌—卡介苗（BCG）生物被膜的体外抗菌效果。方法 采用XTT法与结晶紫染色法确定BCG生物被膜的成熟生长周期。不同浓度的LEV和CL单独或联用处理BCG浮游菌及BCG生物被膜，采用结晶紫染色定量生物被膜中生物量的变化。然后将成熟的BCG生物被膜用CL单独处理24 h，通过SYTO 9染色、Calcofluor White Stain染色后，在共聚焦激光扫描显微镜观察活菌数量、生物被膜中纤维素含量变化，扫描电子显微镜下观察生物被膜的结构变化。结果 采用微量肉汤稀释法检测LEV的MIC、MBC和MBEC值分别为4、8和1024 μg/mL。浓度为2.56、5.12和10.24 U/mL的CL与1/4×MIC抗生素（LEV）的组合会导致生物被膜生物量减少（P<0.001）。CL在10.24、5.12和2.56 U/mL3种浓度下对成熟BCG生物被膜均表现出明显的分散效应（P<0.001）。结论 当左氧氟沙星与纤维素酶联合使用时，抗生素能有效根除BCG生物被膜感染。

关键词: 生物被膜；左氧氟沙星；纤维素酶；卡介苗；体外抗菌

Abstract: Objective To investigate the inhibitory effects of levofloxacin (LEV) combined with cellulase against bacille Calmette-Guerin (BCG) biofilms in vitro. Methods The mature growth cycle of BCG biofilms was determined using the XTT method and crystal violet staining. BCG planktonic bacteria and BCG biofilms were treated with different concentrations of LEV and cellulose alone or jointly, and the changes in biofilm biomass were quantified with crystal violet staining. The mature BCG biofilm was then treated with cellulase alone for 24 h, and after staining with SYTO 9 and Calcofluor White Stain, the number of viable bacteria and the change in cellulose content in the biofilm were observed with confocal laser scanning microscopy. The structural changes of the treated biofilm were observed under scanning electron microscopy. Results The MIC, MBC and MBEC values of LEV determined by broth microdilution method were 4 μg/mL, 8 μg/mL and 1024 μg/mL, respectively. The combined treatment with 1/4 ×MIC LEV and 2.56, 5.12 or 10.24 U/mL cellulase resulted in a significant reduction in biofilm biomass (P<0.001). Cellulase treatments at the concentrations of 10.24, 5.12 and 2.56 U/mL all produced significant dispersion effects on mature BCG biofilms (P<0.001). Conclusion LEV combined with cellulose can effectively eradicate BCG biofilm infections, suggesting the potential of glycoside hydrolase therapy for improving the efficacy of antibiotics against biofilm-associated infections caused by Mycobacterium tuberculosis.

Key words: biofilm inhibition; levofloxacin; cellulase; bacille Calmette-Guerin; antibiotics