氨溴索联合左氧氟沙星对肺炎克雷伯菌生物膜抑制和清除能力的研究

目的：研究氨溴索联合左氧氟沙星对肺炎克雷伯菌生物被膜的抑制和清除作用，旨在为临床提供拮抗肺炎克雷伯菌生物膜形成和治疗的新策略。方法：收集不同耐药性肺炎克雷伯菌各15株，将其分为敏感组（野生菌组）、超广谱β-内酰胺酶（extended spectrum β-Lactamases, ESBLs）组、耐碳青霉烯类肺炎克雷伯菌（carbapenem resistant Klebsiella pneumoniae, CRKP）组，采用结晶紫染色法进行生物被膜半定量检测，再从各组中选取3株生物被膜产量相近的菌株，采用微量肉汤稀释法测定氨溴索和左氧氟沙星对肺炎克雷伯菌的最低抑菌浓度（MIC）；棋盘稀释法测定不同浓度氨溴索对左氧氟沙星MIC的影响以及通过计算部分抑菌浓度指数（FIC）判断联合效果和选择最佳协同浓度；采用结晶紫法联合激光扫描共聚焦荧光显微镜研究不同浓度药物对肺炎克雷伯菌生物被膜的形成抑制试验和清除试验。结果：3组细菌生物被膜均在第5天达到成熟，两两比较发现敏感组较ESBLs组和CRKP组更易形成且产量更多（F=3.725，P=0.032）；棋盘稀释法显示，随着氨溴索浓度的增加，3组细菌左氧氟沙星MIC值的几何均数均明显下降，呈显著的负相关，且联合药敏结果显示，两药组合均呈现协同作用；在生物被膜形成抑制试验中，随着氨溴索浓度的增加，其抑制率均达到了75%以上，但其生物被膜清除率却未达到70%。 结论：氨溴索联合左氧氟沙星对临床早期抑制肺炎克雷伯菌生物被膜形成具有指导意义，且其最佳协同浓度为0.49 mg/mL氨溴索+4 μg/mL左氧氟沙星。

Inhibitory and scavenging ability of ambroxol combined with levofloxacin on the bacterial biofilm of Klebsiella pneumoniae

AIM: To study the inhibitory and scavenging effects of ambroxol combined with levofloxacin on the bacterial biofilm of Klebsiella pneumoniae, and to provide a new strategy to treat and antagonize the formation of the biofilm. METHODS: We collected Klebsiella pneumoniae of different resistance and divided them into sensitive group (wild bacteria group), ESBLs group and CRKP group with 15 strains in each group and performed biological semi-quantitative detection of its biofilm by crystalline violet staining method. After selecting 3 strains with similar membrane yields from each group, we determined the minimum inhibitory concentration (MIC) of ambroxol and levofloxacin against Klebsiella pneumoniae by micro broth dilution method.In addition, we determined the effects of ambroxol in different concentrations on the MIC of levfloxacin by the micrdilution checkerboard techniques and calculated the partial inhibitory concentration index (FIC) to determine the joint effect and select the best synergistic concentration. Finally, the effects of ambroxol and levofloxacin in different concentrations on the inhibition formation test and removal test of Klebsiella pneumoniae biofilm were observed by crystal violet method combined with confocal laser scanning microscopy. RESULTS: We found that all three groups of bacterial biofilms became mature on the 5th day, and the sensitive group was easier to form and produce biofilm more than the ESBLs and CRKP groups (F=3.725, P=0.032). It was showed that the geometric average of levofloxacin MIC value in the three groups decreased significantly. And the selected strains all showed a synergistic effect on the two-drug combination.In the biofilm test, as the concentration of ambroxol increased, its inhibition rate reached more than 75%, but its biofilm removal rate did not reach 70%. CONCLUSION: Our study support that ambroxol combined with levofloxacin can inhibit the bacterial biofilm of Klebsiella pneumoniae early, and its optimal synergistic concentration is 0.49 mg/mL (ambroxol) + 4 μg/mL (levofloxacin).