【摘要】 目的：研究不同ST型肺炎克雷伯菌生物膜的形成能力及阿奇霉素联合左氧氟沙星对不同ST型菌株生物被膜的抑制和清除作用，旨为预防治疗产生物被膜肺炎克雷伯菌感染提供新思路。方法：随机收集皖南医学院第一附属医院2019年8月至2021年11月从住院患者标本分离的全敏感组肺炎克雷伯菌9株、产超广谱β-内酰胺酶（extended spectrum β-Lactamases,ESBLs）肺炎克雷伯菌19株、耐碳青霉烯类肺炎克雷伯菌（carbapenem resistant Klebsiella pneumoniae,CRKP）37株，细菌均采用VITEK MS IVD KB V3.2质谱仪鉴定并使用VITEK 2-compact 60全自动微生物鉴定及药敏系统对细菌进行药敏试验。对各菌株进行多位点序列分型（multilocus sequence typing,MLST）分析其同源性；结晶紫染色法进行生物被膜半定量检测，比较不同ST型肺炎克雷伯菌之间生物膜形成能力的差异；选取不同ST型菌株，采用微量肉汤稀释法计算部分抑菌浓度指数（FICI）判断联合效果并选择最佳联合浓度；结晶紫染色法探究阿奇霉素联合左氧氟沙星对不同ST型肺炎克雷伯菌生物膜的抑制及清除效果；激光扫描共聚焦荧光显微镜观察抗菌药物作用前后肺炎克雷伯菌生物膜的结构变化。结果：MLST分型结果显示敏感组肺炎克雷伯菌株有ST86、ST727等8个序列，ESBLs组菌株属于ST15、ST37、ST11等14个序列型，其中ST15占26.32%(5/19);CRKP组菌株属于ST11、ST15、ST656等9个序列型，其中ST11占48.65%(18/37),ST15占27.03%(10/37);ST15(ESBLs)型、ST11(CRKP)型、ST15(CRKP)型肺炎克雷伯菌生物膜均在第5天达到成熟，ST15(ESBLs)组较ST15(CRKP)组产生物被膜能力强，ST11(CRKP)组较ST15(CRKP)组形成生物膜能力强（P<0.01）。阿奇霉素联合左氧氟沙星药敏结果显示对不同ST型肺炎克雷伯菌均具有相加作用；在生物膜形成的抑制及清除试验中，2×MIC阿奇霉素组及联合浓度组均对不同ST型肺炎克雷伯菌生物膜形成具有较强的抑制作用，且联合组的抑制能力优于单药组，抑制率最高可达到89.93%；清除效果均为联合药物组>阿奇霉素>左氧氟沙星，清除率最高为44.79%。结论：不同ST型肺炎克雷伯菌之间生物膜形成能力存在差异，阿奇霉素联合左氧氟沙星对不同ST型肺炎克雷伯菌生物膜具有较好的抑制作用，可在早期联合用药进行抗生物膜菌感染的治疗。更多还原

【Abstract】 AIM: Given the biofilm formation abili-ty of different ST-type Klebsiella pneumoniae, our study was aimed at exploring the inhibitory and clearance of azithromycin combined with levofloxacin on the biofilm of Klebsiella pneumoniae of different ST-types and providing a new strategy for the prevention of biofilm formation in the treatment of post-infectious Klebsiella pneumoniae.METHODS: 9 strains of Klebsiella pneumoniae from all susceptibility groups, 19 strains of Klebsiella pneumoniae producing extended-spectrum β-lactamases(ESBLs), and 37 strains of Carbapenem-resistant Klebsiella pneumoniae(CRKP) were randomly collected from the samples of patients hospitalized in the First Affiliated Hospital of Wannan Medical College from August 2019 to November 2021.The isolates were identified using VITEK MS IVD KB V3.2 and VITEK 2-Compact 60. Multilocus sequence typing(MLST) was performed to analyze the homology of each strain; crystal violet staining was used for semi-quantitative detection of biofilm to compare the differences in biofilm formation ability between different ST-type Klebsiella pneumoniae. Different ST-type strains were selected, and the partial inhibitory concentration index(FICI) was calculated by micro broth dilution method to judge the combination effect and select the optimal combination concentration; crystalline violet staining method was used to investigate the inhibition and clearance effect of azithromycin combined with levofloxacin on the biofilm of different ST-type Klebsiella pneumoniae; laser scanning confocal fluorescence microscopy was used to observe the structural changes of the biofilm of Klebsiella pneumoniae before and after the effect of the antibacterial drugs.RESULTS: MLST typing results showed that the sensitive group of Klebsiella pneumoniae strains had 8sequences such as ST86, ST727, etc., the ESBLs group strains belonged to 14 sequence types such as ST15, ST37, ST11, etc., of which ST15 accounted for 26.32%(5/19). The CRKP group strains belonged to 9 sequence types such as ST11, ST15,ST656, etc., of which ST11 accounted for 48.65%(18/37), ST15 accounted for 27.03%(10/37); ST15(ESBLs), ST11(CRKP), ST15(CRKP) type Klebsiella pneumoniae biofilms all reached maturity on the5th day, the ST15(ESBLs) group had a stronger ability to produce material to be membranous than the ST15(CRKP) group. The ST11(CRKP) group had a stronger ability to produce material to be membranous than the ST15(CRKP) group(P<0.01); the results of azithromycin combined with levofloxacin drug sensitivity showed that it had an additive effect on different ST-type Klebsiella pneumoniae bacteria; in the inhibition of biofilm formation and clearance test, the 2 × MIC azithromycin group and the combined concentration group had a stronger inhibition of biofilm formation of different ST-type Klebsiella pneumoniae bacteria, and the inhibitory ability of the combined group was better than that of the single-drug group, and the highest inhibition rate could reach 89.93%; the clearance effects were all combined drug group>azithromycin>levofloxacin, and the highest clearance rate was44.79%. CONCLUSION: There are differences in biofilm formation ability between different ST-type Klebsiella pneumoniae, and azithromycin combined with levofloxacin has a better inhibitory effect on different ST-type Klebsiella pneumoniae biofilm,conbined application can be used in the treatment of biofilm infections early stage.更多还原