临床分离肺炎克雷伯菌生物膜形成能力及基因相关性研究  

Relationship between biofilm formation ability and related genes in Klebsiella pneumoniae

作　　者：田学斌 刘海洋[2] 李佳慧[2] 董郭枫 周铁丽[2] 陈栎江[2] 曹建明[1] TIAN Xuebin;LIU Haiyang;LI Jiahui(School of Laboratory Medicine and Life Sciences,Wenzhou Medical University,Wenzhou 325035,China)

机构地区：[1]温州医科大学检验医学院,生命科学学院,325035 [2]温州医科大学附属第一医院医学检验中心

出　　处：《浙江医学》2019年第1期6-9,107,共5页Zhejiang Medical Journal

基　　金：国家自然科学基金(81741059);浙江省卫生高层次创新人才培养工程项目资助浙卫发[2012] 241号

摘　　要：目的研究肺炎克雷伯菌生物膜形成能力及其与luxS、oqxB、rarA等基因的相关性。方法通过96孔板结晶紫染色法测定200株对亚胺培南、头孢他啶、环丙沙星和庆大霉素敏感的肺炎克雷伯菌(敏感组)和300株对上述药物耐药的肺炎克雷伯菌(耐药组)体外生物膜形成能力;采用扫描电镜观察不同生物膜形成能力肺炎克雷伯菌的生物膜形态特点;通过实时荧光定量PCR分析生物膜形成相关基因(luxS、oqxB、rarA、acrB、ramA、ompK36和micF)表达量。结果强生物膜形成能力组的平均生物膜形成量是弱形成能力组的16.8倍,敏感组和耐药组肺炎克雷伯菌生物膜OD590的平均值分别为0.783±0.642和0.672±0.534,差异无统计学意义;扫描电镜显示生物膜形成能力强的菌株团块内包含大量的生物膜,形态完整且细菌密集排列;强生物膜形成能力组中luxS表达水平分别为中等和弱形成能力组的5.98倍和3.37倍;外排泵、外膜蛋白编码基因及其调控基因的表达水平有统计学差异。结论肺炎克雷伯菌生物膜形成能力与luxS基因的表达量有关,luxS作为一个高阶调控基因,其与ramA、ompK36和micF等基因构成错综复杂的关系网,共同影响生物膜的形成。Objective To investigate the relationship between the biofilm formation ability and luxS,oqxB,rarA and other related genes in Klebsiella pneumoniae.Methods The biofilm formation ability was detected by 96-well crystal violet staining method in 200 clinical isolates of Klebsiella pneumoniae which were sensitive to imipenem,ceftazidime,ciprofloxacin and gentamycin(sensitive group)and 300 isolated of Klebsiella pneumoniae which were resistant to those antimicrobials(resistant group);the biofilm features of Klebsiella pneumoniae was ocserved with scanning electron microscopy(SEM);the expression levels of biofilm-related genes(luxS,oqxB,rarA,acrB,ramA,ompK36 and micF)were analyzed by qRT-PCR.Results The average capacity of biofilm formation in the strong biofilm formation group was 16.8 times than that in the weak formation group,however,no significant difference was obtained between the sensitive group(0.783±0.642)and the resistant group(0.672±0.534)for biofilm formation ability.The SEM showed that quantity of the strains with strong ability of biofilm formation contained a large number of biofilms in their clumps,the morphology of the strains was integrated and the arrangement was dense.The level of luxS expression in the strong biofilm forming group was 5.98 and 3.37 times higher than that in the medium and weak forming groups.The expression levels of efflux pump and outer membrane protein coding genes and their regulatory genes were also different.Conclusion The biofilm formation ability is associated with the gene expression level of luxS in Klebsiella pneumoniae.As a high-order regulatory gene,luxS is involved in the complex regulatory network with ramA,ompK36 and micF,which affects the biofilm formation simultaneously.

关 键 词：肺炎克雷伯菌 生物膜 LUXS 实时荧光定量PCR 

分 类 号：R446.5[医药卫生—诊断学]