Objective To investigate the relationship between the biofilm formation ability and the OqxAB efflux pump of carbapenem-resistant K.pneumoniae isolates (CRKPs).Methods Thirty-seven CRKPs were collected from the First Affiliated Hospital of Wenzhou Medical University in China from 2008 to 2013.The minimum inhibition concentrations (MICs) of imipenem,ertapenem and meropenem were tested by the broth microdilution method.The biofilm formation ability was detected by crystal violet staining method.oqxA and oqxB for all strains were screened by PCR methods and the effect of subinhibitory concentration of CCCP (sub-MIC of CCCP) on biofilm formation were also analysed.To measure the expression level of oqxB in the planktonic and biofilm lifestyle,quantitative real-time PCR (qRT-PCR) was conducted in the presence or absence of CCCP (0.5 ×MIC).Experimental data was analyzed by using the SPSS 17.0 statistical software.Results Thirty-seven strains exhibited decreased susceptibility to one kind of carbapenems at least and all strains could form biofilm in different levels (A590 is 0.044～1.113).Among thirty-seven CRKPs,only seven strains did not carry oqxA or oqxB gene and their biofilm formation ability was lower than strains with complete efflux pump.Efflux pump inhibition test was assessed and result revealed subinhibitory concentration (sub-MIC) of CCCP had a positive role in promoting biofilm formation to different extent.Furthermore,qRT-PCR demonstrated strains in biofilm lifestyle contained a higher level of oqxB expression than strains which in planktonic condition.Conclusion The over-expression of OqxAB efflux pump may contribute to the biofilm formation of K.pneumoniae isolates.%目的 分析耐碳青霉烯类肺炎克雷伯菌(CRKPs)的生物膜形成能力与OqxAB外排泵的相关性.方法 收集2008-2013年间温州医科大学附属第一医院临床分离的碳青霉烯类耐药肺炎克雷伯菌37株.采用微量肉汤稀释法检测其对亚胺培南、厄他培南以及美罗培南的最低抑菌浓度(minimal inhibitory concentration,MIC),结晶紫染色法测定其生物膜形成能力,PCR方法筛查外排泵基因oqxA和oqxB的携带情况并分析亚抑菌浓度的外排泵抑制剂羰基氰氯苯腙(CCCP)对生物膜形成的影响;实时荧光定量PCR(qRT-PCR)检测CCCP存在与不存在条件下生物膜状态与浮游状态oqxB外排泵基因的表达量.用统计学软件SPSS 17.0分析实验数据.结果 37株肺炎克雷伯菌均对至少1种碳青霉烯类药物的敏感性降低,37株菌都具有不同程度的生物膜形成能力(A590范围为0.044～1.113);在所有菌株中,只有7株菌存在oqxA或oqxB外排泵基因的缺失,与外排泵基因完整的试验菌株相比,这7株菌形成生物膜的能力较低;外排泵抑制试验发现亚抑菌浓度的CCCP对实验菌株的生物膜形成能力都有不同程度的促进作用;qRT-PCR结果证明,生物膜状态下菌株的外排泵基因的表达量要高于浮游状态下的表达量.结论 OqxAB外排泵过表达可能会促进肺炎克雷伯菌生物膜的形成.
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