耐碳青霉烯类鲍曼不动杆菌生物膜形成与外排泵基因AdeABC表达的相关性研究

Correlation between biofilm formation and efflux pump gene AdeABC expression in carbapenem resistant Acinetobacter baumannii

目的 研究鲍曼不动杆菌的生物膜形成,以及浮游态和生物膜状态下对外排泵基因AdeABC的相对表达量作用于碳青霉烯类抗生素耐药机制的影响。方法 将菌株经药物敏感性试验分为碳青霉烯类耐药鲍曼不动杆菌(CRAB)组和碳青霉烯类敏感鲍曼不动杆菌(CSAB)组,采用结晶紫染色法检测两组生物膜形成能力;通过微量肉汤稀释法分别测定两组对亚胺培南的MIC、MBIC值,并计算MBIC/MIC的比值进一步比较分析生物膜菌和浮游菌的耐药性;同时提取CRAB组浮游态和生物膜态的RNA,通过RT-qPCR检测外排泵基因AdeABC相对表达量。结果 35株标本的生物膜阳性率为91.43%,其中CRAB组生物膜的阳性检出率为92%,生物膜阴性、弱阳性、阳性和强阳性分别占8%、48%、36%和8%。MBIC/MIC比值显示,形成生物膜后,鲍曼不动杆菌的耐药能力呈现不同程度的增加,CSAB组的耐药增加幅度以2倍的菌株最多;CRAB组的增加幅度以4倍的菌株最多。同时CRAB组RT-qPCR结果显示,生物膜较浮游态相比,外排泵基因adeA和adeB的相对表达量明显增高(P<0.01),外排泵基因adeC相对表达量则无差异(P>0.05)。结论 鲍曼不动杆菌碳青霉烯类耐药与生物膜密切相关,且生物膜态的鲍曼不动杆菌较浮游态可促进外排泵基因AdeABC的表达从而增强碳青霉烯类抗生素的耐药性。

Objective To study the formation of biofilm in Acinetobacter baumannii and the effect of the relative expression level of the efflux pump gene AdeABC on the resistance mechanism of carbapenem antibiotics in both planktonic and biofilm states.Methods The strains were divided into the carbapenem-resistant Acinetobacter baumannii(CRAB)group and the carbapenem-sensitive Acinetobacter baumannii(CSAB)group by drug sensitivity testing,and the biofilm formation ability of the two groups was detected using the crystal violet staining method.The MIC and MBIC values of two groups of imipenem using the micro broth dilution method were measured,and the MBIC/MIC ratio was calculated to further compare and analyze the drug resistance of biofilm bacteria compared to planktonic bacteria.Simultaneously,RNA from both planktonic and biofilm states of the CRAB group was extracted,and the relative expression level of the efflux pump gene AdeABC through RT-qPCR was detected.Results The positive rate of biofilm in 35 specimens was 91.43%,of which the positive detection rate of biofilm in the CRAB group was 92%.The negative,weakly positive,positive,and strongly positive biofilms accounted for 8%,48%,36%,and 8%,respectively.The MBIC/MIC ratio showed that after the formation of biofilm,the resistance of Acinetobacter baumannii increased to varying degrees,with the CSAB group showing the most significant increase in resistance compared to strains with a 2-fold increase;The increase in CRAB group was highest among strains with a 4-fold increase.At the same time,the RT-qPCR results of the CRAB group showed that the relative expression levels of the efflux pump genes adeA and adeB were significantly increased compared to the planktonic state of the biofilm(P<0.01),while the relative expression levels of the efflux pump gene adeC were not significantly different(P>0.05).Conclusion The carbapenem resistance of Acinetobacter baumannii was closely related to biofilm,and the biofilm state of Acinetobacter baumannii could promote the expression of the efflux pump gene AdeABC compared to the floating state,thereby enhancing the resistance of carbapenem antibiotics.