携带NDM-1基因合并膜孔蛋白OmpK36缺失肺炎克雷伯菌耐药性分析

Analysis of drug resistance of Klebsiella pneumoniae carrying NDM-1 gene and OmpK36 deletion

目的对产新德里金属β-内酰胺酶(NDM-1)肺炎克雷伯菌耐药情况、分子分型特点,携带耐药基因、传播方式,及其膜孔蛋白表达情况进行分析,以探讨产NDM-1酶肺炎克雷伯菌耐药机制。方法实验菌分离自前列腺增生患者尿液的标本,对其进行细菌鉴定和药敏试验、改良Hodge试验、EDTA协同试验和多位点序列分型(MLST);采用聚合酶链技术检测β-内酰胺酶相关耐药基因和膜孔蛋白OmpK35、OmpK36基因,并对NDM-1和膜孔蛋白OmpK35、OmpK36基因进行序列分析;利用SDS-PAGE电泳检测膜孔蛋白OmpK35、OmpK36;菌株质粒接合实验分析传播方式,对受体菌和接合子进行药敏结果比较。结果实验菌改良Hodge试验阴性、EDTA协同试验阳性,鉴定为耐碳青霉烯类肺炎克雷伯菌(CRKP)株,对13种常见抗生素耐药,MLST基因分子分型为ST15;PCR检测其携带NDM-1基因,经序列测定比对确认,同时该菌株还携带KPC基因及SHV基因,未检测到VIM、IMP、OXA-48、GES、GIM基因;OmpK35、OmpK36基因存正点突变及片段缺失的现象,SDS-PAGE分析发现膜孔蛋白OmpK36缺失;质粒接合实验阳性,接合子E.coliJ53对3种碳青霉烯类药物的抑菌圈明显减小。结论了解NDM-1肺炎克雷伯菌携带耐药基因类型及传播方式、膜孔蛋白是否缺失以及分子分型,为指导临床正确使用抗菌药物及流行病学调查具有重要意义。

Objective Drug resistance and molecular typing of Klebsiella pneumoniae producing NDM⁃1 were analyzed.Drug resistance genes,transmission modes and expression of membrane pore proteins were carried out to explore the drug resistance mechanism of Klebsiella pneumoniae producing NDM⁃1 enzyme.Methods The bacterial identification and drug susceptibility test,modified Hodge test,EDTA synergistic test and multilocus sequencing typing(MLST)were performed.The bacteria were isolated from urine samples of patients with benign prostatic hyperplasia(BPH)in the Department of Urology in affiliated hospital of Putian University in 2017.The bacterial identification and drug susceptibility test,modified Hodge test,EDTA synergistic test and multilocus sequencing typing were performed.Polymerase chain reaction was used to detect the resistance genes related to beta⁃lactamase and membrane porin OmpK35,OmpK36 genes,and the sequences of NDM⁃1,membrane porin OmpK35 and OmpK36 genes were analyzed.Membrane porin OmpK35 and OmpK36 were detected by SDS⁃PAGE electrophoresis.Plasmid conjugation assay was used to analyze the mode of transmission of the strain.The sensitivity of the receptor and the zygote were compared.Results The strain was identified as carbapenem⁃resistant Klebsiella pneumoniae with negative modified Hodge test and positive EDTA synergistic test.It resistant to 13 common antibiotics.MLST genotype was ST15.The NDM⁃1,KPC and SHV gene were detected by PCR.VIM,IMP,OXA⁃48,GES and GIM genes were not detected.The NDM⁃1 gene was confirmed and the existence of positive mutation and deletion of OmpK35 and Ompk36 genes were found by Sequencing analysis.Membrane porin OmpK36 was deleted by SDS⁃PAGE,plasmid conjugation test was positive,and the inhibitory circle of conjugator E.coli J53 on three carbapenems was reduced significantly.Conclusion To understand the type and mode of transmission of drug⁃resistant genes,the deletion of membrane porin and molecular typing of NDM⁃1 producing Klebsiella pneumonia,it is great significance to guide the correct use of antibiotics in clinical and epidemiological investigation.