碳青霉烯类耐药肠杆菌科细菌分子生物学研究

Molecular biology of carbapenem-resistant Enterobacteriaceae isolates

目的对海南地区耐碳青霉烯类肠杆菌科细菌进行碳青霉烯酶基因检测,为院内感染控制和流行病学研究提供依据。方法收集海南地区四家医院2012年8月-2013年8月临床分离的碳青霉烯类耐药肠杆菌科细菌33株,用VITEK 2 compact进行细菌鉴定和药敏试验,采用改良Hodge试验(MHT)和双纸片增效试验分别进行碳青霉烯酶表型确认,对于阳性菌株,设计通用引物行PCR检测KPC、NDM-1、IMP、VIM、SME、OXA-23和NMC 7种基因,并对扩增产物进行测序和BLAST分析。结果 33株耐碳青霉烯类肠杆菌科细菌中MHT试验有22株阳性,双纸片增效试验有18株阳性,经PCR方法对阳性菌株进行基因检测,11株携带NDM-1基因,3株携带KPC-2基因,1株携带IMP-4基因;其中有一株产酸克雷伯菌同时携带KPC-2、NDM-1和IMP-4三种基因,携带碳青霉烯酶基因的菌株对β-内酰胺类抗菌药物呈高度耐药,仅对阿米卡星的敏感性较高。未检测出VIM、SME、NMC和OXA-23基因。结论海南地区肠杆菌科细菌耐碳青霉烯类抗菌药物主要机制为产生NDM-1、KPC-2和IMP-4基因,临床上应引起重视,要严格做好预防和控制工作。

[ Objective ] To provide the basis for health-care associated infection control and epidemiological re- search by investigation of the resistance gene of carbapenem-resistant Enterobacteriaceae. [Methods] A total of 33 carbapenem-resistant Enterobacteriaceae isolates were collected from August 2012 to August 2013 in Hainan area. Identification and sensitivity test were performed on VITEK-2 compact automatic bacterial test systems. Double-disc synergy test and modified Hodge test （MHT） were performed for earbapenemase phenotype screening. PCR amplifica- tion and DNA sequencing were used to detect KPC, NDM-1, IMP, VIM, SME, OXA-23 and NMC genes. [Results] Modified Hodge test showed that 22 strains produced carbapenemase while double-disc synergy test showed 18 strains were positive. Through PCR amplification and DNA sequencing, the encoding gene of NDM-1 was identified in 11 clinical isolates, the encoding gene of KPC-2 was identified in 3 clinical isolates, the encoding gene of IMP-4 was identified in 1 clinical isolates. Among these isolates, one isolate carried genes of NDM-1, KPC-2 and IMP-4 at the same time. Most carbapenemase-producing strains demonstrated a high resistance to β-1actam antibiotics be-sides a high sensitivity to Amikacin. The encoding gene of VIM, SME, NMC or OXA-23 was not detected in any isolate. [ Conclusions] Resistance to carbapenems is mainly due to the production of KPC-2, NDM-1 and IMP-4 in these Enterobacteriaceae strains. It is important for clinical staff to pay attention to it and strengthen the prevention and control work.