新德里1型金属β内酰胺酶肺炎克雷伯菌的分子流行病学特征及耐药机制

Resistance mechanism and molecular epidemiology of the New Delhi metallo-β-lactamase-1 possessing Klebsiella pneumoniae

目的：研究南昌大学第一附属医院产新德里金属β内酰胺酶（ NDM）肺炎克雷伯菌的分子表型、流行病学和耐药基因环境特征。方法回顾性研究。收集2011年1月至2012年12月我院住院患者临床分离的非重复碳青霉烯耐药肺炎克雷伯菌110株,使用生物梅里埃公司生产GN13的检测菌株的MIC值,改良Hodge试验进行产碳青霉烯酶确认,同时对试验菌株的blaNDM-1基因及ISAba125-NDM连锁PCR检测,并将扩增产物纯化、克隆后进行基因测序。多位点序列分析（ MLS栽）技术分析耐药菌株间的亲缘性。对阳性株进行质粒接合和消除试验,并对接合子及质粒消除后菌株进行PCR扩增及碳青霉烯类抗生素 MIC 值的检测。采用 Fisher 确切概率法对耐药率进行比较。结果110株碳青霉烯类耐药肺炎克雷伯菌中检出NDM基因阳性率为13%（14/110）,经测序确认为blaNDM-1基因。14株产NDM-1型金属β内酰胺酶菌株厄他培南、亚胺培南及美罗培南的耐药率均为14/14,环丙沙星耐药率为10/14,左氧氟沙星耐药率为9/14,阿米卡星耐药率为5/14,氨曲南耐药率11/14。 ISAba125-NDM连锁检测阳性率为14/14。14株blaNDM-1基因阳性菌质粒接合试验均为成功,接合子对亚胺培南、美罗培南及厄他培南的MIC值增高4~64倍, PCR扩增接合子大肠埃希菌J53 blaNDM-1基因及ISAba125-NDM连锁均为阳性。而质粒分析发现blaNDM-1基因及ISAba125-NDM连锁位于一大小约65000 bp质粒上。结论本地区新德里金属β内酰胺酶肺炎克雷伯菌对氨基糖苷类抗生素及氨曲南的耐药率相对较低,blaNDM-1基因主要位于质粒上可通过接合方式传播,易于在菌株间流行,应当引起临床高度重视；插入序列ISAba125可能参与了blaNDM-1基因的介导。

Objective To investigate the molecule phenotype, epidemiology, and resistance genes of the New Delhi metallo- β-lactamase-1 （ NDM-1 ） producing Klebsiella pneumoniae （ K. pneumoniae ） . Methods Retrospective study was made on one hundred and ten non-repetitive carbepenem-resistant K. pneumoniae clinical isolated strains, which were collected from January 2011 to December 2012 in our hospital. The minimal inhibitory concentrations （ MICs ） of antibiotics were tested by the GN13 cards of BioMerieux Company. Modified Hodge test were used for the detection of carbapenemases. The blaNDM-1 encoding gene and linkage of ISAba125-NDM were detected by PCR method. The purified PCR products were cloned and sequenced. The homology of the K. pneumoniae were analyzed by the multilocus sequence typing （ MLST ） . Plasmid conjugation experiment and curing method were used to study the transfer of bacterial resistance. The Fisher′s exact probability test was used to compare the data. Results 13% NDM-1-producing K. pneumoniae were detected and confirmed as blaNDM-1 by sequencing （14/110）. The resistance rates of the 14 NDM-1-producing K. pneumoniae strains to imipenem, meropenem, ertapenem, ciprofloxacin,＆amp;nbsp;levofloxacin, amikacin, and aztreonam were 14/14, 14/14, 13/14, 10/14, 9/14, 5/14, and 11/14. Meanwhile, the positive rate of ISAba125-NDM linkage of those 14 NDM-1-producing K. pneumoniae strains was 14/14. The E. coli J53 transconjugants, whose MICs of imipenem, meropenem, and ertapenem were increased by 4 to 64 times, were blaNDM-1 gene and ISAba125-NDM linkage positive. In addition, it was showed that the blaNDM-1 gene and ISAba125-NDM linkage were located on a plasmid with a size of approximately 65 000 bp. Conclusions The NDM-1 producing K. pneumoniae strains in this study were resistant to many commonly used antibiotics, however, the resistance rate to aminoglycoside and aztreonam were relatively low. The carbapenemase-resistant genotype spread by blaNDM-1 carried plasmid. Attention should be paid to its easily transmissible feature among the strains in clinic. The insertion sequence ISAba125 may be involved in the blaNDM-1 gene mediated carbapenemase-resistant genotype.