产NDM-1肺炎克雷伯菌中国分离株的初步研究

NDM-1-producing Klebsiella pneumoniae in mainland China

摘要:

目的：探讨湖南地区出现的肺炎克雷伯菌新德里金属β-内酰胺酶1型（NDM-1）基因特征及其与耐药性的关系。方法：临床菌株分离自湖南某综合医院一例重症肺炎并中毒性心肌炎患儿的痰液标本。采用全自动微生物分析系统VITEK-2 compact对菌株进行鉴定及药敏检测；改良Hodge试验筛查碳青霉烯酶；双纸片协同试验及双纸片增效试验检测金属β-内酰胺酶；聚合酶链反应(PCR)特异性扩增NDM-1基因，并对扩增产物进行测序和BLAST分析；质粒接合转移试验分析其耐药机制，并对接合前后的药敏结果进行比较。结果：该菌株经鉴定为肺炎克雷伯菌。改良Hodge试验、双纸片协同试验及双纸片增效试验均为阳性。PCR扩增产物基因序列与GenBank中NDM-1基因FN396876.1同源性为100%。以筛选的接合子DNA为模板扩增NDM-1基因全长并测序，结果与供体菌NDM-1基因扩增产物完全一致。接合子E.coli J53（NDM-1）对所有β-内酰胺类药物的MIC值较受体菌E.coli J53均有很大提高，其中对厄他培南和亚胺培南MIC均提高了8倍以上，头孢他啶及头孢曲松则提高了64倍以上。结论：中国大陆出现了产NDM-1肺炎克雷伯菌；NDM-1基因可在不同菌株之间传递，导致细菌对β-内酰胺类药物广泛耐药。

Abstract:

OBJECTIVE: To investigate the characteristics of New Delhi metallo-beta-lactamase-1 (NDM-1) gene of Klebsiella pneumoniae (K. pneumoniae) emerging in Hunan, and its relationship to antibiotic resistance. METHODS: The clinical strain was isolated from a sputum sample of a child with severe pnemonia and toxic myocarditis who was admitted into a general hospital of Hunan Province. VITEK-2 compact instrument was used for bacterial identification and antibiotic susceptibility test. Modified Hodge test was used for the screening of carbapenemase. EDTA-synergy test and combination disk diffusion test were used for detection of metallo-β-lactamase (MBL). PCR was performed for amplification of NDM-1 genes and the positive products were sequenced and analyzed with BLAST. Conjugation was also performed to analyze mechanisms of antibiotic resistance. The results of antibiotic susceptibility tests were compared before and after conjugation. RESULTS: The isolated strain was identified as K.pneumoniae. Modified Hodge test, EDTA-synergy test and combination disk diffusion test were all positive for the strain. The homology between gene sequence of PCR amplification products and NDM-1 gene FN396876.1 in the GenBank was 100%. Transconjugant DNA was used as template for the amplification of NDM-1 gene. The amplification products were sequenced and found to be the same as the NDM-1 gene amplification product of the donor strain. The MIC of transconjugant E.coli J53 (NDM-1) to all the β-lactams increased significantly compared with the recipient strain E.coli J53. The MIC of ertapenem and imipenem increased by more than 8 times, while the MIC of ceftazidime and ceftriaxone increased by more than 64 times. CONCLUSIONS: This study first identified a strain of K. pneumoniae carrying NDM-1 in mainland China. NDM-1 gene can be transmitted among different strains and causes extensively drug-resistance to β-lactams.