肺炎克雷伯菌介导碳青霉烯类耐药的基因型检测

A study of molecular and epidemical mechanism of carbapenem-resistant Klebsiella Pneumoniae

目的对临床分离的碳青霉烯类抗生素耐药的肺炎克雷伯菌进行耐药基因型分析。方法采用琼脂稀释法检测菌株最低抑菌浓度（MIC），用聚合酶链反应（PCR）、DNA测序、脉冲场琼脂糖凝胶电泳（PFGE）、等电点聚焦电泳（IEF）和质粒分子杂交等技术对实验菌株进行基因组DNA同源性、介导耐药的基因型、耐药酶pI和耐药基因携带状态等研究。结果12株肺炎克雷伯菌的亚胺培南和美罗培南MIC值分别为16～64μg／Inl和8—256μg／ml；对其他被测的β-内酰胺类和喹诺酮类药物广泛耐药，氨基糖苷类耐药型不定。12株菌均扩增出编码碳青霉烯类抗生素耐药的blaKPC-2。和编码β-内酰胺酶的blas SHV基因；部分菌株扩增出blaTEM或／和blaCTX-M基因。IEF和分子杂交揭示KPC-2的等电点为6．8，编码基因被携带在一个约56kb大小的质粒上；PFGE结果显示12株细菌可分为2个克隆型。结论产KPC-2型碳青霉烯酶是本院肺炎克雷伯菌耐碳青霉烯类药物的主要原因，垂直传播以及通过质粒传递耐药基因可能是其在本医院流行的主要方式。

Objective To investigate the genotype of carbapenem-resistant KlebsieUa pneumoniae isolates. Methods Agar-dilution was used to determine the minimum inhibitory concentrations （ MIC）, specific PCR assays and DNA sequence analysis, pulsed-field gel electrophoresis （PFGE）, isoelectric focusing （IEF） and molecular hybridization were used to confirm the presence of carbapenem-re- sistance gene, genetic relatedness and isoelectric point （pI） of resistant enzyme. Results The MICs of both imipenem and meropenem were from 16 to 64 μg/ml and 8 to 256 μg/ml. All the 12 clinical isolates were resistant to other tested β-1actam and fluoroquinolones agents, but uncertain to aminoglycosides antimicrobial agents. The genes, Blanc_2 encoding Klebsiella pneumoniae carbapenem-hydrolyzing enzyme and blaSHV encoding lactamase, were amplified in all the 12 isolates, blaTEM and blacTx.M were simultaneously present in some strains. IEF and molecular hybridization showed that the encoding gene of KPC-2 with a pI of 6.8 was carried in a plasmid with approximately 56 kb. PFGE showed that the 12 isolates belonged to two clones. Conclusions Production of carbapenemase KPC-2 mainly contributes to reduced carbapenem susceptibility of Klebsiella pneumoniae isolated from our hospital, and vertical transmission or plasmid-mediated transmission may be the principal epidemical mechanism.