下呼吸道感染产AmpC酶和超广谱β-内酰胺酶肺炎克雷伯菌耐药基因检测及亲缘性分析

Multidrug resistant genotypes and phylogenetics of Klebsiella pneumoniae producing AmpC and extendedspectrum β-lactamases （ESBLs） in lower respiratory tract infections

目的分析下呼吸道感染患者气道分泌物或痰液标本分离的产AmpC酶和超广谱β-内酰胺酶（ESBL）肺炎克雷伯菌耐药基因的特点及亲缘性。方法2009年1月至2010年8月由本院呼吸病区住院的下呼吸道感染患者气道分泌物或痰液标本分离得到53株肺炎克雷伯菌，采用K-B纸片扩散法测定其对常用抗菌药物的敏感性；三维实验检测AmpC酶；PCR检测ESBL、质粒介导的AmpC酶、喹诺酮类耐药基因、耐消毒剂．磺胺基因qacE△1-sull、Ⅰ类整合酶基因IntⅠ1的携带情况，并进行聚类分析。结果53株肺炎克雷伯菌未发现对亚胺培南、美罗培南、厄他培南耐药；对环丙沙星、左旋氧氟沙星耐药率均〉80％；对其他抗菌药物也有不同程度的耐药。53株肺炎克雷伯菌中IntI1基因阳性率60．4％（32／53），qnrA基因阳性率54．7％（29／53），qnrS基因阳性率13．2％（7／53），qnrB基因阳性率5．7％（3／53），qacE△1．sull基因阳性率71．7％（38／53），TEM基因阳性率92．5％（49／53），CTX—M基因阳性率100％（53／53），SHV基因阳性率9．4％（5／53），AmpC基因阳性率92．5％（49／53）；其中4株同时携带qnrA、qnr$、intI1、qacE△1．sull、AmpC、CTX—M基因。聚类分析显示40、41、10与18号，25与42号，8与35号菌株有亲缘关系。结论产ESBL肺炎克雷伯菌的多重耐药与整合子相关，且携带多种耐药基因，聚类分析显示存在克隆传播和医院内感染。

Objective To analyze the drug-resistant genotypes and phylogenetics of resistance genes in AmpC and extended-spectrum 13-1actamases （ESBLs） producing Klebsiella pneumoniae isolated from airway secretion or sputum of patients with lower respiratory tract infections （LRTI）. Methods Between January 2009 and August 2010, 53 strains of Klebsiella pneumoniae were isolated from airway secretion or sputum specimens of LRTI patients in respiratory wards of our hospital. Of the Klebsiella pneumoniae strains, Kirby-Bauer method was used to assess the sensitivity to common antibacterial drugs, three-dimensional extract test to determine AmpC, and PCR to detect the genes encoding ESBLs （CTX-M, TEM and SHV） and plasmid-mediated AmpC, quinolones resistance （qnrA, qnrB and qnrS） , antiseptics and sulfanilamide resistance （qacEA1- sull） and class 1 integrase （Int Ⅰ 1）. Cluster analysis of bacteria was performed. Results Of the 53 strains of Klebsiella pneumoniae, none showed resistance to imipenem, meropenem and ertabenem, but over 80% was resistant to ciprofloxacin and levofloxacin. Moreover, a varying range of resistance to other antibacterial agents was also shown. Of 53 strains of Klebsiella pneumoniae, 60.4% （32/53） was positive for Int I 1, 54.7%（29/53） for qnrA, 13.2%（7/53） for qnrS, 5.7%（3/53） for qnrB, 71.7%（38/53） for qacE△1-sull, 92.5%（49/53） for TEM, 100%（53/53） for CTX-M, 9.4%（5/53） for SHV and 92.5% （49/53） for AmpC. Notably, 4 strains were found to simultaneously carry qnrA, qnrS, int I 1, qacE△1-sull, AmpC and CTX-M genes. Cluster analysis demonstrated the phylogenetic relationship among number 40, 41, 10 and 18 strains, between number 25 and 42 strains, and between number 8 and 35 strains. Conclusions ESBLs- producing Klebsiella pneumoniae may have integronrelated multi- drug resistance, and carry multiple drug-resistant genes. Cluster analysis reveals evidences of clonal transmission and hospital-acquired infections.