摘要
目的研究碳青霉烯类耐药肺炎克雷伯菌临床儿童分离株的耐药特点及分子流行病学特征。方法收集温州医学院附属第二医院2010年7月-2011年6月从儿童标本中分离的耐碳青霉烯类肺炎克雷伯菌12株，所有菌株为非重复菌株，菌种鉴定采用全自动微生物分析仪。改良的Hodge试验筛选产碳青霉烯酶阳性菌株，采用PCR法检测KPC、IMP、bla（s）、VIM、SPM和整合酶基因，测序确定基因型。对菌株进行质粒结合试验、质粒消除试验检测质粒的转移性。脉冲场凝胶电泳（PFGE）分析耐药菌株的同源性。结果12株耐碳青霉烯类肺炎克雷伯菌对庆大霉素、妥布霉素、阿米卡星、环丙沙星、左氧氟沙星、复方磺胺甲嗯唑的敏感率分别为8．3％、41．7％、58．3％、8．3％、8．3％、33．3％；12株菌均携带有KPC-2基因，且同时携带有TEM-1和SHV型β-内酰胺酶基因，其中SHV-11-like和SHV·12-like各6株；11株携带CTX-M型基因，其中4株为CTX—M-14-like基因，6株CTX—M-15-like基因；2株携带有OXA-10型基因，1株携带有PER-1基因。未检出NDM-1、GIM、SPM、SIM、VIM型碳青霉烯酶基因。12株均为I类整合酶基因（intl）阳性。2株通过接合试验把质粒传递给受体菌EC600。所有接合子blaTEM-1基因阳性、超广谱B．内酰胺酶（ESBL）基因阳性及对亚胺培南、庆大霉素、阿米卡星、妥布霉素和头孢噻肟耐药，接合子ESBL基因型与供菌-致。2株菌经质粒消除后对亚胺培南的MIC值均有较大程度降低，消除后KPC-2基因扩增为阴性。12株KPC-2基因阳性菌株经PFGE分成5个基因型，主要为B型和C型。结论KPC-2型碳青霉烯酶基因已经在儿童肺炎克雷伯菌中播散，常伴随携带多种类型的ESBL基因和I类整合酶基因，部分耐药基因可通过质粒播散。
Objective To investigate molecular epidemiology and antimicrobial susceptibility of carbapenem-resistant strains of KlebsieUa pneumoniae isolated from children. Methods From July 2010 to June 2011, twelve non-replicate clinical isolates of carbapenem-resistant Klebsiella pneumoniae were consecutively collected from children inpatients in the Second Hospital of Wenzhou Medical Colloge. All of the isolates were identified by the automated microbiology systems. Modified Hodge test was used to screen strains producing carbapenemases. Pulsed field gel electrophoresis （PFGE） was performed to analyze the homogeneity of genomic DNA of Klebsiella pneumoniae. KPC, IMP, GIM, SPM, SME, OXA-10, bla（ s） , VIM gene and integrase gene were amplified by PCR and then sequenced to cofirm the genotypes;Plasmid conjugation experiment was used to study the transfer method of bacterial resistance. Plasmid-curing test were used to initally locate the resistant genes. Results 0ne（8.3%）, 5（41.7%）, 7（58.3%）, 1（8.3%）, 1（8.3%） and4（33.3%） ofl2isolates were susceptible to gentamiein, tobramycin, amikaein, ciprofloxacin, levofloxacin and trimoxazole, respectively. All isolates carried KPC-2, TEM-1 and SHV genes （six for SHV-1 1-like, six for SHV-12-1ike）. Eleven of twelve isolates with KPC-2 gene carried CTX-M genes（4 for CTX-M-14-1ike, 6 for CTX-M-15-1ike）. Two isolates carried OXA-10 geneS, and one isolates carried PER-1 gene. None of NDM-1, GIM, SPM, SIM and VIM carbapenemase genes was detected in 12 isolates. All of 12 isolates carried Int 1 genes. The plasmids of 2 isolates were transgerred into the recipients E. coli EC600. PCR and sequence analysis revealed that blaTEM.1 and blaCTX-M-15like, were co-transferred with the KPC-2 gene to the recipients. Elimination of KPC-2-encoding plasmid from Kp7 and Kpl2 resulted in imipenem susceptibility in the two isolates. Amplification revealed that KPC-2 gene was lost by the plasmid-curing test. Of the 12 isolates, 5 patterns were obtained by PFGE. Pattern B
出处
《中华微生物学和免疫学杂志》
CAS
CSCD
北大核心
2012年第10期861-865,共5页
Chinese Journal of Microbiology and Immunology
