医院获得性肺炎患者耐碳青霉烯类肺炎克雷伯菌分离株耐药基因及毒力因子研究  

Study on resistance genes and virulence factors of carbapenem resistant Klebsiella pneumoniae isolates from hospital acquired pneumonia patients

作　　者：韩晶[1] 刘晶[1] 杨自坚 Han Jing;Liu Jing;Yang Zijian(Medical Laboratory Center,the People's Hospital of Anyang,Anyang 455000,China)

机构地区：[1]河南省安阳市人民医院医学检验中心,455000

出　　处：《临床医学》2024年第8期21-25,共5页Clinical Medicine

摘　　要：目的分析医院获得性肺炎患者耐碳青霉烯类肺炎克雷伯菌(CRKP)分离株的耐药基因和毒力因子。方法选取2021年1月至2022年12月安阳市人民医院收治的96例医院获得性肺炎患者为研究对象,分离得到100株耐碳青霉烯类肺炎克雷伯菌分离株。采用微生物鉴定仪和药敏检测法鉴定其菌种,统计分析患者的临床资料(包括性别、年龄、科室、基础性疾病、标本类型、ICU入住情况、侵袭性操作情况、抗生素暴露情况及预后情况等);采用优化的Hodge法、碳青霉烯酶灭活法和拉丝实验对菌株进行表型检测;采用PCR检测19种β-内酰胺类(碳青霉烯酶、AmpC酶和ESBL)和喹诺酮类耐药基因和毒力基因,选取阳性产物并通过双向测序鉴定其基因型;采用多位点序列分析耐碳青霉烯类肺炎克雷伯菌的流行病学,采用脉冲场凝胶电泳分析耐碳青霉烯类肺炎克雷伯菌的克隆情况。结果本研究100株肺炎克雷伯菌进行体外药敏试验表明,其头孢菌素类、酶抑制剂类、单环类和碳青霉烯类抗生素的耐药率最高,均在95.0%以上,氨基糖苷类抗生素和替加环素的耐药率均较低,均在60.0%左右。分布最广的科室是ICU。菌种主要来源于痰液,其次是血液和尿液。采用优化的Hodge试验进行表型检测检出84阳性菌株,而采用mCIM法检出93株阳性菌株。对耐药基因进行扩增结果表明,NDM、KPC、SHV、IMP、TEM、CTX-M和qnrS 7条基因均可扩增出相应位置大小的阳性条带,而SME、VIM、GIM、GES、OXA-23、OXA-48、VEB、PER、DHA、AAC、qnrA和qnrB这12种耐药基因均未扩增出相应的条带。从中随机选取20株KPC阳性DNA产物进行测序,经序列比对发现其均为KPC-2基因型,而NDM阳性DNA产物经测序比对发现4株为NDM-1基因型,2株为NDM-5基因型。经多序列比对发现NDM-5和NDM-1基因有两处基因发生变异。携带4种不同耐药基因的菌株占比最多(53.0%),其次是携带5种(24.0%)Objective To analyze the drug resistance genes and virulence factors of carbapenem-resistant Klebsiella pneumoniae isolates from patients with hospital-acquired pneumonia.Methods Ninety-six hospital acquired pneumonia patients admitted to Anyang People's Hospital from January 2021 to December 2022 were selected as the research subjects,and 100 carbapenem-resistant Klebsiella pneumoniae isolates were isolated.Microbial identification instrument and drug sensitivity test were used to identify the strains,and clinical data of the patients were statistically analyzed(including gender,age,department,underlying disease,specimen type,ICU admission,invasive operation,antibiotic exposure and prognosis,etc.).The strain phenotype was detected by Hodge method,carbapenase inactivation method and wire drawing test.PCR was used to detect 19β-lactam(carbapenemase,AmpC enzyme and ESBL)and quinolone resistance genes and virulence genes,and the positive products were selected and their genotypes were identified by bidirectional sequencing.The epidemiology of carbapenem-resistant Klebsiella pneumoniae was analyzed by multi-site sequence analysis,and the cloning of carbapenem-resistant Klebsiella pneumoniae was analyzed by pulsed field gel electrophoresis.Results In this study,100 strains of Klebsiella pneumoniae were tested in vitro,and the drug resistance rates of cephalosporins,enzyme inhibitors,monocyclic antibiotics and carbapenems were the highest,all higher than 95.0%,while the drug resistance rates of aminoglycoside antibiotics and tigacycline were lower,all about 60.0%.The most widespread department was the ICU.The main source of bacteria was sputum,followed by blood and urine.Eighty-four positive strains were detected by Hodge test and 93 positive strains were detected by mCIM method.The results showed that NDM,KPC,SHV,IMP,TEM,CTX-M and qnrS could be amplified with positive bands of corresponding location size.However,no corresponding bands were amplified for 12 resistance genes:SME,VIM,GIM,GES,OXA-23,OXA-48,VEB,PER,DHA,AA

关 键 词：医院获得性肺炎 肺炎克雷伯菌 碳青霉烯酶 耐药基因 毒力因子 

分 类 号：R563.1[医药卫生—呼吸系统]