儿科住院患者耐碳青霉烯类肺炎克雷伯菌耐药性监测及分子流行病学特征

Resistance surveillance and molecular epidemiological characteristics of carbapenem-resistant Klebsiella pneumonia in pediatric department

目的探讨儿科患者耐碳青霉烯类肺炎克雷伯菌(CRKP)的耐药性和分子流行病学特点。方法收集2015年1月至2018年4月我院儿科临床分离的肺炎克雷伯菌,用全自动细菌鉴定仪、纸片扩散法及琼脂扩散梯度法进行鉴定及药敏试验;以改良碳青霉烯类失活法(m CIM)和乙二胺四乙酸(EDTA)碳青霉烯类失活法(e CIM)检测碳青霉烯酶;以聚合酶链反应(PCR)检测KPC、NDM、IMP、OXA-48、VIM 5种耐药基因;用多位点序列分型(MLST)对CRKP进行分子遗传学分析;用随机扩增多态性DNA技术(RAPD)分析其同源性;用WHONET 5. 6进行数据分析。结果该研究期间共分离肺炎克雷伯菌189株,其中CRKP占14. 8%(28株);CRKP对氨苄西林/舒巴坦、头孢唑啉、头孢呋辛、头孢他啶、头孢曲松、美洛培南的耐药率均为100%,对其他β内酰胺类耐药率也较高(>85%);对喹诺酮类及阿米卡星耐药率较低(<50%);替加环素对大部分CRKP有较强体外活性,耐药率仅为3. 6%。遗传学分析显示,28株CRKP均为碳青霉烯酶产生株,其中以KPC-2(15/28)为优势基因,其次为NDM-1(6/28),IMP-4 (3/28),NDM-5 (2/28),另外,2株菌同时携带KPC-和NDM-1基因。28株CRKP经MLST分出7种ST型,其中以ST11 (57. 1%,16/28),ST20(17. 9%,5/28),ST17(7. 1%,3/28)为主,ST163、ST299、ST401、ST1446各占3. 6%(1/28)。所有CRKP分为8种RAPD类型,最常见的类型为A型(18/28),值得注意的是,所有的ST11菌株都属于A型。结论近4年来,我院儿科病房中以含有KPC-2的ST11型CRKP菌株为主。菌株之间存在一定的基因多态性,某种克隆株可能存在小范围流行,这表明迫切需要加强临床意识和感染控制措施。

Objective To investigate drug - resistance profiles and mo- lecular epidemiological characteristics of carbapenem resistant Klebsiella pneumoniae( CRKP) strains in the pediatric department. Methods All Klebsiella pneumonia isolates were collected from pediatric wards in Pe- king University First Hospital between January 2015 to April 2018. Anti- microbial susceptibility test was performed by VITEK2 Compact,disk dif- fusion and gradient diffusion methods. Carbapenemases were screened by modified carbapenem inactivation method( mCIM) and EDTA - carba- penem inactivation method( eCIM) test,polymerase chain reaction( PCR) amplification were performed to detect carbapenemase genes KPC,NDM, IMP,OXA -48 and VIM. All isolates were analyzed for sequence type ( ST) by multilocus sequence typing ( MLST) and clone relationships by randomly amplified polymorphic DNA ( RAPD). WHONET 5. 6 carries on the data analysis. Results A total of 189 strains of Klebsiella pneumoniae were isolated,of which CRKP strains accounted for 14. 8%( 28 strains). The resistance rates of CRKP isolates to ampicillin / sulbac- tam,cefazolin,cefuroxime,ceftazidime,ceftriaxone,meropenem were all 100%,other β- lactam resistance rates were also high (> 85%),while quinolones and amikacin resistance rates were relatively low(< 50%),tigecycline exhibited potent activity against most tested strains,only one tigecycline reisitant strain was detected( 3. 6%). In this study,28 CRKP strains were recovered for genetic analysis. All of them were carbapenemase - producing strains,the most dominant gene was KPC -2 ( 15/28),followed by NDM -1 ( 6/28),IMP -4 ( 3/28) and NDM -5 ( 2/28). Moreover,2 strains harbored simultaneously both KPC and NDM. MLST alleles were distributed in 7 STs,of which, ST11( 57. 1%,16/28),ST20( 17. 9%,5/28),ST17( 7. 1%,3/28) were the predominant clones,ST163,ST299, ST401 and ST1446 accounted for 3. 6 %( 1/28) of each. All CRKP were categorized into 8 RAPD types,and the most frequent type was type A ( 18/28). Of note,all of ST11 strains belongs to type A. Conclusion ST11 type CRKP strains harboring KPC are predominant in pediatric wards of this hospital in recently 4 years. The genetic polymorphism is diverse in different strains. The cetrain clones might cause a small scale prevalence,suggesting an urgent need to enhance clinical awareness and infection control measures.