多重耐药肺炎克雷伯菌62种耐药基因元件的检测与gyrA基因突变的发现  

Combined detection of 62 drug-resistant gene elements and detection of gyrA gene mutation in multidrug-resistant Klebsiella pneumoniae

作　　者：丘江 张洁 孙杨 糜祖煌[2] QIU Jiang;ZHANG Jie;SUN Yang;MI Zu-huang(Department of Medical Laboratory,Hubei Jianghan Oil Field General Hospital,Qianjiang 433121,China;Wuxi Clon-Gen Techonology Institution,Wuxi 214026,China)

机构地区：[1]湖北江汉油田总医院检验科,潜江433121 [2]无锡市克隆遗传技术研究所,无锡214026

出　　处：《中国人兽共患病学报》2019年第6期539-544,共6页Chinese Journal of Zoonoses

摘　　要：目的 调查一组临床检出的肺炎克雷伯菌可能存在的耐药基因状况,以及各菌株间可能存在的亲缘关系。方法 25株肺炎克雷伯菌均分离自2016-2017年江汉油田总医院医院住院病人标本。菌种鉴定为gyrA基因测序上网BLASTn比对法,用PCR方法检测35种β-内酰胺酶基因,15种氨基糖苷类药物耐药基因,1种喹诺酮类药物耐药相关基因和11种可移动遗传元件。再用DNA测序法分析基因突变。最后对62种耐药元件基因的检测结果作样本聚类分析(UPGMA法)。结果 25株耐药肺炎克雷伯菌对碳青霉烯类耐药率低(亚胺培南、美罗培南均只有8%),对其它8种抗菌药物的耐药率较高(40%~96%)。每株均检出β-内酰胺类药物耐药相关基因(总阳性率100.00%);有24株菌检出氨基糖苷类药物耐药相关基因(总阳性率96.00%);有20株菌检出喹诺酮类药物耐药相关基因gyrA突变(即突变率为80.00%);每株均检出可移动遗传元件遗传标记基因(总阳性率100.00%)。共检出5种β-内酰胺酶基因(bla TEM、bla LAP、bla KPC、bla DHA群、bla OXA-1群),6种氨基糖苷类药物耐药基因(aac(3)-Ⅱ、aac(6’)-Ⅰb、ant(3”)-Ⅰ、aph 3’-Ⅰ、aad A5、rmt B),20株存在喹诺酮类药物耐药相关基因gyrA第83位密码子发生了同样的突变,且均无第87位密码子的突变。检出8种可移动遗传元件(intⅠ1、tnp U、tnp 513、ISEcp1、IS26、IS903、ISKpn6、trbC),且检出率较高。样本聚类分析显示本组菌可分A与B 2个簇群,2个簇群中均有克隆传播。A簇群中有2个克隆,分别为2-3-5-16等4株、13-14等2株;B簇群中有1个克隆,为19-20号2株。结论 多种β-内酰胺类药物耐药基因、氨基糖苷类药物耐药基因,gyrA第83位密码子突变和可移动遗传元件共同导致本组肺炎克雷伯菌对10种抗菌药物的耐药。本组肺炎克雷伯菌3个克隆传播提示可能有医院感染的存在。We investigated the distribution of resistant determinants in a group of drug-resistant Klebsiella pneumoniae(K.pneumoniae),and the relationships of strains.Twenty-five strains of K.pneumoniae were collected from Hubei Jianghan Oil Field General Hospital in 2016-2017.gyrA sequencing and BLASTn algorithm were used to identify K.pneumonia,35 kinds of beta-lactamase genes,15 kinds of resistant genes to aminoglycosides,a kind of resistant genes to quinolones,11 kins of genetic markers of mobile genetic elements were analyzed by PCR.Then,gene mutations were analyzed by DNA sequencing.At last,the results were analyzed by cluster analysis.In addition,sample cluster analysis was performed(UPGMA method)by 62 kinds of resistant determinants.Results showed that resistance rates of 25 K.pneumoniae to carbapenems were low,but resistance rates to other 8 kinds of antibiotics were higher.Beta lactamase genes were positive in every strain(total positive rate 100%);resistant genes to aminoglycosides were positive in 24 strains(total positive rate 96%);resistant gene to quinolones,gyrA mutation,were positive in 20 strains(mutation rate 80%);genetic markers of mobile genetic elements were positive in every strain(total positive rate 100%).In addition,5 kinds of beta-lactamase genes(bla TEM,bla LAP,bla KPC,bla DHA group,bla OXA-1 group),6 kinds of aminoglycoside resistance genes(aac(3)-Ⅱ,aac(6’)-Ⅰb,ant(3”)-Ⅰ,aph 3’-Ⅰ,aad A5,rmt B),were positive.The same kind of 83 rd codon of gyrA mutation existed in 20 strains,but no 87th codon of gyrA mutation existed.And 8 kinds of mobile genetic elements(intⅠ1,tnp U,tnp 513,ISEcp1,IS26,IS903,ISKpn6,trb C were positive.),and positive rates were high.Sample cluster analysis showed that 25 strains might be divided into cluster A and cluster B,and clone transmission existed.The 2 clones were in cluster A:strain No.2-3-5-16,13-14,while a clone was in cluster B:strain No.19-20.It is suggested that a variety of beta-lactamase genes,aminoglycoside resistance genes,gyrA mutation and mobil

关 键 词：肺炎克雷伯菌 Β-内酰胺类 氨基糖苷类 GYRA 突变 可移动遗传元件 聚类分析 耐药 

分 类 号：R378[医药卫生—病原生物学]