产ESBLs大肠埃希菌对氨基糖苷类药物的耐药性及其耐药基因序列分析

摘要

目的：研究云南玉溪市江川地区产超广谱β-内酰胺酶（ESBLs）大肠埃希菌对氨基糖苷类抗菌药物的耐药性及其耐药基因表达的频率，并对其耐药基因进行测序分析。方法采用纸片扩散法检测32株产ESBLs大肠埃希菌对庆大霉素、阿米卡星、卡那霉素、妥布霉素和奈替米星的耐药性。采用聚合酶链反应（PCR）检测7种氨基糖苷类修饰酶（AME）基因[aac（3）-Ⅰ、aac（3）-Ⅱ、aac（6′）-Ⅰad、aac（6′）-Ⅰb、aac （6′）-Ⅱ、ant（3″）-Ⅰ、ant（2″）-Ⅰ]和6种16S rRNA甲基化酶基因（armA、rmtA、rmtB、rmtC、rmtD、npmA），采用双脱氧末端终止法对aac（3）-Ⅱ基因进行测序分析。结果32株产ESBLs大肠埃希菌对庆大霉素、阿米卡星、奈替米星、卡那霉素和妥布霉素的耐药率分别为100%、0%、0%、18%和87%。有6株菌株同时携带2种AME基因。从32株产ESBLs大肠埃希菌中检出aac（3）-Ⅱ、aac（6′）-Ⅰb、ant（3″）-Ⅰ、ant （2″）-Ⅰ4种AME基因，阳性率分别为100%、9.4%、6.2%和3.1%；未检出16S rRNA甲基化酶基因。32株产ESBLs大肠埃希菌中有4株发生相同的aac（3）-Ⅱ突变，分别为G232A、T251C、G580A和T612A。8株不产ESBLs大肠埃希菌均未检出AME基因及16S rRNA甲基化酶基因。结论云南玉溪市江川地区产ESBLs大肠埃希菌耐氨基糖苷类药物与AME基因表达有关，奈替米星的耐药率增加可能与aac（3）-Ⅱ基因突变有关。%Objective To study the drug resistance of extended-spectrum beta-lactamases(ESBLs)-producing Escherichia coli to aminoglycosides and the expression frequencies and sequences of drug resistant genes in Jiangchuan District,Yuxi City,Yunnan Province. Methods The drug resistance of 32 isolates of ESBLs-producing Escherichia coli to gentamycin,amikacin,kanamycin,tobramycin and netilmicin was determined by disk diffusion test. A total of 7 aminoglycoside-modifying enzyme(AME) genes [aac(3)-Ⅰ,aac(3)-Ⅱ,aac(6′)-Ⅰad,aac(6′)-Ⅰb,aac(6′)-Ⅱ,ant(3″)-Ⅰand ant(2″)-Ⅰ] and 6 genes of 16S rRNA methylases(armA,rmtA,rmtB, rmtC,rmtD and npmA) were determined by polymerase chain reaction(PCR),and the sequence of aac(3)-Ⅱgene was analyzed by double deoxidizing terminal cessation method. Results The drug resistant rates of 32 isolates of ESBLs-producing Escherichia coli to gentamycin,amikacin,netilmicin,kanamycin and tobramycin were 100%,0%,0%,18%and 87%,respectively. It was found that 2 AME genes existed simultaneously in 6 isolates. The positive rates of 4 AME genes,aac(3)-Ⅱ,aac(6′)-Ⅰb,ant(3″)-Ⅰand ant(2″)-Ⅰ,were 100%, 9.4%,6.2%and 3.1%in 32 isolates. There was no gene of 16S rRNA methylases. The aac(3)-Ⅱmutations existedin 4 out of 32 isolates,which were G232A,T251C,G580A and T612A,respectively. There was no gene of AME and 16S rRNA methylases in 8 isolates of ESBLs-producing Escherichia coli. Conclusions The drug resistance of ESBLs-producing Escherichia coli to aminoglycosides was correlated with AME gene expressions,and the mutations of aac(3)-Ⅱmay contribute to the increased drug resistance to netilmicin.