多药耐药铜绿假单胞菌氨基糖苷类修饰酶和16S rRNA甲基化酶基因分析

目的了解临床分离的铜绿假单胞菌对氨基糖苷类、β-内酰胺类、喹诺酮类抗菌药物耐药情况、氨基糖苷类耐药相关基因和16S rRNA甲基化酶基因存在情况以及菌株之间的亲缘性。方法采用琼脂稀释法测定临床分离的30株铜绿假单胞菌对7种临床常用于治疗铜绿假单胞菌感染的抗菌药物的敏感性,采用聚合酶链反应分析氨基糖苷类修饰酶、16S rRNA甲基化酶基因型及其他基因型,运用SPSS统计分析软件对菌株样本亲缘性做聚类分析。结果30株铜绿假单胞菌对临床常用抗生素的耐药率分别是奈替米星70%、妥布霉素63.3%、庆大霉素63.3%、环丙沙星53.3%、亚氨培南40%和阿米卡星13.3%,而多黏菌素B的耐药率为0。21株氨基糖苷类耐药菌株中（其中20株为多药耐药菌株）,氨基糖苷类耐药基因型aac（6＇）-Ⅰ阳性13株（61.9%）、aac（6＇）-Ⅱ阳性13株（61.9%）、ant（2＇＇）-Ⅰ阳性10株（47.6%）、ant（3＇＇）-Ⅰ阳性9株（42.9%）、aac（3）-Ⅱ阳性1株（4.8%）,另有1株菌oprD2基因缺失,未检出基因型aac（6＇）-Ⅰae、aph（3＇）-Ⅲ、aac（6＇）-aph（2＇＇）和ant（4＇）-Ⅰ;16S rRNA甲基化酶基因rmtA基因型阳性19株（90.4%）、armA基因型阳性有8株（38.1%）,未检出基因型rmtC、rmtD。聚类分析结果显示分离的菌株中存在克隆传播。结论大部分测试的铜绿假单胞菌对临床常用的铜绿假单胞菌抗感染药物已产生广泛耐药,尤其对氨基糖苷类抗生素。这些菌株的氨基糖苷类修饰酶常见耐药基因型检出率高,16SrRNA甲基化酶基因型rmtA和armA的检出率亦较高。30株测试菌株中存在克隆传播。

Analysis on gene types of aminoglycoside modifying enzyme and 16S rRNA methylases in multidrug-resistant Pseudomonas aeruginosa

Objective To explore the drug resistance against aminoglycosides,β-lactamases and quinolones,drug-resistant genes encoding aminoglycoside modifying enzymes and 16S rRNA methylases in Pseudomonas aeurginosa isolates,and perform phylogenetic analysis.Method 30 strains of Pseudomonas aerudinosa were isolated from clinical specimens by susceptibility test with agar dilution.Aminoglycoside modifying enzyme genes, 16S rRNA methylases and other genes were detected by polymerase chain reaction (PCR) method, and Cluster analysis was conducted to explore the phylogenetic relationship of sample stains. Result In 30 strains of Pseudomonas aeurginosa,the drug-resistant rates were 70%(netilmicin), 63.3%(tobramycin), 63.3%(gentamicin), 53.3%(ciprofloxacin), 40%(im ipenem), 13.3%(amikacin) respectively,while the drug resistance rate to polymycin B was zero. In 21 aminoglycoside-resistant strains (20 strains of which were multi-drug resistant),the positive rates of aminoglycoside modifying enzyme genes,I.e as aac(6,)-Ⅰ,aac(6,)-Ⅱ,ant(2,,)-Ⅰ,ant(3,,)-Ⅰ and aac(3)- Ⅱ,were 61.9%,61.9%,47.6%,42.9%,and 4.8% respectively.The gene of oprD2 was not detected in only 1 strain.Some of the gene types,such as aac(6,)-Ⅰae,aph(3,)-Ⅲ,aac(6,)-aph(2,,) and ant(4,)-Ⅰ were not detected. The positive rates of 16S rRNA methylase genotypes,were 38.1% (armA) and 90.4%(rmtA) respectively. Genotypes rmtC and rmtD were not detected. Cluster analysis showed clone spread in Pseud-omonas aeurginosa isolates.Conclusion Most of Pseudo-monas aerudinosa isolates were multidrug-resistant, especial-ly to aminolycosides. The positive rate of aminoglycoside modifying enzyme genes was higher,and the result of Cluster analysis indicated that there exists clone spread among the 30 strains.