多药耐药铜绿假单胞菌产超广谱β-内酰胺酶检测及同源性分析

Study on extended-spectrum β-lactamases produced by multidrug-resistant Pseudomonas aeruginosa

目的对多药耐药铜绿假单胞菌产超广谱β-内酰胺酶(ESBLs)进行分子流行病学分析,以明确其分子特征,更有效地控制感染。方法通过药敏试验和三相水解试验分析细菌的耐药表型及产ESBLs表型,通过PCR扩增和克隆测序方法确定其产ESBLs的基因型,应用重复一致序列PCR(rep-PCR)方法分析菌株间的同源性。结果分离的85株铜绿假单胞菌对第三、四代头孢菌素、氨曲南以及氨基糖苷类和喹诺酮类抗菌药物的耐药率均>60.0%,对亚胺培南的耐药率达到23.6%,而且有17株为全耐药菌株;对17株全耐药菌株进行三相水解试验分析,有14株显示产ESBLs表型;β-内酰胺酶基因PCR扩增结果显示,85株铜绿假单胞菌中TEM阳性率最高为77.6%,其次为PER-1阳性率为48.2%,SHV阳性率为5.9%;经rep-PCR进行菌株同源性分析显示多数产PER-1型ESBLs铜绿假单胞菌来源于同一克隆株。结论海南地区铜绿假单胞菌多药耐药状况严重,产ES-BLs是其对β-内酰胺类抗菌药物耐药的重要原因;首次在海南地区检出产PER-1型ESBLs的铜绿假单胞菌,且该院存在产PER-1型ESBLs铜绿假单胞菌同一克隆株引起的医院感染的暴发流行。

OBJECTIVE To investigate the molecular epidemiological characteristics of multiple resistant clinical Pseudomonas aeruginosa producing extended spectrum β-lactamases（ESBLs） ao as to determine its moleeular and to effectineg control infections.METHODS The antibiotics susceptibility testing and three-dimensional hydrolysis test were performed to analyze the phenotypes of resistance and ESBLs.PCR and sequencing methods were used to detect the genotypes of β-lactamases.Rep-PCR was carried out for DNA typing of clonal strains.RESULTS The resistance rates of 85 P.aeruginosa clinical isolates against the third and fourth generation cephalosporins,aztreonam,aminoglycosides and quinolones were all more than 60.0%,the resistance rate to imipenem reached to 23.6% and 17 pan-resistant strains were found.Three-dimensional hydrolysis test showed that 14 strains appeared ESBLs-producing phenotypes in 17 pan-resistant strains.PCR amplification and sequencing results showed that among 85 PA strains,the positive rate of TEM was 77.6%,PER-1 was as high as 48.2% and SHV was 5.9%.The homology analysis by rep-PCR confirmed that PER-1 type ESBLs-producing P.aeruginosa were almost from the same clonal strain.CONCLUSION The resistance condition of P.aeruginosa isolated from Hai Nan province is considerably serious.The producing of ESBLs is the main cause of the resistance to β-lactams.PER-1-producing isolates have been detected for the fist time in Hainan area.The prevalence of PER-1 type of homologous EBBLs-producing P.aeruginosa does exist in the hospital.