革兰阴性杆菌TEM型β-内酰胺酶耐药基因研究

Study on drug resistance genes of TEM type β-lactamases produced by gram-negative bacteria

目的了解中南大学3所附属医院产超广谱β-内酰胺酶(ESBLs)革兰阴性杆菌TEM型耐药基因的检出及流行情况。方法收集254株多重耐药革兰阴性杆菌,采用双纸片协同法(DDS)进行ESBLs表型确证,多重PCR法扩增blaTEM、blaSHV、blaOXA-1基因,TEM特异性引物对其整个开放阅读框进行扩增并对PCR产物测序,通过BLAST分析确定基因型;并对携带有TEM基因的革兰阴性菌株用随机扩增多态DNA(RAPD)方法进行菌株的同源性分析。结果经多重PCR扩增,105株肠杆菌获得阳性结果,其中85株菌携带TEM基因,26株菌携带SHV基因,10株菌携带OXA-1基因;6株非发酵菌获得阳性结果,均携带TEM基因,且全部为TEM-1型广谱β-内酰胺酶。携带blaTEM耐药基因的菌株所含RAPD类型:大肠埃希菌12种,肺炎克雷伯菌6种,阴沟肠杆菌7种。结论 TEM-1型β-内酰胺酶是湖南地区主要流行的TEM型酶;大肠埃希菌产TEM酶情况尤为突出;在同一医院以及同一病区存在TEM-1型β-内酰胺酶的克隆传播。

[ Objective] To investigate the prevalence and the subtype identification of TEM encoding gene of TEM-producing gram-negative bacilli isolated from three affiliated hospitals of Central South University in Hunan. [Methods] A total of 254 clinical isolates of multidrug resistant gram-negative bacteria were collected. The phenotype of ESBLs was screened by double-disk synergy test. The genotype of TEM enzyme was detected by multiplex PCR, amplifying the whole ORF （open reading frame） of TEM enzyme was conducted by specific primers and the PCR products were sequenced by automatic sequencer and the subtype of the gene was determined by BLAST searching. The homology of TEM-produeing strains were detected by RAPD （Random Amplified Polymorphie DNA） typing method. [Results] Of 254 clinical isolates of multidrug resistant gram-negative bacteria, 104 isolates were confirmed to obtain positive results by multiplex PCR. 85 isolates were found to produce blares genes, 26 isolates produced blasw genes, and 10 isolates produced blaOXA-1 genes. 6 of 11 nonfermenter isolates were confirmed to produce blaTEM genes by multiplex PCR. By TEM specific primers amplifying, only one subtype of TEM enzyme was detected： TEM-1. Among TEM-producing strains, there were 12 RAPD types in E.coli, 6 types in K.pneumoniae, 7 types in E. cloacae. [ Conclusions ] TEM-1 is the predominant genotype in Hunan province. TEM-type β-lactamase is detected among a wide range of clinical bacteria, especially in Escherichiae coli. The same TEM-1 type β-1actamase clone spreads in the same hospital and the same ward.