高耐药洛菲不动杆菌分离株超广谱β内酰胺酶的基因变异

Differentiation of bla-TEM genes in highly resistant Acinetobacter lwoffi strains

目的:探讨高度耐药的洛菲不动杆菌超广谱β内酰胺酶基因型及基因变异特征,分析基因变异对氨基酸序列及对超广谱β内酰胺酶可能产生的影响。方法:从下呼吸道感染的患者标本中分离得到3株高度耐药的洛菲不动杆菌超广谱β内酰胺酶阳性菌,PCR检测TEM、SHV、OXA、IMP、CTX-M耐药基因,并对耐药基因进行全序列测定,比较不同菌株耐药基因及其编码氨基酸序列的变异特征。结果:2株耐药基因为TEM型,SHV、OXA、IMP、CTX-M耐药基因均未检出,另1株5种耐药基因均未检出。2株TEM基因扩增序列分别长1012 bp、1007 bp,2株同源性为98.2%,在17个位点上存在碱基差异,TEM基因为832 bp,编码277个氨基酸,两株间有8个氨基酸出现差异,差异主要位于天冬氨酸、甘氨酸和丝氨酸,这些氨基酸分别被组氨酸、精氨酸/丙氨酸、半胱氨酸取代。软件分析显示,2株TEM基因编码TEM型超广谱β内酰胺酶的等电点分别为5.479、5.083。结论:山东济南地区高度耐药洛菲不动杆菌ESBLs耐药基因可见TEM型,不同临床分离株耐药基因存在不同程度的变异,这种变异使其编码的氨基酸序列发生改变,可能影响超广谱β内酰胺酶的某些特性,给耐药菌的检测带来影响。

Objective： To discover the extended spectrum beta-laetamases （ESBLs） gene types and the characteristics of Acinetinbacter lwoffi strains which were highly resistant to most kinds of antibiotics and to compare the differentiation in nucleotide and amino acid sequences to show the possible influence in the laboratory. Methods： Three strains of A. lwoffi with ESBLs phenomenon were isolated from the sputum of patients suffering from respiratory infection. The genes of blaTEM, SHV, OXA, IMP and CTX-M were determined by PCR. Nueleotide sequences were detected by the ABI automated sequencer and analyzed by DNA star software to compare the differentiation in nueleotide and amino acid sequences. Results： The bla-TEM gene type was determined in two strains, but the blaTEM, SHV, OXA, IMP and CTX-M genes were not detemained in another strain. Amplified nueleotide sequences of TEM genes were 1 012 bp and 1 007 bp, respectively. The identical rate was 98.2%. There were 17 pair differences in them. The blaTEM gene, 832 bp coded for 277 amino acids. Among them, 8 amino acids were different, which mainly happened on Asp, Gly and Ser, substituted by His, Arg/Ala and Cys. They had pI values of 5.479 and 5.083, respectively. Coclusion： The bla-TEM gene presented in A. lwoffi has highly resistant strains in Shandong Province. However, they produce some differentiations in nucleotide and amino acid sequences. These differentiations influence conditions of laboratory methods to pick up the resistant strains.