耐亚胺培南铜绿假单胞菌外膜蛋白D基因突变检测

Mutations of Pseudomonas aeruginosa oprD gene in Imipenem-resistant clinical isolates

目的探讨耐亚胺培南铜绿假单胞菌临床分离株外膜蛋白D（oprD）基因突变与耐药关系.方法应用随机扩增DNA多态性分析（RAPD）技术对10株耐亚胺培南铜绿假单胞菌临床分离株进行DNA分型,并对不同克隆耐药株oprD基因编码区进行全基因扩增测序和金属酶检测.结果 10株耐亚胺培南铜绿假单胞菌均不产金属酶,可分为4个不同克隆.与X63152序列比较,所有耐药菌株oprD基因发生显著变异,变异率大于50%.30、11、9、20、31号克隆株编码区276～387 bp之间发生多处点突变,其中308 bp G→C突变使其编码的苏氨酸被丝氨酸代替,344 bp C→A突变使其编码的赖氨酸被苏氨酸代替,393～412位有20 bp DNA片段缺失,其后的核苷酸序列发生移码突变.13、21号克隆株编码区264～273位有10 bp DNA片段缺失,产生移码突变,形成终止密码子TAA（319～321 bp）.1号克隆株和22号克隆株oprD基因分别发生大片段的碱基置换和多处点突变,变异率分别为54.03%和74.89%.结论耐亚胺培南铜绿假单胞菌临床分离株oprD基因变异具有多样性.

Objective To study the mutations of Pseudomonas aeruginosa oprD genc in Imipenem-resistant clinical isolates. Methods Random amplified polymorphic DNA typing（RAPD） was carried out to analyse the homology in 10 Imipenem resistant clinical isolates. Pscudomonas aeruginosa oprD gene in 10 Imipenem resistant clinical isolates were amplified by polymerase chain reaction and sequenced. Results Ten Imipenem-resistant clinical isolates were divided into four different clones which can not produce metallo β- laetamase. As compared with sequence X63152, oprD gene of Pseudomonas aeruginosa varied greally. The aberration rates exceed 50 %. There were multiple point mutations within276-387 bp coding region of 30, 11, 9, 20, 31 strains . Due to the mutations of 308bp G→C and 344 bp C→A, thrconinc and diaminocaproic acid were replaced by serine and threoninc re spectively. The DNA deletion of 393-412 bp in oprD gene contributes to the frame shift mutation in the following nucleotide sequence. The deletion of 264-273 bp in the coding region of oprD gene in 13 and 21 clone strains leads to frame shift mutations forming terminal eodon TAA（319-321 bp）. Base substitutions and multiple point mutations were obvious in the coding regions of 1 and 22 clone strains. Their aberration rates were 54.03% and 74.89% respectively. Conclusions The mutations of Pseudomonas aeruginosa oprD gene in imipenem-resistant clinical isolates are various.