耐亚胺培南铜绿假单胞菌OprD2基因多态性的研究  

Study of OprD2 gene polymorphism in imipenem-resistant Pseucionionas aeruginosa

作　　者：白婷婷 王丹[1] 范英子 杨向贵 周琴[1] 许颖[1] BAI Tingting;WANG Dan;FAN Yingzi;YANG Xianggui;ZHOU Qin;XU Ying(Department of Clinical Laboratory,the First Affiliated Hospital of Chengdu Medical College,Chengdu,Sichuan 610500,P.R.China)

机构地区：[1]成都医学院第一附属医院检验科,成都610500

出　　处：《华西医学》2020年第8期924-929,共6页West China Medical Journal

基　　金：成都医学院校基金(CYZ18-30)。

摘　　要：目的探讨临床分离的耐亚胺培南铜绿假单胞菌膜孔蛋白OprD2基因突变与耐药的关系。方法收集2018年12月—2019年12月分离自成都医学院第一附属医院临床标本中的亚胺培南耐药与敏感的铜绿假单胞菌株各30株,采用VITEK 2全自动细菌药物敏感性鉴定系统结合纸片扩散法进行菌株鉴定和药物敏感性试验,利用实时荧光定量聚合酶链反应法检测亚胺培南耐药组与敏感组OprD2基因的表达量,挑选OprD2基因表达显著下降的耐药株进行编码区的聚合酶链反应扩增测序。结果耐药组OprD2基因的表达量较敏感组显著下降(P=0.048)。与X63152序列比较,11株OprD2表达量显著下降的菌株都存在基因变异,变异率为100%,均发生在编码区,变异位点呈现多样性。其中c.308C→G、c.344A→C、c.379G→C、c.471G→C、c.508T→C、c.553G→C、c.556-558CCG→GGC与c.565-566TG→AC的错义突变引起膜孔蛋白L2、L3茎环结构上的氨基酸改变,将会影响亚胺培南的结合。另外还发现127、169-171、175、177、604、628-630、688、719、785、826、828、842-843、886、901、928-930、934、936、944-945、1039、1041、1274这些位点的碱基突变均引起氨基酸的改变,还检测到c.1114-1115delAT以及其他的无义突变,其中12号菌株发生了OprD2基因的大片段缺失。结论OprD2基因的突变和缺失会引起膜孔蛋白OprD2表达量下降,从而导致铜绿假单胞菌对亚胺培南产生耐药。临床分离的耐亚胺培南铜绿假单胞菌菌株的OprD2基因变异具有多样性。Objective To explore the relationship between imipenem-resistant Pseudomonas aeruginosa(IRPA) and outer membrane porin protein OprD2 gene mutation.Methods IRPA strains(n=30)and imipenem-sensitive Pseudomonas aeruginosa strains(n=30)isolated from the clinical specimens in the First Affiliated Hospital of Chengdu Medical College from December 2018 to December 2019 were collected.Bacteria identification and drug sensitivity experiments were performed by VITEK-2 Compact combined with Kirby-Bauer method.Quantitative real-time polymerase chain reaction was used to detect the expression levels of OprD2 gene in the imipenem-resistant group and the imipenem-sensitive group,and then the strains with decreased expression were sequenced.Results The expression level of OprD2 gene in the imipenem-resistant group was significantly lower than that in the imipenem-sensitive group(P=0.048).Compared with the X63152 sequence,all the 11 Pseudomonas aeruginosa strains with significantly decreased OprD2 expression carried genetic variation,which occurred in coding regions.The variation sites presented diversity.The missense mutation of c.308 C→G,c.344 A→C,c.379 G→C,c.471 G→C,c.508 T→C,c.553 G→C,c.556-558 CCG→GGC and c.565-566 TG→AC caused amino acid change in the loop L2 and L3 of OprD2 porin,which affected the binding to imipenem.In addition,the mutations at 127,169-171,175,177,604,628-630,688,719,785,826,828,842-843,886,901,928-930,934,936,944-945,1039,1041 and 1274 all resulted in the changes of amino acid.We also detected a deletion(c.1114-1115 delAT)and other nonsense mutations.Large fragment deletion of OprD2 gene occurred in Strain 12.Conclusions The mutation and deletion of OprD2 gene can reduce the expression lever of OprD2 gene,leading to the resistance to imipenem of Pseudomonas aeruginosa.The variation of OprD2 gene of IRPA from clinical strains is diverse.

关 键 词：铜绿假单胞菌 亚胺培南 OprD2基因 耐药机制 

分 类 号：R446.5[医药卫生—诊断学]