耐亚胺培南铜绿假单胞菌膜孔蛋白OprD2基因突变与耐药分析

Mutations in the membrane pore-protein OprD2 gene and drug resistance analysis of imipenem resistant Pseudomonas aeruginosa

目的分析耐亚胺培南铜绿假单胞菌膜孔蛋白OprD2基因突变与耐药情况。方法收集2019年1月至2021年12月南京市高淳人民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共90株,均进行细菌鉴定、药敏试验和实时荧光定量PCR检测,对膜孔蛋白OprD2表达量降低的耐亚胺培南铜绿假单胞菌株进行基因序列检测。分析细菌鉴定、药敏试验结果和标本来源情况,比较耐药组与敏感组OprD2的mRNA表达量,分析耐亚胺培南铜绿假单胞菌的抗菌药敏感性结果、OprD2基因突变类型。结果90株亚胺培南耐药与敏感铜绿假单胞菌中,仪器法最低抑菌浓度≥8μg/ml、纸片抑菌圈≤15mm共45株,纳为耐药组;仪器法最低抑菌浓度≤2μg/ml、纸片抑菌圈≥19mm共45株,纳入敏感组。90株亚胺培南耐药与敏感铜绿假单胞菌主要分布于呼吸内科(40.00%)、神经内科(33.33%),标本类型主要为痰(41.11%)。耐药组铜绿假单胞菌OprD2基因的mRNA表达量为0.32(0.03,1.23),低于敏感组的0.70(0.06,2.68),差异有统计学意义(P<0.05)。耐亚胺培南铜绿假单胞菌株均对亚胺培南、哌拉西林完全耐药,对头孢他啶、哌拉西林/他唑巴坦、美罗培南耐药率较低,对阿米卡星、庆大霉素完全敏感,而无耐药性。45株耐亚胺培南株菌中,检出OprD2基因突变或缺失40株,占比88.89%;其中终止密码提前7株、非中断突变及PCR扩增阴性均5株、移码突变13株。结论OprD2基因突变或缺失,会导致膜孔蛋白OprD2表达量明显下降,促使铜绿假单胞菌对亚胺培南产生耐药。

Objective To analyze the mutation in the membrane pore-protein OprD2 gene and drug resistance of imipenem resistant Pseudomo-nas aeruginosa.Methods A total of 90 imipenem resistant and sensitive Pseudomonas aeruginosa strains were collected from clinical samples of Nanjing Gaochun People's Hospital from January 2019 to December 2021,bacterial identification,drug sensitivity test,real-time fluorescence quan-titative PCR detection were performed for all strains,gene sequence detection was performed for the imipenem resistant pseudomonas aeruginosa strains with reduced expression of membrane porin OprD2.Bacterial identification,drug susceptibility test results and specimen sources were ana-lyzed,the mRNA expression of OprD2 was compared between the drug resistance group and the sensitive group,the antimicrobial susceptibility re-sults of imipenem-resistant Pseudomonas aeruginosa and the type of OprD2 gene mutation were analyzed.Results Among the 90 imipenem resis-tant and sensitive Pseudomonas aeruginosa strains,45 strains with minimum inhibitory concentration≥8μg/ml by instrument method and disk inhibi-tion zone≤15 mm by paper method were included in the drug resistance group;45 strains with minimum inhibition concentration≤2μg/ml by instru-ment method and disk inhibition zone≥19 mm by paper method were included in the sensitive group.The 90 strains of imipenem resistant and sensi-tive Pseudomonas aeruginosa were mainly distributed in respiratory department(40.00%)and neurology department(33.33%),and the main speci-men type was sputum(41.11%).The mRNA expression levels of OprD2 gene of Pseudomonas aeruginosa in the drug resistant group was 0.32(0.03,1.23),which was lower than 0.70(0.06,2.68)in the sensitive group,and the difference was statistically significant(P<0.05).Imipenem resistant Pseudomonas aeruginosa strains are completely resistant to imipenem and piperacillin,and had low resistance rates to ceftazidime,piperacillin/tazo-bactam,and meropenem,and were completely sensitive to amikacin and gentamicin,but had no resistance.Among 45 imipenem-resistant strains,40 strains with OprD2 gene mutation or deletion were detected,accounting for 88.89%;among them,there were 7 stopped code advanced,5 non-inter-rupted mutations and negative PCR amplification,and 13 frameshift mutations.Conclusion The mutation and deletion of OprD2 gene can signifi-cantly decrease the expression of membrane pore protein OprD2,and promote the resistance of Pseudomonas aeruginosa to imipenem.