Polymyxin NP实验筛查肠杆菌科多黏菌素耐药菌株临床评价

Clinical evaluation of Polymyxin NP test for screening polymyxin-resistant Enterobacteriaceae

目的评价Polymyxin NP实验筛查多黏菌素耐药肠杆菌科细菌的临床价值。方法采用Polymyxin NP实验对461株肠杆菌科临床分离株进行初步筛查,采用微量肉汤稀释法测定菌株对多黏菌素的最小抑菌浓度(MIC),采用纸片扩散法进行临床常用抗菌药物的体外药物敏感性试验,采用聚合酶链反应(PCR)扩增多黏菌素耐药相关基因pmrA、pmrB、phoP、phoQ、mgrB和mcr,并进行测序,采用实时定量聚合酶链反应(qRT-PCR)检测多黏菌素耐药相关双组分调控元件pmrA/pmrB、pmrD/pmrF、PhoP/PhoQ表达水平。结果461株肠杆菌科细菌中,13株多黏菌素耐药株Polymyxin NP实验在2 h内均表现为阳性,多黏菌素敏感菌株表现为阴性。13株多黏菌素耐药菌株中,10株肺炎克雷伯菌主要由mgrB基因插入突变及双组分调控系统pmrA/pmrB和PhoP/PhoQ突变或表达上调引起,1株大肠埃希菌由质粒携带的mcr-1基因介导,2株阴沟肠杆菌的耐药机制尚不清楚。多黏菌素耐药菌株和敏感菌株对多种常用抗菌药物的耐药率差异无统计学意义(P>0.05)。结论Polymyxin NP实验操作简便、结果可靠,可用于各种机制介导的多黏菌素耐药肠杆菌科细菌的临床筛查,为临床抗感染治疗和耐药防控提供依据。

Objective To evaluate the clinical role of Polymyxin NP test for screening polymyxin-resistant Enterobacteriaceae.Methods A total of 461 isolates of Enterobacteriaceae were screened by Polymyxin NP test preliminarily.Broth microdilution method was performed to determine minimum inhibitory concentration(MIC)of polymyxin,and Kirby-Bauer method was used to determine the in vitro antimicrobial susceptibility of common antibiotics.Polymyxin-related genes,including pmrA,pmrB,phoP,phoQ,mgrB and mcr,were determined by polymerase chain reaction(PCR)and sequencing.Real-time quantitative polymerase chain reaction(qRTPCR)was performed to determine the expression levels of genes associated with pmrA/pmrB,pmrD/pmrF and PhoP/PhoQ regulatory systems.Results Among the 461 Enterobacteriaceae isolates,within 2 h,13 polymyxinresistant isolates were positive in Polymyxin NP test,and polymyxin-susceptible isolates were negative.Totally,10 of 13 polymyxin-resistant isolates belonging to Klebsiella pneumoniae resulted from insertion of mgrB as well as mutants and expression increasing of pmrA/pmrB and PhoP/PhoQ regulatory systems,and 1 isolate belonging to Escherichia coli was mediated by mcr-1 in plasmid.The resistance mechanisms of 2 isolates of Enterobacter cloacae were still unclear.There was no statistical significance between polymyxin-resistant and polymyxin-susceptible isolates(P>0.05).Conclusions Polymyxin NP test is easy to perform and reliable.It could be used in screening polymyxin-resistant Enterobacteriaceae mediated by various mechanisms,which can provide a reference for treating infectious diseases and preventing the prevalence of resistance.