基质辅助激光解析／电离飞行时间质谱仪检测KPC型碳青霉烯酶的研究

Detection of KPC carbapenem by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry

目的用基质辅助激光解析／电离飞行时间质谱仪（matrix—assisted laser desorption／ion-ization time—of-flight mass spectrometry，MALDI—TOFMS）比较产KPC型碳青霉烯酶肠杆菌科细菌在不同药物浓度下水解厄他培南的能力。方法收集浙江大学医学院附属第二医院临床分离的19株单产KPC肠杆菌科细菌，包括10株奇异变形杆菌，3株产气肠杆菌，2株黏质沙雷菌，2株弗劳地柠檬酸杆菌，1株肺炎克雷伯菌，1株阴沟肠杆菌；杭州市中医院分离的7株产KPC摩根摩根菌，以及上述7株摩根摩根菌和部分奇异变形杆菌（4株）的大肠埃希菌转移接合子。用MALDI．TOFMS检测产KPC肠杆菌科细菌水解厄他培南的能力。结果当厄他培南浓度为0．1g／L时，37株单产KPC型碳青霉烯酶的肠杆菌科细菌在1．5h内全部水解厄他培南，质谱图显示厄他培南所呈现的3个峰全部消失，灵敏度高达100％；当厄他培南浓度为0．3g／L时，1．5h组水解厄他培南的灵敏度为70．3％（26／37），2．5h组为89．2％（33／37），3．5h组为94．6％（35／37）；当厄他培南浓度为0．5g／L时，1．5h组水解厄他培南的灵敏度为48．6％（18／37），2．5h组为83．8％（31／37），3．5h组为94．6％（35／37）。两独立样本非参数检验统计显示：厄他培南0．1g／L组与0．3g／L、0．5g/L组之间的水解时间差异有统计学意义，而0．3g／L与0．5g／L组之间的水解时间差异无统计学意义；细菌组内数据统计显示，除奇异变形杆菌组与大肠埃希菌组之间差异有统计学意义外，其他各细菌组之间差异没有统计学意义。结论MALDI—TOFMS操作简便，可快速检测产KPC型碳青霉烯酶的肠杆菌科细菌，敏感率高，假阳性率低，适合微生物检验中用于产KPC型碳青霉烯酶的检测，推荐使用0．1g／L的厄他培南作为水解底物来检测产KPC型肠杆菌科细菌。

Objective To compare the capability of ertapenem-hydrolyzing in various concentra- tions in KPC-producing Enterobacteriaceae by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry（ MALDI-TOF MS）. Methods Nineteen KPC-producing Enterobaeteriaceae including ten Pro- teus rnirabilis isolates, three Enterobacter aerogenes isolates, two Serratia marcescens isolates, two Citrobacter freundii isolates, one Klebsiella pneumoniae isolate and one Enterobacter cloacae isolate were isolated from 2nd Affiliated Hospital of Zhejiang University; Seven KPC-producing Morganella morganii were isolated from Hangzhou Traditional Chinese Medicine Hospital; Eleven Escherichia coli transeonjugants produced the KPC- 2 carbapenemase gene were conjugated from seven M. rnorganii and four P. mirabilis. MALDI-TOF MS was used to detect the ertapenem-hydrolyzing capability in different KPC-producing Enterobacteriaceae. Results When the concentration of ertapenem was 0.1 g/L, ertapenem can be hydrolyzed by KPC earbapenem within 1.5 h and the sensitivity was 100% ,all the three peaks produced by ertapenem disappeared; when the con- eentration of ertapenem raised to 0.3 g/L, the sensitivity fell to 70.3 % （ 26/37 ） within 1.5 h, followed with 89.2% （33/37） within 2.5 h and 94.6% （35/37） within 3.5 h;when the concentration of ertapenem raised to 0.5 g/L, the sensitivity was 48.6% （18/37） within 1.5 h, 83.8% （31/37） within 2.5 h, 94.6% （35/ 37 ） within 3.5 h. Two independent samples tests indicated that 0.1 g/L of ertapenem group has significant difference with 0.3 g,/L and 0.5 g/L group, while there was no significant difference between 0.3 g/L group