应用基质辅助激光解析电离飞行时间质谱检测产碳青霉烯酶肠杆菌科细菌对厄他培南的水解能力

Application of MALDI-TOF mass spectrometry to determine the ertapenemhydrolyzing ability in carbapenemases-producing Enterobacteriaceae

目的应用基质辅助激光解析电离飞行时间质谱(MALDI-TOF MS)检测产KPC-2、NDM-1、VIM-2、IMP-1与IMP-4型碳青霉烯酶肠杆菌科细菌水解厄他培南的能力。方法收集2009年6月-2013年12月上海交通大学医学院附属新华医院各种临床标本中分离的耐碳青霉烯类肠杆菌科细菌(CRE),用改良Hodge试验进行产碳青霉烯酶的表型筛选,用PCR扩增方法检测其碳青霉烯酶基因,并经测序确认。应用MALDI-TOF MS进行厄他培南水解预试验以确定最适厄他培南浓度及孵育时间,随后采用预试验中的最适条件,应用MALDI-TOF MS检测CRE水解厄他培南的能力。结果 108株CRE中,有102株改良Hodge试验阳性,其中90株产KPC-2、4株产NDM-1、3株产VIM-2、2株产IMP-1、2株产IMP-4、1株同时产KPC-2和IMP-4型碳青霉烯酶,且在2 h内均能水解厄他培南;另外6株CRE改良Hodge试验阴性,未检测出上述碳青霉烯酶基因,且不水解厄他培南。结论临床微生物实验室应用MALDI-TOF MS能够快速准确检测CRE水解厄他培南的能力,筛查产碳青霉烯酶肠杆菌科细菌,为临床早期合理使用碳青霉烯类抗生素提供依据。

Objective To determine the ability of KPC-2, NDM-1, VIM-2, IMP-1 and IMP-4 producing Enterobacteriaceae to hydrolyze ertapenem using matrix assisted laser desorption ionization time of flight mass spectrometry （MALDI-TOF MS） system. Methods Carbapenem-resistant Enterobacteriaceae （CRE） isolates were collected from a variety of clinical specimens in Xinhua Hospital Affiliated to Shanghai Jiaotong University School of Medicine, from June 2009 to December 2013. Modified Hodge test was performed to screen phenotype of carbapenemase production, and carbapenemase genes were detected by PCR amplification with sequencing analysis. A MALD1-TOF MS-based assay was set up to investigate the ability of CRE to hydrolyze ertapenem under the optimal concentration of ertapenem and incubation time, which were determined in preliminary experiment. Results Of the 108 CRE isolates, 102 were positive for modified Hodge test, including 90 strains producing KPC-2, 4 strains NDM-1, 3 strains VIM-2, 2 strains IMP-1, 2 strains IMP-4, 1 strains both KPC-2 and IMP-4 carbapenemase. All the carbapenemase-producing Enterobacteriaceae strains could hydrolyze ertapenem in 2 hours. Other 6 CRE strains were negative for modified Hodge test, in which carbapenemase gene were not detected and they did not hydrolyze ertapenem. Conclusions In clinical microbiology laboratory, MALDI-TOF MS is rapid and accurate for determining ertapenem-hydrolyzing ability, which is useful for screening carbapenemases-producing Enterobacteriaceae, and guiding rational carbapenems use at early stage of infection.