MALDI-TOF MS用于碳青霉烯耐药肺炎克雷伯菌同源分析的应用评价

Study on the relatedness of homology of carbapenem-resistant Klebsiella pneumoniae using MALDI-TOF MS

目的评价基质辅助激光解吸电离飞行时间质谱(MALDI-TOFMS)技术在碳青霉烯耐药肺炎克雷伯菌(CRKp)同源性分析中应用的可行性。方法回顾性分析。收集2017年1月至12月全军细菌耐药监测网上海长海医院临床标本分离的肺炎克雷伯菌非重复菌株57株,新疆乌鲁木齐市第一医院临床标本分离的肺炎克雷伯菌非重复菌株36株;纳入标准:对亚胺培南、美罗培南或厄他培南当中任一药物耐药菌株并鉴定质谱蛋白峰数大于100的菌株,共计39株;以脉冲场凝胶电泳(PFGE)结果为依据,将待测菌株分为以下三组:高度同源组(包括产OXA-48型CRKp菌株17株)、中度同源组(产KPC型CRKp菌株16株)和非同源性组(包括携带其他耐药机制的CRKp菌株8株)。所有待测菌株经MALDI-TOFMS分析后,所获结果分别采用BioNumeric软件和质谱仪自带的同源性分析软件(SARAMIS数据库软件)的两种分析方法进行同源性分析。以PFGE分析结果为参考标准,评价其他三种分析方法所获结果的可靠性。结果对高度同源组菌株,采用SARAMIS两种分析方法分析KA-A型质谱数据Identicalmass值高于80%,相似度高于85%,能够获得与PFGE分析结果较一致的结果;对非同源组的菌株,SARAMIS两种分析方法分析质谱数据Identicalmass值低于80%,相似度不足85%,所获分型结果与PFGE结果有较好的一致性,能够反映菌株间的差异性;对中度同源组菌株,BioNumeric软件和SARAMIS数据库软件分析质谱数据KC-A型的identitymass值不足70%;相似度不足90%,所获得的结果与PFGE结果之间均存在着较大的差异,无法获得与PFGE结果相一致的分析结果。结论MALDI-TOFMS结果采用BioNumeric软件和SARAMIS数据库软件进行同源性分析可作为具有高度同源性和非同源菌株间同源关系的初筛方法,但仍需要其他方法加以验证;对同源关系多样的菌株,上述方法仍缺乏稳定性,不适于进行菌株间同源关系分析。

Objective To evaluate the ability of Matrix-assisted laser desorption ionization-time of flight mass spectrum (MALDI-TOF MS) on homology analysis of carbapenem-resistant Klebsiella pneumoniae(Carbapenemase-Resistant K. pneumoniae, CRKp). Methods During January 2017 to December 2017, a total of 57 strains isolatedfromChanghai Hospital, Shanghai and 36 strainsfrom Urumqi First Hospital, Xinjiang were included for retrospective study. Inclusion criteria: strains which resistant to one of the carbapenems and with more than 100 peaks in identification mass spectrum. A total of 39 isolates were selected and divided into three groups according to the PFGE typing results:(1) Highly homologous group (17 strains with blaOXA-48 gene);(2) Moderate homology group (16 strains with blaKPC gene);(3)The non-homology group (8 strains with other resistance mechanisms). Mass spectrometry results were analyzed using BioNumeric and the two classification methods in SARAMIS, respectively.The PFGE analysis results were regarded as standard when evaluate the reliability of the other three analytical methods. Results Forhighly homologous group, the analysisresults of KA-A type dientical mass by two classification methods in SARAMIS were accord with PFGE,and the identity mass value is>80%, the similarity is>85%. However, for non-homologous group, the identity mass value is<80%, the similarity is<85% when compared with PFGE, which indicated that the method based on MALDI-TOF MS was not premium for this group of strains. For moderate homologous strains,the results of two SARAMIS methods for KC-A type havepoor consistency with PFGE, for the identity mass value is<70%, the similarity is<90%. Conclusions Based on MALDI-TOF MS using BioNumeric software and SARAMIS database software could be regarded as a screening method for phylogenetic analysis among CRKp strains with high homology. While, it should not be applied to evaluate homology of the strains with genetic diversity for lacking stability.