应用MALDI-TOF MS快速鉴定bla_(KPC-2)基因型肺炎克雷伯菌

Rapid identification of bla_(KPC-2) genotype Klebsiella pneumoniae by MALDI-TOF MS

目的探讨基质辅助激光解析/电离飞行时间质谱(MALDI-TOF MS)对携带bla_(KPC-2)基因型肺炎克雷伯菌的快速鉴定能力。方法收集2018年9月—2020年11月蚌埠医学院第一附属医院临床住院患者分离的肺炎克雷伯菌,采用纸片扩散法进行药敏试验。应用聚合酶链反应(PCR)方法筛选携带bla_(KPC-2)基因型肺炎克雷伯菌。MALDI-TOF MS鉴定菌株并收集携带bla_(KPC-2)基因型和碳青霉烯类敏感肺炎克雷伯菌(CSKP)的质谱图,各自选取其中70株菌株图谱,建立携带bla_(KPC-2)基因型和CSKP的超级图库(Super-Spectra)。采用超级图库鉴定除建库以外的肺炎克雷伯菌,根据耐药表型和PCR结果,判断鉴定结果是否准确。结果共收集295株肺炎克雷伯菌,经耐药表型筛选耐碳青霉烯类肺炎克雷伯菌(CRKP)143株和CSKP 152株。CRKP中鉴定出140株携带碳青霉烯酶基因(134株检出bla_(KPC-2)基因,3株检出blaKPC-18基因,2株检出blaNDM-1基因,1株检出blaIMP基因),3株不携带碳青霉烯酶基因;其中2株同时检出bla_(KPC-2)基因和blaNDM-1基因。根据建库要求,建立携带bla_(KPC-2)基因型和CSKP的超级图库(Super-Spectra);设置error值<0.5,二者重合率达80%;对比图发现,4154.4、8310.7、10880.8、3579、10079.3 m/z五个峰可作为区分携带bla_(KPC-2)基因肺炎克雷伯菌和CSKP的特征峰。选取除建库以外的155株肺炎克雷伯菌进行验证,准确率为92.90%(144/155);其中携带bla_(KPC-2)基因肺炎克雷伯菌准确率为94.52%(69/73),CSKP准确率为91.46%(75/82)。结论通过MALDI-TOF MS建立Super-Spectra,可快速预测bla_(KPC-2)基因型肺炎克雷伯菌,为临床CRKP感染的诊疗及医院感染防控提供可靠的实验室依据。

Objective To investigate the rapid identification of bla_(KPC-2)-harboring Klebsiella pneumoniae(K.pneumoniae)by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry(MALDI-TOF MS).Methods K.pneumoniae isolated from hospitalized patients in the First Affiliated Hospital of Bengbu Medical College from September 2018 to November 2020 were collected,antimicrobial susceptibility testing was performed by disk diffusion method.bla_(KPC-2)-harboring K.pneumoniae was screened by polymerase chain reaction(PCR).MALDI-TOF MS was used to identify the strains,the mass spectra of bla_(KPC-2)-harboring K.pneumoniae and carbapenems-sensitive K.pneumoniae(CSKP)were collected,70 strains were selected respectively to establish the Super-Spectra of bla_(KPC-2)-harboring K.pneumoniae and CSKP.K.pneumoniae except strains in Super-Spectra were also identified for judging whether the identification results were accurate according to antimicrobial resistance phenotype and PCR results.Results A total of 143 carbapenems-resistant K.pneumoniae(CRKP)strains and 152 CSKP strains were screened out from 295 K.pneumoniae strains.140 CRKP strains were identified carrying carbapenemase gene(134,3,2,and 1 strains were found bla_(KPC-2),blaKPC-18,blaNDM-1 and blaIMP respectively),3 strains didn’t harbor carbapenemase gene,2 strains were found both bla_(KPC-2)gene and blaNDM-1 gene.According to the require-ment of database establishment,a Super-Spectra of bla_(KPC-2)-harboring genotypes of K.pneumoniae and CSKP were established;the coincidence rate of the two was 80%when the error value was<0.5;according to the comparison map,the five peaks of 4154.4m/z,8310.7m/z,10880.8m/z,3579m/z and 10079.3m/z could be used as the characteristic peaks to distinguish the bla_(KPC-2)-harboring K.pneumoniae and CSKP.155 strains of K.pneumoniae except Super-Spectra were selected for verification,the accuracy rate was 92.90%(144/155);the accuracy of bla_(KPC-2)-harboring K.pneumoniae was 94.52%(69/73),and that of CSKP was 91.46%(75/82).Conclusion The establishment of Super-Spectra through MALDI-TOF MS can rapidly predict bla_(KPC-2)genotype K.pneumoniae,which is helpful to provide a reliable laboratory basis for the diagnosis and treatment of CRKP infection as well as prevention and control of healthcare-associated infection.