【作者】 张杰；

【导师】 汪华学；

【作者基本信息】 蚌埠医学院 ， 急诊医学（专业学位）， 2021， 硕士

【摘要】 第一部分MALDI-TOF MS在耐碳青霉烯类肺炎克雷伯菌同源性分析的应用研究目的:探究基质辅助激光解析电离飞行时间质谱(matrix-assisted laser desorption/ionization-time of flight mass spectrometry,MALDI-TOF MS)分析耐碳青霉烯类肺炎克雷伯菌(carbapenem-resistant Klebsiella pneumoniae,CRKP)同源性在临床中的应用价值。方法:收取我院2019年12月~2020年12月临床分离的肺炎克雷伯菌非重复菌株150株,经耐药表型筛选出CRKP;利用MALDI-TOF MS采集CRKP的质谱图,分别运用SARAMIS软件中的相似性分型和identical masses分型进行同源性分析;以脉冲场凝胶电泳(PFGE)为参考标准,将CRKP分为高度、中度、低度同源性三组,评价MALDI-TOF MS在分析CRKP同源性分析中的应用价值。结果:经耐药表型筛选出55株CRKP;三组同源性分析显示:高度同源组中,应用SARAMIS软件自带的identical mass分型和相似性分型分析,大多identical mass集中分布(60%~80%),相似性>80%,与PFGE结果较为一致;中度同源组中,identical mass>70%,相似性>85%,显示菌株间高度集中,跟PFGE结果存在较大的差异;低度同源组中,identical mass<70%,相似性<80%,与PFGE结果类似,能较好体现菌株间的差别。结论:MALDI-TOF MS自带的SARAMIS软件目前仅在高度同源和低度同源菌株中,可作为分析同源性的初筛方式。第二部分MALDI-TOF MS在耐碳青霉烯类肺炎克雷伯菌早期鉴定的应用研究目的:探讨MALDI-TOF MS对耐碳青霉烯类肺炎克雷伯菌的早期鉴定能力。方法:收集2019年12月~2020年12月我院临床分离的肺炎克雷伯菌150株,采用纸片扩散法进行药敏实验,结果参照2020版CLSI M100 30th药敏标准执行。PCR作为耐药初筛的参考标准。采用MALDI-TOF MS收集CRKP和碳青霉烯酶敏感肺炎克雷伯菌(carbapenem-sensitive Klebsiella pneumoniae,CSKP)的质谱图,各自选取其中20株菌株图谱,建立产CRKP和CSKP的超级图库(Super-spectra)。选择除建库以外的KPN菌株验证是否耐药,并根据耐药表型和PCR方法,判断验证结果是否准确。结果:150株KPN经耐药表型筛选出CRKP 55株和CSKP 95株。根据建库要求,选取建库质谱图,建立出CRKP和CSKP的超级图库(Super-spectra);设置error值<0.5,二者重合率达80%,并根据对比图发现,4154.4m/z、8310.7m/z、10880.8m/z、3579m/z及10079.3m/z这五个峰可作为区分CRKP和CSKP的特征峰。选取除建库以外的110株KPN进行验证,CRKP准确率91.43%(32/35),CSKP准确率90.67%(68/75),新建的Super-spectra鉴定KPN是否对碳青霉烯酶耐药的准确率为90.91%(100/110)。结论:通过MALDI-TOF MS建立Super-spectra,可快速鉴定CRKP,有助于提高临床诊疗和院内感染控制水平。更多还原

【Abstract】 Part Ⅰ Application in homology analysis of carbapenem-resistant Klebsiella pneumoniae by MALDI-TOF MSObjective : To explore the clinical applicability of matrix assisted laser desorption/ionization time of flight mass spectrometry(MALDI-TOF-MS)in the analysis of carbapenem-resistant Klebsiella pneumoniae(CRKP).Methods:A total of 150 Klebsiella pneumoniae non repetitive strains were collected from a hospital from December 2019 to December 2020,and CRKP was screened by drug resistance phenotype;The mass spectra of CRKP were collected by MALDI-TOF MS,the similarity and identity masses in SARAMIS software were used to analyze the homology;Using pulsed field gel electrophoresis(PFGE)as reference standard,CRKP were divided into three groups: high,medium and low degree of homology,and MALDI-TOF MS was applied in CRKP homology analysis.Results:55 CRKP strains were screened by drug resistance phenotype;three groups of homology analysis showed that: in the highly homologous group,most of them were distributed in a centralized manner(60%~80%)with similarity>80%,which was consistent with PFGE results;in the moderate homology group,the identity mass value was more than 70%,and the similarity was more than 85%.It showed that the strains were highly concentrated,which was different from the results of PFGE;in the low degree homology group,the identity mass was less than 70%,and the similarity was less than 80%,which was similar to that of PFGE.Conclusion:At present,the SARAMIS software provided by MALDI-TOF MS is only used in high homologous and low homologous strains,which can be used as the primary screening method for analyzing the homology.Part Ⅱ Application in early identification of carbapenem-resistant Klebsiella pneumoniae by MALDI-TOF MSObjective: To investigate the early identification ability of MALDI-TOF MS for carbapenem-resistant Klebsiella pneumoniae.Methods: 150 clinical isolates of Klebsiella pneumoniae from December 2019 to December 2020 were collected.The disk diffusion method was used for drug sensitivity test.The results were in accordance with CLSI M100 30 th standard 2020.PCR method was used as the reference standard for drug resistance screening.The mass spectra of CRKP and carbapenem-sensitive Klebsiella pneumoniae(CSKP)were collected by MALDI-TOF MS.Twenty strains of CRKP and CSKP were selected to establish a Super-spectra library.KPN strains were selected to verify whether they were resistant or not,and the results were judged to be accurate according to the resistant phenotype and PCR method.Results:55 strains of CRKP and 95 strains of CSKP were screened from 150 KPN strains.According to the requirements of the database,the Super-spectra of CRKP and CSKP was established by selecting the mass spectra of CRKP and CSKP.Setting the error value < 0.5,the coincidence rate of CRKP and CSKP was 80%.According to the comparison chart,the characteristic peaks of 4154.4m/z,8310.7m/z,10880.8m/z,3579m/z and 10079.3m/z could be used to distinguish CRKP and CSKP.The accuracy rate of CRKP was 91.43%(32/35)and that of CSKP was 90.67%(68/75).The accuracy rate of KPN to carbapenemase was 90.91%(100/110).Conclusion: The establishment of Super-spectra by MALDI-TOF MS can identify CRKP rapidly,which is helpful to improve the level of clinical diagnosis and treatment and nosocomial infection control.更多还原