MALDI-TOF MS对肺炎链球菌鉴定与分型的应用
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摘要
目的探讨MALDI-TOF MS对肺炎链球菌鉴定和质谱分型的应用价值。方法收集2009年1月至2013年5月温州医科大学附属第二医院临床分离的112株肺炎链球菌标本,采用Optochin敏感试验和全自动细菌分析仪对收集的菌株进行鉴定验证,并用Microflex MALDI-TOF质谱仪进行分析鉴定。根据质谱图的相似性进行细菌同源聚类树分析并构建质谱分型模型,采用荚膜肿胀试验对参与分型的菌株进行血清型比较。结果除20株不符合检测条件之外,92株临床菌株和1株标准株经质谱分析均为肺炎链球菌,选取的60株菌株以0.5的差异水平,将60株肺炎链球菌分为18个质谱型别,在这些菌株的血清分型中有19F、19A、23F、23A、3和14六个血清型别,分布于不同的MALDI-TOF MS分型中,其中19F有18株,占30%(18/60),分布在6种不同的MALDI-TOF MS分型中,也有3型血清型较为集中地分布于相应的MALDI-TOF MS一个型别里。结论 MALDI-TOF MS能快速、准确、简便地鉴定肺炎链球菌,且能达到种的水平。对比血清型,按照0.5差异水平,建立的18个质谱分型部分的型别与血清型有一致性,但也存有差异。
Objective To evaluate the application value of MALDI-TOF MS in identification and typing of Streptococcus pneumoniae.Methods 112 clinical strains isolated from the Second Affiliated Hospital of Wenzhou Medical University from January 2009 to May 2013 were collected.The strains were identified and validated using the Optochin sensitivity test and a fully automated bacterial analyzer,and analyzed using the Microflex MALDI-TOF.Based on the similarity of the mass spectrum,the homology cluster tree analysis of bacteria was performed and a mass spectrometry typing model was constructed.The serotypes of the strains used for typing were determined using a capsular swelling test.Results Except the 20 strains that did not meet the detection conditions,92 strains and one standard strain were identified as S.pneumoniae by MALDI-TOF MS.Sixty selected strains were divided into 18 mass spectrotypes with a difference level of 0.5.Among the serotypes of these strains,there were 6 serotypes(19 F,19 A,23 F,23 A,3 and 14)distributing among different MALDI-TOF MS types;19 Faccount for 30%(18/60),distributing among 6 different MALDI-TOF MS types.Meanwhile,type 3 distributed more intensively in only one type.Conclusion MALDI-TOF MS can identify S.pneumoniae quickly,accurately and easily at the level of species.Comparing with serotypes,the 18 mass spectrotypes were established based on the 0.5 difference level,and some types were consistent with serotypes,but there were differences.
Objective To evaluate the application value of MALDI-TOF MS in identification and typing of Streptococcus pneumoniae.Methods 112 clinical strains isolated from the Second Affiliated Hospital of Wenzhou Medical University from January 2009 to May 2013 were collected.The strains were identified and validated using the Optochin sensitivity test and a fully automated bacterial analyzer,and analyzed using the Microflex MALDI-TOF.Based on the similarity of the mass spectrum,the homology cluster tree analysis of bacteria was performed and a mass spectrometry typing model was constructed.The serotypes of the strains used for typing were determined using a capsular swelling test.Results Except the 20 strains that did not meet the detection conditions,92 strains and one standard strain were identified as S.pneumoniae by MALDI-TOF MS.Sixty selected strains were divided into 18 mass spectrotypes with a difference level of 0.5.Among the serotypes of these strains,there were 6 serotypes(19 F,19 A,23 F,23 A,3 and 14)distributing among different MALDI-TOF MS types;19 Faccount for 30%(18/60),distributing among 6 different MALDI-TOF MS types.Meanwhile,type 3 distributed more intensively in only one type.Conclusion MALDI-TOF MS can identify S.pneumoniae quickly,accurately and easily at the level of species.Comparing with serotypes,the 18 mass spectrotypes were established based on the 0.5 difference level,and some types were consistent with serotypes,but there were differences.
引文
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[15] Trappetti C,van der Maten E,Amin Z,et al.Site of isolation determines biofilm formation and virulence phenotypes of Streptococcus pneumoniae serotype 3clinical isolates[J].Infect Immun,2013,81(2):505-513.
[16] ZHOU Kai,XIE Guojin,WANG Xiaowei,et al.Clinical characteristics and serotype distribution of invasive pneumococcal disease[J].Chin J Nosocomiol,2015,25(15):3392-3394.(in Chinese)周凯,谢国锦,王晓卫,等.侵袭性肺炎链球菌病临床特征与血清型分布[J].中华医院感染学杂志,2015,25(15):3392-3394.
[2] Ortqvist A,Hedlund J,Kalin M.Streptococcus pneumoniae:Epidemiology,risk factors,and clinical features[J].Semin Respir Crit Care Med,2005,26(6):563-574.
[3] Pavlovic M,Wudy C,Zeller-Peronnet V,et al.Identification of bacteria isolated from veterinary clinical specimens using MALDI-TOF MS[J].Berl Munch Tierarztl Wochenschr,2015,128(1/2):24-30.
[4] Friedrichs C,Rodloff AC,Chhatwal GS,et al.Rapid identification of viridans streptococci by mass spectrometric discrimination[J].J Clin Microbiol,2007,45(8):2392-2397.
[5] ZHENG Qiuyue,ZHAN Xiaowei,XIAO Shanshan,et al.Establishment of Salmonella MALDI-TOF MS typing method[J].Chin Public Health,2014,30(10):1344-1347.(in Chinese)郑秋月,战晓微,肖珊珊,等.沙门氏菌MALDI-TOF MS分型方法建立[J].中国公共卫生,2014,30(10):1344-1347.
[6] Sorensen UB.Typing of Pneumococci by using 12pooled antisera[J].J Clin Microbiol,1993,31(8):2097-2100
[7] Wolters M,Rohde H,Maier T.MALDI-TOF MS fingerprinting allows for discrimination of major methicillin-resistant Staphylococcus aureus lineages[J].Int J Med Microbiol,2011,301(1):64-68.
[8] van Belkum A,Chatellier S,Girard V,et al.Progress in proteomics for clinical microbiology:MALDI-TOF MS for microbial species identification and more[J].Expert Rev Proteomics,2015,12(6):595-605.
[9] LI Ruxin,YANG Jing,ZHAO Jianhong.Application of matrixassisted laser desorption/ionization time-of-flight mass spectrometry in bacterial typing[J].Chin J Lab Med,2017,40(11):890-893.(in Chinese)李茹鑫,杨靖,赵建宏.基质辅助激光解析电离飞行时间质谱在细菌分型的应用进展[J].中华检验医学杂志,2017,40(11):890-893.
[10] QIU Bingfeng,ZHANG Xiaofei,GU Danxia,et al.Application evaluation of VITEK MS matrix-assisted laser desorption/ionization time-of-flight mass spectrometry in pathogen identification[J].Chin J Nosocomiol,2016,26(10):2199-2202.(in Chinese)邱炳峰,张晓飞,顾丹霞,等.VITEK MS基质辅助激光解析电离飞行时间质谱在病原菌鉴定中的应用评价[J].中华医院感染学杂志,2016,26(10):2199-2202.
[11] XIAO Shali,LI Shuo,HE Wenxiu,et al.Evaluation of the ability of matrix-assisted laser desorption/ionization time-offlight mass spectrometry to identify Staphylococcus[J].Chin J Nosocomiol,2014,24(20):5192-5194.(in Chinese)肖莎丽,李硕,何文秀,等.评价基质辅助激光解析电离飞行时间质谱对葡萄球菌属的鉴定能力[J].中华医院感染学杂志,2014,24(20):5192-5194.
[12] LI Dongju,ZHU Yuanqi,LIANG Bing.Preliminary study on MALDI-TOF MS for homology analysis of Klebsiella pneumoniae[J].Chin J Microecol,2016,28(5):528-532.(in Chinese)李东菊,朱元祺,梁冰.MALDI-TOF MS用于肺炎克雷伯菌同源性分析的初步研究[J].中国微生态学杂志,2016,28(5):528-532.
[13] Chen JH,She KK,Wong OY,et al.Use of MALDI biotyper plus ClinProTools mass spectra analysis for correct identification of Streptococcus pneumoniae and Streptococcus mitis/oralis[J].J Clin Pathol,2015,68(8):652-656.
[14] LIU Chunlin,ZHAO Chunjiang,LIU Yudong,et al.Study on serotype,drug resistance and molecular typing of 148strains of invasive Streptococcus pneumoniae[J].Chin J Med,2010,90(22):1565-1570.(in Chinese)刘春林,赵春江,刘昱东,等.侵袭性肺炎链球菌148株血清型、耐药性及分子分型研究[J].中华医学杂志,2010,90(22):1565-1570.
[15] Trappetti C,van der Maten E,Amin Z,et al.Site of isolation determines biofilm formation and virulence phenotypes of Streptococcus pneumoniae serotype 3clinical isolates[J].Infect Immun,2013,81(2):505-513.
[16] ZHOU Kai,XIE Guojin,WANG Xiaowei,et al.Clinical characteristics and serotype distribution of invasive pneumococcal disease[J].Chin J Nosocomiol,2015,25(15):3392-3394.(in Chinese)周凯,谢国锦,王晓卫,等.侵袭性肺炎链球菌病临床特征与血清型分布[J].中华医院感染学杂志,2015,25(15):3392-3394.