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中华实验和临床感染病杂志(电子版) ›› 2021, Vol. 15 ›› Issue (03) : 209 -215. doi: 10.3877/cma.j.issn.1674-1358.2021.03.011

论著

B族链球菌基质辅助激光解析电离飞行时间质谱蛋白指纹特征肽峰验证研究
陈光炼1, 刘海英1, 高坎坎1, 关小珊1, 钟华敏1, 邓秋连1, 谌亚星1, 梁文怡2, 蒋莹2, 谢永强1, 黄莲芬1,†()   
  1. 1. 510120　广州市，广州医科大学广州市妇女儿童医疗中心检验科
    2. 511483　广州市，广州医科大学儿科学院
  • 收稿日期:2020-05-10 出版日期:2021-06-15
  • 通信作者: 黄莲芬
  • 基金资助:
    广州市科技创新委员会项目(No. 201804010447); 广州医科大学大学生科技创新项目(No. 2019AEK01); 广州市妇女儿童医疗中心院内课题(No. YIP-2019-051)

Validation of peptide peaks specific for Group B Streptococcus with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry

Guanglian Chen1, Haiying Liu1, Kankan Gao1, Xiaoshan Guan1, Huamin Zhong1, Qiulian Deng1, Yaxing Chen1, Wenyi Liang2, Ying Jiang2, Yongqiang Xie1, Lianfen Huang1,†()   

  1. 1. Department of Clinical Laboratory, Guangzhou Women and Children’s Medical Center, Guangzhou 510120, China
    2. Academy of Pediatrics, Guangzhou Medical University, Guangzhou 511483, China
  • Received:2020-05-10 Published:2021-06-15
  • Corresponding author: Lianfen Huang
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引用本文：

陈光炼, 刘海英, 高坎坎, 关小珊, 钟华敏, 邓秋连, 谌亚星, 梁文怡, 蒋莹, 谢永强, 黄莲芬. B族链球菌基质辅助激光解析电离飞行时间质谱蛋白指纹特征肽峰验证研究[J]. 中华实验和临床感染病杂志(电子版), 2021, 15(03): 209-215.

Guanglian Chen, Haiying Liu, Kankan Gao, Xiaoshan Guan, Huamin Zhong, Qiulian Deng, Yaxing Chen, Wenyi Liang, Ying Jiang, Yongqiang Xie, Lianfen Huang. Validation of peptide peaks specific for Group B Streptococcus with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry[J]. Chinese Journal of Experimental and Clinical Infectious Diseases(Electronic Edition), 2021, 15(03): 209-215.

目的

基质辅助激光解析电离飞行时间质谱（MALDI-TOF MS）分析验证既往发现的B族链球菌（GBS）序列分型（ST）、血清型蛋白指纹特征肽峰及D试验阳性相关肽峰，为临床GBS快速分型和有效诊疗提供依据。

方法

选取2015年1月至2017年12月全国多中心新生儿侵袭感染分离的237株GBS，均已知血清分型及多序列分型（MLST），运用Bruker微生物质谱仪采集菌体蛋白的肽指纹图谱，纸片扩散法分析菌株对红霉素和克林霉素耐药表型及红霉素诱导克林霉素的耐药发生率，用FlexAnalysis 3.0软件分析和验证特征肽峰。

结果

分子分型相关特征肽峰分析示，237株GBS均未见m/z 7 625肽峰，但48.3% ST17型菌株中（41/85）有m/z 7 620肽峰；所有ST1型菌株（2/2）均检出m/z 6 250肽峰且无m/z 6 888肽峰，即肽峰对（m/z 6 250+/6 888-），该峰对亦见于所有ST10菌株（31株）及ST2（2株）、ST4（1株）、ST156（1株）和ST579（1株）菌株。血清分型相关特征肽峰示，m/z 6 891在血清Ⅲ型（96.4%、133/138）和Ⅰb型（81.2%、56/69）常见，但在Ⅰa（90.5%、19/21）和Ⅴ型（87.5%、7/8）亦常见，故m/z 6 891肽峰无血清型特异性；m/z 7 620处小肽峰可见于29.7% Ⅲ型菌株（41/138），且均为ST17型，于其他血清型中偶见，包括Ⅴ型（12.5%、1/8）、Ⅰa型（9.5%、2/21）和Ⅰb（2.9%、2/69）。m/z 7620肽峰对Ⅲ/ST17型GBS判断的灵敏度和特异性分别可达48.2%和93.1%。D-试验阳性耐药相关特征峰，红霉素为中介或耐药，克林霉素敏感的菌株占33.76%（80/237），其中D-试验阳性占77.5%（62/80）。主要克隆亚型D试验阳性率从高到低依次为ST10/Ⅰb（100%、9/9）和ST12/Ⅰb或ST12/Ⅲ（100%、13/13）、ST17/Ⅲ（90%、27/30）、ST19/Ⅲ（40%、4/10）、ST23/Ⅰa（16.67%、1/6），ST10、ST12和ST17亚型D试验阳性率高达94.23%（49/52）。

结论

肽峰对（m/z 6 250+/6 888-）为ST1、ST10及少许罕见ST型GBS菌共有特征峰，m/z 6 891为不同血清型共有肽峰，而非Ⅲ/Ⅰb特异；m/z 7 620则为Ⅲ/ST17型菌特征肽峰，提示其可用于高毒力GBS的快速鉴定；100% ST10、ST12和90% ST17 D-试验阳性，其ST型特征肽峰可用于预测GBS D实验阳性。

关键词: B族链球菌, 基质辅助激光解析电离飞行时间质谱, 肽峰, D-试验
Objective

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) analysis was applied to verify the characteristic peptide peaks of group B Streptococcus (GBS) protein fingerprints for sequence types (ST), serotypes and positive "D-zone test" , providing evidence for rapid GBS subtyping and effective diagnosis and treatment.

Methods

Total of 237 GBS isolates were collected from multicenter neonatal invasive infections in China from January 2015 to December 2017 with known STs and serotypes, whose GBS peptide fingerprints were collected by Bruker microbial mass spectrometer, and the characteristic peaks were analyzed by FlexAnalysis 3.0, also the disk diffusion method was used for analysis of erythromycin and clindamycin resistance as well as erythromycin-induced clindamycin resistance.

Results

Molecular type-correlated characteristic peptide peaks: m/z 7 625 was not found in all 237 strains of GBS, but 48.3% (41/85) strains of ST17 showed a peptide peak around m/z 7 620; all strains of ST1 (2 strains) showed presence of m/z 6 250 and absence of m/z 6 888 (m/z 6 250+/6 888-), which was also found in all strains of ST10 (31 strains), ST2 (2 strains), ST4 (1 strain), ST156 (1 strain) and ST579 (1 strain). Serotype-correlated peptide peaks: m/z 6 891 was not only present in serotype Ⅲ (96.4%, 133/138) and Ⅰb (81.2%, 56/69), but also common inⅠa (90.5%, 19/21) and Ⅴ strains (87.5%, 7/8), suggesting m/z 6 891 peptide peak had no specificity for serotypes; m/z 7 620 was detected with low intensity in 29.7% Ⅲ GBS (41/138), all of which belonged to ST17 subclone, m/z 7 620 was occasionally existed in other serotypes including Ⅴ (12.5%, 1/8), Ⅰa (9.5%, 2/21) and Ⅰb (2.9%, 2/69), with a sensitivity of 48.2% and specificity of 96.71% for Ⅲ/ST17 GBS. Positive "D-zone test" -associated peptide peaks: 33.76% (80/237) strains were sensitive to clindamycin with resistance or intermediate-resistance to erythromycin, of which 77.5% (62/ 80) were D-test positive. D-test positive rates of the main subtype clone were ST10/Ⅰb (100%, 9/9), ST12/Ⅰb or ST12/Ⅲ (100%, 13/13), ST17/Ⅲ (90%, 27/30), ST19/Ⅲ (40%, 4/10), ST23/Ⅰa (16.67%, 1/6) sequentially, and the D-test positive rate of ST10, ST12 and ST17 subtypes was high (94.23%, 49/52).

Conclusions

Peptide peak pairs (m/z 6 250+/ 6 888-) was specific to STs including ST1, ST10 and a few rare ST types, while m/z 6 891 peptide peak was common in different serotypes but not specific for Ⅲ/Ⅰb; m/z 7 620 was the characteristic peptide peak for Ⅲ/ST17 lineage, suggesting it could be used for rapid identification of hypervirulent GBS. D test was positive in all ST10 and ST12, as well as 90% ST17 GBS strains, indicating their ST characteristic peptide peaks could be taken for prediction of positive D-test.

Key words: Group B Streptococcus, Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry, Peptide peak, D-test
图1 ST17型GBS菌m/z 7 625肽峰表达指纹图
图2 ST1型和ST10型GBS菌m/z 6 250与6 888肽峰图
图3 Ⅰa型、Ⅰb型、Ⅲ型和Ⅴ型菌株m/z 6 891肽峰图
图4 血清Ⅲ型GBS菌m/z 7 620处肽峰图
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