MALDI-TOF MS快速鉴定耐万古霉素屎肠球菌及其流行病学分型性能分析

doi:10.3969/j.issn.1673-8640.2020.05.017

摘要/Abstract

摘要：

目的利用基质辅助激光解析电离飞行时间质谱（MALDI-TOF MS）快速鉴别van A型万古霉素耐药屎肠球菌（VREfm）和万古霉素敏感屎肠球菌（VSEfm）,并对VREfm进行流行病学相关性分析。方法收集139株屎肠球菌临床分离株,其中VREfm66株、VSEfm73株。采用聚合酶链反应（PCR）确定66株VREfm的万古霉素耐药基因。采用MALDI-TOF MS鉴定屎肠球菌。随机选取45株VREfm和50株VSEfm,采用ClinProTools 3.0软件对质谱峰进行分析并建立模型,用最佳模型验证另外的21株VREfm和23株VSEfm。随机选取21株VREfm进行多位点序列分型（MLST）分析,并采用MALDI-TOF MS对其进行层次聚类分析。结果 MALDI-TOF MS鉴定出所有菌株均为屎肠球菌,鉴定分值为2.321~2.602。66株VREfm均为vanA基因型。采用ClinProTools 3.0软件建立的最佳模型进行外部验证时,敏感性和特异性分别为95.2%和95.7%。21株VREfm共有7种序列分型（ST）型别,以ST78最常见。MALDI-TOF MS将21株VREfm分为4个集群,绝大多数ST78菌株被归于同一集群,与ST78高度相关的其他ST型别也被归于此集群。结论在严格控制实验条件的前提下,MALDI-TOF MS可快速区分vanA型VREfm和VSEfm,但无法区分与VREfm高度相关的ST型别。

关键词: 基质辅助激光解析电离飞行时间质谱, 万古霉素耐药屎肠球菌, 万古霉素敏感屎肠球菌, 多位点序列分型

Abstract:

Objective To identify vanA-positive vancomycin-resistant Enterococcus faecium（VREfm） and vancomycin-susceptible Enterococcus faecium（VSEfm） and determine the epidemiological correlation of VREfm by matrix-assisted laser desorption ionization-time of flight mass spectrometry（MALDI-TOF MS）. Methods A total of 139 Enterococcus faecium isolates were collected,and there were 66 VREfm and 73 VSEfm. Vancomycin resistance genes of 66 VREfm were confirmed by polymerase chain reaction（PCR）. Enterococcus faecium isolates were identified by MALDI-TOF MS. Models with ClinProTools 3.0 software were generated using the spectra of 45 VREfm and 50 VSEfm randomly selected. The remaining 21 VREfm and 23 VSEfm were validated with optimal model. Totally,21 VREfm randomly selected were analyzed by multilocus sequence typing（MLST） and subjected to hierarchical cluster analysis by MALDI-TOF MS. Results A total of 139 Enterococcus faeciumisolates were identified by MALDI-TOF MS,and the score range was 2.321-2.602. All the 66 VREfm carried vanA gene. External validation of the optimal model generated produced a sensitivity of 95.2% and a specificity of 95.7%. There were 7 sequence types（ST） of 21 VREfm,with most isolates identified as ST78. Hierarchical cluster analysis showed that 21 VREfm were classified into 4 clusters. Most of the ST78 isolates were classified into the same cluster,and highly-ST78-related ST were also classified into the ST78 cluster. Conclusions MALDI-TOF MS can rapidly differentiate vanA-positive VREfm from VSEfm with strict control of experimental conditions. Highly-related ST of VREfm are distinguishable.

Key words: Matrix-assisted laser desorption ionization-time of flight mass spectrometry, Vancomycin-resistant Enterococcus faecium, Vancomycin-susceptible Enterococcus faecium, Multilocus sequence typing

中图分类号:

R446.1

吴思颖, 王晓庆, 刘山泉, 肖玉玲, 何超, 马莹, 谢轶, 康梅. MALDI-TOF MS快速鉴定耐万古霉素屎肠球菌及其流行病学分型性能分析[J]. 检验医学, 2020, 35(5): 470-475.

WU Siying, WANG Xiaoqing, LIU Shanquan, XIAO Yuling, HE Chao, MA Ying, XIE Yi, KANG Mei. Identification of vancomycin-resistant Enterococcus faecium by MALDI-TOF MS and epidemiological typing performance[J]. Laboratory Medicine, 2020, 35(5): 470-475.

图/表 4

参考文献 17

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质谱峰	质荷比/ （m/z）	DAVE	PTTA	PWKW	PAD	Ave1	Ave2	StdDev1	StdDev2	VREfm组变异系数/%	VSEfm组变异系数/%
97	6 699.6	2.78	<0.000 001	<0.000 001	0.000 003 87	2.52	5.30	0.73	1.83	29.04	34.48
20	2 428.17	4.63	<0.000 001	<0.000 001	<0.000 001	6.91	2.27	3.19	1.25	46.12	54.89
117	8 119.98	1.4	<0.000 001	<0.000 001	0.000 441	1.97	3.37	0.54	0.98	27.48	28.94
135	10 225.33	3.05	<0.000 001	<0.000 001	0.000 001 83	2.04	5.09	0.85	2.30	41.48	45.10
85	6 341.92	7.48	<0.000 001	<0.000 001	0.007 05	9.66	17.14	2.68	5.67	27.74	33.08

质谱峰	质荷比/ （m/z）	DAVE	PTTA	PWKW	PAD	Ave1	Ave2	StdDev1	StdDev2	VREfm组变异系数/%	VSEfm组变异系数/%
97	6 699.6	2.78	<0.000 001	<0.000 001	0.000 003 87	2.52	5.30	0.73	1.83	29.04	34.48
20	2 428.17	4.63	<0.000 001	<0.000 001	<0.000 001	6.91	2.27	3.19	1.25	46.12	54.89
117	8 119.98	1.4	<0.000 001	<0.000 001	0.000 441	1.97	3.37	0.54	0.98	27.48	28.94
135	10 225.33	3.05	<0.000 001	<0.000 001	0.000 001 83	2.04	5.09	0.85	2.30	41.48	45.10
85	6 341.92	7.48	<0.000 001	<0.000 001	0.007 05	9.66	17.14	2.68	5.67	27.74	33.08