MALDI-TOF MS在耐万古霉素屎肠球菌同源性分析中的价值

doi:10.3969/j.issn.1673-8640.2026.09.011

摘要/Abstract

摘要：

目的分析基质辅助激光解吸电离飞行时间质谱（MALDI-TOF MS）在耐万古霉素屎肠球菌（VREFm）同源性分析中的价值。方法收集2017年11月—2024年11月空军军医大学第一附属医院31株非重复VREFm临床分离株，采用VITEK2 Compact全自动细菌鉴定药敏仪进行细菌鉴定和体外药物敏感性试验，采用纸片扩散法和微量肉汤稀释法对万古霉素耐药性进行复核。采用荧光定量聚合酶链反应（qPCR）检测万古霉素的耐药基因，采用MALDI-TOF MS进行同源性分析。采用VITEK-MS SARAMIS Premium软件进行聚类分析，并通过多位点序列分型（MLST）进行验证。结果 31株VREFm对万古霉素、替考拉宁、青霉素、氨苄西林、左氧氟沙星、环丙沙星和红霉素均耐药，耐药基因型均为vanA基因。蛋白质质量峰聚类分析结果显示，31株VREFm可分为五大簇；MLST验证结果显示，31株VREFm中，有27株为MALDI-TOF MS第1簇，包括ST78型19株、ST80型4株、ST17型4株；第2、3、4、5簇各1株，分别为ST389、ST192、ST78、ST80型。结论 MALDI-TOF MS在菌株VREFm同源性分析方面存在一定的局限性，其分析结果与MLST结果的一致性较差，分辨能力也较弱，无法区分与VREFm高度相关的ST型别。

关键词: 基质辅助激光解吸电离飞行时间质谱, 耐万古霉素屎肠球菌, 多位点序列分型, 同源性分析

Abstract:

Objective To analyze the role of matrix-assisted laser desorption/ionization time-of-flight mass spectrometry （MALDI-TOF MS） in the homology analysis of vancomycin-resistant Enterococcus faecium （VREFm）. Methods Totally，31 non-repetitive clinical isolates of VREFm from the First Affiliated Hospital of Airforce Medical University from November 2017 to November 2024 were collected. Bacterial identification and in vitro drug susceptibility tests were performed using VITEK Compact automatic bacterial identification and drug susceptibility testing instrument. The vancomycin resistance was rechecked using disk diffusion method and microbroth dilution method. The vancomycin resistance genes were detected by fluorescence quantitative polymerase chain reaction （qPCR），and homology analysis was conducted using MALDI-TOF MS. Cluster analysis was performed using VITEK-MS SARAMIS Premium software and validated by multilocus sequence typing （MLST）. Results All the 31 VREFm isolates were resistant to vancomycin，teicoplanin，penicillin，ampicillin，levofloxacin，ciprofloxacin and erythromycin. The resistance genotypes were all vanA genes. Protein mass peak clustering analysis showed that the 31 VREFm isolates could be classified into 5 major clusters. Compared with the validation results，the 27 of 31 VREFm isolates belonged to the first cluster of MALDI-TOF MS，including 19 ST78 types，4 ST80 types and 4 ST17 types；1 isolate each in the second，third，fourth and fifth clusters，namely ST389，ST192，ST78 and ST80 types. Conclusions MALDI-TOF MS has certain limitations in the homology analysis of VREFm，the consistency with MLST results is poor，and its resolution ability is weak，unable to distinguish ST types highly related to VREFm.

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

中图分类号:

R446

康蓓佩, 郑恬, 付晓蕊, 杨佩红, 贺文芳, 周珊, 周柯. MALDI-TOF MS在耐万古霉素屎肠球菌同源性分析中的价值[J]. 检验医学, 2026, 41(9): 891-895.

KANG Beipei, ZHENG Tian, FU Xiaorui, YANG Peihong, HE Wenfang, ZHOU Shan, ZHOU Ke. Role of MALDI-TOF MS in the homology analysis of vancomycin-resistant Enterococcus faecium[J]. Laboratory Medicine, 2026, 41(9): 891-895.

图/表 4

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基因名称	引物序列（5'~3'）	产物长度/bp
vanA	F：GCTATTCAGCTGTACTC	783
	R：CAGCGGCCATCATACGG
vanB	F：ATGGGAAGCCGATAGTC	635
	R：GATTTCGTTCCTCGACC
vanC1	F：GGTATCAAGGAAACCTC	822
	R：CTTCCGCCATCATAGCT
vanC2/C3	F：CTCCTACGATTCTCTTG	439
	R：CGAGCAAGACCTTTAAG

基因名称	引物序列（5'~3'）	产物长度/bp
vanA	F：GCTATTCAGCTGTACTC	783
	R：CAGCGGCCATCATACGG
vanB	F：ATGGGAAGCCGATAGTC	635
	R：GATTTCGTTCCTCGACC
vanC1	F：GGTATCAAGGAAACCTC	822
	R：CTTCCGCCATCATAGCT
vanC2/C3	F：CTCCTACGATTCTCTTG	439
	R：CGAGCAAGACCTTTAAG

基因名称	引物序列（5'~3'）	产物长度/bp
adk	F：GAACCTCATTTTAATGGGG	437
	R：TGATGTTGATAGCCAGACG
atpA	F：TTCAAATGGCTCATACGG	649
	R：AGTTCACGATAAGCAACAGC
ddl	F：GAGACATTGAATATGCCTTATG	560
	R：AAAAAGAAATCGCACCG
gdh	F：GGCGCACTAAAAGATATGGT	530
	R：CCAAGATTGGGCAACTTCGTCCCA
gyd	F：CAAACTGCTTAGCTCCAATGGC	395
	R：CATTTCGTTGTCATACCAAGC
purK	F：CAGATTGGCACATTGAAAG	492
	R：TTCATTCACATATAGCCCG
pstS	F：TTGAGCCAAGTCGAAGCTGGAG	583
	R：CGTGATCACGTTCTACTTCC

基因名称	引物序列（5'~3'）	产物长度/bp
adk	F：GAACCTCATTTTAATGGGG	437
	R：TGATGTTGATAGCCAGACG
atpA	F：TTCAAATGGCTCATACGG	649
	R：AGTTCACGATAAGCAACAGC
ddl	F：GAGACATTGAATATGCCTTATG	560
	R：AAAAAGAAATCGCACCG
gdh	F：GGCGCACTAAAAGATATGGT	530
	R：CCAAGATTGGGCAACTTCGTCCCA
gyd	F：CAAACTGCTTAGCTCCAATGGC	395
	R：CATTTCGTTGTCATACCAAGC
purK	F：CAGATTGGCACATTGAAAG	492
	R：TTCATTCACATATAGCCCG
pstS	F：TTGAGCCAAGTCGAAGCTGGAG	583
	R：CGTGATCACGTTCTACTTCC

抗菌药物	VSEFm			VREFm
抗菌药物	耐药	中介	敏感	耐药	中介	敏感
青霉素	94.2	0	5.8	100.0	0	0
氨苄西林	93.1	0	6.9	100.0	0	0
环丙沙星	93.6	2.3	4.1	100.0	0	0
左氧氟沙星	92.5	2.6	4.9	100.0	0	0
红霉素	91.4	5.8	2.8	100.0	0	0
利奈唑胺	0.1	0	99.9	0	0	100.0
万古霉素	0	0	100.0	100.0	0	0
替考拉宁	0	0	100.0	100.0	0	0
四环素	54.5	0.2	45.3	81.8	0	18.2
替加环素	0	0	100.0	0	0	100.0