耐万古霉素肠球菌耐药基因、转座子结构和多位点序列分析

doi:10.3969/j.issn.1673-8640.2021.09.001

摘要/Abstract

摘要：

目的分析耐万古霉素肠球菌（VRE）耐药基因和多位点序列分型（MLST）情况。方法收集三亚中心医院临床分离的耐万古霉素的80株屎肠球菌和12株粪肠球菌,采用VITEK MS微生物鉴定系统进行菌种鉴定。采用VITEK 2 Compact自动化鉴定药敏仪测定VRE对14种常用抗菌药物的敏感性。采用微量肉汤稀释法测定VRE对万古霉素和替考拉宁的最小抑菌浓度（MIC）,并确定耐药表型;采用聚合酶链反应（PCR）筛选VRE的毒力基因（esp、hyl、gelE、asal、cy1A、efaA、ace）和转座子结构;对VRE进行MLST。结果质谱鉴定结果为80株屎肠球菌和12株粪肠球菌。体外药物敏感性试验结果表明,屎肠球菌和粪肠球菌对万方霉素的耐药率均为100%,对氨苄西林的耐药率分别为97.5%和50.0%,但对呋喃妥因的耐药率较低。92株VRE对万古霉素和替考拉宁的MIC分别为192~256和32~256 μg/mL,均为VanA表型;92株VRE基因型均为vanA,以esp毒力基因所占比例最高（82.5%）;屎肠球菌以esp-hyl基因组合最常见,粪肠球菌以多基因组合为主;通过Tn1546基因转座子结构分析可分为A~E 5个型别;92株VRE的MLST包括7个ST型别,分别为ST17（43.5%）、ST78（32.6%）、ST203（6.5%）、ST363（5.4%）、ST555（4.3%）、ST1392（4.3%）和ST1394（3.4%）,其中ST17和ST78均属于CC17克隆复合体。结论 92株VRE均为多重耐药株、vanA基因型和CC17克隆复合体,耐药基因和毒力基因携带率均较高,临床应引起重视。

关键词: 耐万古霉素肠球菌, 菌种鉴定, 耐药性, 基因型, 多位点序列分析

Abstract:

Objective To study vancomycin-resistant Enterococcus（VRE） drug resistance genes and multilocus sequence typing（MLST）. Methods Totally,80 isolates of VAN-resistant Enterococcus faecium and 12 isolates of Enterococcus faecalis isolated from the clinical specimens in Sanya Central Hospital were collected and were identified by VITEK MS microbial identification system. The susceptibilities of VRE to 14 kinds of commonly used antibiotics were determined by VITEK 2 Compact automatic drug sensitivity test system. The minimum inhibitory concentrations（MIC）of vancomycin and tekoranin were determined by microdilution broth method. Virulence genotypes（esp,hyl,gelE,asal,cy1A,efaA and ace） and transposon structure of VRE isolates were screened by polymerase chain reaction（PCR）. MLST was used to analyze the multi-site sequence typing of VRE. Results A total of 80 isolates of Enterococcus faecium and 12 isolates of Enterococcus faecalis were identified by mass spectrometry. Both Enterococcus faecium and Enterococcus faecalis showed 100.0% drug resistance to vancomycin in in vitro drug susceptibility test,and the drug resistance rates to ampicillin were 97.5% and 50.0%,respectively,but the drug resistance rates to furantoin were low. All the isolates of VRE were resistant to vancomycin and tekoranin,and MIC were 192-256 and 32-256 μg/mL,respectively,representing VanA phenotype. All the VRE isolates were vanA genotype,and the proportion of esp virulence genotype was the highest（82.5%）,Enterococcus faecium was common in esp-hy1 gene combination,and Enterococcus faecalis was mainly in multi-gene combination. The Tn1546 transposon structure analysis could be classified into A-E types. MLST showed that 92 isolates of VRE could be classified into 7 ST types,including ST17（43.5%）,ST78（32.6%）,ST203（6.5%）,ST363（5.4%）,ST555（4.3%）,ST1392（4.3%） and ST1394（3.4%）,among which ST17 and ST78 were the most common types,belonging to CC17 clone complex. Conclusions The 92 VRE isolates are all multi-drug resistant isolates,vanA genotype and CC17 clone complex,and they are highly carrying drug-resistant genes and virulence genes.

Key words: Vancomycin-resistant Enterococcus, Isolate identification, Drug resistance, Genotype, Multilocus sequence typing

中图分类号:

R446.1

王婧婧, 吴林清, 陈如寿, 王玉丰. 耐万古霉素肠球菌耐药基因、转座子结构和多位点序列分析[J]. 检验医学, 2021, 36(9): 891-895.

WANG Jingjing, WU Linqing, CHEN Rushou, WANG Yufeng. Drug resistance genotype,transposon structure and MLSA of vancomycin-resistant Enterococcus[J]. Laboratory Medicine, 2021, 36(9): 891-895.

图/表 1

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抗菌药物	屎肠球菌（80株）		粪肠球菌（12株）
抗菌药物	耐药菌/ 株	耐药率/%	耐药菌/株	耐药率/%
万古霉素	80	100.0	12	100.0
氨苄西林	78	97.5	6	50.0
青霉素	76	95.0	10	83.3
左氧氟沙星	75	93.8	10	83.3
环丙沙星	73	91.3	10	83.3
红霉素	72	90.0	9	75.0
克林霉素	74	92.3	10	83.3
四环素	67	83.8	9	75.0
替加环素	75	93.8	8	66.7
利奈唑胺	76	95.0	9	75.0
链霉素	68	85.0	8	66.7
庆大霉素	33	41.3	7	58.3
奎奴普丁-达福普汀	74	92.5	7	58.3
呋喃妥因	4	5.0	0	0

抗菌药物	屎肠球菌（80株）		粪肠球菌（12株）
抗菌药物	耐药菌/ 株	耐药率/%	耐药菌/株	耐药率/%
万古霉素	80	100.0	12	100.0
氨苄西林	78	97.5	6	50.0
青霉素	76	95.0	10	83.3
左氧氟沙星	75	93.8	10	83.3
环丙沙星	73	91.3	10	83.3
红霉素	72	90.0	9	75.0
克林霉素	74	92.3	10	83.3
四环素	67	83.8	9	75.0
替加环素	75	93.8	8	66.7
利奈唑胺	76	95.0	9	75.0
链霉素	68	85.0	8	66.7
庆大霉素	33	41.3	7	58.3
奎奴普丁-达福普汀	74	92.5	7	58.3
呋喃妥因	4	5.0	0	0