宁夏地区耐甲氧西林金黄色葡萄球菌分子流行病学研究

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张楠, 赵志军, 贾伟, 师志云, 杨晓燕, 李刚, 魏军. 宁夏地区耐甲氧西林金黄色葡萄球菌分子流行病学研究. 2011, 26(5): 287-290
ZHANG Nan, ZHAO Zhijun, JIA Wei, SHI Zhiyun, YANG Xiaoyan, LI Gang, WEI Jun. Molecular epidemiology research of methicillin-resistant Staphylococcus aureus in Ningxia. Labratory Medicine, 2011, 26(5): 287-290 复制到剪切板

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《检验医学》编辑部

宁夏地区耐甲氧西林金黄色葡萄球菌分子流行病学研究

张楠¹, 赵志军², 贾伟², 师志云², 杨晓燕², 李刚², 魏军²

1. 宁夏医科大学,宁夏银川 750004

2. 宁夏医科大学附属医院医学实验中心,宁夏银川 750004

作者简介：张楠,女,1984年生,学士,主要从事病原微生物与分子生物学研究。

通讯作者：魏军,联系电话：0951-6743191。

基金:宁夏回族自治区重大科技攻关计划项目（2008）; 宁夏自然科学基金资助项目（NZ10132）;

摘要

目的

调查临床分离的耐甲氧西林金黄色葡萄球菌(MRSA)的染色体mec基因盒(SCCmec)分型及杀白细胞素(PVL)的pvl基因携带率,初步了解宁夏地区MRSA分子流行病学特点。

方法

对2005年9月至2008年1月从临床标本中分离的MRSA菌株进行pvl基因检测,并应用多重聚合酶链反应(PCR)对MRSA菌株进行SCCmec分型。

结果

88株MRSA菌株中pvl基因阳性菌株8株(9.1%),SCCmec分型结果为Ⅲ型86株(97.7%)、Ⅱ型2株(2.3%)。

结论

宁夏地区分离的MRSA菌株SCCmec分型以Ⅲ型为主,SCCmec分型是探索MRSA多重耐药性有效的分子生物学手段。

关键词: 耐甲氧西林金黄色葡萄球菌; 分子流行病学; 染色体mec基因盒分型; 杀白细胞素基因

中图分类号:R378.1 文献标志码:A 文章编号:1673-8640(2011)05-0287-04

Molecular epidemiology research of methicillin-resistant Staphylococcus aureus in Ningxia

ZHANG Nan¹, ZHAO Zhijun², JIA Wei², SHI Zhiyun², YANG Xiaoyan², LI Gang², WEI Jun²

1. Ningxia Medical University,Ningxia Yinchuan 750004, China

2. Medical Experimental Center, Affiliated Hospital of Ningxia Medical University,Ningxia Yinchuan 750004, China

Fund:

Abstract

Objective

To investigate the genotype and carry over rate of staphylococcal chromosomal cassette mec (SCCmec) and panton-valentine leukocidin(PVL) pvl gene in clinical methicillin-resistant Staphylococcus aureus(MRSA)isolates, and primarily find out the characteristic of molecular epidemiology of MRSA isolates in Ningxia.

Methods

The pvl gene of the MRSA strains isolated from September 2005 to January 2008 were determined, and the genotypes of SCCmec were determined by multiplex polymerase chain reaction (PCR).

Results

Among 88 MRSA isolates,8 isolates were pvl gene positive(9.1%). SCCmec genotyping results showed that 86 isolates were Ⅲ type (97.7%), and 2 isolates were Ⅱ type (2.3%).

Conclusions

The most isolates of MRSA in Ningxia are SCCmec Ⅲ. SCCmec typing is an effective molecular epidemiology method to research the multiresistant characteristic of MRSA isolates.

Keyword: Methicillin-resistant Staphylococcus aureus; Molecular epidemiology; Chromosomal cassette mec genotyping; Panton-valentine leukocidin gene

Show Figures

耐甲氧西林金黄色葡萄球菌(methicillin-resistant Staphylococcus aureus,MRSA)是院内感染的重要病原菌之一,对多种抗菌药物耐药,致病性强且治疗效果差,其造成的直接后果是被感染者由于长期住院治疗导致医疗费用增加1倍以上,且病死率高。在美国每年MRSA导致大约19 000例住院患者死亡,相当于获得性免疫缺陷综合征(AIDS)、结核病和病毒性肝炎患者死亡的总数。在我国MRSA同样有很高的检出率,部分地区甚至达到70%以上^{[ 1]}。本研究利用多重聚合酶链反应(PCR)检测MRSA的杀白细胞素(panton-valentine leukocidin, PVL)的pvl基因和葡萄球菌染色体mec基因盒(SCCmec)型别,旨在了解宁夏医科大学附属医院分离的MRSA的分子流行状况,为临床及时预防和控制MRSA所致院内感染提供依据。

材料和方法

一、材料

1. 菌株来源 2005年9月至2008年1月186株经VITEK-32全自动细菌鉴定仪鉴定的金黄色葡萄球菌,来自宁夏医科大学附属医院住院患者送检的各种标本。

2. 主要试剂与仪器引物由上海生工生物工程有限公司合成,2×Taq Plus PCR MasterMix(含染料)为北京天根公司产品。MasterCycler PCR扩增仪(Eppendorf,德国),Gel Doc2000凝胶图像分析系统(BIO-RAD,美国),核酸蛋白测定仪(NanoDrop,美国)。

二、方法

1. MRSA的检测应用mecA-femB双重PCR验证,mecA与femB均为阳性者判定为MRSA。金黄色葡萄球菌(ATCC 25923)作为质控菌株。

2. pvl基因的检测 pvl基因扩增引物和反应体系参照文献[2],稍作修改, PCR反应条件为:94 ℃预变性5 min,94 ℃变性30 s,55 ℃退火45 s,72 ℃延伸1 min,进行25个循环,最后72 ℃延伸5 min。质控菌株为金黄色葡萄球菌(ATCC 49775)。

3. SCCmec分型多重PCR引物参照文献[3]。引物序列见表1。反应体系为25 μL, 2×Taq Plus PCR MasterMix混合体系12.5 μL, DNA模板2 μL,去离子水补足25 μL。PCR反应条件为:94 ℃预变性5 min,94 ℃变性45 s,45 ℃退火45 s,72 ℃延伸90 s,进行10个循环;再94 ℃变性45 s,52 ℃退火45 s,72 ℃延伸90 s,进行30个循环,最后72 ℃延伸10 min。PCR产物直接用凝胶成像仪拍照及保存结果。SCCmec分型的阳性质控株为中国协和医院王辉博士惠赠。

表1 SCCmec多重扩增引物

结果

一、MRSA检测试验

在经VITEK-32全自动细菌鉴定仪鉴定的186株金黄色葡萄球菌中,经过双重扩增mecA、femB基因,结果显示有88株分离株为mecA、femB基因均阳性,该88株双阳性菌株被确认为MRSA。MRSA分离率为47.3%,见图1。

	Figure Option View Download New Window
	图1 mecA、femB基因PCR扩增电泳图

二、pvl基因PCR检测

88株MRSA分离株中有8株经过pvl基因检测为阳性,分离率为9.1%。电泳结果见图2。

	Figure Option View Download New Window
	图2 pvl基因PCR扩增电泳图

三、SCCmec分型

88株MRSA分离株中SCCmecⅡ型2株,分离率2.3%;SCCmecⅢ型86株,分离率97.7%。未发现Ⅰ、Ⅳ、Ⅴ型。多重PCR对SCCmec基因分型电泳结果见图3。

	Figure Option View Download New Window
	图3 SCCmec基因PCR扩增电泳图

讨论

MRSA获得甲氧西林耐药性的主要结构基因是mecA基因,但在凝固酶阴性葡萄球菌(CNS)中也可见mecA基因的表达。因此为了区别MRSA和其他mecA基因阳性的葡萄球菌属,特别是CNS,应同时检测femB基因。femB基因是甲氧西林耐药的辅助表达基因,在金黄色葡萄球菌中高度保守,被认为是其特异的表达基因,在CNS中不表达。因此本研究把mecA和femB基因表达均为阳性者确认为MRSA。

在MRSA中,SCCmec是一种新型的可移动元件,大小为21~67 kb,能作为载体在葡萄球菌属中交换基因信息。作为MRSA的耐药性基因岛,SCCmec除携带mecA基因以外还整合、编码多种耐药基因,这是导致MRSA多重耐药出现的主要原因。目前,SCCmec分型作为MRSA最常用的分型方法之一被广泛应用于MRSA的分子流行病学研究。SCCmec主要分为5个型别,Ⅰ、Ⅱ和Ⅲ型见于医院获得性MRSA(HA-MRSA),Ⅳ、Ⅴ型多见于社区获得性MRSA(CA-MRSA)。SCCmec的基因型与MRSA的流行背景有关。在欧洲,挪威的流行株主要是SCCmecⅢ型^{[ 4]},荷兰报道的流行株主要是SCCmecⅣ和SCCmecⅤ型^{[ 5]},而在南美洲的阿根廷,流行株则是SCCmecⅡ型^{[ 6]}。亚洲除分离自日本和韩国的MRSA以SCCmec Ⅱ型为主外,其他国家和地区多以SCCmec Ⅲ型为主^{[ 7]}。在我国北京、上海、浙江及四川等地的MRSA流行株均为SCCmec Ⅲ型^{[ 8]} ,而香港主要为SCCmecⅣ型^{[ 9]}。本研究结果表明,宁夏医科大学附属医院88株MRSA经SCCmec基因分型检测得出SCCmec Ⅲ型86株(97.7%),与我国其他地区报道的研究结果相近。表明在宁夏医科大学附属医院MRSA以SCCmecⅢ型为主,对于此型MRSA所致感染临床应首选万古霉素、去甲万古霉素、替考拉宁等糖肽类抗菌药物进行治疗。本研究未发现Ⅳ和Ⅴ型,可能是由于标本来源均为患者住院后采集所致。

PVL是坏死性细胞毒素,其毒性因子可以导致皮肤及软组织感染,并且可引起急性坏死性肺炎,造成肺泡膜大量坏死,病死率达75%。国外有报道称,在CA-MRSA中pvl基因的阳性率>80%^{[ 10]},而在HA-MRSA中阳性率<0.5%。因此pvl基因可作为CA-MRSA菌株的重要分子生物标记。本研究中pvl基因的阳性率为9.1%,高于国外的研究结果,但较我国部分地区报道的15.7%^{[ 11]}阳性率低,这说明MRSA的pvl基因携带情况可能和地域性有一定的关系,不同地区、不同时期的阳性率也可能不同。同时pvl基因阳性菌株SCCmec分型显示均为Ⅲ型,这也提示我们不能以pvl基因的检出与否作为CA-MRSA的惟一判定标准。

本研究首次通过对MRSA的pvl基因检测和SCCmec基因分型来探讨宁夏地区MRSA菌株的分子流行病学特征,了解了宁夏地区MRSA的主要流行株,并为临床正确使用抗菌药物治疗MRSA感染以及更好地控制MRSA引起的院内感染提供了科学的指导依据。

The authors have declared that no competing interests exist.

参考文献

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2009

0.0

... 在我国MRSA同样有很高的检出率,部分地区甚至达到70%以上^[1] ...

1999

9.374

0.0

2005

4.068

0.0

2006

4.578

0.0

. 2006, 12(7):627-633

Increased incidence of methicillin-resistant Staphylococcus aureus ST80, novel ST125 and SCCmecIV in the south-eastern part of Norway during a 12-year period

A. E. Fossum andG. Bukholm

Institute of Clinical Epidemiology and Molecular Biology, University of Oslo, Akershus University Hospital, Lørenskog, Norway * Corresponding author and reprint requests: G. Bukholm, Epi-Gen Institute, Akershus University Hospital, University of Oslo, N-1478 Lørenskog, Norway E-mail: geir.bukholm@ahus.no

> A retrospective population-based study of genotypes of methicillin-resistant Staphylococcus aureus (MRSA) was performed during the period 1991–2003 in two counties in the south-eastern part of Norway. Isolates of MRSA from all individuals in the two counties in whom MRSA was detected were genotyped by means of multilocus sequence typing (MLST), staphylococcal cassette chromosome mec (SCC mec ) typing, staphylococcal protein A gene ( spa ) typing and amplified fragment length polymorphism (AFLP) analysis. Until 1999, only sporadic cases of MRSA infection were reported in these counties, but the incidence increased during the following years. Nine new MLST types were identified in this study. The predominant strains were ST239-MRSA-III, the novel ST125-MRSA-IV, and the central European community-acquired strain ST80-MRSA-IV reported previously. ST80-MRSA-IV was introduced into the two counties in 1997, and the incidence of infections has increased since 2000, so that ST80-MRSA-IV is now the commonest MRSA strain in the region. An increase in MRSA clones carrying SCC mec IV has occurred during recent years, which could indicate a shift in the MRSA population in Norway from hospital-acquired MRSA to community-acquired-MRSA.

... 型^[4],荷兰报道的流行株主要是SCCmec#cod#x02163 ...

2006

4.068

0.0

... 型^[5],而在南美洲的阿根廷,流行株则是SCCmec#cod#x02161 ...

2006

4.068

0.0

... 型^[6] ...

2005

4.068

0.0

... 型为主^[7] ...

2006

0.0

... 型^[8] ,而香港主要为SCCmec#cod#x02163 ...

2002

9.737

0.0

PNAS. 2002, 99(11):7687-7692

The evolutionary history of methicillin-resistant Staphylococcus aureus (MRSA)

Mark C. Enright*†, D. Ashley Robinson*, Gaynor Randle‡, Edward J. Feil*, Hajo Grundmann§, and Brian G. Spratt¶

* Department of Biology and Biochemistry, University of Bath, Bath BA2 7AY, United Kingdom; ‡ Institute of Molecular Medicine, John Radcliffe Hospital, Oxford, OX3 9DU, United Kingdom; § Division of Microbiology and Infectious Diseases, University Hospital Nottingham, Nottingham, NG7 2UH, United Kingdom; and ¶ Department of Infectious Disease Epidemiology, Faculty of Medicine, Imperial College, St. Mary's Hospital, London W2 1PG,United Kingdom

Methicillin-resistant Staphylococcus aureus (MRSA) is a major cause of hospital-acquired infections that are becoming increasingly difficult to combat because of emergingresistance to all current antibiotic classes. The evolutionary origins of MRSA are poorly understood, no rational nomenclatureexists, and there is no consensus on the number of major MRSA clones or the relatedness of clones described from differentcountries. We resolve all of these issues and provide a more thorough and precise analysis of the evolution of MRSA clonesthan has previously been possible. Using multilocus sequence typing and an algorithm, burst , we analyzed an international collection of 912 MRSA and methicillin-susceptible S. aureus (MSSA) isolates. We identified 11 major MRSA clones within five groups of related genotypes. The putative ancestral genotypeof each group and the most parsimonious patterns of descent of isolates from each ancestor were inferred by using burst , which, together with analysis of the methicillin resistance genes, established the likely evolutionary origins of each majorMRSA clone, the genotype of the original MRSA clone and its MSSA progenitor, and the extent of acquisition and horizontalmovement of the methicillin resistance genes. Major MRSA clones have arisen repeatedly from successful epidemic MSSA strains,and isolates with decreased susceptibility to vancomycin, the antibiotic of last resort, are arising from some of these majorMRSA clones, highlighting a depressing progression of increasing drug resistance within a small number of ecologically successful S. aureus genotypes.

... 型^[9] ...

2008

0.0

... 80%^[10],而在HA-MRSA中阳性率#cod#x0003C ...

2006

0.0

... 7%^[11]阳性率低,这说明MRSA的pvl基因携带情况可能和地域性有一定的关系,不同地区、不同时期的阳性率也可能不同 ...