一起金黄色葡萄球菌食物中毒检测与分析

目的查明食物中毒致病菌,分析菌株间的亲缘关系与毒力,为食物中毒诊断提供依据。方法致病菌分离采用国家标准和文献方法;金黄色葡萄球菌鉴定采用仪器法;同源性分析采用脉冲肠凝胶电泳法;肠毒素检测采用PCR法;药敏试验采用VITEK 2 Compact法。结果从22份标本中检出12株金黄色葡萄球菌,阳性检出率为54.55%;检出SEa、SEb、SEd、SEe传统肠毒素基因4种和SEh、SEj新型肠毒素基因2种;食品株与患者株金黄色葡萄球菌的脉冲肠凝胶电泳带型一致,具有高度同源性;菌株对大多数常用抗生素敏感。结论本起食物中毒由金黄色葡萄球菌及其肠毒素污染鸡肉卷所致;菌株携带多种肠毒素基因,致病性较强;鸡肉卷与患者检出的金黄色葡萄球菌脉冲肠凝胶电泳显示其来源为同一克隆群,从分子生物学上证明了食物中毒病原菌的相关性。     

两起金黄色葡萄球菌所致食物中毒的病原学研究及溯源分析

目的探讨两起同地区发生的金黄色葡萄球菌食物中毒分离株之间的分子流行病学关系。方法参照国家标准进行金黄色葡萄球菌分离培养与鉴定,用PCR方法和脉冲场凝胶电泳技术(PFGE)分析分离菌株的分子流行病学特征,采用微量肉汤稀释法进行药物敏感试验。结果共检出15株金黄色葡萄球菌,其中病人生物标本检出6株,剩余食品中9株,烤鸡肉中金黄色葡萄球菌含量最高;葡萄球菌肠毒素均为SEA型,同一事件的菌株为同一克隆株;两起事件的菌株PFGE型别不一致,药敏结果也有差异。结论两起SEA型葡萄球菌肠毒素引起的食物中毒,由不同来源的金黄色葡萄球菌污染所导致。     

一起食物中毒事件金黄色葡萄球菌肠毒素及肠毒素基因检测与分析

目的了解分离自食物中毒事件的金黄色葡萄球菌肠毒素的型别,并对肠毒素基因携带情况及菌株分子分型进行分析。方法对分离自某起食物中毒的12株金色葡萄球菌分离株进行生化鉴定,采用微孔板酶联免疫法(ELISA)、酶联荧光免疫法(ELFA)、聚合酶链反应(PCR)进行肠毒素型别和肠毒素基因检测,并应用脉冲场凝胶电泳(PFGE)法对菌株进行分子分型,应用Bio Numerics软件对不同来源的菌株进行比对,分析菌株之间的相关性。结果 12株来源于患者和食物样本的菌株经ELFA、ELISA法检测,结果均为阳性,其中1株来源于某患者呕吐物样本的菌株为肠毒素B型,其余11株来源于该患者的肛拭样本和其他样本的菌株均为肠毒素A型。肠毒素基因检测结果显示,除1株未能检出肠毒素基因外,1株检出肠毒素基因seb,10株检出肠毒素基因sea,其中8株同时检出肠毒素基因see。PFGE分型结果显示,11株菌为1型,1株菌为2型。结论金黄色葡萄球菌肠毒素检测对明确引起食物中毒的原因十分重要,PFGE分型技术有助于食物中毒的溯源研究。     

金黄色葡萄球菌食品和病人分离株肠毒素基因和耐药性比较

目的：对金黄色葡萄球菌食品（不含生牛奶）分离株和病人分离株的肠毒素基因进行分型和耐药性检测,比较两种分离菌株的肠毒素基因分布和耐药性差异。方法：用国标方法分离鉴定金黄色葡萄球菌,VITEK32生化验证,PCR检测金黄色葡萄球菌肠毒素基因（肠毒素SEA-SEJ基因）,VITEK32测定分离菌株抗生素的耐药性。结果：从病人分离的80株金黄色葡萄球菌检测到9种基因。肠毒素基因携带率为85.0%,同时携带2种及以上毒素基因的菌株占76.3%,含传统肠毒素基因（SEA-SEE）的菌株占43.8%,有新发现肠毒素基因（SEG-SEJ）的菌株占71.3%。从食品中分离的51株金黄色葡萄球菌检测到7种肠毒素基因。肠毒素基因携带率为68.7%,同时携带2种及以上毒素基因的菌株占21.6%。有SEA基因的占27.5%,含其它传统的毒素基因类型（SEB-SEE）基因的菌株占23.5%,携带新发现的毒素基因SEG、SEH、SEI的菌株只占9.8%。金黄色葡萄球菌病人分离株与食品分离株的耐药谱和耐药率有较大差异。结论：金黄色葡萄球菌食品分离株和病人分离株的肠毒素基因分布不同,其耐药性有明显区别。     

一起由金黄色葡萄球菌肠毒素引起超市食物中毒的病原学分析

目的 了解一起疑似金黄色葡萄球菌肠毒素引起食物中毒事件的病原学及溯源分析。 方法 对采集的样本进行分离培养鉴定,所得金黄色葡萄球菌进行肠毒素胶体金法快速初判定, PCR法检测肠毒素基因,酶联免疫法检测葡萄球菌肠毒素,同时进行耐药监测,并对不同来源分离株进行脉冲场凝胶电泳(pulse field gel electrophoresis,PFGE)分子分型,分析菌株之间的相关性。 结果 对患者、食品及相关加工工具,从业人员共检出13株金黄色葡萄球菌;13株分离株肠毒素基因为sea+see;肠毒素为SEA+SEE混合型;PCR法和酶联免疫法结果一致,PFGE对不同来源分离株同源性准确锁定,显示13株分离株分子分型同源性为100%;药敏结果显示,13株菌均对青霉素和四环素耐药。 结论 本起食物中毒由产肠毒素SEA+SEE混合型的金黄色葡萄球菌污染食物所致,PCR法与酶联免疫法同时检测葡萄球菌肠毒素,对食物中毒病因准确定位,PFGE分子分型对不同来源菌株溯源分析起到关键作用。相关部门应加大监管力度,加强从业人员健康教育。     

一起食物中毒事件的金黄色葡萄球菌分子分型研究

目的 运用分子分型方法分析一起在广州发生的由金黄色葡萄球菌所致的重大食物中毒事件,并进行溯源.方法 在常规分离的基础上,采用荧光定量PCR检测所获得的金黄色葡萄球菌特异耐热核酸酶基因(nuc)、耐甲氧西林决定基因(mecA)和其他5种肠毒素基因(sea,seb,sec,sed,see),并对16 S rRNA核苷酸进行序列扩增和使用DNAStar MegAlign 5.0软件分析,同时应用脉冲场凝胶电泳(PFGE)和BioNumerics Version 4.0软件进行聚类分析.结果 10株金黄色葡萄球菌特异性nuc基因均为阳性,其中7株分离菌的肠毒素sea,seb基因为阳性,分离菌可以分为4个16 SrRNA型别和5个PFGE型别.结论 本次食物中毒事件至少由3株以上亲缘关系相近的产毒金黄色葡萄球菌污染食物所致,分子分型方法可以为疫情溯源提供分子流行病学证据和支持.     

丹东口岸出口食品中金黄色葡萄球菌及肠毒素检测分析

〔目的〕了解丹东口岸出口食品中金黄色葡萄球菌的污染状况,检测食品中金黄色葡萄球菌携带肠毒素的情况,为口岸加强食品卫生监管提供科学依据。〔方法〕参照标准GB/T4789.10-2008,进行金黄色葡萄球菌检测,同时用全自动荧光酶标免疫测试系统(mini-VIDAS)检测分离菌株携带肠毒素SEA-SEE情况。〔结果〕885份样本中检出金黄色葡萄球菌15株,检出率1.69%;对15份金黄色葡萄球菌肠毒素进行检测,14株呈阳性,阳性率为93.33%。〔结论〕丹东口岸出口食品存在金黄色葡萄球菌污染,并且产肠毒素的金黄色葡萄球菌占很高的比例,对出口食品应加强金黄色葡萄球菌的检测,检出金黄色葡萄球菌的食品一定要按有关规定严格处理。     

一起食源性疾病的病原学分析

目的对一起食源性疾病的样本进行相关微生物学检验,确定该起疾病的致病菌。方法采集患者的呕吐物、粪便和剩余食物样本,依据GB 4789和WS/T 9—1996方法进行病原菌分离、鉴定后,应用酶联免疫吸附法(ELISA)对金黄色葡萄球菌分离株进行肠毒素检测,应用脉冲场凝胶电泳(PFGE)方法对奇异变形杆菌分离株进行检测分析,确定是否为同源菌株。结果在剩余食物和3位患者的粪便中检出金黄色葡萄球菌和奇异变形杆菌。ELISA结果表明剩余食物与所有患者的粪便样本检出的金黄色葡萄球菌肠毒素类型相同,为A型、D型、E型肠毒素。剩余食物与3号患者的粪便样本检出的奇异变形杆菌PFGE图谱相似,具有同源性,与另两位患者有差异。结论该起食源性疾病由金黄色葡球菌和奇异变形杆菌共同引起。PFGE方法可有效应用于食源性疾病的溯源分析及分子流行病学调查。     

西藏酥油中金黄色葡萄球菌肠毒素A基因检测及脉冲场凝胶电泳分型研究

目的旨在了解西藏酥油中金黄色葡萄球菌携带肠毒素A(sea)毒力基因及脉冲场凝胶电泳(PFGE)分型情况。方法通过聚合酶链式反应(PCR)检测35株酥油来源的金黄色葡萄球菌的sea毒力基因并利用PFGE分型技术对上述金黄色葡萄球菌进行全基因组分析,DNA酶切图谱用Bio Numerics软件进行聚类分析。结果 35株金黄色葡萄球菌中sea毒力基因检出率为8.6%(3株);PFGE图谱聚类分析共得到5大簇和25种PFGE型别。结论建立的金黄色葡萄球菌PFGE分型数据库分子型别较多,对西藏地区酥油中由金黄色葡萄球菌引起的食物中毒的防控与溯源工作具有重要的意义。     

一起由金黄色葡萄球菌引起幼儿园食源性疾病暴发事件的病原学分析

目的 对一起疑似为金黄色葡萄球菌导致的幼儿园食源性疾病暴发事件进行葡萄球菌肠毒素检测,结合病原学分析,为明确食源性疾病诊断提供依据。方法 对分离自某起食源性疾病暴发事件的6株金色葡萄球菌分离株进行生化鉴定,采用微孔板酶联免疫法(enzyme-linked immunosorbent assay, ELISA)进行肠毒素型别检测,并应用脉冲场凝胶电泳(pulsed field gel electrophoresis,PFGE)法对菌株进行分子分型,应用BioNumerics软件对不同来源的菌株进行比对,分析菌株之间的相关性。结果 34份样品检出6株金黄色葡萄球菌,肠毒素检测均为阳性,其中3株(分别来源于患者呕吐物、患儿粪便、切菜台)检出SEA、SEC、SEE等3种肠毒素,2株(分别来源于食物样本、生活老师粪便)检出SEE 1种肠毒素,1株(来源于食物样本)检出SEA、SEC等2种肠毒素。PFGE分型结果显示,1株来源于患者呕吐物的株菌和1株来源于切菜台的菌株具有高度同源性(96.8%)。结论 本起食源性疾病暴发事件由具有肠毒素的金黄色葡萄球菌污染切菜台导致,葡萄球菌肠毒素检测对明确食源性疾病的病因十分重要,PFGE分型技术有助于食源性疾病的溯源分析。关键词: 金黄色葡萄球菌; 肠毒素; 食源性疾病     

2013-2017年西安市食物中毒中金黄色葡萄球菌分型分析

  目的  了解2013-2017年西安市食物中毒中分离的金黄色葡萄球菌（staphylococcus aureus,S.aureus）分子流行特点。  方法  通过脉冲场凝胶电泳（pulsed field gel electrophoresis,PFGE）、多位点序列分型（multilocus sequence typing,MLST）和spa分型方法对八起食物中毒标本的26株金黄色葡萄球菌进行分子分型,运用聚合酶链式反应（polymerase chain reaction,PCR）检测相关肠毒素基因。  结果  26株S.aureus PFGE可分为7种型别（A-G）;MLST可分为3种序列型（ST6,ST5和ST59）。其中,ST6占65.38%,为主要型别;spa可分为4种型别（t437,t701,t11363和t5923）,优势型别为t701,占65.38%。肠毒素基因检测中,sea基因占76.92%（20/26）;sed-seg基因占23.08%（6/26）;其它肠毒素基因未检出。  结论  2013-2017年西安市食物中毒标本中分离培养的S.aureus的主要流行型别为ST6-t701-sea,并首次检出含有seg新型肠毒素基因。PFGE、MLST和spa分型三种方法相结合全面的了解菌株的分子流行特点。     

Prevalence of enterotoxin and toxic shock syndrome toxin genes in Staphylococcus aureus isolated from milk of cows with mastitis

Staphylococcus aureus isolated from milk of cows with mastitis were evaluated for the prevalence of 16 enterotoxin genes (sea-see and seg-seq) and toxic shock syndrome toxin gene (tsst-1). Of 78 S. aureus examined, 73 (93.6%) were positive for one or more enterotoxin genes and these were divided into 36 groups by the presence of different enterotoxin genes. Enterotoxin genes including sen (84.6%), sem (71.8%), sei (60.3%) and sed (52.6%) were found frequently, while seg (24.4%), seq (16.7%), seo (12.8%), and seb (1.3%) were found at lower frequencies. Toxic shock syndrome toxin (tsst-1) gene was detected in 20 (25.6%) isolates and was always found in combination with other enterotoxin genes. The majority (88.5%) harbored more than one enterotoxin gene in different combinations. Eight S. aureus isolates (10.3%) were positive for sed, sei, sem, and sen; six (7.7%) possessed sed, seg, sei, sem, sen, and tsst-1; five (6.4%) had sei, sem, and sen; and four (5.1%) had sei, and sen. One isolate was positive for seb along with other SE genes including sed, seh, sem, sen, seq, and tsst-1. None of the isolates carried other enterotoxin genes (sea, sec, see, sej, sek, sel, and sep). PFGE profiles revealed 15 distinct pulsotypes among the 78 S. aureus isolates evaluated. PFGE and enterotoxin gene profiles did not match with each other because a single pulsotype carried different combinations of enterotoxin genes. The majority of S. aureus isolated from milk of mastitic cows carried newly described SE genes sem, sen and sei along with classical SE genes, sed and tsst-1. This is the first report describing the high prevalence of newly described enterotoxin genes, sem and sen in S. aureus from bovine mastitis. The high prevalence of enterotoxin genes and tsst-1 in S. aureus may be important as it is relevant to udder pathogenicity and food hygiene.     

Molecular subtyping of Staphylococcus aureus from an outbreak associated with a food handler

On 6 May 2000, a staphylococcal food poisoning outbreak occurred at a high school, affecting 10 of the 356 students who attended the breakfast. Twenty-seven Staphylococcus aureus isolates, producing enterotoxin A (SEA), SEB-, or non-SEA-E, were recovered from 7 patients, 2 food handlers and left-overs. To investigate the outbreak, we genotyped the isolates by using pulsed-field gel electrophoresis (PFGE) and three PCR-based techniques: inter-IS256 PCR typing, protein A gene (spa) typing, and coagulase gene restriction profile (CRP) analysis. Our results show that PFGE was the most discriminatory technique, whereas the three PCR-based techniques were insufficient in the discriminatory power to distinguish the S. aureus isolates from the outbreak. Based on the enterotoxin-producing types and the results of genotyping, three distinct types of strains (A1111, B2221 and N3221) were designated. Both the A1111 and B2221 strains were found in the specimens from the patients and a hand lesion of a food handler, suggesting that the source of contamination for the outbreak was most likely originated from a food handler.     

应用实时荧光PCR技术检测金黄色葡萄球菌肠毒素基因

目的:建立一种简便、特异的荧光PCR检测方法,用于金黄色葡萄球菌肠毒素的检测。方法:按金黄色葡萄球菌SEA～SEE型肠毒素基因序列设计引物,在普通PCR检测体系中,加入SYBR Green I荧光染料,建立荧光PCR检测体系。结果:46株金黄色葡萄球菌中检出22株携带肠毒素基因,阳性率为47.83%。以SEA、SEB检出率较高,分别为31.82%和27.27%;不同来源的分离株携带肠毒素基因的比例不同,同时携带2种及以上毒素基因的菌株占27.27%。结论:荧光PCR检测金黄色葡萄球菌肠毒素的方法具有快速、敏感、特异性高的特点,适用于肠毒素基因的分型与分布的研究,适合基层疾控部门使用。     

Phenotypic characterization and prevalence of enterotoxin genes in Staphylococcus aureus isolates from outbreaks of illness in Chengdu City

Staphylococcus aureus produces a spectrum of enterotoxin that is recognized as the main reason for causing staphylococcal food poisoning. The aim of the current study was to investigate the phenotypic characteristics and enterotoxin genotypes of S. aureus isolated from food poisoning sufferers. On the basis of the amplification of 16S rRNA and nuc gene specific to S. aureus assay and the phenotype (hemolytic activity, thermal stable nuclease [Tnase] test, and biofilm formation), all isolates were identified as S. aureus. To genotypically characterize S. aureus isolates, genes encoding staphylococcal enterotoxin (sea, seb, sec, sed, see, seg, seh, sei, sej, sek, sem, sen, ser, and seu) were investigated by using polymerase chain reaction technique. The results showed that the eight isolates of S. aureus had different enterotoxin genotypic characteristics, which was the main cause of food poisoning. One isolate contained 10 enterotoxin genes, and the other 7 isolates carried 3 or more enterotoxin genes. The frequency of the newly identified enterotoxin genes (seg-seu) was higher than classical genes (sea-see). Overall, multi-gene detection rates were 75% (for sek, ser, and seu); 50% (for sea and sem); 37.5% (for sen, seg, and sei); and 12.5% (for seb, sec, sed, and sej), respectively. The see and seh gene were not detected in any isolates. The current study provided the exact distribution of enterotoxin genes in eight S. aureus strains from food poisoning sufferers, which indicated that the pathogenicity of the newly identified enterotoxin should be highlighted. The need for prevention of food poisoning occurrences caused by enterotoxin of S. aureus should be reinforced.     

食源性金黄色葡萄球菌流行特征、产肠毒素特性及耐药性研究

目的：了解食源性金黄色葡萄球菌流行特点及产肠毒素特性及其耐药性，为制定HACCP以防止金黄色葡萄球菌的食源性疾病提供依据。方法：采集8类食品1243件分离金黄色葡萄球菌并进行产肠毒素A—E试验及药物敏感试验。结果：从生肉、熟食、生奶、及水产品中分别分离出40株（10．96％）、12株（3．13％）、34株（16．27％）及1株（1．12％）金黄色葡萄球菌，相应的产肠毒素率分别达到52．50％、58．33％、61．76％、0．oo％；对青霉素、四环素、凝固酶阴性苯唑西林耐药率分别高达93．10％、49．43％、37．93％；79．31％菌株对2种以上的药物多重耐药，最多耐药种类达7种，表现出30种耐药谱。结论：扬州市食品中存在着较严重的金黄色葡萄球菌潜在的危害，主要来源于生肉、生奶及熟食；食源性金黄色葡萄球菌产毒素能力较强。金黄色葡萄球菌对多种药物的耐药性提示安全应用治疗药物的重要性。     

多重PCR技术在检测食源性金黄色葡萄球菌 肠毒素基因中的应用

摘要:目的　金黄色葡萄球菌A、B、C、D和E型肠毒素是引起食源性疾病的主要类型,采用多重PCR技术分析食源性金黄色葡萄球菌分离株肠毒素基因携带状况。方法　以19株食源性金黄色葡萄球菌分离株为研究对象,利用多重PCR技术对核酸酶基因以及肠毒素A、B、C、D和E型基因进行检测。结果　19株菌中均检出金黄色葡萄球菌特异性核酸酶基因,与传统菌落分离培养鉴定结果一致;9株菌含肠毒素SEA基因,1株同时含SEA和SEB基因,没有检测到肠毒素C、D和E基因。结论　在19株食源性金黄色葡萄球菌分离株中,10株携带肠毒素基因,以携带肠毒素A基因为主。     

MRSA分子流行病学研究 FREE

目的 采用脉冲场凝胶电泳（PFGE）分型及多重聚合酶链反应（PCR）SCCmec分型技术对耐甲氧西林金黄色葡萄球菌（MRSA）进行基因分型,以了解医院感染MRSA的流行情况。方法 收集天津市南开医院2007年1月-2008年12月住院患者送检标本分离的MRSA 40株,进行耐药谱分析、PFGE分型和SCCmec分型。结果40株菌耐药谱高度相似,可被分为A-E 5个PFGE型,其中A型21株,B型8株,C型4株,D型6株,E型1株;SCCmec分型：Ⅰ型1株,Ⅲ型33株,Ⅳ型1株,V型5株。通过对医院内菌株发生的时间和空间分析,明确了感染的发生和传播情况,即在2007年1月-2008年12月存在MRSA散发感染。结论 PFGE可有效鉴定菌株间的亲缘关系,SCCmec分型对是否为医院感染菌株有提示作用,两种分型方法相结合可更好地鉴定流行菌株的特征,为医院感染的监控提供依据。     

Enterotoxin and toxic shock syndrome toxin-1 production of methicillin resistant and methicillin sensitive Staphylococcus aureus strains

In this study the production of enterotoxin A-D and toxic shock syndrome toxin-1 (TSST-1) of 181 methicillin resistant (MRSA) and 100 methicillin sensitive (MSSA) Staphylococcus aureus first isolates from different patients was investigated. All the MRSA- and MSSA isolates in the study were collected in a period between 1993 and 1995 from specimens sent from 11 different acute care hospitals in the greater Düsseldorf area. As far as possible the isolates were matched according to ward and hospital. The isolates were collected in the same time period and matched for specimen from which isolated. Furthermore, only first isolates were analysed in both groups. No significant difference in the production of toxin of any type between MRSA and MSSA could be detected (51 and 40% respectively). When the individual toxins were analysed, again no significant difference between MRSA and MSSA was demonstrable (enterotoxin production by MRSA 40% and MSSA 36%, and TSST-1 16% and 8% respectively). Despite this, a slight tendency for MRSA to produce enterotoxin A and B and for MSSA to produce enterotoxin C was observed. In addition, generation of TSST-1 by both groups was independent of enterotoxin A-D production. Interestingly, no increase in the proportion of TSST-1- or enterotoxin-producing MRSA and MSSA isolates was observed in strains isolated from blood cultures from patients with a clinical diagnosis of sepsis. Genotypical pulsed-field-gel-electrophoresis (PFGE) and phenotypical (bacteriophage typing, lysotyping) characterization of the 181 MRSA isolates resulted in 28 different PFGE patterns (of which 19 were toxin producers) and 22 lysotyping groups (18 of which produced toxin). In summary, the investigated clinical S. aureus isolates showed no difference in their ability to produce toxin and this was independent of their sensitivity to methicillin.