基于环介导等温扩增法(LAMP)检测上海市售贝类产品中副溶血性弧菌的毒力菌株

基于环介导等温扩增法(LAMP)对上海市8-10月市售贝类产品中副溶血性弧菌毒力菌株(tdh和trh毒力基因)进行检测分析,共检测贝类样品180份,6个常规品种,实验同时采用PCR测定方法进行对比。结果表明,含tdh和trh毒力基因的副溶血性弧菌在市售贝类中的检出率分别是12.77%和11.66%,PCR的分析结果为11.11%和7.78%。对分离的毒力菌株进行血清型分型后发现了2株O3:K6型副溶血性弧菌,其中1株为毒力基因双阳性菌(tdh+/trh+)。2株O3:K6型副溶血性弧菌的PFGE条带型相似度较高(相似度90%)。这些结果表明上海市售贝类产品中副溶血性弧菌毒力菌株存在一定的污染,应引起足够重视。双阳性O3:K6型副溶血性弧菌的出现值得关注,应对各血清型菌株尤其是O3:K6型副溶血性弧菌的流行情况加强监测。PCR检测结果对比分析表明,LAMP方法适用于贝类产品中副溶血性弧菌毒力菌株的检测分析。     

烟台地区食源性疾病中副溶血性弧菌的病原特征及溯源分析

目的了解烟台地区引发食物中毒的副溶血性弧菌分离菌株的主要血清型、抗生素耐药情况、致病力的强弱以及传播流行趋势。方法对2017～2019年11起食物中毒爆发事件中分离的14株副溶血性弧菌进行血清分型;采用微量肉汤稀释法进行药敏试验;采用PCR技术检测毒力基因不耐热溶血素(tld)、耐药直接溶血素(tdh)和溶血相关溶血素(trh);采用脉冲场电泳(pulse-field gel electrophoresis,PFGE)进行分子分型的溯源分析。结果引发烟台地区食物中毒爆发的副溶血性弧菌血清型多样,但以O3:K6型为主(28.6%),分离菌株中11株(78.6%)表现为tdh阳性和trh阴性,3株(21.4%)未携带tdh和trh基因,对头孢唑啉(71.4%)、多粘菌素E(57.1%)、美罗培南和阿莫西林/克拉维酸(7.1%)均出现耐药情况,对氨苄西林(21.4%)、多粘菌素B(14.3%)为中度敏感,耐药谱显示没有出现对3种以上同时耐药的情况,但是42.9%的分离菌株对2种同时耐药情况。结论烟台地区引发食物中毒爆发的副溶血性弧菌以O3:K6型为主,主要携带tdh,对头孢唑啉耐药和多粘菌素耐药普遍存在,具有聚集性爆发的风险,因此加强腹泻病的监测,充分利用国家致病菌识别网集中进行本地区分离菌株的病原特征研究是防控食物中毒爆发的有效手段。     

两起副溶血性弧菌食物中毒血清学溯源结果分析

目的 对两起副溶血性弧菌(VP)引起的食物中毒进行血清学溯源,分析可疑食品和病人样品中菌株血清型之间的关系.方法 依据GB/T4789.7-2008方法,对检出的VP做血清分型、溶血素试验;PCR扩增VP直接耐热溶血素基因(tdh)、tdh相关溶血素基因(trh)和毒素调控基因(toxR).结果 通过增加样品中可疑菌落数量的鉴定,两起食物中毒共检出9种VP血清型,主要有O3∶K6型13株,O2∶K28型6株,O1∶K56型2株,其它各1株;两起食物中毒中分离的27株VP有17株tdh基因检测阳性,与溶血试验结果一致.结论 增加可疑菌落数鉴定,有助于VP食物中毒的溯源;虽然O3∶ K6血清型是引起食物中毒的主要病原菌,但不同样品来源的VP血清型呈现多样性;副溶血性弧菌tdh基因检测等同于溶血试验来鉴定VP致病性.     

一起多型别副溶血性弧菌引起农村喜宴食物中毒的溯源分析

目的通过对腹泻患者肛拭子标本和外环境样品的病原菌检测,利用脉冲场凝胶电泳(PFGE)分子分型技术对一起多型别副溶血性弧菌引起农村喜宴食物中毒原因开展溯源分析。方法对采集的共20份肛拭子标本和外环境样品进行病原学检测,并对检出的副溶血性弧菌进行血清分型、PFGE分型及tdh、trh、tlh、toxR基因检测。结果 20份样品/标本中15份检出副溶血性弧菌,11株为tdh+trh-tlh+toxR+基因型,4株为tdh-trh-tlh+toxR+基因型。其中12份患者肛拭子标本中有9份检出副溶血性弧菌,7份为O3∶K6血清型,2份为O2群;3份墩板涂抹样品中有2份检出O3∶K6血清型,1份为O1群;3份剩余食物样品中有2份检出副溶血性弧菌,分别为O1群和O10群;厨师肛拭子标本检出O11群副溶血性弧菌;PFGE聚类分析显示7份患者肛拭子标本和2份墩板涂抹样品中的O3∶K6型副溶血性弧菌带型高度同源,剩余食物来源的副溶血性弧菌核酸降解未能显示PFGE条带。结论本次事件是可疑食物交叉污染切菜墩板引起的以O3∶K6血清型为主的多种血清型别副溶血性弧菌的食物中毒。     

江苏及周边地区副溶血性弧菌分子流行病学特征研究

目的了解江苏及周边地区副溶血性弧菌的分子流行病学特征。方法对2010年江苏及周边地区食物中毒事件中分离的、经API生化鉴定的63株副溶血性弧菌进行毒力基因(tdh、trh)测定、血清学分型和脉冲场凝胶电泳(PFGE)分型。结果 tdh阳性率79.4%(50/63),trh阳性率1.6%(1/63);病人株血清型以O3∶K6为主(26/57),食品株以O1∶KUT为主(4/6);PFGE分型显示A-E群间相似度大于60%。结论江苏及周边地区中不同地区的副溶血性弧菌具有高度同源性,该地区副溶血性弧菌引起的食物中毒可能具有相同的污染源。     

副溶血弧菌临床分离株的血清分型及毒力基因分析

副溶血弧菌是一种以鱼类、贝类等海产品为主要传播载体的嗜盐性食源性致病菌。近年来,由该菌引起的食物中毒病例呈明显上升趋势。本文采用11种O抗体和8种K抗体,针对来自舟山、宁波、上海浦东和金山的58株副溶血弧菌食物中毒临床分离株(2006-2009年)进行血清型及毒力基因分析。菌株血清分型结果表明:上述4地致病性副溶血弧菌以O3群和K6型为主,共分为22个血清型。其中主要流行株为O3:K6型,占67.72%(107/158);其次为O1:K6,O3:K68,O1:K25,O1:K56,O2:K3等。菌株的来源地分析结果表明:上海金山临床分离株的血清型最复杂,表现出明显的多样性;舟山血清型较单一。利用PCR方法对上述158株副溶血弧菌的主要毒力因子——耐热溶血素基因(tdh)和耐热相关溶血素基因(trh)进行检测,结果发现,其阳性率分别为96.84%(153/158)和3.80%(6/158)。其中(tdh+/trh+)6株,(tdh+/trh-)147株和(tdh-/trh-)5株。结论:杭州湾4地致病性副溶血弧菌流行菌株血清型为O3:K6,且不同地区血清型差异较大;除极少数菌株外,绝大部分菌株都携带tdh或者trh。     

珠江三角洲地区副溶血性弧菌遗传多样性和致病性相关研究

副溶血性弧菌(Vibiro parahaemolyticus)是一种常见的食源性致病菌,本文研究了珠江三角洲地区副溶血性弧菌的遗传多样性。从54株副溶血性弧菌出发,研究了它们的API20E生化反应、抗生素耐药性、O抗原血清型,进行了ERIC-PCR分子分型,并检测了两种毒力基因tdh和trh的分布。54株副溶血性弧菌可被分为6个生化反应类群,主要类群为Biochem-A;菌株对萘啶酮酸、环丙沙星、氯霉素均不耐药,而对氨苄青霉素耐药率最高,耐药率0.88;O抗原血清型分别为O1、O2、O3、O4、O5、O6、O8、O10、O11,O2为主要血清型,O3为临床主要血清型;ERIC-PCR分子分型将54株菌分成47个型别,ERIC-PCR图谱相似性大于0.80的类群有12个,没有明显的优势类群;有12株副溶血性弧菌为tdh阳性,阳性率为0.22,其中10株为临床来源菌株;有2株副溶血性弧菌为trh阳性,阳性率为0.04,均为食品分离株。珠三角地区食品和临床来源的副溶血性弧菌具有丰富的遗传多样性。     

北京市水产品污染与感染病例中副溶血性弧菌血清型和毒力基因型的比较研究

目的对北京市夏季市售水产品污染与感染病例中副溶血性弧菌血清型和毒力基因型进行比较研究,为评估食品安全风险监测的目的与意义提供思路,为北京市水产品副溶血性弧菌污染与临床感染病例的关联性研究提供技术支持。方法对采集的水产品样品和哨点医院腹泻患者粪便样本进行副溶血性弧菌的分离鉴定,采用血清玻片凝集法对分离出的副溶血性弧菌进行血清分型,PCR方法检测菌株的tlh、tdh、trh基因。结果 2014年7~9月共采集水产品样品164份,检出副溶血性弧菌80份,总污染率为48.78%;其中淡水产品污染率为38.78%(19/49),平均菌量浓度为66.63 MPN/g;海水产品污染率为53.04%(61/115),平均菌量浓度为38.14 MPN/g。80株副溶血性弧菌分属于9个血清群,其中O2群28株,占35.00%,O1群11株,占13.75%,O5群10株,占12.50%。80株菌tlh基因均为阳性,只有1株菌携带tdh毒力基因,所有菌株trh毒力基因均为阴性。哨点医院腹泻病人粪便样本中分离鉴定副溶血性弧菌21株,血清型O3∶K6占61.90%(13/21),O4:K8占28.57%(6/21);毒力基因型tdh(+)/trh(-)占95.24%(20/21),tdh(-)/trh(-)占4.76%(1/21)。结论来源于食品样品的副溶血性弧菌绝大部分不具备致病性,而导致消费者腹泻的副溶血性弧菌绝大部分携带致病性毒力基因,表明目前的食品安全风险监测结果不能作为评估副溶血弧菌导致的食源性疾病暴发和散发的依据。     

食品来源副溶血性弧菌毒力基因分布及分子分型研究

分析食品中分离获得的副溶血性弧菌主要毒力基因的分布特征、血清型及分子分型特点,为食源性副溶血性弧菌感染暴发的早期预警和疫情溯源提供实验室数据。提取40株副溶血性弧菌基因组,分别用PCR检测VcrD1、Vp1680、VopP和VcrD2基因,荧光PCR检测tdh、trh和tlh基因。对40株菌株进行血清型分型并采用脉冲场凝胶电泳(PFGE)对其进行分子分型。利用BioNumerics软件对图谱进行聚类分析,建立PFGE分子分型数据库。40株菌株均未检测到tdh、trh、Vop P和VcrD2基因,tlh、VcrD1和Vp1680基因阳性检测率为100%。40株菌株中含有16种血清型,其中9株未分型,主要血清型为O_2∶K_(28)和O_5∶K_(17),血清型分布呈多样性。40株菌株共获得39个不同的PFGE带型,PFGE呈现遗传多样性,无优势带型。本实验食品环境中分离的副溶血性弧菌未发现毒力因子,副溶血性弧菌血清型呈分散趋势且菌株之间亲缘关系较远。     

一起由副溶血性弧菌引起的聚集性腹泻事件的病原特征分析

目的 对2021年湖州市南浔区一起聚集性腹泻事件分离的副溶血性弧菌进行病原特征分析，为及时采取控制措施提供依据。方法 通过询问患者、医院核查等方法搜索到疑似病例59例，对采集的29份厨师、服务员和病例的肛拭子标本以及18份留存餐食进行病原学检测，采用血清学鉴定、脉冲场凝胶电泳（PFGE）、多重荧光聚合酶链式反应和微量肉汤稀释法分别对副溶血性弧菌分离株进行血清分型、分子分型、tlh/tdh/trh毒力基因检测和药敏试验。结果 从3份厨师和6例病例的肛拭子标本中分离到副溶血性弧菌，检出率为15.25%；分离株血清型均为O10:K4。所有菌株tlh、tdh基因均为阳性，trh基因均为阴性。9株分离株仅对氨苄西林耐药，对其他12种抗生素均敏感。经PFGE聚类分析，菌株间相似系数为100.0%，厨师分离株和病例分离株带型一致。结论 这是一起由O10:K4副溶血性弧菌引起的聚集性腹泻事件，分离株均携带tdh毒力基因，分离株之间的同源性高。     

Rapid detection of Vibrio parahaemolyticus strains and virulent factors by loop-mediated isothermal amplification assays

A loop-mediated isothermal amplification (LAMP) method for rapid detection of the foodborne Vibrio parahaemolyticus strains and related virulent factors had been developed and evaluated in this study. Six primers, including outer primers, inner primers, and loop primers, were specially designed for recognizing 8 distinct sequences on 3 target genes, which were tlh, tdh, and trh. The detection limits were found to be 100, 100 fg, and 1 pg DNA/tube for tlh, tdh, and trh, respectively. Application of LAMP assays were performed on 368 foodborne V. parahaemolyticus strains, the sensitivities of LAMP assays for the tlh, tdh, and trh were 100, 95.6, and 96.4%, and the negative predictive values (NPV) were 100, 84.7, and 93.1%, respectively; with a 100% specificity and positive predictive value (PPV) for all 3 target genes.     

Presence of pathogenic Vibrio parahaemolyticus strains in mussels from the Adriatic Sea, Italy

Samples of mussels (Mytilus galloprovincialis) collected from approved coastal sites located on the Adriatic Sea (Central Italy) were examined for the presence of Vibrio parahaemolyticus and the occurrence of pathogenic strains. The isolation of the micro-organisms was performed using a standard method. A biochemical protocol was applied for the identification of the isolates. Polymerase chain reaction (PCR) was used to confirm the identification of the strains and to detect the tdh and trh genes. The Kanagawa phenomenon was assayed as phenotypic marker of thermostable direct hemolysin (TDH) toxin. The urease activity was assayed as phenotypic marker of trh gene. The protease activity and the cytotoxicity of strains were examined to identify other potential virulence factors. Thirty-five V. parahaemolyticus strains were isolated out of 144 samples. The tdh and trh genes were in one and three isolates, respectively. All strains, independent of the presence of tdh and trh genes, showed protease activity and cytotoxicity. Due to the occurrence of pathogenic V. parahaemolyticus, the potential risk of eating raw or undercooked mussels is envisaged.     

Isolation and molecular characterization of Vibrio parahaemolyticus from fresh, low-temperature preserved, dried, and salted seafood products in two coastal areas of eastern China

A total of 1293 seafood samples from fishing farm, retail markets, restaurants and cooking rooms of hotels in Jiangsu province and Shanghai city of China were collected and analyzed for the prevalence of Vibrio parahaemolyticus during July to October in 2007. Two hundred and fifty one isolates of V. parahaemolyticus were identified, of which 8 isolates were positive for tdh and 2 were positive for trh gene. Three tdh positive isolates were identified from low-temperature preserved seafood samples and 5 isolates from fresh seafood samples, of these tdh positive isolates, 3 were positive in ORF8-PCR test. The genetic diversity among V. parahaemolyticus isolates was assessed using random amplified polymorphic DNA (RAPD)-PCR and the results showed that there were 33 different genetic patterns that were clustered into nine groups (groups A to I) at 82% similarity level. About 31.9% of the isolates belong to type III9d that were widely distributed in fresh, iced, frozen, dried and salted seafood samples. Seven tdh positive isolates belonged to group A and one belonged to group C, 2 trh positive isolates were type I10d belonging to group F, which was identical to that of reference strains isolated from patients. This study demonstrated genetic variability within V. parahaemolyticus isolates from seafood in Chinese markets and confirmed the presence of toxigenic V. parahaemolyticus not only in fresh but also in iced and frozen seafood products indicating that low-temperature preserved seafood might be also a vehicle for transmitting pathogenic V. parahaemolyticus.     

2016年江苏省淡水养殖环节常见致病性弧菌污染状况调查

目的 了解江苏省淡水动物性水产品养殖环节中常见弧菌(副溶血性弧菌、霍乱弧菌等)的污染程度。 方法 参照《2016年国家食品安全风险监测淡水动物性水产品养殖环节中常见弧菌专项监测工作手册》进行样品的采集和检测。 结果 从淡水养殖场中采集水产品、水体、水底沉积物样品共507份, 检出副溶血性弧菌17株(3.4%); 霍乱弧菌4株(0.8%); 溶藻弧菌及创伤弧菌均未检出。检出的副溶血性弧菌均不携带毒力基因(trh-;tdh-), 主要血清型为O5:H7(41.2%), 主要耐受的抗生素为头孢唑林(94.1%), 其中3株PFGE带型相似性系数100%; 4株霍乱弧菌均为非O1/O139群, 未发现耐药株。结论 江苏省淡水养殖环节中副溶血性弧菌环境污染程度低, 但在淡水养殖环节中检出致病性弧菌, 提示淡食用水产品依然可能存在引发食物中毒的风险。     

Incidence and Molecular Typing of Vibrio parahaemolyticus from Tiger Shrimp Culture Environments along the Southwest Coast of India

Vibrio parahaemolyticus is one of the most prevalent food-borne pathogens along the southwest coast of India, where marine foods are frequently consumed. Shrimp (Penaeus monodon) and environmental samples were collected from aquaculture farms located in and around Cochin. Confirmation of the biochemically identified strains with species-specific toxR gene and detection of virulent genes viz., tdh and trh was performed by polymerase chain reaction (PCR). The phenotypic markers for the presence of tdh and trh genes were assayed by Kanagawa phenomenon and urease activity, respectively. Protease activity was examined to identify other potential virulence factors. After phenotypic characterization of bacterial strains fingerprinting of genomic DNA was carried by various typing methods, viz., random amplified polymorphic DNA (RAPD), enterobacterial repetitive intergenic consensus sequence (ERIC), repetitive extragenic palindromic sequence (REP), and ribosomal gene spacer sequence (RS) PCR methods to assess the genetic diversity within the isolates. Eighteen percent of the samples were found positive for the incidence of V. parahaemolyticus by biochemical protocols and toxR (368 bp) targeted PCR. PCR analyses revealed 1% of the samples positive for tdh (269 bp) and trh (500 bp) gene. RAPD analysis revealed clustering of toxigenic strains into a single group. Cluster analysis revealed the conglomeration of isolates into two, five, and seven major groups using RS, ERIC, and REP PCR methods, respectively. RS PCR generated fewer amplified bands compared to REP and ERIC PCR methods, thus giving scope for higher discrimination. Moreover, RS PCR patterns were more discernible visually from other patterns, suggesting RS PCR as a considerably practical method for routine use.     

Detection and molecular characterization of Vibrio parahaemolyticus isolated from seafood harvested along the southwest coast of India

The levels of total and tdh⁺ Vibrio parahaemolyticus were estimated in 83 seafood samples from southwest coast of India by colony hybridization. Conventional enrichment and isolation technique was also used to study the prevalence. Polymerase chain reaction (PCR) was performed on bacterial cell lyates for detection of total and pathogenic V. parahaemolyticus by amplification of specific genes. Of 83 samples tested, V. parahaemolyticus could be detected in 74 (89.2%) samples and tdh⁺ V. parahaemolyticus in 5 (6.0%) samples by colony hybridization. V. parahaemolyticus was detected in 68 (81.9%) of 83 samples after 18 h of enrichment by PCR, and isolated from 63 (75.9%) of 83 samples by conventional isolation. The virulence genes tdh and trh could be detected in 8.4% and 25.3%, respectively, in the sample enrichment broths by PCR. Use of colony hybridization following enrichment to achieve sensitive detection of tdh⁺ V. parahaemolyticus in seafood was evaluated using another set of 58 seafood samples. Thirty pathogenic V. parahaemolyticus strains isolated during the study were screened by PCR for genetic markers to be specific for the detection of the pandemic clone. Results of this study suggest that the GS-PCR may serve as a reliable genetic marker for the pandemic clone of V. parahaemolyticus.