一起副溶血性弧菌致食源性疾病暴发疫情的流行病学调查

目的查明S饭店群体性食源性疾病暴发的原因,找出可疑食物,防止类似事件再次发生。方法制定病例定义,开展病例搜索,对病例信息进行描述性分析和病例对照研究,并采集病例粪便标本、剩余食物样品等进行实验室检测。结果暴发病例162例,罹患率22.2%(162/730),临床表现主要为腹泻(100%,162/162)、腹痛(96.3%,156/162)、乏力(77.2%,125/162)、恶心(68.5%,111/162)、呕吐(53.1%,86/162),平均潜伏期为12 h,病例对照结果显示扇贝(OR=1.74,95%CI=1.00~3.02)和牛仔骨(OR=2.87,95%CI=1.38~5.99)为可疑危险因素,34份粪便标本、1份甲鱼切块和1份砧板上检出副溶血性弧菌。结论本次事件为一起副溶血性弧菌污染宴席食物导致群体性食源性疾病暴发事件,交叉污染和食品未加工熟透很可能是发病的主要原因,建议餐饮机构要规范操作流程,加强自我监管,防止此类事件再次发生。     

同时检测海产品中副溶血弧菌和霍乱弧菌的合检方法评价研究

目的建立水产品中副溶血弧菌和霍乱弧菌合检方法,并对合检方法对比进行评价。方法利用副溶血弧菌和霍乱弧菌阳性菌株,制备纯菌液、不同浓度梯度的人工污染样品。通过对30份纯菌液、30份人工污染样品和250份实际样品的检测,将建立的合检方法与行业标准(SN/T 1022-2010进出口食品中霍乱弧菌检验方法和SN/T 0173-2010进出口食品中副溶血性弧菌检验方法)进行比较,对合检方法进行效果评价。结果实验结果表明副溶血弧菌和霍乱弧菌合检方法,与行业标准检测结果完全一致。结论该方法可靠,对实验仪器和操作人员的要求低,具有良好的实用性,适合基础检测实验室。     

海产品中副溶血性弧菌防控技术研究进展

副溶血性弧菌是沿海省份引起食物中毒的一种主要的食源性致病菌,因此必须采取有效措施预防控制副溶血性弧菌在海产品中的污染。随着近些年人们对副溶血性弧菌防控技术研究的深入,总结出了大量的经验,开发出了许多新方法。本文主要从监管、净化、物理、化学及生物控制等方面对现有的副溶血性弧菌防控技术进行系统阐述,旨在为这些方法的应用和发展提供参考,进而为海产品的质量安全控制提供有利保障。     

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.     

海产品副溶血性弧菌控制研究进展

副溶血性弧菌是引起海产品食物中毒的主要致病因子。文章拟从物理、化学和生物三个方面对海产品中的副溶血性弧菌现有的控制措施进行概述,并展望未来的发展方向,以期为相关技术的应用和后续研究提供借鉴。     

大连市不同海产品中副溶血性弧菌污染的健康风险分级研究

目的对大连市不同海产品中副溶血性弧菌(Vibrio parahaemolyticus, VP)污染的健康风险进行分级和评价。方法 2017年1～10月,在大连市10个县市区分层随机采集4类944份常见海产品。依据GB 4789.7—2013《食品安全国家标准食品微生物学检验副溶血性弧菌检验》进行海产品中VP定量检测,使用实时荧光定量聚合酶链式反应(PCR)方法进行VP毒力基因检测;采用食物频率法获得各类海产品的消费量数据;利用专家咨询法获得海产品交叉污染和烹调习惯参数;采用快速微生物定量风险评估(sQMRA)方法,对4类海产品中致病性VP的健康风险进行分级。结果甲壳类导致人体感染致病性VP发病风险和年发病例数最高,分别为3.5×10~(-6)和2 799.3例。鱼类导致人体感染致病性VP发病风险和年发病例数居第二位,分别为1.1×10~(-6)和1 304.4例。海产品导致人群VP发病的主要途径为交叉污染。结论应关注大连市甲壳类中VP对人群的致病风险,重点控制海产品在加工处理过程中VP的交叉污染。     

副溶血性弧菌食源性疾病暴发分离株的血清型、核糖型及毒力基因研究

目的 了解目前食源性副溶血性弧菌暴发菌株在上海地区的血清型分布、毒力相关基因的携带情况,及Ribo分型.方法 收集上海市27起食源性副溶血性弧菌暴发事件分离的98株副溶血性弧菌,采用副溶血性弧菌分型血清对所收集的菌株进行血清学分型.利用双重PCR方法扩增tdh,trh两个重要的毒力基因以了解其毒力基因的携带情况.应用Riboprinter系统检测上述菌株的酶切片段杂交多态性.结果 引起27起暴发的98株副溶血性弧菌分为11个血清型,5个Ribo型.优势血清型为03:K6,优势Ribo型为vp-EcorI-002型.全部分离菌株均携带toxR基因,97株携带tdh基因,1株携带trh基因.有多起暴发事件分离到多个血清型的副溶血性弧菌.结论 血清分型和PCR方法检测毒力基因,是副溶血性孤菌暴发事件实验室诊断的有用方法.对于从一起暴发事件中检出的不同血清型的副溶血性弧菌需要确认各菌株与暴发的关系.     

Evaluation of antibacterial resistance in Vibrio strains isolated from imported seafood and Italian aquaculture settings

Ninety-two Vibrio strains isolated over a period of 9 years from different sources (national and imported fishery products, shellfish, seawater from aquaculture settings, etc.) and belonging mostly to two species relevant for human health and fish pathology, V. parahaemolyticus and V. alginolyticus, were tested for resistance to different antibacterials using the standard disk diffusion test (five antibacterials: ampicillin, sulfamethoxazole, tetracycline, trimethoprim/sulfamethoxazole and chloramphenicol) and the Minimum Inhibitory Concentrations method (six antibacterial families: β-lactams, tetracyclines, chloramphenicol, aminoglycosides, quinolones, trimethoprim/sulfamethoxazole) providing consistent results. The analysis performed by standard disk diffusion test showed resistance to ampicillin in 82% of the strains, whereas 7% were resistant to sulfamethoxazole, 3% to tetracycline, and 1% to trimethoprim/sulfamethoxazole; no strain was found to be resistant to chloramphenicol. The results obtained by the MIC tests, because of the absence of established breakpoints for some antibacterials, were not readily interpretable for all substances, but showed no statistically significant difference to the results obtained by the standard disk diffusion test. Beside these results, high MIC₉₀ values (128 μg ml⁻¹ and above) were obtained for kanamycin and streptomycin, indicating that high concentrations of these antibacterials were required to inhibit the growth of the strains. Strains exhibiting resistance or intermediate resistance to two or more antibacterials represented 15% of the total and included, beside resistance to ampicillin, resistance to sulfamethoxazole or, for V. parahaemolyticus isolates, tetracycline. Interestingly, differences in the mechanism of resistance to β-lactams were detected between the species V. parahaemolyticus and V. alginolyticus.     

Growth inhibition of Vibrio parahaemolyticus in seafood by tabletop dry ice cooler

Tabletop dry ice coolers (three types; dome model, cap model and tripod model), which are used in kitchens and hotel banquet halls to refrigerate fresh seafood, were investigated to determine whether growth of Vibrio parahaemolyticus was inhibited by their use. On TSA plates containing 1.8% NaCl and fresh seafood (fillets of squid, pink shrimp and yellowtail), V. parahaemolyticus (O3:K6, TDH+) inoculated at 4 to 5 log CFU/sample and left at ambient temperature (25 degrees C) grew by 1.0 to 2.8 orders in 4 hours. In contrast, with tabletop coolers no significant increase in viable count occurred in 3 to 4 hours, confirming that tabletop coolers inhibited the growth of V. parahaemolyticus. The temperature in each tabletop cooler was kept below 10 degrees C for 80 to 135 min, though the CO2 gas concentration in them remained high for only a short time (0 to 75 min). It was presumed that the refrigeration function mainly contributed to growth inhibition. Our results indicate that tabletop dry ice coolers are helpful for prevention of food-borne disease due to V. parahaemolyticus in food-service locations, such as kitchens and banquet halls.     

Characterization of Vibrio parahaemolyticus isolates obtained from foodborne illness outbreaks during 1992 through 1995 in Taiwan

Vibrio parahaemolyticus is an important foodborne pathogen in Taiwan and many other Asian countries. A total of 371 isolates of V. parahaemolyticus collected from patients involved in foodborne illness outbreaks in Taiwan from 1992 to 1995 were characterized. These isolates had typical biochemical characteristics and only 4% were urease positive. The most frequently isolated serovars were O5:K15 (18.5%), O4:K8 (16.2%), O3:K29 (12.5%), O1:K56 (8.3%), O2:K3 (6.5%), and O4:K12 (6.0%). Most of the isolates were susceptible to nalidixic acid, tetracycline, tobramycin, cephalothin, and gentamicin. About 10% of the isolates were resistant to seven or more antibiotics. Approximately 92.4% of these V. parahaemolyticus showed beta-hemolysis on Wagatsuma blood agar plate and approximately 62.1% of these isolates exhibited detectable amounts of thermostable direct hemolysin. Most of the isolates examined exhibited two copies of tdh genes on the 1.3- and 2.5-kb HindIII-digested chromosome fragments with several variations on other fragments. A pulsed-field gel electrophoresis (PFGE) subspecies typing scheme was used to analyze these domestic isolates and the O3:K6 strains from Japan, Korea, and Taiwan. Fifty seven patterns were differentiated with A, B, C, E, and H being the major domestic types (cumulatively 76% of isolates), while O3:K6 strains (PFGE type I), abruptly occurring since 1996, were genetically distant from the major domestic types.     

Antimicrobial resistance of Vibrio parahaemolyticus and Vibrio alginolyticus strains isolated from farmed fish in Korea from 2005 through 2007

The antimicrobial resistance patterns to 15 antimicrobial agents of Vibrio parahaemolyticus and Vibrio alginolyticus isolated from farmed fishes, including olive flounder (Paralichthys olivaceus), black rockfish (Sebastes schlegeli), red sea bream (Pagrus major), and sea bass (Lateolabrax japonicus), were investigated from 2005 through 2007. A total of 218 V. parahaemolyticus isolates and 153 V. alginolyticus isolates were obtained from the 180 fish samples collected from fish farms located along the southern coast of Korea. We found that 65.1% of V. parahaemolyticus and 85.6% of V. alginolyticus isolates showed antimicrobial resistance against more than one antimicrobial agent. The prevalence of resistance in V. parahaemolyticus isolates to ampicillin was highest (57.8%), followed by resistance to rifampin (11.9%), streptomycin (8.7%), and trimethoprim (6.4%). V. alginolyticus isolates were also most resistant to ampicillin (75.2%), followed by tetracycline (15.0%), trimethoprim (12.4%), and rifampin (9.8%). The prevalence of multiresistance to four or more antimicrobials was higher in V. alginolyticus (11.1%) than in V. parahaemolyticus (5%). Antimicrobial resistance rates per isolate of V. parahaemolyticus and V. alginolyticus possessing virulence genes were not different from those of the rest of the isolates.     

Incidence of highly genetically diversified Vibrio parahaemolyticus in seafood imported from Asian countries

Vibrio parahaemolyticus is an important pathogen of humans and aquacultured animals, especially in Asian countries. In this study, we examined 686 samples of seafood imported from Hong Kong, Indonesia, Thailand and Vietnam for V. parahaemolyticus. V. parahaemolyticus was recovered from 315 (45.9%) samples. The incidence of V. parahaemolyticus in products from Hong Kong and Thailand was markedly higher than the incidence in products from Indonesia and Vietnam. The incidence rates in shrimp, crab, snail, lobster, sand crab, fish and crawfish were 75.8, 73.3, 44.3, 44.1, 32.5, 29.3 and 21.1%, respectively. None of the isolates possessed the hemolysin genes (tdh, trh). The chromosomal DNA of 121 randomly selected imported isolates and three local environmental strains was digested with SfiI and analyzed by pulsed-field gel electrophoresis (PFGE). These isolates were then grouped into 96 different but mostly unique PFGE patterns. After hierarchical cluster analysis, these patterns could be arbitrarily grouped into twenty-two PFGE types (type A to V). A wide range of PFGE types were identified in isolates from different origin. Moreover, the PFGE types were not specifically associated with the origin or kind of seafood. These results reveal the high genetic diversity in V. parahaemolyticus isolated from seafood.     

Characteristics of a sharp decrease in Vibrio parahaemolyticus infections and seafood contamination in Japan

Vibrio parahaemolyticus has been one of the most important foodborne pathogens in Japan since the 1960s, and a large epidemic was caused by the pandemic serotype O3:K6 from 1997 to 2001. V. parahaemolyticus infections, however, have sharply declined since that time. Data on serotypes isolated from 977 outbreaks were collected and analysed. Total and pathogenic, thermostable direct hemolysin (TDH) gene-positive V. parahaemolyticus were qualitatively and quantitatively detected in 842 seafood samples from wholesale markets in 2007-2009. Strains isolated from patients and seafood were analysed by serotyping, tdh-PCR, group-specific PCR for pandemic strains, and pulsed-field gel electrophoresis (PFGE). The sharp decrease in the infections from 1999 onwards was noted not only for O3:K6 infections but also for other serotypes. The change in the seafood contamination situation from 2001 to 2007-2009 was characterised by a decrease to three-fourths in the frequency of tdh-positive samples, although that decrease was small compared to the 18-fold decrease in the cases of V. parahaemolyticus outbreaks. PFGE detected the pandemic O3:K6 serotype in the same profile in seafood and patients from 1998 to the present. Because of no large decrease in seafood contamination by V. parahaemolyticus from the production to distribution stages and the presence of pandemic O3:K6 serotype in seafood to the present, it was suggested that the change of seafood contamination was unrelated to the sharp decrease in V. parahaemolyticus infections. V. parahaemolyticus infections might be prevented at the stages after the distribution stage.     

成都市海产品中副溶血性弧菌耐药特征及多位点序列分型

目的了解成都市不同种类的海产品中副溶血性弧菌的污染程度、耐药情况、毒力基因分布、基因分型情况,为成都市食源性副溶血性弧菌流行及其风险评估提供基础数据。方法参照GB 4789. 7—2013《食品安全国家标准食品微生物学检验副溶血性弧菌检验》,从不同种类的海产品中分离副溶血性弧菌疑似菌株,通过生化试验及16S r DNA测序进行准确鉴定;采用纸片扩散法对分离株进行药敏试验,通过聚合酶链式反应(PCR)检测与其致病性相关的2个毒力基因,对分离株进行多位点序列分型分析。结果从采集的380份海产品中共104份样品检出副溶血性弧菌,总检出率为27. 4%。药敏试验表明,97. 1%(101/104)的分离株具有耐药性,其中对氨苄西林的耐药率最高(95. 2%,99/104)。分离株trh基因携带率为12. 5%(13/104),tdh基因携带率为1. 0%(1/104); 104株分离株共分为38个ST型,其中ST1801、ST392、ST413型分离率较高,分离株未出现流行克隆群。结论流通过程中不同种类海产品副溶血性弧菌污染率、耐药情况、毒力基因分布存在差异,可能与养殖环境、运输条件等有关。     

Vibrio species involved in seafood-borne outbreaks (Vibrio cholerae,V. parahaemolyticus and V. vulnificus): Review of microbiological versus recent molecular detection methods in seafood products

Seafood products are widely consumed all around the world and play a significant role on the economic market. Bacteria of the Vibrio genus can contaminate seafood and thus pose a risk to human health. Three main Vibrio species, V. cholerae, V. parahaemolyticus and V. vulnificus, are potentially pathogenic to humans. These species are responsible for a dramatic increase of seafood-borne infections worldwide. Hence, early detection of total and pathogenic Vibrio is needed and should rely on quick and effective methods. This review aims to present the standard methods FDA-BAM, ISO/TS 21872–1:2007 and TS 21872–2:2007 and compare them to recent molecular biology methods including endpoint PCR, quantitative real-time PCR (qPCR) and PCR-derived methods with a focus on LAMP (loop-mediated isothermal amplification). The available methods presented here are dedicated to the detection and identification of the Vibrio species of interest in seafood.     

Increased sensitivity in PCR detection of tdh-positive Vibrio parahaemolyticus in seafood with purified template DNA

PCR is an important method for the detection of thermostable direct hemolysin gene (tdh)-positive (pathogenic hemolysin-producing) strains of Vibrio parahaemolyticus in seafood because tdh-negative (nonpathogenic) V. parahaemolyticus strains often contaminate seafood and interfere with the direct isolation of tdh-positive V. parahaemolyticus. In this study, the use of PCR to detect the tdh gene of V. parahaemolyticus in various seafoods artificially contaminated with tdh-positive V. parahaemolyticus was examined. PCR was inhibited by substances in oysters, squid, mackerel, and yellowtail but not by cod, sea bream, scallop, short-necked clam, and shrimp. To improve detection, DNA was purified by either the silica membrane method, the glass fiber method, or the magnetic separation method, and the purified DNA was used as the PCR primer template. For all samples, the use of the silica membrane method and the glass fiber method increased detection sensitivity. The results of this study demonstrate that the use of properly purified template DNA for PCR markedly increases the effectiveness of the method in detecting pathogenic tdh-positive V. parahaemolyticus in contaminated seafood.     

上海地区副溶血性弧菌大流行菌株血清型及分子特征研究

了解上海地区大流行副溶血性弧菌(VP)大流行菌株血清型分布及分子特征。方法 对2010—2012年分离自腹泻患者和食品中的VP菌株进行血清分型,以GS-PCR辨别大流行株,以PCR检测菌株的毒力基因tdh、trh和大流行株分子标识f237噬菌体orf8基因,以PFGE分型来分析菌株间的遗传关系。结果 1136株VP可分为52个血清型(群),其中64.5%的菌株GS-PCR阳性,判定为大流行菌株,其血清型有11种,主要集中于O3∶K6(76.8%)、O4∶K68(9.4%)、O1∶K25(6.8%)、O1∶K36(4.5%)4种血清型。相对于非流行菌株,O10∶K60、O3∶K3、O1∶K33三种血清型的菌株其PFGE图谱与已报道的大流行株更为接近,为新检测到的大流行血清型变种。大流行产毒株中仅能检测到tdh基因,未检测到trh基因,94.3%的大流行菌株携带噬菌体f237的orf8基因,但有5.7%的大流行株orf8基因缺失。相同血清型的大流行菌株其PFGE图谱存在差异,且orf8基因的携带与否不能以PFGE进行区分。结论 上海地区VP大流行菌株血清型相对集中,且不断有新的血清型变种出现。从PFGE图谱的差异和orf8基因的携带与否上来看,大流行菌株仍在演变。     

Development of monoclonal antibody based sandwich ELISA for the rapid detection of pathogenic Vibrio parahaemolyticus in seafood

Thermostable direct hemolysin (TDH) and TDH-related hemolysin (TRH) are considered important virulence factors of Vibrio parahaemolyticus and strains producing either of these or both are considered pathogenic. In this study, we generated monoclonal antibodies (mAbs) against purified TRH recombinant protein of pathogenic V. parahaemolyticus. Sandwich enzyme-linked immunosorbent assays (ELISA) using the hybridoma clone 4B10 showed higher sensitivity of detection compared to other clones. Using mAb 4B10 based sandwich ELISA, we could detect pathogenic V. parahaemolyticus in 41.18% (14 out of 34) of the seafood samples analyzed. PCR targeting the toxR gene showed the presence of V. parahaemolyticus in 64.7% (22 out of 34) seafood samples. Further, PCR targeting the virulence genes showed that 6 seafood samples harboured the tdh gene while 9 harboured the trh gene indicating the presence of pathogenic V. parahaemolyticus. Our results show that mAb 4B10 sandwich ELISA developed in this study could be used as a rapid method for screening seafood samples for the presence of pathogenic V. parahaemolyticus.     

风险分级矩阵在贝类海产品中副溶血性弧菌风险评估的应用研究

目的 研究风险分级矩阵方法学应用以及我国沿海地区居民贝类海产品中副溶血性弧菌污染的健康风险等级.方法 利用贝类海产品消费量以及副溶血性弧菌污染等数据,计算危害严重性(5分制)和疾病发生可能性(5分制)参数,导入风险分级模型矩阵,对我国沿海地区不同人群副溶血性弧菌健康风险进行赋值和等级评价.结果 我国沿海地区全人群和贝类...