Building PulseNet Latin America and Caribbean Salmonella regional database: First conclusions of genetic subtypes of S. Typhi,S. Typhimurium and S. Enteritidis circulating in six countries of the region

PulseNet Latin America and Caribbean Network (PulseNet LA and C) works together with PulseNet International sharing molecular epidemiologic information for the recognition and investigation of foodborne disease outbreaks.The participants of PulseNet LA and C perform standardized pulsed field gel electrophoresis (PFGE) protocols and analysis generating data that is incorporated into Regional Databases. In this study we present the relationship and distribution of genetic subtypes of Salmonella enterica ser. Typhimurium (STM), Salmonella enterica ser. Enteritidis (SE), and Salmonella enterica ser. Typhi (ST) human isolates circulating in six countries of the Region between 2005 and 2009, from the analysis of the Salmonella Database.The 70 ST isolates analyzed were diverse and none of the countries shared the same PFGE profiles with XbaI enzyme. These results show a high genetic diversity among the strains studied and provide background to trace future outbreaks and travel related cases. In the analysis of 550 STM isolates, we found 10 patterns shared at least between two countries, suggesting the need of further studies of attribution to the source of origin. Only one of these PFGE patterns was associated with a known outbreak. Among 225 SE isolates, a predominant subtype was identified, that grouped 83.5% of the isolates and was associated with foodborne outbreaks in five of the six countries; showing the need to use other subtyping techniques for this serovar.The continuous update of PulseNet LA and C Salmonella Regional Database provides an important tool for the laboratory based surveillance of the serovars analyzed, for the prevention and control of foodborne outbreaks, and for the detection of emerging strains in the Region.     

Molecular surveillance of shiga toxigenic Escherichia coli O157 by PulseNet USA

PulseNet USA is the national molecular subtyping network system for foodborne disease surveillance. Sixty-four public health and food regulatory laboratories participate in PulseNet USA and routinely perform pulsed-field gel electrophoresis of Shiga toxigenic Escherichia coli isolated from humans, food, water, and the environment on a real-time basis. Clusters of infection are detected in three ways within this system: through rapidly alerting the participants in the electronic communication forum, the PulseNet Web conference; through cluster analysis by the database administrators at the coordinating center at the Centers for Disease Control and Prevention of the patterns uploaded to the central server by the participants; and by matching profiles of strains from nonhuman sources with recent human uploads to the national server. The strengths, limitations, and scope for future improvements of PulseNet are discussed with examples from 2002. In that year, notices of 30 clusters of Shiga toxigenic E. coli O157 infections were posted on the Web conference, 26 of which represented local outbreaks, whereas four were multistate outbreaks. Another 27 clusters were detected by central cluster detection performed at the Centers for Disease Control and Prevention, of which five represented common source outbreaks confirmed after finding an isolate with the outbreak pattern in the implicated food. Ten food isolates submitted without suspicion of an association to human disease matched human isolates in the database, and an epidemiologic link to human cases was established for six of them.     

Most Common Foodborne Pathogens and Mycotoxins on Fresh Produce: A Review of Recent Outbreaks

Every year millions of people are affected and thousands of them die due to infections and intoxication as a result of foodborne outbreaks, which also cause billions of dollars' worth of damage, public health problems, and agricultural product loss. A considerable portion of these outbreaks is related to fresh produce and caused by foodborne pathogens on fresh produce and mycotoxins. Escherichia coli O104:H4 outbreak, occurred in Germany in 2011, has attracted a great attention on foodborne outbreaks caused by contaminated fresh produce, and especially the vulnerability and gaps in the early warning and notification networks in the surveillance systems in all around the world. In the frame of this paper, we reviewed the most common foodborne pathogens on fresh produce, traceback investigations of the outbreaks caused by these pathogens, and lastly international early warning and notification systems, including PulseNet International and Rapid Alert System for Food and Feed, aiming to detect foodborne outbreaks.     

食源性病原菌分型方法研究进展

目前食源性病原菌常用的分型方法主要包括血清学分型、脉冲场凝胶电泳分型、多位点序列分型、多位点可变数目串联重复序列分型等, 在食源性病原菌的监测、传染源的追踪、流行病监测等方面具有重要意义。本文就食源性病原菌常用的分型方法及其应用情况进行综述。     

Information systems in food safety management

Information systems are concerned with data capture, storage, analysis and retrieval. In the context of food safety management they are vital to assist decision making in a short time frame, potentially allowing decisions to be made and practices to be actioned in real time. Databases with information on microorganisms pertinent to the identification of foodborne pathogens, response of microbial populations to the environment and characteristics of foods and processing conditions are the cornerstone of food safety management systems. Such databases find application in: Identifying pathogens in food at the genus or species level using applied systematics in automated ways. Identifying pathogens below the species level by molecular subtyping, an approach successfully applied in epidemiological investigations of foodborne disease and the basis for national surveillance programs. Predictive modelling software, such as the Pathogen Modeling Program and Growth Predictor (that took over the main functions of Food Micromodel) the raw data of which were combined as the genesis of an international web based searchable database (ComBase). Expert systems combining databases on microbial characteristics, food composition and processing information with the resulting "pattern match" indicating problems that may arise from changes in product formulation or processing conditions. Computer software packages to aid the practical application of HACCP and risk assessment and decision trees to bring logical sequences to establishing and modifying food safety management practices.In addition there are many other uses of information systems that benefit food safety more globally, including: Rapid dissemination of information on foodborne disease outbreaks via websites or list servers carrying commentary from many sources, including the press and interest groups, on the reasons for and consequences of foodborne disease incidents. Active surveillance networks allowing rapid dissemination of molecular subtyping information between public health agencies to detect foodborne outbreaks and limit the spread of human disease. Traceability of individual animals or crops from (or before) conception or germination to the consumer as an integral part of food supply chain management. Provision of high quality, online educational packages to food industry personnel otherwise precluded from access to such courses.     

International Life Sciences Institute North America Listeria monocytogenes strain collection: development of standard Listeria monocytogenes strain sets for research and validation studies

Research and development efforts on bacterial foodborne pathogens, including the development of novel detection and subtyping methods, as well as validation studies for intervention strategies can greatly be enhanced through the availability and use of standardized strain collections. These types of strain collections are available for some foodborne pathogens, such as Salmonella and Escherichia coli. We have developed a standard Listeria monocytogenes strain collection that has not been previously available. The strain collection includes (i) a diversity set of 25 isolates chosen to represent a genetically diverse set of L. monocytogenes isolates as well as a single hemolytic Listeria innocua strain and (ii) an outbreak set, which includes 21 human and food isolates from nine major human listeriosis outbreaks that occurred between 1981 and 2002. The diversity set represents all three genetic L. monocytogenes lineages (I, n = 9; II, n = 9; and III, n = 6) as well as nine different serotypes. Molecular subtyping by EcoRI automated ribotyping and pulsed-field gel electrophoresis (PFGE) with AscI and ApaI separated the 25 isolates in the diversity set into 23 ribotypes and 25 PFGE types, confirming that this isolate set represents considerable genetic diversity. Molecular subtyping of isolates in the outbreak set confirmed that human and food isolates were identical by ribotype and PFGE, except for human and food isolates for two outbreaks, which displayed related but distinct PFGE patterns. Subtype and source data for all isolates in this strain collection are available on the Internet and are linked to the PathogenTracker database (www.pathogentracker.com), which allows the addition of new, relevant information on these isolates, including links to publications that have used isolates from this collection. We have thus developed a core L. monocytogenes strain collection, which will provide a resource for L. monocytogenes research and development efforts with centralized Internet-based data curation and integration.     

Evaluation of a statewide foodborne illness complaint surveillance system in Minnesota, 2000 through 2006

Foodborne outbreaks are detected by recognition of similar illnesses among persons with a common exposure or by identification of case clusters through pathogen-specific surveillance. PulseNet USA has created a national framework for pathogen-specific surveillance, but no comparable effort has been made to improve surveillance of consumer complaints of suspected foodborne illness. The purpose of this study was to characterize the complaint surveillance system in Minnesota and to evaluate its use for detecting outbreaks. Minnesota Department of Health foodborne illness surveillance data from 2000 through 2006 were analyzed for this study. During this period, consumer complaint surveillance led to detection of 79% of confirmed foodborne outbreaks. Most norovirus infection outbreaks were detected through complaints. Complaint surveillance also directly led or contributed to detection of 25% of salmonellosis outbreaks. Eighty-one percent of complainants did not seek medical attention. The number of ill persons in a complainant's party was significantly associated with a complaint ultimately resulting in identification of a foodborne outbreak. Outbreak confirmation was related to a complainant's ability to identify a common exposure and was likely related to the process by which the Minnesota Department of Health chooses complaints to investigate. A significant difference (P < 0.001) was found in incubation periods between complaints that were outbreak associated (median, 27 h) and those that were not outbreak associated (median, 6 h). Complaint systems can be used to detect outbreaks caused by a variety of pathogens. Case detection for foodborne disease surveillance in Minnesota happens through a multitude of mechanisms. The ability to integrate these mechanisms and carry out rapid investigations leads to improved outbreak detection.     

一起食物中毒病原菌斯坦利沙门菌的分子分型及耐药性分析

目的对引起跨区域食物中毒的斯坦利沙门菌分离株进行耐药性检测和分子分型分析,追溯并明确病因食品,为该菌防控工作提供科学依据。方法采用微量肉汤稀释法对15株斯坦利沙门菌进行18种抗生素敏感性试验,同时对菌株基因组DNA经限制性内切酶Xba I酶切后进行脉冲场凝胶电泳(PFGE)分子分型,并与数据库中其他沙门菌比较。结果此次疫情分离菌株中食品分离株和腹泻患者分离株血清型一致,耐药谱基本一致。经PFGE溯源分析后具有100%同源的PFGE带型,数据库中无相同带型的菌株。结论本起跨区域食物中毒是由斯坦利沙门菌污染的煮花生引起,分离菌株对红霉素耐药,对氯霉素处于中介,对其他抗生素敏感。     

Standardization of diagnostic PCR for the detection of foodborne pathogens

In vitro amplification of nucleic acids using the polymerase chain reaction (PCR) has become, since its discovery in the 1980s, a powerful diagnostic tool for the analysis of microbial infections as well as for the analysis of microorganisms in food samples. However, despite its potential, PCR has neither gained wide acceptance in routine diagnostics nor been widely incorporated in standardized methods. Lack of validation and standard protocols, as well as variable quality of reagents and equipment, influence the efficient dissemination of PCR methodology from expert research laboratories to end-user laboratories. Moreover, the food industry understandably requires and expects officially approved standards. Recognizing this, in 1999, the European Commission approved the research project, FOOD-PCR (http://www.PCR.dk), which aims to validate and standardize the use of diagnostic PCR for the detection of pathogenic bacteria in foods. The present review focuses on the harmonization procedure and standardization criteria for detection of foodborne pathogens by PCR. The progress of standardization so far and future perspectives of diagnostic PCR are discussed.     

一起基于脉冲场凝胶电泳技术主动监测发现的韦太夫雷登沙门菌感染暴发调查

目的 通过对广东实验室沙门菌分子分型主动监测系统脉冲场凝胶电泳分子分型数据的分析,核实暴发,进行流行病学调查,探讨并制订预防控制措施.方法 采用描述流行病学方法,对暴发个案资料进行流行病学分析,采集涉事单位的食品和鸸鹋肛拭子样本进行沙门菌分离.结果 主动监测系统发现,广州市海珠区发生了一起由韦太夫雷登血清型沙门菌感染引起的食源性疾病暴发,发病5例,可疑食物载体为未煮熟的鸸鹋肉制食品.结论 这是广东省内首起基于脉冲场凝胶电泳技术建立的主动监测系统发现的食源性疾病暴发,应重视新型监测技术应用,以及由此带来的对目前政府部门应对暴发的协调机制的考验.     

Low prevalence of Listeria monocytogenes in human stool

Listeria monocytogenes is a foodborne pathogen that is found widely in the environment and in a variety of ready-to-eat foods, yet human invasive infection is relatively rare (five cases per million people annually in the United States). Despite wide exposure to this organism, little is known about the prevalence of L. monocytogenes in human stool, and it is not known whether human fecal dispersal contributes to human foodborne transmission. We cultured 827 stool specimens (well formed and loose-watery) from individuals from four large metropolitan areas of New York state for L. monocytogenes and found only 1 (0.12%) positive specimen. L. monocytogenes was also isolated from the blood of the person with the single positive specimen, and the two isolates were indistinguishable by molecular subtyping (both were ribotype DUP-1042B). This provides further evidence that human L. monocytogenes fecal carriage among persons with and without diarrheal disease is remarkably low. Unlike the case for other foodborne pathogens (e.g., Salmonella), human shedders probably do not contribute significantly to L. monocytogenes contamination of foods. However, we observed a single individual with invasive listeriosis that shed the pathogen in feces, indicating the potential for fecal dispersal of L. monocytogenes from persons with listeriosis.     

Comparison of dichloran 18% glycerol (DG18) agar with general purpose mycological media for enumerating food spoilage yeasts

Dichloran 18% glycerol (DG18) agar was originally developed to enumerate xerophilic foodborne moulds. However, some laboratories are using DG18 agar as a general medium to enumerate foodborne moulds and yeasts. A collaborative study, with the participation of seven laboratories, was undertaken to compare DG18 agar with dichloran rose bengal chloramphenicol (DRBC) agar, tryptone glucose yeast extract chloramphenicol (TGYC) agar, and plate count agar supplemented with chloramphenicol (PCAC) for enumerating 14 species of common food spoilage yeasts. Comparison of the mean values of populations of all yeasts recovered on each medium revealed no significant differences among DRBC agar, PCAC, and TGYC agar, while each of these media supported the development of significantly (P < or = 0.05) higher numbers of colonies than DG18 agar. However, differences were only 0.08 to 0.10 log10 cfu/ml, making the practical significance questionable. The overall coefficient of variation (CV) for within laboratory repeatability was 1.71%, while the CV for reproducibility of counts obtained among laboratories was 6.96%. Compared to DRBC agar, TGYC agar, and PCAC, yeast colonies were smaller on DG18 agar. Growth of Brettanomyces anomalus, Cryptococcus albidus, and Rhodotorula mucilaginosa was particularly retarded or inhibited on DG18 agar. Based on the performance of media in supporting colony development and ease of counting colonies, the use of DG18 agar as a general enumeration medium for foodborne yeasts cannot be recommended.     

北京市食源性非伤寒沙门菌的分子分型和耐药性研究

目的了解北京市食源性非伤寒沙门菌的分子特征及耐药情况。方法对2004—2010年北京市食源性致病菌监测网收集的100株沙门菌进行脉冲场凝胶电泳(PFGE)分型和抗生素敏感性检测。结果 100株非伤寒沙门菌通过PFGE分型分为62个不同的带型,每个带型包含1~11株菌。抗生素敏感性结果显示,100株菌中有55株菌表现为对至少1种抗生素耐药,其中多重耐药菌株15株。菌株对各抗生素的耐药率为萘啶酸40%、四环素30%、氯霉素15%、庆大霉素10%、甲氧苄啶/磺胺甲恶唑10%、环丙沙星9%、头孢西丁1%、头孢噻肟0%。结论沙门菌PFGE带型和耐药谱均与血清型存在很高的一致性。提示北京市食源性非伤寒沙门菌的耐药情况比较严重,开展对该菌分子分型与耐药特征分析的联合监测意义重大。     

Listeria monocytogenes: Strain Heterogeneity,Methods, and Challenges of Subtyping

Listeria monocytogenes is a food‐borne bacterial pathogen that is associated with 20% to 30% case fatality rate. L. monocytogenes is a genetically heterogeneous species, with a small fraction of strains (serotypes 1/2a, 1/2b, 4b) implicated in human listeriosis. Monitoring and source tracking of L. monocytogenes involve the use of subtyping methods, with the performance of genetic‐based methods found to be superior to phenotypic‐based ones. Various methods have been used to subtype L. monocytogenes isolates, with the pulsed‐field gel electrophoresis (PFGE) being the gold standard. Although PFGE has had a massive impact on food safety through the establishment of the PulseNet, there is no doubt that whole genome sequence (WGS) typing is accurate, has a discriminatory power superior to any known method, and allows genome‐wide differences between strains to be quantified through the comparison of nucleotide sequences. This review focuses on the different techniques that have been used to type L. monocytogenes strains, their performance challenges, and the tremendous impact WGS typing could have on the food safety landscape.     

Listeria monocytogenes virulence and pathogenicity,a food safety perspective

Several virulence factors of Listeria monocytogenes have been identified and extensively characterized at the molecular and cell biologic levels, including the hemolysin (listeriolysin O), two distinct phospholipases, a protein (ActA), several internalins, and others. Their study has yielded an impressive amount of information on the mechanisms employed by this facultative intracellular pathogen to interact with mammalian host cells, escape the host cell's killing mechanisms, and spread from one infected cell to others. In addition, several molecular subtyping tools have been developed to facilitate the detection of different strain types and lineages of the pathogen, including those implicated in common-source outbreaks of the disease. Despite these spectacular gains in knowledge, the virulence of L. monocytogenes as a foodborne pathogen remains poorly understood. The available pathogenesis and subtyping data generally fail to provide adequate insight about the virulence of field isolates and the likelihood that a given strain will cause illness. Possible mechanisms for the apparent prevalence of three serotypes (1/2a, 1/2b, and 4b) in human foodborne illness remain unidentified. The propensity of certain strain lineages (epidemic clones) to be implicated in common-source outbreaks and the prevalence of serotype 4b among epidemic-associated stains also remain poorly understood. This review first discusses current progress in understanding the general features of virulence and pathogenesis of L. monocytogenes. Emphasis is then placed on areas of special relevance to the organism's involvement in human foodborne illness, including (i) the relative prevalence of different serotypes and serotype-specific features and genetic markers; (ii) the ability of the organism to respond to environmental stresses of relevance to the food industry (cold, salt, iron depletion, and acid); (iii) the specific features of the major known epidemic-associated lineages; and (iv) the possible reservoirs of the organism in animals and the environment and the pronounced impact of environmental contamination in the food processing facilities. Finally, a discussion is provided on the perceived areas of special need for future research of relevance to food safety, including (i) theoretical modeling studies of niche complexity and contamination in the food processing facilities; (ii) strain databases for comprehensive molecular typing; and (iii) contributions from genomic and proteomic tools, including DNA microarrays for genotyping and expression signatures. Virulence-related genomic and proteomic signatures are expected to emerge from analysis of the genomes at the global level, with the support of adequate epidemiologic data and access to relevant strains.     

食品中甲型肝炎病毒RNA提取方法的比较及应用

目的：对食品中甲型肝炎病毒的3 种RNA提取方法进行综合比较,以优选出最佳的核酸提取方法,为各级实验室开展食源性甲型肝炎病毒核酸检验提供技术参考。方法：分别采用ABI AM1836、QIAGEN 74104和 ROCHE11858882001三种商业化核酸提取试剂盒对人工污染甲型肝炎病毒的食品样品进行核酸提取,根据3 种核酸提取方法的提取效率、抑制剂去除效率、检测灵敏度、综合应用指标进行病毒RNA的优化,且采用优化后的提取方法进行实际样品的检测。结果：综合病毒各项评价指标显示,ROCHE 11858882001在食品中提取甲型肝炎病毒RNA效果最优,其次为QIAGEN 74104和ABI AM1836。101 份样品的检测结果显示,一份蓝靛果样品为甲型肝炎病毒核酸阳性,其余样品均为阴性。结论：ROCHE 11858882001是一种快捷有效的病毒RNA提取方法,适用于食品中甲型肝炎病毒的日常检测工作。     

Current Methods for Extraction and Concentration of Enteric Viruses from Fresh Fruit and Vegetables: Towards International Standards

Virus-contaminated soft fruits or vegetables are increasingly identified as causes of foodborne viral illness. Noroviruses and hepatitis A virus are the most common pathogens in viral infections transmitted by these kinds of foods. To improve microbiological detection and monitoring and to increase insights into the contribution of fruits and vegetables to foodborne viral transmission, sensitive, reliable, and standardized methods are needed. More studies on virus detection methods for foods are being published, but validated consensus protocols are not yet available. In this paper, different methodologies are reviewed critically. The use of process controls and internal amplification controls is discussed.     

免疫学方法在食源性副溶血性弧菌检测中的应用研究进展

副溶血性弧菌可污染以海产品为主的各类食品,其引发的食品安全事件数量已经跃升至我国食品中毒事件数首位。相对于传统培养法及现代分子检测技术,基于抗原-抗体特异性识别反应的免疫鉴定及分型策略在检测效率、特异性、现场适用性、前处理简便性等诸多方面显现突出优势。本文在解析当前副溶血性弧菌抗体种类及制备方法的基础上,就免疫磁珠分离、酶联免疫吸附测定、免疫层析测试、免疫传感器、免疫荧光显微术等方法在食源性副溶血性弧菌检测中的应用现状进行综述,以期为该病原菌的快速检测提供借鉴和参考。     

Evaluation of an Extraction Method for the Detection of GI and GII Noroviruses in Fruit and Vegetable Salads

Human norovirus (HuNoV) is a major foodborne virus causing gastroenteritis outbreaks in humans. Salad products can be vectors of transmission for foodborne viruses such as HuNoV when these products are contaminated naturally or through unsanitary food handling. Therefore, development of simple, reliable and sensitive techniques for the detection of HuNoV in salad products is needed to ensure food safety. The purpose of our study was to optimize a method for the detection of HuNoV in artificially contaminated salad products. To this end, 2 different kinds of salads (fruit salads and vegetable salads) were experimentally inoculated with HuNoV GI, HuNoV GII, and MS2 suspensions. The selected method was based on treatment with pectinase followed by Trizol‐chloroform purification, and the recovery efficiencies were 6.07% to 26.52% for HuNoV GI and 5.54% to 37.36% for HuNoV GII. MS2 was used as the process control, and the recovery efficiencies for fruit salad and vegetable salad samples were 38.57% and 41.13%, respectively. The optimized method could be applied in diagnostic laboratories to identify NoV contamination in composite foods, such as salad products, should an event of foodborne outbreak occur.