Improved sample treatment protocol for accurate detection of live Salmonella spp. in food samples by viability PCR.

Culture-based detection is still considered as the standard way for detection of Salmonella in foods, although molecular methods, such as viability PCR (vPCR), have been introduced to overcome some disadvantages of traditional culture methods. Despite the success of the vPCR methodology, the problem of false-positive results is a major drawback, especially when applied to environmental samples, hindering the interpretation of the results. To improve the efficiency of vPCR, many approaches have been introduced by several authors during the last years. In the present work, the combination of PEMAX dye, double tube change, and double photo-activation step was established as a strategy to improve vPCR protocol. By combining these approaches, we developed an improved sample treatment protocol able to neutralize DNA signals of up to 5.0×107 dead cells/sample from both pure culture and artificially contaminated food samples. Our results indicate that vPCR can work reliable and has a potential for high throughput detection of live Salmonella cells in food samples, minimizing false-positive signals.

Differential detection of pathogenic Yersinia spp. by fluorescence in situ hybridization.

Yersinia enterocolitica, Y. pseudotuberculosis and Y. pestis are pathogens of major medical importance, which are responsible for a considerable number of infections every year. The detection of these species still relies on cultural methods, which are slow, labour intensive and often hampered by the presence of high amounts of accompanying flora. In this study, fluorescence in situ hybridization (FISH) was used to develop a fast, sensitive and reliable alternative to detect viable bacteria in food. For this purpose, highly specific probes targeting the 16S and 23S ribosomal RNA were employed to differentially detect each of the three species. In order to enable the differentiation of single nucleotide polymorphisms (SNPs), suitable competitor oligonucleotides and locked nucleic acids (LNAs) were used. Starved cells still showed a strong signal and a direct viable count (DVC) approach combined with FISH optimized live/dead discrimination. Sensitivity of the FISH test was high and even a single cell per gram of spiked minced pork meat could be detected within a day, demonstrating the applicability to identify foodborne hazards at an early stage. In conclusion, the established FISH tests proved to be promising tools to compensate existing drawbacks of the conventional cultural detection of these important zoonotic agents.

Genetic Diversity as Consequence of a Microaerobic and Neutrophilic Lifestyle.

As a neutrophilic bacterium, Helicobacter pylori is growth deficient under extreme acidic conditions. The gastric pathogen is equipped with an acid survival kit, regulating urease activity by a pH-gated urea channel, opening below pH 6.5. After overcoming acid stress, the bacterium's multiplication site is situated at the gastric mucosa with near neutral pH. The pathogen exhibits exceptional genetic variability, mainly due to its capability of natural transformation, termed competence. Using single cell analysis, we show here that competence is highly regulated in H. pylori. DNA uptake complex activity was reversibly shut down below pH 6.5. pH values above 6.5 opened a competence window, in which competence development was triggered by the combination of pH increase and oxidative stress. In contrast, addition of sublethal concentrations of the DNA-damaging agents ciprofloxacin or mitomycin C did not trigger competence development under our conditions. An oxygen-sensitive mutant lacking superoxide dismutase (sodB) displayed a higher competent fraction of cells than the wild type under comparable conditions. In addition, the sodB mutant was dependent on adenine for growth in broth and turned into non-cultivable coccoid forms in its absence, indicating that adenine had radical quenching capacity. Quantification of periplasmically located DNA in competent wild type cells revealed outstanding median imported DNA amounts of around 350 kb per cell within 10 min of import, with maximally a chromosomal equivalent (1.6 Mb) in individual cells, far exceeding previous amounts detected in other Gram-negative bacteria. We conclude that the pathogen's high genetic diversity is a consequence of its enormous DNA uptake capacity, triggered by intrinsic and extrinsic oxidative stress once a neutral pH at the site of chronic host colonization allows competence development.

FoodChain-Lab: A Trace-Back and Trace-Forward Tool Developed and Applied during Food-Borne Disease Outbreak Investigations in Germany and Europe.

FoodChain-Lab is modular open-source software for trace-back and trace-forward analysis in food-borne disease outbreak investigations. Development of FoodChain-Lab has been driven by a need for appropriate software in several food-related outbreaks in Germany since 2011. The software allows integrated data management, data linkage, enrichment and visualization as well as interactive supply chain analyses. Identification of possible outbreak sources or vehicles is facilitated by calculation of tracing scores for food-handling stations (companies or persons) and food products under investigation. The software also supports consideration of station-specific cross-contamination, analysis of geographical relationships, and topological clustering of the tracing network structure. FoodChain-Lab has been applied successfully in previous outbreak investigations, for example during the 2011 EHEC outbreak and the 2013/14 European hepatitis A outbreak. The software is most useful in complex, multi-area outbreak investigations where epidemiological evidence may be insufficient to discriminate between multiple implicated food products. The automated analysis and visualization components would be of greater value if trading information on food ingredients and compound products was more easily available.

Sampling and Homogenization Strategies Significantly Influence the Detection of Foodborne Pathogens in Meat.

Efficient preparation of food samples, comprising sampling and homogenization, for microbiological testing is an essential, yet largely neglected, component of foodstuff control. Salmonella enterica spiked chicken breasts were used as a surface contamination model whereas salami and meat paste acted as models of inner-matrix contamination. A systematic comparison of different homogenization approaches, namely, stomaching, sonication, and milling by FastPrep-24 or SpeedMill, revealed that for surface contamination a broad range of sample pretreatment steps is applicable and loss of culturability due to the homogenization procedure is marginal. In contrast, for inner-matrix contamination long treatments up to 8 min are required and only FastPrep-24 as a large-volume milling device produced consistently good recovery rates. In addition, sampling of different regions of the spiked sausages showed that pathogens are not necessarily homogenously distributed throughout the entire matrix. Instead, in meat paste the core region contained considerably more pathogens compared to the rim, whereas in the salamis the distribution was more even with an increased concentration within the intermediate region of the sausages. Our results indicate that sampling and homogenization as integral parts of food microbiology and monitoring deserve more attention to further improve food safety.

Towards a Food Safety Knowledge Base Applicable in Crisis Situations and Beyond.

In case of contamination in the food chain, fast action is required in order to reduce the numbers of affected people. In such situations, being able to predict the fate of agents in foods would help risk assessors and decision makers in assessing the potential effects of a specific contamination event and thus enable them to deduce the appropriate mitigation measures. One efficient strategy supporting this is using model based simulations. However, application in crisis situations requires ready-to-use and easy-to-adapt models to be available from the so-called food safety knowledge bases. Here, we illustrate this concept and its benefits by applying the modular open source software tools PMM-Lab and FoodProcess-Lab. As a fictitious sample scenario, an intentional ricin contamination at a beef salami production facility was modelled. Predictive models describing the inactivation of ricin were reviewed, relevant models were implemented with PMM-Lab, and simulations on residual toxin amounts in the final product were performed with FoodProcess-Lab. Due to the generic and modular modelling concept implemented in these tools, they can be applied to simulate virtually any food safety contamination scenario. Apart from the application in crisis situations, the food safety knowledge base concept will also be useful in food quality and safety investigations.

FISHing for bacteria in food--a promising tool for the reliable detection of pathogenic bacteria?

Foodborne pathogens cause millions of infections every year and are responsible for considerable economic losses worldwide. The current gold standard for the detection of bacterial pathogens in food is still the conventional cultivation following standardized and generally accepted protocols. However, these methods are time-consuming and do not provide fast information about food contaminations and thus are limited in their ability to protect consumers in time from potential microbial hazards. Fluorescence in situ hybridization (FISH) represents a rapid and highly specific technique for whole-cell detection. This review aims to summarize the current data on FISH-testing for the detection of pathogenic bacteria in different food matrices and to evaluate its suitability for the implementation in routine testing. In this context, the use of FISH in different matrices and their pretreatment will be presented, the sensitivity and specificity of FISH tests will be considered and the need for automation shall be discussed as well as the use of technological improvements to overcome current hurdles for a broad application in monitoring food safety. In addition, the overall economical feasibility will be assessed in a rough calculation of costs, and strengths and weaknesses of FISH are considered in comparison with traditional and well-established detection methods.

Characterization of illegal food items and identification of foodborne pathogens brought into the European Union via two major German airports.

Foods of animal origin brought illegally from third party countries into the European Community pose a risk for the introduction of diseases. This can lead to animal disease outbreaks with significant economic and social costs and subsequent severe trade restrictions. Further, disease outbreaks in humans due to illegally imported foods of animal origin have been described, yet, there are very few studies examining the potential human health impact. Passenger baggage is the most likely route by which illegal products enter a country. Therefore, the volume and geographic origin of foods of animal origin introduced illegally into Germany via the Frankfurt International Airport and Berlin-Schönefeld Airport by passenger luggage were characterized. Further, the occurrence of foodborne zoonotic bacteria such as Salmonella spp., Listeria spp., Campylobacter spp., Yersinia spp., Verocytotoxin-producing Escherichia coli (VTEC) and Brucella spp. and the microbial quality of the foods were analysed by total bacterial count. Between 2012 and 2013, a total of 663 food items were seized from 296 passengers arriving in Germany from 35 different departure countries. The majority of confiscates (51%) originated from Turkey and Russia. A selection of 474 samples was subjected to microbiological analyses. Twenty-three food products tested positive for at least one of the pathogens analysed. The majority of the contaminated foods were meat (33%) or meat products (42%), and milk products (21%). Considering that only a small fraction of arriving passengers is subjected to airport custom controls and only a small number of confiscated foods could be analysed during this study, further investigations are needed to understand the public health risks posed by illegally introduced food items.

Subgrouping of ESBL-producing Escherichia coli from animal and human sources: an approach to quantify the distribution of ESBL types between different reservoirs.

Escherichia (E.) coli producing extended-spectrum beta-lactamases (ESBLs) are an increasing problem for public health. The success of ESBLs may be due to spread of ESBL-producing bacterial clones, transfer of ESBL gene-carrying plasmids or exchange of ESBL encoding genes on mobile elements. This makes it difficult to identify transmission routes and sources for ESBL-producing bacteria. The objectives of this study were to compare the distribution of genotypic and phenotypic properties of E. coli isolates from different animal and human sources collected in studies in the scope of the national research project RESET. ESBL-producing E. coli from two longitudinal and four cross-sectional studies in broiler, swine and cattle farms, a cross-sectional and a case-control study in humans and diagnostic isolates from humans and animals were used. In the RESET consortium, all laboratories followed harmonized methodologies for antimicrobial susceptibility testing, confirmation of the ESBL phenotype, specific PCR assays for the detection of bla(TEM), bla(CTX), and bla(SHV) genes and sequence analysis of the complete ESBL gene as well as a multiplex PCR for the detection of the four major phylogenetic groups of E. coli. Most ESBL genes were found in both, human and non-human populations but quantitative differences for distinct ESBL-types were detectable. The enzymes CTX-M-1 (63.3% of all animal isolates, 29.3% of all human isolates), CTX-M-15 (17.7% vs. 48.0%) and CTX-M-14 (5.3% vs. 8.7%) were the most common ones. More than 70% of the animal isolates and more than 50% of the human isolates contained the broadly distributed ESBL genes bla(CTX-M-1), bla(CTX-M-15), or the combinations bla(SHV-12)+bla(TEM) or bla(CTX-M-1)+bla(TEM). While the majority of animal isolates carried bla(CTX-M-1) (37.5%) or the combination bla(CTX-M-1)+bla(TEM) (25.8%), this was the case for only 16.7% and 12.6%, respectively, of the human isolates. In contrast, 28.2% of the human isolates carried bla(CTX-M-15) compared to 10.8% of the animal isolates. When grouping data by ESBL types and phylogroups bla(CTX-M-1) genes, mostly combined with phylogroup A or B1, were detected frequently in all settings. In contrast, bla(CTX-M-15) genes common in human and animal populations were mainly combined with phylogroup A, but not with the more virulent phylogroup B2 with the exception of companion animals, where a few isolates were detectable. When E. coli subtype definition included ESBL types, phylogenetic grouping and antimicrobial susceptibility data, the proportion of isolates allocated to common clusters was markedly reduced. Nevertheless, relevant proportions of same subtypes were detected in isolates from the human and livestock and companion animal populations included in this study, suggesting exchange of bacteria or bacterial genes between these populations or a common reservoir. In addition, these results clearly showed that there is some similarity between ESBL genes, and bacterial properties in isolates from the different populations. Finally, our current approach provides good insight into common and population-specific clusters, which can be used as a basis for the selection of ESBL-producing isolates from interesting clusters for further detailed characterizations, e.g. by whole genome sequencing.

Pathogenic vibrios in environmental, seafood and clinical sources in Germany.

Bacteria of the family Vibrionaceae naturally occur in marine and estuarine environments. Only few species of Vibrionaceae are associated with human cases of gastroenteritis, ear and wound infections, caused by ingestion of seafood or contact with Vibrio containing water. Increasing consumption of seafood (fish, fishery products and shellfish) poses a possible source of Vibrio infections in Germany. Additionally, there is a growing concern that abundances of pathogenic vibrios may increase in German coastal waters as a result of e.g. climate change resulting in probably rising surface water temperatures. According to the One Health concept the VibrioNet consortium started in 2010 to investigate the occurrence and relevance of non-cholera vibrios of human concern in Germany. Vibrios from environmental, seafood and clinical sources were analyzed with the aim to find connections between different reservoirs or sources and to identify potential ways of transmission of these pathogens to assess the risk of infections associated with them. Potentially pathogenic strains mostly belong to the species Vibrio parahaemolyticus, Vibrio vulnificus and non-O1/non-O139 Vibrio cholerae. Investigations on imported seafood and mussels from primary production areas confirmed the frequent occurrence of these species. Moreover, studies of German coastal waters and sediments showed the presence and seasonality of these marine bacteria. So far the incidence of clinical cases of vibriosis in Germany is low. Between 1994 and 2013 thirteen cases of Vibrio spp. associated wound infections and/or septicaemia have been reported. However, the high prevalence of vibrios in aquatic environments and aquatic organisms is of concern and demands continued control of food and surveillance for clinical infections with pathogenic vibrios.

A likelihood-based approach to identifying contaminated food products using sales data: performance and challenges.

Foodborne disease outbreaks of recent years demonstrate that due to increasingly interconnected supply chains these type of crisis situations have the potential to affect thousands of people, leading to significant healthcare costs, loss of revenue for food companies, and--in the worst cases--death. When a disease outbreak is detected, identifying the contaminated food quickly is vital to minimize suffering and limit economic losses. Here we present a likelihood-based approach that has the potential to accelerate the time needed to identify possibly contaminated food products, which is based on exploitation of food products sales data and the distribution of foodborne illness case reports. Using a real world food sales data set and artificially generated outbreak scenarios, we show that this method performs very well for contamination scenarios originating from a single "guilty" food product. As it is neither always possible nor necessary to identify the single offending product, the method has been extended such that it can be used as a binary classifier. With this extension it is possible to generate a set of potentially "guilty" products that contains the real outbreak source with very high accuracy. Furthermore we explore the patterns of food distributions that lead to "hard-to-identify" foods, the possibility of identifying these food groups a priori, and the extent to which the likelihood-based method can be used to quantify uncertainty. We find that high spatial correlation of sales data between products may be a useful indicator for "hard-to-identify" products.

"Limits of control"--crucial parameters for a reliable quantification of viable campylobacter by real-time PCR.

The unsuitability of the "CFU" parameter and the usefulness of cultivation-independent quantification of Campylobacter on chicken products, reflecting the actual risk for infection, is increasingly becoming obvious. Recently, real-time PCR methods in combination with the use of DNA intercalators, which block DNA amplification from dead bacteria, have seen wide application. However, much confusion exists in the correct interpretation of such assays. Campylobacter is confronted by oxidative and cold stress outside the intestine. Hence, damage caused by oxidative stress probably represents the most frequent natural death of Campylobacter on food products. Treatment of Campylobacter with peroxide led to complete loss of CFU and to significant entry of any tested DNA intercalator, indicating disruption of membrane integrity. When we transiently altered the metabolic state of Campylobacter by abolishing the proton-motive force or by inhibiting active efflux, CFU was constant but enhanced entry of ethidium bromide (EtBr) was observed. Consistently, ethidium monoazide (EMA) also entered viable Campylobacter, in particular when nutrients for bacterial energization were lacking (in PBS) or when the cells were less metabolically active (in stationary phase). In contrast, propidium iodide (PI) and propidium monoazide (PMA) were excluded from viable bacterial cells, irrespective of their metabolic state. As expected for a diffusion-limited process, the extent of signal reduction from dead cells depended on the temperature, incubation time and concentration of the dyes during staining, prior to crosslinking. Consistently, free protein and/or DNA present in varying amounts in the heterogeneous matrix lowered the concentration of the DNA dyes at the bacterial membrane and led to considerable variation of the residual signal from dead cells. In conclusion, we propose an improved approach, taking into account principles of method variability and recommend the implementation of process sample controls for reliable quantification of intact and potentially infectious units (IPIU) of Campylobacter by real-time PCR.

F1 and tbilisi are closely related brucellaphages exhibiting some distinct nucleotide variations which determine the host specificity.

We report on the 41,143-bp genome of brucellaphage F1, a podovirus that infects several Brucella species. The F1 genome is almost identical to the genome of brucellaphage Tb. However, some structural proteins of the phages exhibit extensive polymorphisms and might be responsible for their different host ranges.

[Increase in antimicrobial resistance of Salmonella from food to fluoroquinolones and cephalosporins--a review of data from ten years]. / Anstieg der Resistenz von Salmonellen aus Lebensmitteln gegenüber Fluorchinolonen und Cephalosporinen--eine Ubersicht uber zehn Jahre.

Animal derived food is a relevant source of human infections with Salmonella enterica. In this paper we analyse the presence of Salmonella in meat with respect to the observed serovars and their resistance to the fluoroquinolone ciprofloxacin and 3rd generation cephalosporins in the years 2003 to 2012. Data originated from 8176 isolates that were isolated from meat, characterized in the National Reference Laboratory for Salmonella and tested for antimicrobial resistance in the National Reference Laboratory for antimicrobial resistance in this time period. The analysis reveals substantial differences in resistance patterns between isolates from different types of meat and different serovars. Frequent serovars were mostly associated with one type of meat, suggesting an additional influence of specific characteristics of the serovars besides the effect of selection pressure excerted by antimicrobial treatments. Results show a clear increase in resistance to fluoroquinolones and 3rd generation cephalosporins that was most prominent in isolates from poultry meat. Although the number of human infections with Salmonella in Germany decreased sharply in recent years, results indicate a substantial exposure of consumers to Salmonella that are resistant to important antimicrobials via meat.

[Estimation of the transfer of ESBL-producing Escherichia coli to humans in Germany]. / Abschätzung des Transfers von ESBL-bildenden Escherichia coli zum Menschen für Deutschland.

In 2011 EFSA has evaluated the risk for the consumer caused by ESBL-/AmpC-producing bacteria in food of animal origin and in livestock animals. Human-to-human transfer in hospitals.and in the community was considered as the most relevant route of transmission for ESBL-producing E. coli. ESBL-/AmpC-producing E. coli are in Germany, as in many other Member States of the European Union, widely spread in food of animal origin and in livestock animals. In an assessment of the relevance of livestock animals as reservoir for ESBL-/AmpC-producing E. coli as well as for ESBL-coding resistance genes the heterogeneity of the resistance genes, plasmids and bacteria in animals, foods and humans needs to be considered. In this context, both, the clonal spread of bacteria as well as horizontal transfer of resistance genes, e. g. by plasmids, have to be analyzed. Whereas studies in The Netherlands identified poultry as the most relevant reservoir, the transfer of ESBL-gene carrying plasmids from pigs to the farmers was demonstrated in Denmark. First attempts to quantify the relevance of livestock animals as reservoir for ESBL-producing E. coli in Germany showed, that the proportions of the most frequent ESBL-resistance genes are quite different between animal and human derived E. coli isolates. If in addition properties of the bacterial cells, e.g. resistance to several antibiotic classes are considered, only a small proportion of human isolates showed the same patterns as animal isolates. The results achieved so far demonstrate that certain ESBL-types are prevalent in all livestock populations investigated. Currently, the majority of cases of colonizations with ESBL-producing E. coli among humans cannot be directly linked to livestock and food-producing animals as reservoirs. This reflects that transmission routes are more complex and other reservoirs and sources including human-human interactions have to be taken into consideration.

Post-harvest application of lytic bacteriophages for biocontrol of foodborne pathogens and spoilage bacteria.

Bacteriophages are a promising tool to combat pathogenic bacteria along the food chain. In this review we give a comprehensive overview about studies that have been carried out to appraise the potential of phages to reduce the number of foodborne pathogens and spoilage bacteria in food products. The published data demonstrate that phages are generally suited for such applications, albeit high numbers of phages had to be applied in most experiments. The level of attainable reduction largely depended on the food matrix and the physiochemical conditions. However, microbial reductions of more than two orders of magnitude have been achieved in many studies. This review summarizes a vast quantity of papers describing the reduction of pathogens on food products by phages and demonstrates where phage applications are more or less promising. Based on these data we discuss the merits and drawbacks of post-harvest applications of phages.

Decontamination of high-risk animal and zoonotic pathogens.

Preparedness for the decontamination of affected environments, premises, facilities, and products is one prerequisite for an immediate response to an animal disease outbreak. Various information sources provide recommendations on how to proceed in an outbreak situation to eliminate biological contaminants and to stop the spread of the disease. In order to facilitate the identification of the right decontamination strategy, we present an overview of relevant references for a collection of pathogenic agents. The choice of pathogens is based on a survey of lists containing highly pathogenic agents and/or biological agents considered to be potential vehicles for deliberate contamination of food, feed, or farm animals. European legislation and guidelines from national and international institutions were screened to find decontamination protocols for each of the agents. Identified recommendations were evaluated with regard to their area of application, which could be facilities and equipment, wastes, food, and other animal products. The requirements of a disinfectant for large-scale incidents were gathered, and important characteristics (eg, inactivating spectrum, temperature range, toxicity to environment) of the main recommended disinfectants were summarized to assist in the choice of a suitable and efficient approach in a crisis situation induced by a specific high-risk animal or zoonotic pathogen. The literature search revealed numerous relevant recommendations but also legal gaps for certain diseases, such as Q fever or brucellosis, and legal difficulties for the use of recommended disinfectants. A lack of information about effective disinfectants was identified for some agents.

A generic open-source software framework supporting scenario simulations in bioterrorist crises.

Since the 2001 anthrax attack in the United States, awareness of threats originating from bioterrorism has grown. This led internationally to increased research efforts to improve knowledge of and approaches to protecting human and animal populations against the threat from such attacks. A collaborative effort in this context is the extension of the open-source Spatiotemporal Epidemiological Modeler (STEM) simulation and modeling software for agro- or bioterrorist crisis scenarios. STEM, originally designed to enable community-driven public health disease models and simulations, was extended with new features that enable integration of proprietary data as well as visualization of agent spread along supply and production chains. STEM now provides a fully developed open-source software infrastructure supporting critical modeling tasks such as ad hoc model generation, parameter estimation, simulation of scenario evolution, estimation of effects of mitigation or management measures, and documentation. This open-source software resource can be used free of charge. Additionally, STEM provides critical features like built-in worldwide data on administrative boundaries, transportation networks, or environmental conditions (eg, rainfall, temperature, elevation, vegetation). Users can easily combine their own confidential data with built-in public data to create customized models of desired resolution. STEM also supports collaborative and joint efforts in crisis situations by extended import and export functionalities. In this article we demonstrate specifically those new software features implemented to accomplish STEM application in agro- or bioterrorist crisis scenarios.

Historical perspective on agroterrorism: lessons learned from 1945 to 2012.

This article presents a historical perspective on agroterrorism cases from 1945 until 2012. The threat groups and perpetrators associated with bio- and agroterrorism are clustered into several groups: apocalyptic sects, lone wolves, political groups, and religious groups. We used open-source information, and 4 biological agroterrorism cases are described: (1) in 1952, Mau Mau poisoned cattle in Kenya by using a plant toxin from the African milk bush plant; (2) in 1985, the USDA claimed that Mexican contract workers were involved in deliberately spreading screwworm (Cochliomyia hominivorax) among livestock; (3) in 2000, Palestinian media reported that Israeli settlers released sewer water into Palestinian agricultural fields; and (4) in 2011, a person was sentenced to prison after threatening US and UK livestock with the deliberate spread of foot-and-mouth disease virus. All 4 cases can be assigned to political groups. These cases have not attracted much attention in literature nor in the public media, and the credibility of the sources of information varies. We concluded that agroterrorism has not been a problem during the period studied. Lessons learned from the few cases have generated awareness about the fact that nontypical biological weapons and non-high-risk agents, such as African milk bush, screwworm, and sewer water, have been used by attackers to influence local decision makers. This review will be useful in improving future preparedness planning and developing countermeasures.