Background • VibrioNet Europe

The current knowledge of the occurrence of vibrioses and agents thereof in Germany is fragmentary. At present human gastrointestinal infections in Germany are not investigated for non-cholera Vibrio spp., as mandatory notification exists only for cholera, which is caused by V. cholerae O1 and O139 strains (cholera toxin producing strains are not in the focus of this project). By the systematic isolation of strains from the environment, food and clinical sources, the VibrioNet will collect information on prevalence of human pathogenic Vibrio spp. in bivalve molluscs from natural and retail sources and in seasonal cases of human gastroenteritis and wound infections in Germany. 

There is circumstantial evidence that V. parahaemolyticus, V. vulnificus, V. alginolyticus and V. cholerae (non-O1, non-O139) occur bivalve molluscs and plankton in German coastal waters. In order to gain knowledge about the seasonal occurrence of pathogenic and pandemic Vibrio spp. in German coastal waters, the Alfred-Wegner-Institut (AWI) will monitor plankton, shellfish and seawater in the North Sea and Baltic Sea (Project C-2). This effort will be complemented by the Institut für Fische und Fischereierzeugnisse (IFF, LAVES), which monitors shellfish harvested from the production areas of the German Wadden Sea, thus representing the primary production for retail seafood (Project C-3).

Increasing global consumption and trade of seafood produce are predicted to raise the risk of a significant increase in V. parahaemolyticus and V. vulnificus infections in the coming decades. The occurrence of Vibrio spp. in retail seafood consumed in Germany will be investigated by the Institute of Food Hygiene of the FU Berlin (IFH). Food will be obtained from food producers, shops etc. and analysed for the occurrence of pathogenic Vibrio spp. (Project C-4).

Besides diarrheal illnesses, an increasing number of vibrioses are cases of wound infections that have been reported in Germany after bathing in the Baltic Sea, when the water temperatures were higher than 20°C. The Robert-Koch-Institut (RKI) will initiate a surveillance system to collect data about wound and diarrhoeal infections caused by Vibrio spp. and set up contacts to hospitals in the coastal regions of Germany and relevant diagnostic laboratories (Project C-7). All epidemiological data will be collected and consolidated in databases which will be at the disposal of all participating laboratories.

It is likely that under-reporting, particularly of V. parahaemolyticus infections which are generally self-limiting and transient, greatly masks the true clinical burden associated with these pathogens in Europe. The project of the Institut für Medizinische Mikrobiologie und Hygiene der Technischen Universität Dresden (TUD) aims to evaluate the seasonal  incidence of Vibrio spp. in stool samples from patients of a university hospital (Project C-8) Additionally, we will also cooperate with the RKI and hospitals in coastal regions of Germany, trying to estimate the incidence of Vibrio spp. in wound and diarrheal infections in these areas.

Rising seawater temperatures are thought to favour the proliferation and a more northerly geographic distribution of pathogenic Vibrio spp. All isolates of Vibrio spp. from human clinical cases (TUD, RKI) and food sources (IFH) will be added to the BfR collection as part of the national reference laboratory for monitoring bacterial contaminations of bivalve molluscs, while environmental strains (IFF, AWI) will be collected by the AWI. Data on the occurrence and the seasonal variations of Vibrio spp. pathotypes in natural ecosystems and seafood will be subject for multivariate statistical analysis to identify correlations with climate-associated parameters (AWI, BfR). Potentially climate-driven effects will be compared with observation from international partners in Asia and South America (Project C-1).

Aetiology, Pathogenesis

It is recognized that the majority of Vibrio spp. strains in the natural environment and in seafood does not pose a risk to humans. Hence, it is of uttermost importance to understand the adaptive mechanisms and driving forces leading to the seasonal accumulation, the evolution of human pathogenic Vibrio spp. and their transmission.

The majority of clinical isolates of Vibrio spp. show hemolysis due to the production of cytotoxins. The BfR will study the toxigenic potential of Vibrio strains in established cell culture systems and performing haemolytic assays (Project C-5). The BfR has established mussel and oyster cultures which can be used for testing toxin production under different environmental conditions (e.g. temperature) thus addressing the safety of seafood with respect to contaminations by V. parahaemolyticus, V. vulnificus, V. alginolyticus and V. cholerae (non-O1, non-O139).

Genome sequence analyses of Vibrio spp. pathotypes suggest the existence of other, as-yet-unidentified virulence determinants. The Project C-6 (BfR, IFH, TUD) will use a functional genomics approach to identify novel colonization and virulence factors in bivalve and mammalian models. Moreover, the project will experimentally test the hypothesis that adaptations to the bivalve host drive the evolution of virulence traits and enhance the human-pathogenic potential of Vibrio spp. The system is expected to unravel the adaptive mechanisms and driving forces leading to the seasonal accumulation of Vibrio spp. numbers in bivalves and the seasonal occurrence of disease.

Proliferation of Vibrio spp. in seafood is favoured by improper chilling, temperature abuse and extended storage time. The project of the IFH will quantify the growth dynamics of pathogenic Vibrio spp. in artificially contaminated seafood samples at different chilling and storage temperatures (Project C-4). In collaboration with the BfR, IFH and TUD (Project C-6) this project will the effect of storage temperature on proliferation, transmission rates and the underlying molecular investigate mechanisms.

Progress in the comprehensive analysis of virulence factors in Vibrio spp. is hampered by the lack of adequate mammalian infection models. TUD will develop an in vivo mouse model to evaluate the virulence well characterized Vibrio spp. (Project C-8). This will be followed by the functional characterization of selected virulence determinants from V. parahaemolyticus in this infection model. V. parahaemolyticus mutants of relevant virulence markers and functional genomics tools will be provided by the BfR, IFH and TUD (Project C-6).

Diagnostics, typing

The lack of a legal basis for systematic surveillance in clinics, seafood and environment as well as the lack of universal virulence markers have hampered the use of standardized, validated, fit-for-purpose methodologies. A major effort of the consortium will be to develop diagnostic tools for the qualitative and quantitative detection and characterization of Vibrio spp. pathotypes in environmental, seafood and clinical samples from Germany and international partners in Asia and South America.

Due to the large number of non-pathogenic Vibrio spp. in environmental and seafood samples, it requires an accurate taxonomic classification of isolates as well as the discrimination between non-pathogenic and potentially pathogenic strains. AWI and BfR will make use of modern mass spectrometry for the rapid taxonomic classification of their strain collections and the establishment of a MALDI-TOF database (Projects C-2, C-5).

In collaboration with the international partners in Asia and South America (Project C-1), the BfR, IFH and TUD aim to develop a culture-based virulotyping scheme to discriminate pathogenic and non-pathogenic Vibrio spp. strains (Projects C-5, C-6). This will be done by assessing the pathogenic potential of environmental vs. clinical isolates across defined geographical regions.

In order to characterize the Vibrio spp. pathotypes collected within the consortium, TUD will develop and validate a custom tailored DNA subset array for the presence of currently known pathogenicity markers (Project C-8), and those newly identified by the functional genomics studies in collaboration with the BfR, IFH and TUD  (Project C-6).

Transmission of Vibrio spp. pathotypes and clinical manifestation depends on the infectious dose which is a direct function of season, origin and sanitary conditions of harvest and processing. In collaboration with Q-Bioanalytik GmbH (QBA), IFH aims to develop real-time PCR assays as a tool for the rapid quantification of bacteria in different seafood matrices (Projects C-4, C-9).

A main goal will be the development of a robust and cost-effective “on-site” detection system for pathogenic Vibrio spp. which can be used outside laboratories and could be used in developing countries.

QBA will develop a nucleic acid lateral flow immunoassay, which in combination with PCR detection kits could be a favoured solution for implementing molecular techniques in developing countries by circumventing the use of expensive and labor-intensive equipment (Project C-9). In collaboration with the international partners in Asia and South America (Project C-1), the assays will be validated in food testing and clinical samples.