Ecology, diversity and pathogenicity of Vibrio cholerae

Vibrio cholerae is both a human pathogen and natural inhabitant of the aquatic environment. Of more than 200 serogroups, only O1 and O139 are associated with epidemic cholera. In most developed countries nonO1/nonO139 strains are responsible for less severe watery diarrhoea and infections of blood, wound, ear and the respiratory tract. In the past five years, 13 cases of V. cholerae nonO1/nonO139 infections were documented in Austria, of which eight had a local history. Five cases could be explicitly associated with recreational activities in an alkaline lake area in Eastern Austria, with four cases of otitis and one of lethal septicemia. Similar cases are frequently reported from Sweden, Germany, Poland, and the Netherlands. Epidemic cholera is mainly restricted to developing countries in Asia, Africa and South America, but in the 1990s also in Europe several countries were plagued by cholera. Travel activities and temperature increase due to global warming enhances the probability of the establishment of V. cholerae O1/O139 strains in temperate aquatic ecosystems. In addition, virulence genes can be transferred to V. cholerae nonO1/nonO139, transforming a benign strain into a pathogen. Thus, as a basis of a future risk assessment, it is of prime importance to understand the ecology of endemic nonO1/nonO139 V. cholerae strains and the environmental performance of "foreign" pathogenic strains in saline waters of developed countries. Several investigations have traced the potential niches of V. cholerae in the aquatic environment, but the ecology of this human pathogen is still poorly understood. V. cholerae has been shown to live in association with crustacean zooplankton and algae where it degrades polymeric substances. In contrast, it has also been reported that V. cholerae grow rapidly in water as free-floating organisms. Thus, the existence of at least two main growth strategies can be assumed, which has important consequences for mechanisms controlling population size and survival. Understanding the influence of key biotic and abiotic factors on V. cholerae is thus a prerequisite for predicting the fate of this pathogen in the aquatic environment. A series of about 40 alkaline lakes are located in Eastern Austria. They exhibit steep gradients in environmental characteristics and are thus ideal model ecosystems for testing ecological hypotheses. V. cholerae has been detected in these lakes since 2001. To elucidate the ecology of V. cholerae populations in these lakes two strategies will be adopted. First, the in situ abundance of V. cholerae will be monitored over the seasons along the environmental gradients and their significance will be assessed via statistical analyses. Second, the influence of selected factors on the autochthonous V. cholerae strains will be investigated in specifically designed microcosm experiments. In similar experiments the environmental performance of selected O1/O139 strains will be tested to allow for risk assessment, whether such cholera-toxigenic strains are capable of establishing stable populations in the investigated saline lakes. In addition, the genetic diversity and pathogenic potential of endemic V. cholerae strains and strains imported by birds will be determined to detect relationships of different V. cholerae clones to changing environmental conditions and whether these environmental strains are identical with clinical strains isolated from patients. The proposed research will significantly enhance our knowledge of the threat potential of V. cholerae for human health, not only on a regional but also on a global basis, because it will elucidate general principles of V. cholerae ecology, the potential import of cholera-toxigenic strains to developed countries, and the survival performance of toxic strains in saline inland waters.

Due to climate change, (re-)emerging aquatic pathogens like Vibrio cholerae are an increasing public health concern in the temperate climate zone. Since the beginning of this century, increasing numbers of Vibrio related infections have been experienced by visitors of European marine and saline inland waters. To assess the potential health risk, it is thus of prime importance to develop tools and strategies to investigate the abundance, distribution and environmental behaviour of aquatic pathogens like V.cholerae. In Austria, the Lake Neusiedler See and adjacent smaller saline lakes are ideal model ecosystems, as they harbour abundant V.cholerae populations and are hot spots of migratory bird associated microbial import in Europe. In this project, two novel culture-independent methods were developed as tools to quantify V.cholerae in aquatic environments. By the application of a mass-balanced approach and by integrating a comprehensive set of environmental variables it was possible to identify the main habitats of V.cholerae and the main environmental predictors of V.cholerae abundance. We could show that a specific set of ecological parameters control V.cholerae numbers in a specific type of environment. In addition, we could demonstrate that a highly specific relationship of V.cholerae to the dominant crustacean zooplankton species in the lakes exists and that crustaceans are the main habitat for V.cholerae only during a short period in summer. During the major part of the year V.cholerae were detected in the planktonic state, even in winter, when V.cholerae were not detectable by culture, but were still able to cause ear infections. The V.cholerae populations in the lake exhibited an extreme genetic diversity that was highest in the structured habitat of the reed-stand, in contrast to a much lower diversity in the open lake area. Interestingly, several strains from other European countries (France, Netherlands, Romania, and Sweden) displayed the same genotyping pattern as strains isolated from the lake. This indicates frequent intra- and intercontinental transport of V.cholerae strains, most probably via birds. In fact, we were able to isolate V.cholerae strains from freshly collected migratory bird faeces, including a non-cholera-toxigenic O1 strain, while not a single V.cholerae O1/O139 was isolated from water, sediment or zooplankton (n > 3000). As lab experiments showed, V.cholerae O1 ElTor strain was not able to proliferate in lake water under optimal growth conditions. Thus, the survival and invasion potential of imported V.cholerae strains may be highly strains specific, which has to be considered in future hazard scenarios. Based on the developed methodical tools and the holistic strategy, this study may serve as a basic model for investigating pathogenic Vibrios and other waterborne health-relevant bacteria in the aquatic environment.