Incidence of pathogenic Vibrio species in marine samples from the Mediterranean area and its correlation with environmental factors The VibrioSea Consortium: Centre National d’Etudes Spatiales France (CNES), MEDIAS-France, Collecte Localisation Satellites (CLS), Institut Pasteur (IP) Paris-France, IP Marocco, IP Algeria and IP Tunisia, Institut Agronomique et Vétérinaire Hassan II du Maroc, Institut Français de Recherche pour l'exploitation de la Mer (IFREMER), Department of Biology, University of Genova, Department of Pathology, University of Verona Studies regarding emergence and spread of waterborne diseases (WBD) on a changing climate scenario delineating in the Mediterranean area are of increasing interest as concern predictive and preventive measures to avoid disease outbreaks. It has been proposed that there are some environmental/climatic parameters influencing distribution, abundance and persistence of auchthoctonous bacteria of medical interest such as pathogenic vibrios. The establishment of clear correlations between specific parameters, measured in situ and remotely, and quantity and distribution of Vibrio species would be the basis for early warning system, taking advantage of satellite tools, on vibrios- and other WBD-related health risks. The partners of the VibrioSea consortium (Fig 1) have collected samples from different sites in the Mediterranean area. The objectives were to study the occurrence of the three Vibrio species pathogenic for human in marine samples and their distribution in different areas in the Mediterranean Basin, and to analyze possible correlations between the presence of the pathogenic vibrios and a number of environmental factors. INTRODUCTION CONCLUSIONS Human pathogenic strains have been isolated in all the monitored areas with similar frequencies either in a freeform in water or adhered to sediment, plankton and shellfish. V. parahaemolyticus shows the highest occurrence in all the studied areas while V.vulnificus has been hardly isolated. Some V. cholerae strains have been isolated too. Pathogenic species have been isolated with a higher frequency in the sites located close to the coast. A significant correlation has been established for V.parahaemolyticus and SST which is an environmental parameter accurately measurable by satellite. Further analysis and more data are needed to establish a correlation between the environment and the other human pathogenic species. MATERIALS AND METHODS Sampling sites are located on the map Sampling campaigns took place from June 2006 to November 2007, twice monthly during the summer (June, July, August) and monthly the rest of the year. Environmental/climatic parameters: Sea Surface Temperature (SST), chlorophyll A, salinity, turbidity, were monitored by in situ measurements and remote sensing. Type of samples: surface water (1m from the surface), plankton, sediment and shellfish Enrichment was performed in peptone alkaline water pH 8.5, 3.5% NaCl for 7 hours. Selective media used for Vibrio isolation: TCBS agar All the samples have been processed and used to calculate the total Vibrio count by using the Most Probable Number (MPN) method Analysis of bacterial colonies suspected to be Vibrio pathogenic species (V. cholerae, V. vulnificus, V. parahaemolitycus) was conducted by: biochemical and cultural tests: API 20E and growth in 0-10% NaCl molecular tests: PCR, see Table 1 REFERENCES 1-. Appl. Environ. Microbiol., 1995, 61, Appl. Environ. Microbiol., 1991, 57, Appl. Environ. Microbiol., 1999, 65, J. Clin. Microbiol., 1992, 30, J. Clin. Microbiol., 1993, 31, J. Microbiol. Meth., 1999, 36, RESULTS Location of the partners around the Mediterranean Bassin Fig. 1. Percentage of samples containing pathogenic vibrios (PV) species in relation to the sampling season: cold season (October to March) and warm season (April to September) Table 1: PCR primers for confirmation of V. cholerae, V. parahaemolyticus and V. vulnificus species and for detection of genes encoding virulence factors. * : significant value; NA: not applicable; NS : not significant Fig.2. Percentage of samples positive for pathogenic vibrios relating to different ecological habitats Relationship between presence of Vibrio and environmental parameters Sea Surface Temperature Table 2. Mean comparison between presence of pathogenic Vibrio species and environmental parameters. *. Significance at the level of 0.05 ; **. Significance at the level of 0.01 Fig.3. Pathogenic Vibrios species in different type of samples Fig.4. Vibrios isolation frequencies vs the distance from the coast PROJECT TEAM: CNES: M. Lafaye; Medias France: JP. Lacaux, Y. Toure, E.Oryekhova; CLS: J. Stum, L. Commien, A. Ollivier; IP Paris: ML. Quilic, A. Guénolé, L. Lemée; IP Maroc: N. Cohen, A. Boukhanjer, M. Bennani; IP Algérie: F. Mouffok; IP Tunisie: R. Benaissa; IAV Hassan II: M. Bouslikhane; Ifremer: D. Hervio-Heath; Department of Biology, University of Genova: C. Pruzzo, L. Vezzulli; Department of Pathology, University of Verona: MM. Lleo, G. Caburlotto. Isolation frequency (%)