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

REFERENCES1-. Appl. Environ. Microbiol., 1995, 61, 1311-1317. 2- Appl. Environ. Microbiol., 1991, 57, 2651-2655 3- Appl. Environ. Microbiol. , 1999, 65, 2202-22084- J. Clin. Microbiol., 1992, 30, 2118-2121.5- J. Clin. Microbiol., 1993, 31, 22-25.6- J. Microbiol. Meth., 1999, 36, 215-225.

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)

Target sequence Size of the amplicon Vibrio speciesR72H1 320 ou 387 bp V. parahaemolyticusHly2 388 bp V. vulnificus

16 S-23S RNA 300 bp V. cholerae

ctxA4 564 bp V. choleraectxB5 460 bp V. choleraetdh6 269 bp V. parahaemolyticustrh6 500 bp V. parahaemolyticus

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 significantFig.2. Percentage of samples positive for pathogenic vibrios relating to different ecological habitats

Relationship between presence of Vibrio and environmental parameters

Sea Surface Temperature

Global VPP_Present VPP_Absent Two tailed test fordifference

Mean Mean P-value SignificanceSST 20,7 17 0,001 **pH 7,85 8 0,150 NSSal 35,82 37 0,030 NS

Torb 3,42 2 0,010 *Cond 76,94 78 0,210 NSSM 35,35 47 0,190 NS

Chlo.A 1,3 1 0,006 *

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

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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.

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