Transcript Incidence of pathogenic Vibrio species in marine

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
INTRODUCTION
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.
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
Table 1: PCR primers for confirmation of V. cholerae, V. parahaemolyticus and
V. vulnificus species and for detection of genes encoding virulence factors.
Target sequence
R72H1
Hly 2
16 S-23S RNA
ctxA4
ctxB5
tdh6
trh6
Size of the amplicon
320 ou 387 bp
388 bp
300 bp
564 bp
460 bp
269 bp
500 bp
Vibrio species
V. parahaemolyticus
V. vulnificus
V. cholerae
V. cholerae
V. cholerae
V. parahaemolyticus
V. parahaemolyticus
Location of the partners around the Mediterranean Bassin
RESULTS
Relationship between presence of Vibrio and environmental parameters
Sea Surface Temperature
Global
SST
pH
Sal
Torb
Cond
SM
Chlo.A
VPP_Present VPP_Abse nt
Mean
20,7
7,85
35,82
3,42
76,94
35,35
1,3
Mean
17
8
37
2
78
47
1
Two tailed test f or
difference
P-value
0,001
0,150
0,030
0,010
0,210
0,190
0,006
Significance
**
NS
NS
*
NS
NS
*
*. Significance at the level of 0.05 ; **. Significance at the level of 0.01
Table 2. Mean comparison between presence of pathogenic Vibrio species and
environmental parameters.
* : significant value; NA: not applicable; NS : not significant
Fig.2. Percentage of samples positive for pathogenic vibrios relating to
different ecological habitats
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)
100%
Isolation frequency (%)
90%
80%
70%
60%
50%
40%
30%
20%
10%
Fig.4. Vibrios isolation frequencies vs the distance from the coast
Po
si
tiv
e
sa
m
pl
es
0%
Fig.3. Pathogenic Vibrios species in different type of samples
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.
REFERENCES
1-. Appl. Environ. Microbiol., 1995, 61, 1311-1317.
2- Appl. Environ. Microbiol., 1991, 57, 2651-2655
3- Appl. Environ. Microbiol. , 1999, 65, 2202-2208
4- J. Clin. Microbiol., 1992, 30, 2118-2121.
5- J. Clin. Microbiol., 1993, 31, 22-25.
6- J. Microbiol. Meth., 1999, 36, 215-225.
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.