The present study focused on the isolation of culturable bacteria from mussels and sea water to identify Vibrionaceae potentially pathogenic for humans. Three sites located on the French Atlantic coast were monitored monthly (twice each month during summer) for 1 year. Environmental parameters were surveyed (water temperature, salinity, turbidity, chlorophyll a) and bacteria were detected by culture and identified by API 20E(®) systems (BioMérieux) and PCR. A total of seven species were detected (Grimontia hollisae, Photobacterium damselae, Vibrio alginolyticus, V. cholerae, V. fluvialis, V. vulnificus and V. parahaemolyticus) and species diversity was higher at the end of summer. Surprisingly, V. cholerae non-O1/non-O139 was detected in spring. No site effect was detected. Using Sørensen similarity indices and statistical analyses, we showed that chlorophyll a had a significant influence on the bacterial community detected in mussels and assemblages were more similar to one another when chlorophyll a values were above 20 µg l(-1) . No significant effect of any parameter was found on the community detected in water samples. Such surveys are essential for the understanding of sanitary crises and detection of emerging pathogens.

AIMS:This study was carried out to investigate the occurrence of potentially pathogenic species of Vibrio in French marine and estuarine environments.METHODS AND RESULTS:Samples of coastal waters and mussels collected between July and September 1999 were analysed by culture, using selective media including thiosulphate-citrate-bile salts-sucrose and modified cellobiose-polymixin B-colistin agar. Presumptive Vibrio colonies were isolated and identified using selected biochemical tests. Specific primers based on flanking sequences of the cytolysin, vvhA gene, pR72H DNA fragment and 16S-23S rRNA intergenic spacer region (ISR) were used in a polymerase chain reaction (PCR) to confirm the identification of Vibrio vulnificus, V. parahaemolyticus and V. cholerae, respectively. In this study, V. alginolyticus (99 of 189) was the predominant species, followed by V. parahaemolyticus (41 of 189), V. vulnificus (20 of 189) and non-O1/non-O139 V. cholerae (three of 189). All 20 V. vulnificus isolates showed PCR amplification of the vvhA gene, 16 of which had been isolated from estuarine water. The PCR amplification of the pR72H DNA fragment in 41 V. parahaemolyticus isolates generated two unique amplicons of 387 and 320 bp. The latter, present in 24.4% of these isolates, had not previously been found in V. parahaemolyticus strains examined to date. Amplification of the trh gene in two of the isolates suggested these to be virulent strains. Three strains identified as V. cholerae by amplification of the 16S-23S rRNA ISR were confirmed to be non-cholera (non-O1/non-O139) strains.CONCLUSIONS:The results of this study demonstrated the presence of pathogenic Vibrio species in French coastal waters. Furthermore, the PCR approach proved useful for the rapid and reliable confirmation of species identification.SIGNIFICANCE AND IMPACT OF THE STUDY:These findings indicate the potential sanitary risk associated with the presence of pathogenic Vibrio spp. in cultivated mussels and in the aquatic environment. The PCR can be used to detect pathogenic vibrios directly in environmental samples.

Des bacteries fecales telles que les Escherichia coli ou les coliformes thermotolerants ont ete recherchees dans des sediments cotiers vaseux (preleves sur deux sites differents: Morlaix et St Pol de Leon; Finistere). Ces vases, sous influence de rejets polluants, de par leur richesse en matiere organique et en particules de faible taille sont susceptibles d'heberger ces bacteries. Une partie de l'etude a consiste a comparer differentes techniques de relargage des bacteries des particules de sediment decrites dans la litterature.

The effects of different environmental factors (nutrient deprivation, hyperosmotic shock, exposure to light) on enteric bacteria which have been transferred into the marine environment, have been studied experimentally (microcosms) by considering demographic, physiological and genetic responses in Escherichia coli or Salmonella typhimurium populations. Short-term experiments (less than or equal to 48 h) showed that nutrient deprivation induced limited changes in measured bacteriological variables, but when combined with hyperosmotic shock, it results in an energy charge decrease and inactivation of membrane transport. Light exposure mainly affects the colony-forming capacity of bacterial populations. Combining different stress factors confirmed the rapid appearance of a viable, but nonculturable state (VBNC) in populations of E. coli and S. typhimurium. It has been shown that cellular forms other than those previously described in the literature can be generated following incubation in seawater. It was also established that pre-adaptation phenomena may occur, leading to better survival (e.g. pre-incubation in seawater in darkness enhanced survival under light exposure). An explanation concerning these phenomena can be found by looking at the rpoS gene which controls the expression of numerous genes and can trigger a general anti-stress response under different adverse conditions. Although the results provide better comprehension of the fate of enteric bacteria in the marine environment, they also raise numerous questions related to fundamental and applied problems, given in the conclusion of this paper. (C) Elsevier, Paris.

Coastal management and water protection must provide acceptable water quality for shellfishing and tourist activities. To determine the impact of sewage on microbial water quality, engineers need to know the laws of bacterial reduction. In this study, experiments were performed with Escherichia coli in the laboratory and in situ (Atlantic and Mediterranean coast) using diffusion chambers at several depths. T90 was calculated from culturable bacterial counts over time. Establishing an abacus integrating the depth of water impacted by sewage and turbidity enabled us to calculate T90 from daily sunlight illuminations. Results obtained by cultures minimise health risks and yet viability and cellular integrity of E. coli remain, even under intense sunlight stress. When direct viable count is used to calculate a VT90, the results are dramatically higher and show how necessary – and difficult – it is to develop a concept which takes account of better understanding of sanitary risks in impact studies.

The risks of false-positive responses were examined when using the polymerase chain reaction (PCR) method for the detection of Salmonella in the marine environment (water and shellfish). The degradation rates of DNA, both free and from dead Salmonella, were evaluated in natural seawaters maintained at 10 degrees and 20 degrees C, using PCR with Vir and invA primers. The DNA of dead Salmonella was detected up to 55 d in seawater collected in winter and stored at 10 degrees C. But in summer, the persistence was shorter: 10 d or even 2 d for a smaller inoculum (3 x 10(3) Salmonella ml-1). The role of the planktonic organisms present in spring and summer was pinpointed. For free DNA, the persistence times were shorter: from 2 to 4 d at 20 degrees C, and from 3 to 8 d at 10 degrees C showing that the nuclease activity of marine organisms is higher at warm temperatures. These data led us to recommend careful interpretations of direct PCR results, especially during cold periods and for samples collected close to terrestrial discharges of high concentrations of live, dead or lysed Salmonella. PCR is a rapid, specific and sensitive method, but should be applied with care to marine samples, in order to avoid false-positive responses.

The effect of natural sunlight on culturability and persistence of pathogenicity of Escherichia coli was examined in the field, i.e., in the Morlaix Estuary, France, using an enterotoxigenic strain of Escherichia coli H10407. Results showed that E. coli responds to the estuarine diurnal solar cycle by entering the viable but nonculturable state upon exposure to sunlight. That is, direct counts of viable cells remained stable without significant change, but E. coli cells remained fully culturable only when exposed to seawater in control chambers in the dark, i.e., without solar irradiation. The effect of sunlight on the pathogenicity of E. coli H10407 was studied, using both the rabbit intestinal loop assay and ganglioside-enzyme-linked immunosorbent assay (GM1-ELISA), a sensitive procedure for testing for production of enterotoxin. Results of the GM1-ELISA demonstrated that strains of E. coli, after exposure to sunlight and entering the viable but nonculturable state, as well as culturable E. coli, retained pathogenicity, i.e., produced enterotoxin. The GM1-ELISA is concluded to be more sensitive than the rabbit intestinal loop assay for analysis of enterotoxin in natural water samples.

La survie en mer de bactéries d'origine fécale (E. coli et Salmonella spp.) a été étudiée après avoir fait transiter ces bactéries en eaux usées, de façon à rechercher une éventuelle adaptation. L'étude a été réalisée en microcosmes, les bactéries séjournant entre 24 et 72 h en effluents bruts et traités, avant d'être remises en suspension en eau de mer. Le suivi des populations pendant six jours a montré une protection des bactéries, par rapport au passage direct de la souche en eau de mer. Le séjour en eaux usées permet à la bactérie de conserver une viabilité importante. Ce phénomène a été observé pour les sept effluents testés, et semble lié à un stress quel-conque qui permettrait à la bactérie d'adapter son métabolisme à des conditions hostiles dès le transit dans les réseaux d'assainissement et les stations d'épuration.

A 24 h period in waste waters improved the subsequent survival of Salmonella in oligotrophic sea water, at 20 degrees C, compared to a direct input control. The main osmoprotective compound accumulated, investigated by 1H-NMR spectroscopy (nuclear magnetic resonance), after 6 d in sea water was trehalose. Taking into account these observations, this paper put forward the following explanation concerning the survival mechanism: (1) stress in waste waters induces the endogenous synthesis of trehalose via the activation of the gene kat F; (2) when exposed to an osmotic stress, two degradative cytoplasmic enzymes are repressed and the bacteria accumulate trehalose which acts as an osmoprotectant. The succession of the two steps enables Salm. manhattan to immediately resist to the high salinity of oligotrophic seawater.

Sewage treatment plants are designed to purify wastewaters for given parameters, but their microbiological effectiveness is often poor without addition of a specific treatment (disinfection, filtration, etc.). Large amounts of enteric bacteria are thus released into the environment, in rivers and seawater. Is their subsequent survival in these hostile environments influenced by their previous stay in wastewaters? In order to assess this potential adaptation, we studied the behavior of E. coli, S. typhimurium and S. manhattan in seawater microcosms, with and without a previous 24 to 72 h stay in raw and treated wastewaters. The seawater and wastewater media were 0.22 pm filtered and autoclaved. The protocol followed is outlined in Fig. 1. At each step, bacteria were harvested by filtration and re-suspended in the following medium. All microcosms were maintained at 20 degrees C, in the dark. Seven experiments were carried out, testing seven different wastewaters. The survival of the inoculated bacteria in seawater during 6 days was monitored by plate counts on trypticsoy agar and by acridine orange epifluorescence counts (Hobble et al., 1977). In two experiments, the bacterial enzymatic activity was assessed before and after the stay in seawater, using APIZYM kits. S. typhimurium's survival in seawater was increased when cells had previously spent 24 h in raw wastewaters, and 24 and 48 h in treated wastewaters (Fig. 3). The culturable count was about 2 log units higher after 6 days. The same phenomenon was observed for S. manhattan tested in two raw wastewaters (Fig. 4). For this strain, with a concentrated inoculum (7 x 10(6)/ml), survival was quite the same, with or without a previous stay in raw sewage. An adaptation of E. coli while in wastewater to subsequent seawater stress seemed less obvious (Fig. 5). While monitoring the behavior of enteric bacteria in seawater with different previous conditions, we assessed the ratio of culturable cells in the total population. Thus, loss of culturability of tested strains was smaller when bacteria had been put in wastewaters before seawater (Table 1). This could show that the level of cellular metabolism maintained was higher in this case. This hypothesis was confirmed by studying the enzymatic activity of S. typhimurium before and after 6 days in seawater (Fig. 6). A previous stay in sewage could preserve or even induce some enzymatic activity which was then maintained during the entire exposition to seawater. This suggests an active metabolism enabling new protein synthesis. In this study, we showed that Enterobacteriaceae could adapt to seawater stress during their stay in sewage and sewage treatment plants. The actual mechanism is not yet clear. It could be osmoprotection, modification of cellular metabolism, or another. Very few studies have dealt with this topic. Munro et al. (1987, 1989) observed that a previous culture in a saline medium or on a mixed wastewater/seawater agar could allow better E. coli survival in seawater. The osmoregulation mechanisms induced by high osmolarity are probably set up during this phase. This hypothesis cannot explain our results, since the wastewaters we used had very low osmolarity. It seems that adaptation to drastic conditions occurs during the stay in wastewater (lower temperature, lack of easily assimilated substrates, etc.) allowing fdr better survival in more drastic seawater conditions. This conclusion may be of great importance in assessing the sanitary quality of coastal areas used for bathing and shellfishing activities.

Les departements et territoires d'outre mer sont generalement l'objet d'enjeux economiques importants, et de conflits d'activites lies au developpement touristique, aquacole, agricole et industriel. Les etudes font generalement etat d'une lente degradation de l'environnement marin avec diminution des coraux et de la faune associee, au profit d'une augmentation de la couverture algale. On constate parfois un envasement ou un ensablement des sites, la presence dans l'eau de polluants organiques, dont des pesticides, des metaux ainsi que des microorganismes d'origine fecale. En Martinique, ce dernier aspect, lie a l'assainissement des zones rurales, urbaines et touristiques est actuellement considere comme une preoccupation majeure pour les prochaines annees. L'etude realisee sur le site de la baie de Fort de France en Martinique, en 1990 - 1993, avait pour objectif de definir l'impact des activites sur la qualite bacteriologique des eaux de la baie. Ce travail comporte plusieurs aspects : - l'estimation des concentrations en bacteries d'origine fecale des principaux rejets ou rivieres situees autour de la baie, - l'etude de la circulation des masses d'eaux dans la baie pour l'etablissement d'un modele hydrodynamique, - l'estimation des temps de mortalite des bacteries d'origine fecale dans les eaux martiniquaises en fonction des conditions locales (ensoleillement, temperature de l'eau, teneur en matiere en suspension ... ).

The survival of the enterotoxigenic Escherichia coli H 10407 was observed in different marine seawaters, both in situ and in vitro by use of dialysis chambers and microcosms, exposed and not exposed to light, Survival was monitored during several days by measurement of plate counts (culturable bacteria), direct viable counts with yeast extract and nalidixic acid (DVC : viable bacteria), and acridine orange direct counts (total bacteria).Without light, culturable E. coli counts decreased slowly (1.5 log unit), whereas viable and total bacteria counts remained almost the same through 8 days. When light stress emphasized the sea-water stress, the difference between culturable and viable counts increased from the very first two days (4 log units). In all the experiments, the number of viable bacteria remained very high. On the other hand, one experiment using dialysis chambers held in a turbid and brackish estuarine water showed no marked decrease in the initial counts. This better survival was probably due to the presence of organic matter. So culturability and viability of E. coli can change according to its environment. And because of the consequences for sanitary monitoring of seawaters, it must be kept in mind that viability of E. coli can be preserved within several days, in drastic conditions.

In order to improve bacterial water quality of shellfish farming areas, studies were conducted in the English Channel (Morlaix) and on the Mediterranean coast (Toulon). These two areas were chosen in order to compare behaviour of fecal bacteria in two different ecosystems. In the estuary of Morlaix sediments are polluted by way of settlement, but most of the bacteria are mixed with turbid waters and are able to survive a very long time (T90 are in a range of several hours to several days). By measuring the increase in salt tolerance of the strains grown in natural organic matter, it was demonstrated that Salmonella can tolerate coastal water salinities. Moreover, because light penetration is prevented by suspended matter, the solar bactericidal effect is very low. On the contrary, through lack of nutrients and very high sunlight intensity, die-off rates in Mediterranean waters are very high (at the surface T90 are less than 2 hours, and several hours in deep waters). A close relationship was found between the light intensity received by bacteria and the T90. Predicted T90 must be found using these two parameters(including turbidity and deep effect on light intensity). The authors suggest that precautions must be taken to carry out impact studies depending on water quality of the area, especially in turbid areas. The knowledge of these mechanisms is very important to evaluate waste water impact on the quality of shellfish farming areas, and to improve elimination of fccal bacteria in sewage treatment plants.

The production of an enzyme, 4-methylumbelliferyl heptanoate hydrolase, in Escherichia coli exposed to enriched and nonenriched seawater was studied. In all media, except for seawater with no or very small amounts of organic material and seawater enriched with peptone, 4-methylumbelliferyl heptanoate hydrolase activity increased by 2 to 3 orders of magnitude within 2 days. Increased enzyme activity was assumed to be related to cells not undergoing lysis but adapting to conditions of nutrient limitation.