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.

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.

At elevated osmolarity of the mineral medium M63, marine macroalgae constitute important osmoprotectants and nutrients sources for Escherichia coli. Growth of bacterial population (16 strains) was improved by supplementing M63 salts medium with either aqueous or ethanolic algal extracts obtained from Ascophyllum nodosum, Fucus serratus, Enteromorpha ramulosa, Ulva lactuca, and Palmaria palmata. In their presence, growth was still observed even at 1.02 m NaCl. Furthermore, the E. coli ZB400 growth in presence of whole macroalgae thalli in M63/0.85 m NaCI reached its maximum within 24 h (5 × 107 − 5 × 108 colony-forming units [CFU] per milliliter). In the presence of A. nodosum, bacterial growth was inhibited. In the same experimental conditions, ethanolic extracts improved E. coli growth significantly, because the yield reached 1011 CFU per milliliter. Ulva lactuca and P. palmata allowed the better growth. The Dragendorff-positive compounds extracted from bacterial cells growing on each ethanolic extract exhibited an osmoprotective effect as proved by a disk-diffusion assay. On the other hand, the -onium compounds (quaternary ammonium [betaines] and tertiary sulphonium) and total free amino acid contents of U. lactuca ethanolic extracts were higher than in others. Fucaceae extracts demonstrated especially high protein content. Algal extracts constitute not only an appreciable osmoprotection source for E. coli but also nutrient sources.

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.

The aim of this paper is to present some results of bacterial studies which were developed by IFREMER in coastal discharge areas of urban wastewaters; they are focused on the determination of bacterial inputs by wastewater treatment plants, the role of environmental factors on the enteric bacteria survival in the coastal zone, and the modelling of bacteria transport and disappearance in order to provide useful management information for minimizing faecal pollution in the coastal zone.

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 purpose of this paper is to evaluate the pollution introduced into the Morlaix estuary by the city discharge of sewage and to determine its consequences on the microbiological quality of estuarine water and sediment. It has been shown that between 10(13) and 10(14) fecal coliforms and streptococci and between 10(6) and 10(7) enteroviruses are thus conveyed per day. The study of the downstream contamination indicates that the bacterial and viral density is much more significant in the sediment and that the bacterial concentration decreases gradually from upstream to downstream whereas the viral density only varies slightly in the water and shows very irregular fluctuation in the sediment.

The growth of Vibrio parahaemolyticus in a liquid medium was compared with that of human fecal flora and estuarine flora. No marked differences were noted between growth at 25 and 37 degrees C for V. parahaemolyticus. However, the marine organisms were strongly inhibited when incubated at 37 degrees C. Incubation for 8 h in an enrichment broth yielded V. parahaemolyticus growth, even with a small inoculum, whereas the marine and fecal floras were inhibited. Therefore, enrichment for 8 h at 37 degrees C appears to be optimal for isolation of V. parahaemolyticus, permitting more rapid results in seafood analysis.