INFLUENCE OF THE PREVIOUS STAY OF ESCHERICHIA-COLI AND SALMONELLA SPP IN WASTE-WATERS ON THEIR SURVIVAL IN 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.