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Survival of stressed Escherichia coli with or without the rpoS gene was assessed after 2 and 6 days in sterile seawater. Cells were submitted to thermal (48°C), acidic (pH 5.1), oxidative (H2O2 1mM), nutritional (C, N, P starvation), or osmotic (NaCl 0.5M) stresses for periods ranging from 0 to 4 h. We found a stress-mediated cross protection against seawater relative to controls. Viability was higher when cells were acid, oxidatively, nutritionally or osmotically stressed. Survival increased in cells stressed at 37°C as compared with 20°C. With the exception of osmotic stress, we found that this stress-induced cross protection was rpoS dependent.

Using strains with or without the PhoE porin or different components of the phosphate regulon, we determined that maintenance of the culturability of Escherichia coli in seawater depended significantly on the presence of structures allowing access of phosphate ions to the periplasm, then to the cytoplasm of cells. Cells totally deprived of the two main phosphate transport systems (Pit, Pst) exhibited the highest loss of culturability. Most of this effect resulted from the loss of the high-affinity Pst system, and more specifically that of the periplasmic phosphate-binding protein PhoS. Survival was enhanced in seawater supplemented with phosphate (0.5 mM), whether or not these structures were present. From an ecological point of view, it is assumed that the presence of phosphate ions, even at low concentrations, can influence the behavior of E. coli cells in seawater.

In filtered natural seawater supplemented with potassium glutamate, the ability of Escherichia coli MC4100 cells to grow on a complex medium was enhanced as a logarithmic function of the external glutamate concentration. By comparison, a glutamate-respiring strain of E. coli exhibited a greater decline in culturability in seawater, suggesting a protective influence of the accumulated amino acid. Potassium glutamate increased the uptake of 14C-glycinebetaine by E. coli MC4100 cells in seawater and enhanced the protective effects of the betaine against culturability loss, possibly by increasing the expression of the ProU transport system. This bacterium apparently was able to synthesize glutamate because a protective effect (i.e. a lower culturability loss) was observed in seawater when supplemented with precursor compounds (2-oxoglutarate and glutamine). The combination of 2-oxoglutarate and glutamine resulted in the greatest protection of cells, possibly due to the synthesis of glutamate through glutamine 2-oxoglutarate amino transferase activity. The possible influence of glutamate and its precursors on survival of E. coli cells in the natural marine environment is considered, since glutamate, glutamine and betaines have been found in marine coastal waters and sediments.

The contribution of the major outer membrane porins OmpF and OmpC to the maintenance of viability and culturability of Escherichia coli cells in seawater was analyzed using isogenic mutant strains lacking one or both porins. Cells that possessed OmpF and OmpC survived better than those lacking one or both of them. However, the results differed, depending on whether the cells were adapted to high osmolarity or not before transfer to seawater. When cells were grown at low osmolarity, survival was largely influenced by porins, the OmpF+ strains surviving better than those lacking this porin. Addition of an OmpF plasmid to OmpF- OmpC- cells also improved their viability. When grown at high osmolarity, the role of porins was less critical since both the viability and culturability of the cells increased. However, cells that expressed only OmpC showed the most dramatic loss of viability. Cells lacking both OmpF and OmpC exhibited a higher loss of viability and culturability in seawater. Regarding the influence of porins on survival, these results show that the conditions that prevail during the growth of cells before their transfer to seawater are highly influential: cells that express the porin corresponding to the growth conditions they are in at the time of transfer survive better.

The efficiency of stabilization pond treatment of domestic wastewater in removing culturable cells of motile Aeromonas and its influence on the incidence of resistance to seven antibiotics were investigated in this study. Removal efficiency was higher (P < 0.001) in the warm months (98.8%) than in the cold months (97%). Among the 264 isolates, 163 were Aeromonas caviae, 24 were A. hydrophila, and 54 were A. sobria. Twenty-three isolates could not be identified to the species level. In the influent, A. caviae dominated in both cold and warm months. In the water samples originating from the influent, A. sobria was present at higher percentages in the warm period. All the isolates were resistant to amoxicillin and most of them (73%) exhibited resistance to cephalothin. Of the three species tested, A. sobria was more susceptible to antibiotics than either A. caviae or A. hydrophila. The most striking difference among the species was seen in resistance to cephalothin. There were 91 % of A. caviae strains and 96% of A. hydrophila isolates that were resistant to cephalothin. However, only 9% of A. sobria strains exhibited resistance to this drug. The high incidence of resistance in raw sewage was connected with a high proportion of A. caviae, whereas in the water samples collected from the effluent during the warm months, a high proportion of A. sobria decreased the total amount of multiple-resistant bacteria. Results demonstrated the need for identification to the species level.

When grown in a minimal medium and suspended for 2 hours in distilled water, seawater, phosphate buffer or a polyphosphate solution,E. coli MC4100 cells with high alkaline phosphatase activity survived in seawater for longer periods than cells with low or no activity. However, mutant cells totally deprived of alkaline phosphatase activity held in phosphate-containing media before transfer to seawater showed survival almost as high as the wild type strain, indicating that alkaline phosphatase activity is not the only factor influencing survival. Alkaline phosphatase activity also increased the protection of cells provided by glycine betaine. Survival was enhanced when cells were preincubated in the presence of phosphate or polyphosphate. Thus, the transfer of cells in wastewater could influence their subsequent survival in seawater.