Influence of the RpoS (KatF) sigma factor on maintenance of viability and culturability of Escherichia coli and Salmonella typhimurium in seawater.

The sigma factor RpoS is essential for stationary-phase-specific, multiple-stress resistance. We compared the viabilities (direct viable counts) and culturabilities (colony counts) in seawater of Escherichia coli and Salmonella typhimurium strains and those in which rpoS was deleted or which were deficient in guanosine 3',5'-bispyrophosphate (ppGpp) synthesis (relA spoT). RpoS, possibly via ppGpp regulation, positively influenced the culturability of these bacteria in oligotrophic seawater. This influence closely depended, however, upon the growth state of the cells and the conditions under which they were grown prior to their transfer to seawater. The protective effect of RpoS was observed only in stationary-phase cells grown at low osmolarity. A previous exposure of cells to high osmolarity (0.5 M NaCl) also had a strong influence on the effect of RpoS on cell culturability in seawater. Both E. coli and S. typhimurium RpoS mutants lost the ability to acquire a high resistance to seawater, as observed in both logarithmic-phase and stationary-phase RpoS+ cells grown at high osmolarity. A previous growth of S. typhimurium cells under anoxic conditions also modulated the incidence of RpoS on their culturability. When grown anaerobically at high osmolarity, logarithmic-phase S. typhimurium RpoS+ cells partly lost their resistance to seawater through preadaptation to high osmolarity. When grown anaerobically at high osmolarity until stationary phase, both RpoS+ and RpoS- cells retained very high levels of both viability and culturability and then did not enter the viable but nonculturable state for over 8 days in seawater because of an RpoS-independent, unknown mechanism.

Seawater effects on various Vibrio species.

This study compared the effects of sea water on Vibrio cholerae and six other Vibrio spp. Survival in seawater microcosms as well as uptake of a carbonated substrate in marine or non-marine conditions were investigated. Except for V. vulnificus becoming non-culturable, all the other selected species survived in sea water for at least 15 days at 20 degrees C. Depending on the species tested, the substrate was better transported in a high salt medium (V. cholerae, V. fluvialis and V. metschnikovii), than in a low salt medium (V. fluvialis, V. furnissii, V. parahaemolyticus and V. vulnificus). In terms of the response of the species to marine conditions, no correlation was found between survival in sea water and substrate uptake.

Effect of thermal, oxidative, acidic, osmotic, or nutritional stresses on subsequent culturability of Escherichia coli in seawater.

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.

Glutamate uptake and synthesis by Escherichia coli cells in seawater: effects on culturability loss and glycinebetaine transport.

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 loss of culturability by Escherichia coli cells in seawater depends on availability of phosphate ions and phosphate transport systems.

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.

Sensitivity of Escherichia coli cells to seawater closely depends on their growth stage.

Sensitivity of Escherichia coli cells in seawater, considered in terms of culturability loss, was examined after different growth periods in a mineral medium supplemented with glucose (M9) at 37 degrees C under aerobic or anaerobic conditions. Their sensitivity varied considerably during the different growth phases and differed when cells were grown under aerobic or anaerobic conditions. Sensitivity of aerobic cells rapidly increased during the lag phase, then decreased during the exponential phase and became minimal during the stationary phase. Coliforms isolated from human faeces showed a similar sensitivity after incubation in wastewater at 37 degrees C for 3 h. The sensitivity phase was completely eliminated when cells were incubated with chloramphenicol. Variation of sensitivity in anaerobic cells according to their growth phase was comparable with that found for aerobic cells which had been left in seawater for a long period (6 d). However, for shorter periods in this medium (1-2 d), cells grown until the mid-exponential phase remained resistant to seawater. During the second half of the growth phase, they were as sensitive as aerobic cells at lag phase. Escherichia coli cells grown under anaerobic conditions, such as found in the intestine, progressively adapt to aerobic conditions after their transfer into aerated seawater and their sensitivity to seawater increases. On a practical level, these observations show that it is necessary to control accurately the age of cells before inoculation in seawater microcosms to conserve a comparative value in results. The importance of this factor is vital as all variations in sensitivity of cells to seawater according to their prior growth phase proved to be logarithmic functions of time.

OmpC and OmpF porins influence viability and culturability of Escherichia coli cells incubated in seawater.

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.

Intracellular accumulation of potassium and glutamate specifically enhances survival of Escherichia coli in seawater.

The high resistance of Escherichia coli grown in saline media to seawater was suppressed by an osmotic down-shock. The shock released several molecules into the medium, including potassium, glutamate, and glycine betaine when cells were previously grown in the presence of this osmolyte. Incubation of such sensitized cells in a solution containing K+ (80 mM) and glutamate (50 mM) at pH 7.4 restored their resistance to seawater up to a level close to that observed initially. The protective effect was partly due to the rapid accumulation of K+; a significant exponential relationship between intracellular concentration of K+ and resistance to seawater was observed. Glutamate was accumulated more slowly and progressively completed the action of K+. These data emphasize the specific influence of potassium glutamate on osmotically stressed E. coli cells. They confirm that regulation of osmotic pressure and, probably, of intracellular pH strongly enhances survival of E. coli in seawater. Osmotic fluctuations in waters carrying enteric bacteria from intestines to seawater, together with variations in their K+ and amino acid contents, could modify the ability of cells to survive in marine environments. These results demonstrate the need to strictly control conditions (K+ content, temperature) used to wash cells before their transfer to seawater microcosms. They suggest that the K+ and glutamate contents of media in which E. coli cells are transported to the sea can influence their subsequent survival in marine environments.

Influence of phosphate ions and alkaline phosphatase activity of cells on survival ofEscherichia coli in seawater.

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.

Role of transmembrane electrical potential on cadmium fixation by a marine pseudomonad.

The role of cellular energy, and mainly that of electrical transmembrane potential, in cadmium fixation by a marine pseudomonad suspended in a mineral medium was investigated by studying the effects of ionophores. Although fixation of cadmium by cells was generally less when respiratory activity was inhibited, it was not affected by a reduction of the transmembrane electrical potential delta psi in mureinoplasts. These observations strongly suggest that cadmium fixation in this isolate was not the result of a delta psi-dependent active transport.

Myocutaneous flaps in lip reconstruction. Applications of the Karapandzic principle.

Our experiences with the reconstruction of lips by the use of innervated myocutaneous flaps is described. We present the technique and some illustrative cases.

Applications of the Karapandzic principle of lip reconstruction after excision of lip cancer.

The number and variety of technics available for lip reconstruction has suggested to some authors that no method is ideal. In fact, excellent results can be obtained under proper circumstances by any one of several procedures. The principle of innervated myocutaneous flaps adds yet another tool to the surgeon's armamentarium, but it does not relieve him of the responsibility to select the most appropriate procedure in each case. As Karapandzic has stated (or understated), "This obviously is not a technique suitable for all lip defects, but in selected cases it gives a very satisfactory result."

Recognizing oral lesions.

Common benign lesions include hemagniomas and congenital cysts, pyogenic granuloma, aphthous ulcer, fibromas and changes produced by drugs. Many of these are self-limited and of little consequence. The point of the careful examination is the early detection of oral cancer. Only one patient in three with oral cavity cancer is cured. Premalignant and early asymptomatic lesions must be detected. Recognize the high-risk patient and examine the oral cavity meticulously.