Changes in Escherichia coli cells starved in seawater or grown in seawater-wastewater mixtures

Some metabolic modifications of Escherichia coli cells during starvation in seawater were studied in laboratory microcosms. The apparent die-off of this bacterium under such conditions, as observed by comparing the enumeration of CFU in conventional freshwater media and direct epifluorescence counts, was partially prevented when cells were previously grown in salted organic medium or on seawater-wastewater agar. beta-Galactosidase activity of starved cells disappeared gradually with time, even though some other enzymatic activities, such as that of alkaline phosphatase, increased. Moreover, some modifications of sensitivity to antibiotics, heavy metals, and bacteriophages in seawater- and wastewater-grown cells suggested that the cells undergo structural changes under natural marine conditions. These results provide additional experimental data indicating the possible active adaptation of E. coli cells to seawater.     

Changes in Escherichia coli outer membrane subproteome under environmental conditions inducing the viable but nonculturable state

Changes in the outer membrane subproteome of Escherichia coli along the transition to the viable but nonculturable state (VBNC) were studied. The VBNC state was triggered by exposure of E. coli cells to adverse conditions such as aquatic systems, starvation, suboptimal temperature, visible light irradiation and seawater. The subproteome, obtained according to Molloy et al ., was analysed at the beginning of exposure (inoculum, phase 1), after a variable exposure time (95% of population culturable, phase 2) and when populations were mainly in the VBNC state (95% of cells VBNC, phase 3). Proteome changes were dependent on adverse conditions inducing the transition and were detected mainly in phase 2. The permanence of E. coli cells in seawater under illumination conditions entailed a dramatic rearrangement of the outer membrane subproteome involving 106 new spots, some of which could be identified by peptide fingerprinting. However, proteins exclusive to the VBNC state were not detected.     

Modification of the envelope and the protein content of Escherichia coli surviving in sea water

A toxigenic strain of Escherichia coli displayed important structural modifications when placed in seawater which naturally lacked nutritive elements, as observed by electron microscopy. These include cell wall and cell body distortion, modification of the membranes, central segregation of the chromosome, and retraction of the cytoplasm. These modifications were accompanied by a decrease in cell protein content of approximately 40%. Certain cytoplasmic membrane proteins were lost, and new ones appeared. The development of these changes was considerably slower in cells that had previously been grown in a seawater medium. This suggests that osmotic regulation mechanisms, which enable E. coli to survive much longer in marine conditions, may have a protective influence.     

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.     

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.     

Effect of sunlight on the infectivity of Cryptosporidium parvum in seawater

The prevalence of pathogenic microorganisms in seawater can result in waterborne and food borne outbreaks. This study was performed to determine the effect of sunlight and salinity on the die-off of Cryptosporidium parvum. Cryptosporidium parvum oocysts, Escherichia coli, and MS2 coliphage were seeded into tap water and seawater samples and then exposed to sunlight. The die-off of C. parvum in seawater, as measured by infectivity, was greater under sunlight (-3.08 log10) than under dark conditions (-1.31 log10). While, no significant difference was recorded in the die-off of C. parvum, under dark conditions, in tap water as compared to seawater (P < 0.05), indicating that the synergistic effect of salinity and sunlight was responsible for the enhanced die-off in seawater. The die-off of MS2 coliphage and E. coli was greater than that observed for C. parvum under all tested conditions. This indicates that these microorganisms cannot serve as indicators for the presence of C. parvum oocysts in seawaters. The results of the study suggest that C. parvum can persist as infectious oocysts for a long time in seawater and can thus pose a serious hazard by direct and indirect contact with humans.     

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.     

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 DNA supercoiling on the loss of culturability of Escherichia coli cells incubated in seawater

The relationship between the loss of culturability of Escherichia coli cells in seawater and the DNA supercoiling level of a reporter plasmid (pUC8) have been studied under different experimental conditions. Transfer to seawater of cells grown at low osmolarity decreased their ability to grow without apparent modification of the plasmid supercoiling. We found that E. coli cells could be protected against seawater-induced loss of culturability by increasing their DNA-negative supercoiling in response to environmental factors: either a growth at high osmolarity before the transfer to seawater, or addition of organic matter (50-mg/l peptone) in seawater. We further found conditions where a DNA-induced relaxation was accompanied by an increase in seawater sensitivity. Indeed, inactivation of either one of the subunits A and B of DNA gyrase, which leads to important DNA relaxation, was accompanied in both cases by an increased loss of culturability of conditional mutants after transfer to seawater which could not be explained uniquely by the increase in the temperature required to inactivate the gyrase. Similarly, a strain harbouring a mutation in topoisomerase I, compensated by another mutation in subunit B of the gyrase, was more sensitive to seawater than the isogenic wild-type cell and this greater sensitivity was correlated to a relaxation of plasmid DNA. Again, in these different cases, a previous growth at high osmolarity protected against this seawater sensitivity. We thus propose that the ability of E. coli cells to survive in seawater and maintain their ability to grow on culture media could be linked, at least in part, to the topological state of their DNA.(ABSTRACT TRUNCATED AT 250 WORDS)     

Influence of prior growth conditions on low nutrient response of Escherichia coli in seawater

By use of experimental microcosms, it was demonstrated that the survival of Escherichia coli in nutrient-free seawater depended on the age of cells and on some physicochemical conditions during their prior growth. Cells grown in a bacteriological medium, with an acid or an alkaline pH, at high temperature (44 degrees C), or in the absence of oxygen were more sensitive to exposure to seawater of low nutrient content. In contrast, some complex media allowed production of cells adapting more rapidly to seawater. Cells grown in urine were far more sensitive than those grown in all bacteriological media tested. The sensitivity of all cells was highest when they were harvested during the early exponential phase of growth.     

Long-term survival and plasmid maintenance of Escherichia coli in marine microcosms

Abstract The survival pattern and plasmid maintenance of Escherichia coli was examined in an artificial seawater microcosm. It was found that the three strains of E. coli (EK3C, H10407 and 34309) included in the study were able to maintain a portion of cells in the culturable phase for at least 3 years in artificial seawater. Along with retaining culturability, that portion of the cell population also maintained their indigenous plasmids over the 3-year period. It is concluded that cells of E. coli maintaining culturability in seawater are selectively adapted to the salinity of seawater, remaining in a culturable state. The results of the study are significant in that it has been assumed by many public health authorities that E. coli cannot survive, without nutrient addition, in seawater for long periods of time, i.e., years of exposure to seawater.     

DNA oxidation products determined with repair endonucleases in mammalian cells: types, basal levels and influence of cell proliferation

Purified repair endonucleases such as Fpg protein, endonuclease III and IV allow a very sensitive quantification of various types of oxidative DNA modifications in mammalian cells. By means of these assays, the numbers of base modifications sensitive to Fpg protein, which include 8-hydroxyguanine (8-oxoG), were determined to be less than 0.3 per 10⁶ bp in several types of untreated cultured mammalian cells and human lymphocytes and less than 10 per 10⁶ bp in mitochondrial DNA from rat and porcine liver. Oxidative 5,6-dihydropyrimidine derivatives sensitive to endonuclease III and sites of base loss sensitive to endonuclease IV or exonuclease III were much less frequent than Fpg-sensitive modifications. Here, we summarize our indications that all Fpg-sensitive modifications are recognized under the assay conditions and that on the other hand there is no artifactual generation of oxidative damage during the analysis. In addition, we show that the steady-state levels of Fpg-sensitive modifications in human lymphocytes and in two mammalian cell lines were higher in proliferating than in resting (confluent) cells. Only some of the Fpg-sensitive base modifications induced by various oxidants are 8-oxoG residues, as demonstrated for the damage under cell-free conditions. The percentage was dependent on the species ultimately responsible for the DNA damage and was approx. 40% in the case of hydroxyl radicals and peroxynitrite, 75% for type II photosensitizers (reacting via singlet oxygen) and only 20-30% in the case of type I photosensitizers such as riboflavin and acridine orange, which are assumed to react directly with the DNA.     

Ultrastructural features of chloride cells in the gill epithelium of the Atlantic salmon, Salmo salar, and their modifications during smoltification

To elucidate the ultrastructural modifications of the gill epithelium during smoltification, gills of the Atlantic salmon (Salmo salar) were examined by electron microscopy at three stages of this process, which were defined as follows: "parrs" were freshwater fish that had not yet started their transformation; "freshwater smolts" were freshwater fish that were ready to enter seawater; and "seawater smolts" were smolts that had been transferred from fresh water and maintained for 4 days in seawater (35%). In the gill epithelium of parrs, there were two types of chloride cells. The large chloride cells contained deeply stained mitochondria and numerous apical, irregular, dense, membrane-bound bodies that formed 77% of the chloride cell population and were distinguished easily from small chloride cells that have distinctly paler mitochondria and no dense bodies in their apical cytoplasm. In freshwater smolts, the large chloride cells formed 95% of the chloride-cell population. In contrast to the small chloride cells that were not modified, they almost doubled in size. Their tubular system developed extensively to form a tight network with regular meshes significantly smaller than those observed in parr chloride cells. Forty percent of the large chloride cells were associated with a new type of cell, the accessory cell, to which they were bound by shallow apical junctions. Half of these accessory cells were not seen to be in contact with the external medium. In seawater smolts, 80% of the large chloride cells were associated with accessory cells. Most accessory cells reached the external medium and sent numerous cytoplasmic interdigitations within the apical portion of the adjacent chloride cells. As a result, a section through the apical portion of the chloride cells and their associated accessory cells revealed a mosaic of interlocked cell processes bound together by an extended, shallow apical junction. It was concluded that the Atlantic salmon develops in fresh water most of the ultrastructural modifications of the gill epithelium which in most euryhaline fish are triggered by exposure to seawater. The effective transfer into seawater would act only as a final stimulus to achieve some adequacy between the freshwater smolt and its new environment.     

Persistence, dissipation, and activity of Escherichia coli O157:H7 within sand and seawater environments

Runoff from agricultural land into watercourses may transport and deposit animal-derived waste contaminated with Escherichia coli O157:H7 onto beaches, which may in turn lead to human infection. To simulate contamination, freshwater mixed with cattle slurry containing E. coli O157:H7 was added to sand from three recreational beaches. The sand was then maintained in a dry state (nontidal) or subjected to a repeated seawater tidal simulation. The pathogen could still be recovered from all sands by day 5. Although survival of the pathogen did not statistically vary between sands of different origin under nontidal conditions, significant differences in numbers occurred between sands when subject to tidal simulation. In the tidal simulations, a considerable proportion of the E. coli O157:H7 rapidly dissipated from sand into the seawater. In a separate experiment, the activity of bioluminescent (lux-marked) E. coli O157:H7 cells was monitored in various mixtures of contaminated runoff water and seawater over 5 days. Pathogen activity declined with increasing seawater concentration; however, cells remained viable in all treatments over the 5-day period. The addition of nutrients to water rapidly increased pathogen activity in all treatments. Our findings highlight the resilience of E. coli O157:H7 in aquatic and marine environments.     

In situ development of sublethal stress in Escherichia coli: effects on enumeration.

Development of sublethal stress in Escherichia coli exposed in situ to estuarine waters was examined during various seasons. An electrochemical detection technique was utilized to derive a stress index based upon the difference between a predicted electrochemical response time in Trypticase soy broth or EC medium at 44.5 degrees C estimated from a standard curve for unstressed cells and an observed response time for cells exposed to seawater. This stress index was related to recovery efficiencies of seawater-exposed cells, using a variety of standard and resuscitative enumeration procedures. Stress was further studied by determination of the adenylate energy charge. Sublethal stress as measured by the electrochemical detection method was an inverse function of water temperature, with maximum stress occurring after exposure to temperatures below 10 degrees C. Total adenylates and ATP decreased dramatically at low temperatures, although energy charge remained relatively constant under various environmental conditions. Decreases in E. coli ATP suggest that ATP may not be an adequate measure of biomass for in situ stressed cells. Discrepancies in enumeration efficiency were most pronounced at temperatures below 10 degrees C. Resuscitative procedures for solid-media techniques increased the recovery of stressed cells under cold water conditions but were not as effective as the standard most-probable-number procedure.     

Maintenance of plasmids pBR322 and pUC8 in nonculturable Escherichia coli in the marine environment.

Maintenance of plasmids pBR322 and pUC8 in Escherichia coli that was nonculturable after exposure to seawater was studied. E. coli JM83 and JM101, which contained plasmids pBR322 and pUC8, respectively, were placed in sterile artificial seawater for 21 days. Culturability was determined by plating on both nonselective and selective agar, and plasmid maintenance was monitored by direct isolation of plasmid nucleic acid from bacteria collected on Sterivex filters. E. coli JM83 became nonculturable after incubation for 6 days in seawater yet maintained plasmid pBR322 for the entire period of the study, i.e., 21 days. E. coli JM101 was nonculturable after incubation in seawater for 21 days and also maintained plasmid pUC8 throughout the duration of the microcosm experiment. Direct counts of bacterial cells did not change significantly during exposure to seawater, even though plate counts yielded no viable (i.e., platable) cells. We concluded that E. coli cells are capable of maintaining high-copy-number plasmids, even when no longer culturable, after exposure to the estuarine or marine environment.     

Survival strategy of Escherichia coli and Enterococcus faecalis in illuminated fresh and marine systems

Some effects of visible light on Escherichia coli and Enterococcus faecalis in natural freshwater and seawater were studied by plate counts, colony area measurements, and direct counts. A large number of somnicells (non-culturable cells) were noted in illuminated systems as compared with non-illuminated ones. Colony areas were significantly smaller in illuminated systems. Indirect activity measurements were used to test the effects of visible light on the ability of E. coli and Ent. faecalis to metabolize substrates ([14C]glucose) in natural waters. In illuminated systems, a decrease of glucose uptake was observed. When percentages of assimilation and respiration with respect to the total glucose uptake were analysed a decrease of assimilation percentages and an increase of respiration percentages were observed. In addition, differences in glucose uptake, assimilation and respiration by enteric bacteria were detected for E. coli at the beginning of the experiments between fresh- and seawater and these were interpreted as a toxic effect exerted by seawater on E. coli cells. Differences between species, natural waters and parameters studied (excepting glucose assimilation) were detected in the illuminated systems. We concluded, however, that enteric bacteria under visible light illumination show a general survival strategy characterized by reaching progressively a somnicell stage which can be defined in terms of their (1) inability to form colonies on standard bacteriological media, (2) inability to incorporate substrates, and (3) inactivation of biosynthetic processes.     

Maintenance of plasmids pBR322 and pUC8 in nonculturable Escherichia coli in the marine environment

Maintenance of plasmids pBR322 and pUC8 in Escherichia coli that was nonculturable after exposure to seawater was studied. E. coli JM83 and JM101, which contained plasmids pBR322 and pUC8, respectively, were placed in sterile artificial seawater for 21 days. Culturability was determined by plating on both nonselective and selective agar, and plasmid maintenance was monitored by direct isolation of plasmid nucleic acid from bacteria collected on Sterivex filters. E. coli JM83 became nonculturable after incubation for 6 days in seawater yet maintained plasmid pBR322 for the entire period of the study, i.e., 21 days. E. coli JM101 was nonculturable after incubation in seawater for 21 days and also maintained plasmid pUC8 throughout the duration of the microcosm experiment. Direct counts of bacterial cells did not change significantly during exposure to seawater, even though plate counts yielded no viable (i.e., platable) cells. We concluded that E. coli cells are capable of maintaining high-copy-number plasmids, even when no longer culturable, after exposure to the estuarine or marine environment.     

Changes in protein composition and hydrolytic enzyme activity of Escherichia coli and Hafnia alvei grown in human fluids

Growing of Escherichia coli and Hafnia alvei cells in several cell-free human fluids, such as normal serum, serum from diabetic patients, pleural, ascitic and spinal fluid, revealed that various biochemical changes occurred. Protein profile on SDS-PAGE as well as acid and alkaline phosphohydrolytic enzymes on native gels of cell extracts were affected after culturing of bacteria in the above fluids. Gelatinolytic and hyaluronolytic activity was of interest because both of them are histolytic enzymes. Although there was a potential appearance of gelatinolytic bands on gelatin-SDS-PAGE in cells starved in seawater, none of these activities were expressed in cells grown in human fluids. A hyaluronolytic activity of approximately 45 KDa was present in cells cultured in Mueller Hinton broth. This enzyme was decreased either in cells starved in seawater or in cells grown in human fluids to an almost invisible band on hyaluronan-SDS-PAGE.     

Characterization of an oxygen-dependent inducible promoter,the Escherichia coli nar promoter,in gram-negative host strains

The Escherichia coli nar promoter is maximally induced under anaerobic conditions in the presence of nitrate ion or under anaerobic only conditions, depending on the genotype of the E. coli nar promoter. Previously, we found that the E. coli nar promoter has some desirable characteristics as an inducible promoter in the E. coli host strains. In this study, the E. coli nar promoter with lacZ gene at the downstream was cloned onto a broad-host-range Gram-negative vector, pBBR122. It was then induced in some other Gram-negative host strains, such as Agrobacterium, Pseudomonas, and Rhizobium, to determine whether the E. coli nar promoter could be used as an inducible promoter in these strains. From shake-flask experiments it was found that the wild-type E. coli nar promoter cloned onto pBBR122, pNW61, was suppressed under aerobic conditions in an Agrobacterium host strain, was partially induced under microaerobic only conditions, and was maximally induced under microaerobic conditions in the presence of nitrate ion. Whereas the mutant-type E. coli nar promoter cloned onto pBBR122, pNW618, was suppressed under aerobic conditions and was maximally induced under microaerobic conditions, regardless of the presence of nitrate ion. This kind of induction pattern observed for the E. coli nar promoters in the Agrobacterium host strain was similar to that observed for the E. coli nar promoters in the E. coli host strain. On the other hand, it was found that both of the E. coli nar promoters, pNW61 and pNW618, in a Pseudomonas host strain were partially induced under aerobic conditions and were maximally induced under microaerobic conditions, regardless of the presence of nitrate. Finally, it was found that both of the E. coli nar promoters in a Rhizobium host strain were minimally induced, regardless of the presence of oxygen or nitrate ion. Similar induction patterns for the three strains were also observed from fermentor experiments in which the dissolved oxygen (DO) level was tightly controlled. From an evolutionary point of view, the results from the three Gram-negative host strains indicate that the E. coli nar promoter system, including the promoter and regulatory proteins, was best conserved in the Agrobacterium host strain and the least conserved in the Rhizobium host strain. From an industrial point of view, the results indicate that the E. coli nar promoter system can be used as an oxygen-dependent inducible promoter in both Agrobacterium and Pseudomonas host strains.