Alkaline phosphatase activity of Escherichia coli starved in sterile lake water microcosms

Escherichia coli grown in high or low phosphate medium was inoculated into a lake water starvation medium. The viable count decreased at 37°C but not at the lower temperatures over 70 d. Alkaline phosphatase was monitored using a colorimetric assay with pNPP as the substrate. Derepression of the enzyme occurred in cultures starved for > 30 d in the lake water and within 5 d in lake water microcosms supplemented with carbon and nitrogen sources where there was rarely an increase in viable count. Chloramphenicol prevented the synthesis of alkaline phosphatase suggesting that, even under starvation conditions, de novo synthesis of the enzyme occurs.     

The effects of nutrients on the survival of Escherichia coli in lake water

Escherichia coli was shown to survive without decline in viable counts for at least 12 d in filtered-autoclaved lake water. In unfiltered lake water there was a rapid decline in the viable count of E. coli. The addition of synthetic sewage to filtered-autoclaved lake water led to an increase in the viable count of E. coli at 15 degrees C and 37 degrees C and to an increase in the survival time of the E. coli in unfiltered water. The addition of phosphate and carbon sources (glucose, glycerol, succinate, acetate and lactose) did not significantly increase the survival time of E. coli in unfiltered water over the controls. The addition of ammonium sulphate and some amino acids (as nitrogen sources) to the unfiltered lake water did lead to an increase in the survival times for E. coli and this increase was proportional to the concentration of the added nitrogen source.     

The effects of nutrients on the survival of Escherichia coli in lake water

Escherichia coli was shown to survive without decline in viable counts for at least 12 d in filtered-autoclaved lake water. In unfiltered lake water there was a rapid decline in the viable count of E. coli. The addition of synthetic sewage to filtered-autoclaved lake water led to an increase in the viable count of E. coli at 15°C and 37°C and to an increase in the survival time of the E. coli in unfiltered water. The addition of phosphate and carbon sources (glucose, glycerol, succinate, acetate and lactose) did not significantly increase the survival time of E. coli in unfiltered water over the controls. The addition of ammonium sulphate and some amino acids (as nitrogen sources) to the unfiltered lake water did lead to an increase in the survival times for E. coli and this increase was proportional to the concentration of the added nitrogen source.     

Enhanced survival of plasmid-containing Escherichia coli in aquatic microcosms

The survival of Escherichia coli K-12 J62-1 containing the antibiotic-resistance plasmid R1 and an isogenic plasmid-free strain were studied in pond water microcosms. The number of plasmid-containing cells recovered from the microcosms remained constant over a sampling period of 31 days whereas plasmid-free cell numbers declined.     

Molecular genetic and phenotypic alteration of Escherichia coli in natural water microcosms containing toxic chemicals

Abstract Microcosms of sterile Chesapeake Bay water were used to study effects of sub-lethal concentrations (1 μl/l) of nitrobenzene, m -cresol, and dibutyl phthalate on Escherichia coli H10407. E. coli remained viable during the 19-day test period in estuarine water, both in the presence and absence of the chemicals, long after it became non-culturable. Analysis of membrane proteins revealed changes in the protein composition. Carbohydrate and amino acid utilization was affected by these changes. Plasmids in E. coli H10407 could not be detected following microcosm exposure. When the cells were transferred to rich medium without toxic chemicals, growth resumed and plasmid bands were again detectable.     

Survival of Escherichia coli and Campylobacter jejuni in untreated and filtered lake water

L.K. KORHONEN AND P.J. MARTIKAINEN. 1991. The survival of Campylobacter jejuni and Escherichia coli in lake water was studied using viable counts. Escherichia coli survived better than C. jejuni in all the test conditions studied. Both the species survived better in filtered than in untreated water. This suggests that predation and/or competition for nutrients affect the survival of both the species in an aquatic environment. Campylobacter jejuni survived less well in filtered autoclaved water and in 0.9% NaC1 than in filtered water without autoclaving. The lack of some essential nutrients, which may be degraded by autoclaving, might explain these results.     

The effect of starvation stress on the porin protein expression of Escherichia coli in lake water

AIMS: The aim was to identify changes in outer membrane proteins (omps), OmpA, OmpC and OmpF, in Escherichia coli under starvation conditions in lake water microcosms studied at different temperatures. METHODS AND RESULTS: Escherichia coli was incubated in lake water microcosms at a variety of temperatures and the omps studied using quantitative densitometric analysis of protein bands of sodium dodecyl sulphate gels of omp preparations. The amount of OmpF increased over the incubation period relative to that of OmpC whereas the relative abundance of OmpA declined, most notably at 25 and 37 degrees C. This change was linked to changes in peak cell volume as determined by cell measurements. CONCLUSIONS: Major changes to the omps of E. coli accompany the adaptation of the organism to starvation conditions in lake water microcosms. SIGNIFICANCE AND IMPACT OF THE STUDY: Prolonged starvation affects the relative amounts of outer membrane porins. This study furthers the understanding of the role played by changes in the omp composition in E. coli during survival in lake water environments.     

Survival of Escherichia coli and Campylobacter jejuni in untreated and filtered lake water

The survival of Campylobacter jejuni and Escherichia coli in lake water was studied using viable counts. Escherichia coli survived better than C. jejuni in all the test conditions studied. Both the species survived better in filtered than in untreated water. This suggests that predation and/or competition for nutrients affect the survival of both the species in an aquatic environment. Campylobacter jejuni survived less well in filtered autoclaved water and in 0.9% NaCl than in filtered water without autoclaving. The lack of some essential nutrients, which may be degraded by autoclaving, might explain these results.     

Self-maintaining plankton: pelagic Cladocera in small microcosms with lake water

Monospecific and mixed populations of pelagic cladocerans, Daphnia galeata, Bosmina coregoni, and Diaphanosoma brachyurum of Lake Glubokoe (Moscow Region) were isolated in 0.6 litre flasks with their natural medium — epilimnion water. In the laboratory they persisted for more than 100 days without external additions of food until the experiment was stopped. In three-species cultures Diaphanosoma was outcompeted, but in the presence of only Bosmina it persisted. Daphnia coexisted with Bosmina in two- and three-species cultures the whole period of observations in spite of competition. Birth and death rates of animals in microcosms depended on their density indicating that experimental conditions did not spoil natural population regulation. Demographic time lags underlay the observed oscillations in numbers. Because the time of Cladocera persistence in the cultures was comparable with lake vegetational period I conclude that summer zooplankton is capable of existing solely at the expense of nutrient recycling within epilimnion. Density regulation of cladocerans reinforces the stability of the system herbivore — algae, but it is weakened by the demographic time lags.     

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.     

The Escherichia coli aquaporin-Z water channel

The membrane pathway of the rapid fluxes of water by which microorganisms adapt promptly to abrupt changes in environmental osmolality have begun to be understood since the discovery of the Escherichia coli aquaporin-Z water channel, AqpZ. As in animals and plants, aquaporins are variously represented among microorganisms, in which 31 homologous genes have already been identified in eubacteria, Archaea, fungi and protozoa. The AqpZ channel is selectively permeable to water, although other functions are not excluded. Consistent with a conservation over the course of evolution, AqpZ and AQP1, a human counterpart, share similar structures. The aqpZ gene is growth phase and osmotically regulated. AqpZ has a role in both the short- and the long-term osmoregulatory response and is required by rapidly growing cells. AqpZ-like proteins seem to be necessary for the virulence expressed by some pathogenic bacteria. Microbial aquaporins are also likely to be involved in spore formation and/or germination. Additional roles may still be unknown. The use of AqpZ as a model system will continue to provide insight into the understanding of the importance of aquaporins.     

Relationship between respiratory enzymes and survival of Escherichia coli under starvation stress in lake water

Survival, electron transport system (ETS) activity and the activity of NADH and succinate dehydrogenase of Escherichia coli ML30 were studied under starvation stress at different temperatures in a filtered-autoclaved lake water microcosm. ETS activity in E. coli declined rapidly at 30 degrees C but more slowly at 4 degrees and 15 degrees C over a 20 d starvation period. The decrease in ETS activity in E. coli only started after 6 d of incubation at 4 degrees C and 15 degrees C. Viability of E. coli, as determined by plate counts, declined faster at 37 degrees C than at the other temperatures and remained highest at 4 degrees C in filtered-autoclaved lake water. There was also a significant cell size reduction at 37 degrees C in filtered-autoclaved lake water but not at 4 degrees C. ETS activity after up to 16 d of starvation increased after the addition of nutrient broth to the filtered-autoclaved lake water at 15 degrees C and 30 degrees C suggesting that cells were still able to respond to nutrients, even after prolonged starvation. The response to the addition of nutrient broth, however, declined with the length of the starvation period. The activity of both succinate and NADH dehydrogenase declined over a 13 d starvation period. The loss of activity was fastest at 37 degrees C compared to lower incubation temperatures but even at 4 degrees C, a significant proportion of the activity was lost over the 13 d period.     

Survival of Escherichia coli in lake bottom sediment.

The survival of Escherichia coli in bottom sediment (Lake Onalaska, navigation pool no. 7, Mississippi River) was studied by using in situ dialysis culture of sterile (autoclaved) and unsterile sediment samples. Bags made from dialysis tubing were filled with either course sand sediment (28.8% fine) or organic, silty clay sediment (77.2% fine) and placed at the sediment-water interface. Bags representing sterile controls, unsterile uninoculated controls, autoclaved inoculated sediment, and unsterile inoculated sediment were studied during a 5-day period for each sediment type. Daily most-probable-number determinations indicated that E. coli populations in unsterile inoculated sediment fluctuated between 5.3 X 10(2) and 2.2 X 10(3) bacteria per g of silty clay and between 3.0 X 10(3) and 1.4 X 10(4) bacteria per g of sand. Autoclaved silty clay sediment inoculated with 1.0 X 10(6) bacteria per g increased to 2.2 X 10(8) bacteria per g in 3 days. During the same period, autoclaved sand sediment inoculated with 1.2 X 10(5) cells per g increased to 5.4 X 10(7) bacteria per g. By day 5, populations in both cultures had decreased by 1 log. The ability of E. coli to survive for several days in aquatic sediment in situ suggests that fecal coliforms in water may not always indicate recent fecal contamination of that water but rather resuspension of viable sediment-bound bacteria.     

Coexistence of transgenic Escherichia coli strains and natural microorganisms in experimental aquatic microcosms

In experimental aquatic microcosms (AMCs), the population of the Escherichia coli strain Z905 harboring the recombinant plasmid pPHL7 (AprLux+) was found to gradually accumulate AMC-adapted cells, which retained the plasmid but differed from the original cells in some biochemical and physiological characteristics. Both the original and the AMC-adapted E. coli cells could coexist with the native AMC microflora for a year or longer. When introduced into AMCs together with native pseudomonads, the AMC-adapted E. coli Z905-33 (pPHL7) cells were more competitive than nonadapted cells.     

The long-term survival of Escherichia coli in river water

Escherichia coli introduced into autoclaved filtered river water survived for up to 260 d at temperatures from 4° to 25°C with no loss of viability. Survival times were less in water which was only filtered through either a Whatman filter paper or a 0.45 μm Millipore filter or in untreated water, suggesting that competition with the natural microbial flora of the water was the primary factor in the disappearance of the introduced bacteria. Survival was also dependent upon temperature with survival at 4°C > 15°C > 25°C > 37°C for any water sample. Direct counts showed that bacterial cells did not disappear as the viable count decreased. The possession of the antibiotic resistance plasmids, R 1 drd -19 or R144-3, did not enhance survival nor cause a faster rate of decay, indicating that the metabolic burden imposed by a plasmid was not a factor in survival under starvation conditions. There was no evidence of transfer of either plasmid at 15°C or of loss of plasmid function during starvation.     

The long-term survival of Escherichia coli in river water

Escherichia coli introduced into autoclaved filtered river water survived for up to 260 d at temperatures from 4 degrees to 25 degrees C with no loss of viability. Survival times were less in water which was only filtered through either a Whatman filter paper or a 0.45 micron Millipore filter or in untreated water, suggesting that competition with the natural microbial flora of the water was the primary factor in the disappearance of the introduced bacteria. Survival was also dependent upon temperature with survival at 4 degrees C greater than 15 degrees C greater than 25 degrees C greater than 37 degrees C for any water sample. Direct counts showed that bacterial cells did not disappear as the viable count decreased. The possession of the antibiotic resistance plasmids, R1drd-19 or R144-3, did not enhance survival nor cause a faster rate of decay, indicating that the metabolic burden imposed by a plasmid was not a factor in survival under starvation conditions. There was no evidence of transfer of either plasmid at 15 degrees C or of loss of plasmid function during starvation.     

Use of microcosms to determine persistence of Escherichia coli in recreational coastal water and sediment and validation with in situ measurements

AIMS: To determine the persistence of the faecal indicator organism Escherichia coli in recreational coastal water and sediment using laboratory-based microcosms and validation with in situ measurements. METHODS AND RESULTS: Intact sediment cores were taken from three distinct coastal sites. Overlying estuarine water was inoculated with known concentrations of E. coli and decay rates from both overlying water and sediment were determined following enumeration by the membrane filtration method at fixed time intervals over a 28-day period. It was demonstrated that E. coli may persist in coastal sediment for >28 days when incubated at 10 degrees C. Escherichia coli survival was found to have an inverse relationship with temperature in both water and sediment. In general the decay rate for E. coli was greater in water than in sediment. Small particle size and high organic carbon content were found to enhance E. coli survival in coastal sediments in the microcosms. CONCLUSIONS: Results of this microcosm study demonstrated the more prolonged survival of E. coli in coastal sediments compared with overlying water, which may imply an increased risk of exposure because of the possible resuspension of pathogenic micro-organisms during natural turbulence or human recreational activity. SIGNIFICANCE AND IMPACT OF THE STUDY: A more accurate estimate of exposure risk has been described which may subsequently be used in a quantitative microbial risk assessment for recreational coastal waters.     

Determination of plasmid DNA concentration maintained by nonculturable Escherichia coli in marine microcosms.

The concentration of plasmid pBR322 DNA in nonculturable Escherichia coli JM83 was measured to determine whether the plasmid concentration changed during survival of E. coli in marine and estuarine water. E. coli JM83 containing the plasmid pBR322 was placed in both sterile seawater and sterile estuarine water and analyzed for survival (i.e., culturability) and plasmid maintenance. The concentration of pBR322 DNA remained stable in E. coli JM83 for 28 days in an artificial seawater microcosm, even though nonculturability was achieved within 7 days. E. coli JM83 incubated in sterile natural seawater or sterile estuarine water did not reach nonculturability within 30 days. Under all three conditions, plasmid pBR322 DNA was maintained at approximately the initial concentration. Cloning of DNA into the plasmid pUC8 did not alter the ability of E. coli to maintain vector plasmid DNA, even when the culture was in the nonculturable state, but the concentration of plasmid DNA decreased with time in the microcosm. We conclude that E. coli is able to maintain plasmid DNA while in the nonculturable state and that the concentration at which the plasmid is maintained appears to be dependent upon the copy number of the plasmid and/or the presence of foreign DNA.     

Determination of plasmid DNA concentration maintained by nonculturable Escherichia coli in marine microcosms.

The concentration of plasmid pBR322 DNA in nonculturable Escherichia coli JM83 was measured to determine whether the plasmid concentration changed during survival of E. coli in marine and estuarine water. E. coli JM83 containing the plasmid pBR322 was placed in both sterile seawater and sterile estuarine water and analyzed for survival (i.e., culturability) and plasmid maintenance. The concentration of pBR322 DNA remained stable in E. coli JM83 for 28 days in an artificial seawater microcosm, even though nonculturability was achieved within 7 days. E. coli JM83 incubated in sterile natural seawater or sterile estuarine water did not reach nonculturability within 30 days. Under all three conditions, plasmid pBR322 DNA was maintained at approximately the initial concentration. Cloning of DNA into the plasmid pUC8 did not alter the ability of E. coli to maintain vector plasmid DNA, even when the culture was in the nonculturable state, but the concentration of plasmid DNA decreased with time in the microcosm. We conclude that E. coli is able to maintain plasmid DNA while in the nonculturable state and that the concentration at which the plasmid is maintained appears to be dependent upon the copy number of the plasmid and/or the presence of foreign DNA.