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.     

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 nonculturable Aeromonas salmonicida in lake water.

The survival of Aeromonas salmonicida subsp. salmonicida was investigated in sterile and untreated lake water. In sterile lake water (filtered and autoclaved), it was found that cells of A. salmonicida entered a nonculturable but viable condition. Viability was determined by flow cytometry with the dye rhodamine 123, which is taken up and maintained within cells with a membrane potential. For survival studies in untreated lake water, A. salmonicida was marked with the xylE gene by using the plasmid pLV1013. Marked cells were detected by growth on tryptone soy agar and tryptone soy agar supplemented with kanamycin. Cells were also detected by polymerase chain reaction DNA amplification of the xylE gene and a chromosomal DNA fragment specific for A. salmonicida (pLV1013). The results indicated that A. salmonicida entered a nonculturable condition in untreated lake water over a 21-day study. The viability of nonculturable cells could not be determined in mixed samples; however, the presence of nonculturable cells containing both chromosomal and plasmid DNA was confirmed.     

Survival of 2,4-dichlorophenoxyacetic acid degrading Alcaligenes eutrophus AE0106(pR0101) in lake water microcosms

Survival of the 2,4-dichlorophenoxyacetic acid (2,4-D) degrading Alcaligenes eutrophus strain AEO 106 harboring the catabolic plasmid pRO101 was studied in lake water from the eutrophic lake Frederiksborg Slotssø. Survival experiments were performed for periods of 7 days in laboratory microcosms containing filtered (0.2-µm pore size) or natural lake water amended with increasing concentrations of 2,4-D. A. eutrophus AE0106 was detected by combining the fluorescent antibody method with selective and nonselective plating followed by colony blotting and colony hybridization. Comparison of colony blotting and colony hybridization demonstrated that the A. eutrophus AE0106 host organism and the catabolic plasmid pRO101 had similar fates in the model system employed. In all experiments culturable counts of A. eutrophus AE0106 were lower than fluorescent antibody counts and frequently a decline in culturable counts occurred at times when the fluorescent antibody method showed an increasing population size. Amendment with 2,4-D increased survival of A. eutrophus AE0106 both in filtered and in natural lake water. Survival was always poorer in model systems with natural water than in 0.2 µm-filtered water. Send offprint requests to: A. Kandel at Department of Microbiology, Water Quality Institute, Agern Alle 11, DK-2970 Hørsholm, Denmark.     

Fate of Pseudomonas putida after release into lake water mesocosms: Different survival mechanisms in response to environmental conditions

To study the fate of Pseudomonas putida DSM 3931 in an aquatic environment, cultures of the strain were released into lake water mesocosms. P. putida, bearing the TOL-plasmid, was released as a representative xenobiotic-degrading microorganism. The release was carried out in mesocosms with unamended lake water and in lake water with added culture medium to compare the survival of the strain due to the influence of different organic load. As a comparison, the survival of P. putida was followed in microcosms with sterile lake water. Survival and fate of the strain were determined by means of immunofluorescence with highly specific monoclonal antibodies and growth on selective agar medium for up to ten weeks after release. Addition of medium had a pronounced influence on survival in mesocosms. In mesocosms without added medium, the number of P. putida cells decreased within ten days by over 2 orders of magnitude. In mesocosms with medium, cell numbers increased in the first two days by an order of magnitude and were, after ten days, in the same range as at the time of introduction. Over time, cell numbers decreased but remained detectable in both types of mesocosms for up to ten weeks after release. In mesocosms with unamended lake water, the major fraction of the cells was attached to particles after two days. In mesocosms with medium, large aggregates of P. putida cells formed which included algae. The observed decrease in cell numbers in mesocosms was attributed mainly to grazing. Sedimentation was an additional factor contributing to loss of cells out of the water column, which especially affected aggregate-forming cells in mesocosms with medium in the long run (beyond two weeks). These studies demonstrate that experimental tools on a mesoscale are crucial in order to understand the complex processes microorganisms are subjected to after release into a natural environment, and that single cell detection, such as immunofluorescence, is essential to understand mechanisms of survival and elimination.Correspondence to: M.G. Höfle     

Survival and detection of bacteria in an aquatic environment

A genetically engineered plasmid, pPSA131, was used as a DNA probe to detect homologous DNA in Escherichia coli HB101(pPSA131) after it was mixed with aquatic microorganisms from Lake Mead, Nevada, water samples. An isolate from the pLAFR1 chromosomal library of Pseudomonas syringae Cit 7 was used to detect parent P. syringae Cit 7 that had been mixed with Lake Mead water. E. coli(pPSA131) was kept in variously treated samples of lake water or buffer, and its survival was measured by viable cell counting on modified Luria-Bertani (LB) agar. Full-strength LB agar proved better than 0.1 x LB agar at recovering E. coli(pPSA131) after survival in low-nutrient environments. Survival of E. coli(pPSA131) remained high in filtered (0.22-micron pore size) lake water and salts buffer on both selective and nonselective agars but was lower in untreated lake water or lake water filtered with a 0.8-micron-pore-size membrane. Total recoverable colonies grown on LB agar were higher when lake water was filter treated (0.8-micron pore size) than when lake water was untreated. Microorganisms recovered from lake water alone grew rapidly on nonselective media, probably because of the "bottle effect." After being mixed with Lake Mead water, E. coli(pPSA131) and P. syringae were detected by colony blotting with non-radioactively labeled DNA probes. E. coli(pPSA131) were recovered at three times during 48 h from variously treated samples of lake water and from a mixture with Lake Mead water organisms. Colonies were supported on either nonselective or selective agar for comparison.(ABSTRACT TRUNCATED AT 250 WORDS)     

Survival and detection of bacteria in an aquatic environment.

A genetically engineered plasmid, pPSA131, was used as a DNA probe to detect homologous DNA in Escherichia coli HB101(pPSA131) after it was mixed with aquatic microorganisms from Lake Mead, Nevada, water samples. An isolate from the pLAFR1 chromosomal library of Pseudomonas syringae Cit 7 was used to detect parent P. syringae Cit 7 that had been mixed with Lake Mead water. E. coli(pPSA131) was kept in variously treated samples of lake water or buffer, and its survival was measured by viable cell counting on modified Luria-Bertani (LB) agar. Full-strength LB agar proved better than 0.1 x LB agar at recovering E. coli(pPSA131) after survival in low-nutrient environments. Survival of E. coli(pPSA131) remained high in filtered (0.22-micron pore size) lake water and salts buffer on both selective and nonselective agars but was lower in untreated lake water or lake water filtered with a 0.8-micron-pore-size membrane. Total recoverable colonies grown on LB agar were higher when lake water was filter treated (0.8-micron pore size) than when lake water was untreated. Microorganisms recovered from lake water alone grew rapidly on nonselective media, probably because of the "bottle effect." After being mixed with Lake Mead water, E. coli(pPSA131) and P. syringae were detected by colony blotting with non-radioactively labeled DNA probes. E. coli(pPSA131) were recovered at three times during 48 h from variously treated samples of lake water and from a mixture with Lake Mead water organisms. Colonies were supported on either nonselective or selective agar for comparison.(ABSTRACT TRUNCATED AT 250 WORDS)     

Survival of a plasmid-bearing strain of Bacillus subtilis introduced into compost

Survival of Bacillus subtilis strain 168 containing plasmid pAB224, which carries a gene for tetracycline resistance, was studied in mushroom compost under mesophilic and thermophilic conditions. Stable populations of B. subtilis were maintained as spores in both sterile and fresh mushroom compost incubated at 37 degrees C. At 65 degrees C, the introduced B. subtilis populations declined during incubation but spores were still detectable after 28 d. Survival at the higher temperature was greater in fresh than in sterile compost. There was no apparent loss of plasmid pAB224 or plasmid-determined phenotype from the introduced B. subtilis population at either incubation temperature. The frequency of tetracycline resistance in the indigenous Bacillus population was very low (10(-5), but some tetracycline-resistant isolates contained plasmid DNA. Four plasmid DNA profiles were found associated with five Bacillus phenotypes, and some evidence for homology with pAB224 was found. However, pAB224 was found to be a suitable marker for release studies because it was easily recovered, readily distinguished from indigenous plasmids on agarose gels, and was maintained in compost-grown B. subtilis 168 in the absence of any selective pressure.     

Survival of and plasmid stability in Pseudomonas and Klebsiella spp. introduced into agricultural drainage water

Cell survival and plasmid stability in Pseudomonas fluorescens R2f and Pseudomonas putida CYM 318 containing respectively, plasmid RP4 and pRK2501, and Klebsiella aerogenes NCTC 418 harboring plasmid pBR322 were studied in sterile and nonsterile agricultural drainage water under both aerobic and anaerobic conditions and in the absence and presence of added nutrients. Both Pseudomonas strains survived well in sterile drainage water incubated aerobically, with or without added nutrients. However, Klebsiella aerogenes NCTC 418 (pBR322) only survived in the presence of added nutrients. Pseudomonas fluorescens R2f (RP4) and K. aerogenes NCTC 418 (pBR322) did not survive under anerobic conditions without added nutrients, but showed good survival in the presence of nutrients. Survival of all three strains was negatively affected in nonsterile agricultural drainage water when compared with survival in sterile water. Maintenance of the three plasmids was host, plasmid, and environment dependent. Plasmid pBR322 was not stably maintained in K. aerogenes NCTC 418 under all conditions used in the study, and pRK2501 was readily lost from P. putida CYM 318. Maintenance of RP4 by P. fluorescens R2f was markedly influenced by added nutrients, which caused a loss of the plasmid from cells. The results of the present study demonstrate the influence of nutrients, O2, and native microorganisms on the survival of introduced bacterial strains and plasmid stability in agricultural drainage water.     

Influence of ecosystematic factors on survival of Escherichia coli after large-scale release into lake water mesocosms.

Mass cultures of an Escherichia coli K-12 strain were released into exposed mesocosms in a eutrophic lake. The release was performed with and without additional input of the E. coli culture medium to stimulate the scenario of leakage of a production fermenter on one hand and to compare the influence of the added organic nutrients with that of the added strain on the other hand. The survival of the introduced strain and the influence on ecological processes in the mesocosms were monitored for 10 weeks after release. For comparison, survival of the strain in microcosms with sterile lake water was also monitored. Survival of the strain was determined by means of immunofluorescence and growth on selective agar medium. In lake mesocosms, E. coli showed a rapid and constant dieback during the first week. After 4 days, cells were mostly restricted to particles, which seemed to provide niches for survival. From the second week onward, survival was improved in mesocosms with culture medium added. In microcosms with sterile lake water, plate counts of E. coli showed a strong decrease within 2 weeks, while total cell numbers remained approximately the same. The rapid elimination of E. coli from the free-water phase of the mesocosms was probably due to the combined effect of the inability to grow in lake water and grazing. The better survival of E. coli (mainly on particles) in mesocosms with added medium was attributed to the medium-induced enhancement of primary production, which was the source of a large quantity of particles. These particles, in turn, may have functioned as niches for prolonged survival as well as transport vehicles for sedimentation of the E. coli cells.     

Influence of ecosystematic factors on survival of Escherichia coli after large-scale release into lake water mesocosms.

Mass cultures of an Escherichia coli K-12 strain were released into exposed mesocosms in a eutrophic lake. The release was performed with and without additional input of the E. coli culture medium to stimulate the scenario of leakage of a production fermenter on one hand and to compare the influence of the added organic nutrients with that of the added strain on the other hand. The survival of the introduced strain and the influence on ecological processes in the mesocosms were monitored for 10 weeks after release. For comparison, survival of the strain in microcosms with sterile lake water was also monitored. Survival of the strain was determined by means of immunofluorescence and growth on selective agar medium. In lake mesocosms, E. coli showed a rapid and constant dieback during the first week. After 4 days, cells were mostly restricted to particles, which seemed to provide niches for survival. From the second week onward, survival was improved in mesocosms with culture medium added. In microcosms with sterile lake water, plate counts of E. coli showed a strong decrease within 2 weeks, while total cell numbers remained approximately the same. The rapid elimination of E. coli from the free-water phase of the mesocosms was probably due to the combined effect of the inability to grow in lake water and grazing. The better survival of E. coli (mainly on particles) in mesocosms with added medium was attributed to the medium-induced enhancement of primary production, which was the source of a large quantity of particles. These particles, in turn, may have functioned as niches for prolonged survival as well as transport vehicles for sedimentation of the E. coli cells.     

Aeromonas Distribution and Survival in a Thermally Altered Lake

Par Pond is a thermally enriched monomictic southeastern lake which receives heated effluent from a production nuclear reactor. Fish populations in the lake have lesions of epizooty from which Aeromonas spp. are readily isolated. Distribution and population densities of Aeromonas in the water column were measured along an oxygen and temperature gradient as well as seasonally. Greater population densities of Aeromonas occurred below the oxygen chemocline when the lake was stratified. Survival of Aeromonas hydrophila under in situ conditions in both epilimnetic and hypolimnetic waters was determined through the use of polycarbonate membrane diffusion chambers during two separate reactor operating conditions. Survival levels of pure cultures of A. hydrophila corresponded to the distribution patterns of the naturally occurring Aeromonas-like populations. The greater survival of A. hydrophila during full reactor operation suggests that the fish populations may be exposed to Aeromonas for a longer period of time than when the reactor is not operating.     

Survival of Aeromonas salmonicida in lake water.

The survival of Aeromonas salmonicida subsp. salmonicida in lake water was investigated by using a variety of techniques. They included acridine orange epifluorescence, respiration, cell culture, cell revival, flow cytometry, plasmid maintenance, and membrane fatty acid analysis. During a 21-day study, A. salmonicida became nonculturable in sterile lake water samples. Flow cytometry and direct microscopy indicated that cells were present. Although the nonculturable cells could not be revived, the recovery method did indicate that the presence of low numbers of culturable cells within samples could produce misleading results. Plasmid DNA, genomic DNA, and RNA were maintained in the nonculturable cells; in addition, changes in the fatty acid profiles were also detected. Although viability could not be proven, it was shown that the morphological integrity of nonculturable cells was maintained.     

Survival of Aeromonas salmonicida in lake water

The survival of Aeromonas salmonicida subsp. salmonicida in lake water was investigated by using a variety of techniques. They included acridine orange epifluorescence, respiration, cell culture, cell revival, flow cytometry, plasmid maintenance, and membrane fatty acid analysis. During a 21-day study, A. salmonicida became nonculturable in sterile lake water samples. Flow cytometry and direct microscopy indicated that cells were present. Although the nonculturable cells could not be revived, the recovery method did indicate that the presence of low numbers of culturable cells within samples could produce misleading results. Plasmid DNA, genomic DNA, and RNA were maintained in the nonculturable cells; in addition, changes in the fatty acid profiles were also detected. Although viability could not be proven, it was shown that the morphological integrity of nonculturable cells was maintained.     

Bacterioplankton Production in Humic Lake Örträsket in Relation to Input of Bacterial Cells and Input of Allochthonous Organic Carbon

In order to compare riverine bacteria input with lake water bacterial production and grazing loss with output loss, a bacterial cell budget was constructed for humic Lake ?rtr?sket in northern Sweden. The riverine input of bacterial cells in 1997 represented 29% of the number of bacterial cells produced within the layer of the lake affected by inlet water. A large share of the in situ lake bacterial production was consumed by grazers, mainly flagellates, which stresses the importance of bacteria as energy mobilizers for the pelagic food web in the lake. The bacterial production in Lake ?rtr?sket, which is almost entirely dependent on humic material as an energy source, was clearly stimulated by high flow episodes which brought high amounts of little degraded material into the lake. During base flow condition the bacterial production in the inlet rivers was high, which led to an input of more degraded material to the lake. This material did not stimulate the lake bacterial production. Internal factors that determined the utilization of the allochthonous DOC in the lake were the retention time and the exposure to light and high temperatures. Thus, the potential for in situ production of bacteria in Lake ?rtr?sket was to a large extent a function of how precipitation and runoff conditions affected terrestrial losses and river transport of humic material.     

Adaptation of model genetically engineered microorganisms to lake water: growth rate enhancements and plasmid loss.

When a genetically engineered microorganism (GEM) is released into a natural ecosystem, its survival, and hence its potential environmental impact, depends on its genetic stability and potential for growth under highly oligotrophic conditions. In this study, we compared plasmid stability and potential for growth on low concentrations of organic nutrients of strains of Pseudomonas putida serving as model GEMs. Plasmid-free and plasmid-bearing (NAH7) prototrophic isogenic strains and two amino-acid auxotrophs, all containing antibiotic resistance markers, were held physically separate from but in chemical contact with lake water containing the natural bacterium-sized microbial populations. Cells were reisolated at intervals over a 2-month period to determine the percent retaining the plasmid and the specific growth rate on various media. Plasmid stability in lake water was strongly strain specific; the NAH7 plasmid was stably maintained by the prototrophic strain for the duration of the test but was lost within 24 h by both of the auxotrophs. Specific growth rates of reisolates, compared with those of the corresponding non-lake water-exposed strains (i.e., parental strains), were not different when measured in rich medium (Luria-Bertani broth). However, specific growth rates were 42, 55, and 63% higher in reisolates of auxotrophs and the plasmid-free prototroph, respectively, when measured in 10-fold-diluted medium after exposure of 15 days or longer to lake water. Moreover, lake water-exposed strains grew actively when reintroduced into sterile lake water (28- to 33-fold increase in numbers over 7 days), while the corresponding unadapted parental strains exhibited no growth over the same period.(ABSTRACT TRUNCATED AT 250 WORDS)     

Stability of pBR322-derived plasmids

The stability of pBR322-derived plasmids was studied during growth of their Escherichia coli host in the absence of antibiotics. Plasmid pBR322, as well as its delta rom and delta bla derivatives, were lost from their host within 60 generations, but a number of delta tet derivatives were quite stable under the same conditions. An evaluation of the data indicated that primary plasmid loss due to random partitioning corresponds to the generation of a plasmid-free cell about every 10(4) divisions (probability P0; = "intrinsic" instability). Secondary loss of plasmid-carrying cells resulted from a growth advantage of the plasmid-free cells when bacteria die, perhaps due to unrepaired lethal damage in the DNA, under conditions of stationary incubation (= "apparent" instability). This cell death also occurred in the absence of plasmids but was accelerated by the presence of extra plasmid DNA in the cell and further accelerated by a functional tet gene. This was the reason for the differential apparent stabilities of delta bla and delta tet plasmids. There was no indication that an accumulation of plasmid multimers contributed to the plasmid instability, as has been suggested in the literature. The value of P0 = 10(-4) is 14 orders of magnitude greater than expected under the assumption of a random (Poisson) distribution of plasmid copy numbers in a population of cells.     

Adaptation of model genetically engineered microorganisms to lake water: growth rate enhancements and plasmid loss.

When a genetically engineered microorganism (GEM) is released into a natural ecosystem, its survival, and hence its potential environmental impact, depends on its genetic stability and potential for growth under highly oligotrophic conditions. In this study, we compared plasmid stability and potential for growth on low concentrations of organic nutrients of strains of Pseudomonas putida serving as model GEMs. Plasmid-free and plasmid-bearing (NAH7) prototrophic isogenic strains and two amino-acid auxotrophs, all containing antibiotic resistance markers, were held physically separate from but in chemical contact with lake water containing the natural bacterium-sized microbial populations. Cells were reisolated at intervals over a 2-month period to determine the percent retaining the plasmid and the specific growth rate on various media. Plasmid stability in lake water was strongly strain specific; the NAH7 plasmid was stably maintained by the prototrophic strain for the duration of the test but was lost within 24 h by both of the auxotrophs. Specific growth rates of reisolates, compared with those of the corresponding non-lake water-exposed strains (i.e., parental strains), were not different when measured in rich medium (Luria-Bertani broth). However, specific growth rates were 42, 55, and 63% higher in reisolates of auxotrophs and the plasmid-free prototroph, respectively, when measured in 10-fold-diluted medium after exposure of 15 days or longer to lake water. Moreover, lake water-exposed strains grew actively when reintroduced into sterile lake water (28- to 33-fold increase in numbers over 7 days), while the corresponding unadapted parental strains exhibited no growth over the same period.(ABSTRACT TRUNCATED AT 250 WORDS)