Survival of verocytotoxigenic Escherichia coli O157 in soil, water and on surfaces

Cattle and sheep are major reservoirs of Escherichia coli O157 and consequently these and certain other farm animals can pass out large numbers of this organism in their faeces. Thus the ability of the organism to survive in faeces, on pastureland and in associated water systems has important implications for its spread to crops by direct application of manure, by irrigation with infected water or directly to man by contact with animals or contaminated soil. Model systems were used to determine the persistence of the organism in river water, cattle faeces, soil cores and on stainless steel work surfaces. Survival of the organism was found to be greatest in soil cores containing rooted grass. Under these conditions viable numbers were shown to decline from approximately 10(8) g(-1) soil to between 10(6) and 10(7) g(-1) soil after 130 d. When the organism was inoculated into cattle faeces it remained detectable at high levels for more than 50 d. In contrast the organism survived much less readily in cattle slurry and river water where it fell in numbers from more than 10(6) ml(-1) to undetectable levels in 10 and 27 d, respectively. The survival of E. coli O157 was also investigated on stainless steel surfaces, where as air-dried deposits, it was shown to survive for periods in excess of 60 d. It was most stable at chill temperatures (4 degrees C) and viability was only partially reduced at 18 degrees C. In addition to stainless steel, the organism was shown to survive for extended periods on domestic (plastic) cutting boards, both at room and chill temperatures. Sanitizing agents, such as hypochlorites and a compound comprising both cationic and anionic-based active ingredients were found to be effective in killing various VTEC on stainless steel surfaces.     

Death of the Escherichia coli K-12 strain W3110 in soil and water.

Whether Escherichia coli K-12 strain W3110 can enter the "viable but nonculturable" state was studied with sterile and nonsterile water and soil at various temperatures. In nonsterile river water, the plate counts of added E. coli cells dropped to less than 10 CFU/ml in less than 10 days. Acridine orange direct counts, direct viable counts, most-probable-number estimates, and PCR analyses indicated that the added E. coli cells were disappearing from the water in parallel with the number of CFU. Similar results were obtained with nonsterile soil, although the decline of the added E. coli was slower. In sterile water or soil, the added E. coli persisted for much longer, often without any decline in the plate counts even after 50 days. In sterile river water at 37 degrees C and sterile artificial seawater at 20 and 37 degrees C, the plate counts declined by 3 to 5 orders of magnitude, while the acridine orange direct counts remained unchanged. However, direct viable counts and various resuscitation studies all indicated that the nonculturable cells were nonviable. Thus, in either sterile or nonsterile water and soil, the decline in plate counts of E. coli K-12 strain W3110 is not due to the cells entering the viable but nonculturable state, but is simply due to their death.     

Survival of the phytopathogen Erwinia carotovora subsp. carotovora in sterile and non-sterile soil, sand and their admixture

Survival of phytopathogens in irrigation water and irrigated soil is of major concern to the agricultural community. In the present study, an Erwinia carotovora subsp. carotovora strain was tested for survival capability in three non-sterile and heat-sterilized soil matrices (soil, sand and soil + sand) over 35 d. In all non-sterile soil matrices, Erw. carotovora subsp. carotovora numbers declined below the detection limit over 35 d, the main variance being the decline rate related to nutrients available in the different matrices (soil, soil + sand and sand respectively). In heat-sterilized soil and soil + sand matrices Erw. carotovora subsp. carotovora revealed a regrowth, while in sterile sand matrix its decline was lower over the same time period. In previous published reports, when soil was sterilized by irradiation, such a regrowth was not observed. Application of an initial single load of sodium nitrate solution (70 mg l⁻¹) was found to extend bacteria survival rate in non-sterile and sterile soil columns. In sterile soil columns supplemented with sodium nitrate, Erw. carotovora subsp. carotovora did survive well for up to 60 d, with a major regrowth over the first 12 d and decline up to day 60, reaching initial loading numbers. The information on the potential survival of Erw. carotovora subsp. carotovora in soil for up to 35 d and regrowth in sterile soil should be of concern, especially when irrigation is performed with poor quality water.     

Composition and Biophysical Properties of Lipids in Xenorhabdus nematophilus and Photorhabdus luminescens, Symbiotic Bacteria Associated with Entomopathogenic Nematodes

Primary and secondary forms of Photorhabdus luminescens Hm and Xenorhabdus nematophilus N2-4 were grown at 18 and 28(deg)C for 24 to 96 h, and we made determinations of the fatty-acid compositions of total lipids and of the fluidity measured by 5-doxyl-stearic acid embedded in liposomes made from total lipids. The levels of the unsaturated fatty acids 16:1 and 18:1 (those with chain lengths of 16 or 18 and one double bond) generally were higher in primary-phase variants of P. luminescens grown at 18(deg)C than in those grown at 28(deg)C. Prolonged culture at 18(deg)C caused the level of 18:1 to fall and reach that observed at 28(deg)C. The ratio of saturated to unsaturated fatty acids rose with prolonged culture times in variants of each species at both phases. When grown at 18(deg)C, the proportion of 16:1 in X. nematophilus was lower than in P. luminescens; the patterns of temperature-induced changes were similar in these species. X. nematophilus contained a greater percentage of short-chain fatty acids (i.e., with chain lengths of <14.0) than P. luminescens. Lipid liposomes from primary and secondary cultures of both bacterial species grown at 18(deg)C were more ordered (i.e., less fluid) than those grown at 28(deg)C. This result suggests the surprising absence of homeoviscous adaptation of membranes to temperature. Also, liposomes from primary cultures were more ordered than those from secondary cultures and membranes from primary cultures of P. luminescens were more ordered at both culture temperatures than membranes from X. nematophilus. The biological significance of the effect of growth conditions on membrane biophysical properties in these bacteria is discussed.     

Lysogeny and bacteriocinogeny in Xenorhabdus nematophilus and other Xenorhabdus spp.

Induction by mitomycin or high-temperature treatment resulted in the production of bacteriocins and phages in both phases of Xenorhabdus nematophilus A24, indicating lysogeny. Phage DNA purified from X. nematophilus A24 hybridized to several fragments of DraI-digested A24 chromosomal DNA, confirming that the phage genome was incorporated into the bacterial chromosome. Bacteriocins and phages were detected in cultures of most other Xenorhabdus spp. after mitomycin or high-temperature treatment. Xenorhabdus luminescens K80 was not lysed by these treatments, and no phages were seen associated with this strain. However, bacteriocins were detected in limited quantities in all Xenorhabdus cultures, including X. luminescens K80, without any induction. X. nematophilus A24 bacteriocins were antagonistic for other Xenorhabdus species but not for A24 or other strains of X. nematophilus.     

Lysogeny and bacteriocinogeny in Xenorhabdus nematophilus and other Xenorhabdus spp.

Induction by mitomycin or high-temperature treatment resulted in the production of bacteriocins and phages in both phases of Xenorhabdus nematophilus A24, indicating lysogeny. Phage DNA purified from X. nematophilus A24 hybridized to several fragments of DraI-digested A24 chromosomal DNA, confirming that the phage genome was incorporated into the bacterial chromosome. Bacteriocins and phages were detected in cultures of most other Xenorhabdus spp. after mitomycin or high-temperature treatment. Xenorhabdus luminescens K80 was not lysed by these treatments, and no phages were seen associated with this strain. However, bacteriocins were detected in limited quantities in all Xenorhabdus cultures, including X. luminescens K80, without any induction. X. nematophilus A24 bacteriocins were antagonistic for other Xenorhabdus species but not for A24 or other strains of X. nematophilus.     

Intraspecific competition as a regulating factor limiting the number of transconjugants in soil

A laboratory study was carried out to determine survival of transconjugant cells ofPseudomonas fluorescens intro duced into sterile soil. The transconjugant survived significantly better when it was the only strain inoculated into the soil; when introduced into soil pre-colonized by the recipient strain, the transconjugant was undetectable. These results indicate that intraspecific competition is a regulating factor limiting the number of transconjugants in soil.     

Enumeration of viable E. coli in rivers and wastewaters by fluorescent in situ hybridization

A combination of direct viable count (DVC) and fluorescent in situ hybridization (FISH) procedures was used to enumerate viable Escherichia coli in river waters and wastewaters. A probe specific for the 16S rRNA of E. coli labeled with the CY3 dye was used; enumeration of hybridized cells was performed by epifluorescence microscopy. Data showed that the method was able to accurately enumerate a minimum of 3000 viable E. coli among a large number of non-fecal bacteria. When applied to river water and wastewater samples, the DVC-FISH method gave systematically higher E. coli counts than a reference culture-based method (miniaturized MPN method). The ratio between both counts (DVC-FISH/MPN) increased with decreasing abundance of culturable E. coli indicating that the proportion of viable but non-culturable (VBNC) E. coli (detectable by the DVC-FISH procedure and not by a culture-based method) was higher in low contaminated environments. We hypothesized that the more stressing conditions, i.e. nutritional stress and sunlight effect, met in low contaminated environments were responsible for the larger fraction of VBNC E. coli. A survival experiment, in which sterile mineral water was inoculated with a pure E. coli strain and incubated, confirmed that stressing conditions induced the apparition of non-culturable E. coli detectable by the DVC-FISH procedure. The analysis of the E. coli concentration along a Seine river longitudinal profile downstream a large input of fecal bacteria by a WWTP outfall showed an increasing fraction of VBNC E. coli with increasing residence time of the E. coli in the river after release. These data suggest that the DVC-FISH method is useful tool to analyze the dynamics of fecal bacteria in river water.     

Cloning and heterologous expression of a novel insecticidal gene (tccC1) from Xenorhabdus nematophilus strain

We have identified and cloned a novel toxin gene (tccC1/xptB1) from Xenorhabdus nematophilus strain isolated from Korea-specific entomophagous nematode Steinernema glaseri MK. The DNA sequence of cloned toxin gene (3048 bp) has an open reading frame encoding 1016 amino acids with a predicted molecular mass of 111058 Da. The toxin sequence shares 50-96% identical amino acid residues with the previously reported tccC1 cloned from X. nematophilus, Photorhabdus luminescens W14 P. luminescens TTO1, and Yersinia pestis CO92. The toxin gene was successfully expressed in Escherichia coli, and the recombinant toxin protein caused a rapid cessation in mortality of Galleria mellonella larvae (80% death of larvae within 2 days). Conclusively, the heterologous expression of the novel gene tccC1 cloned into E. coli plasmid vector produced recombinant toxin with high insecticidal activity.     

Geosmin and 2-methylisoborneol from cyanobacteria in three water supply systems

The changes in populations of Staphylococcus aureus, Bacillus subtilis, Salmonella typhimurium, Klebsiella pneumoniae, Agrobacterium tumefaciens, Rhizobium meliloti, and Saccharomyces cerevisiae were measured after their introduction into samples of sewage, lake water, and soil. Enumeration of small populations was possible because the strains used were resistant to antibiotics in concentrations and combinations such that few species native to these ecosystems were able to grow on agar containing the inhibitors. Fewer than 2 cells per ml of sewage or lake water and 25 cells per g of soil could be detected. A. tumefaciens and R. meliloti persisted in significant numbers with little decline, but S. aureus, K. pneumoniae, S. typhimurium, S. cerevisiae, and vegetative cells of B. subtilis failed to survive in samples of sewage and lake water. In sterile sewage, however, K. pneumoniae, B. subtilis, S. typhimurium, A. tumefaciens, and R. meliloti grew; S. cerevisiae populations were maintained at the levels used for inoculation; and S. aureus died rapidly. In sterile lake water, the population of S. aureus and K. pneumoniae and the number of vegetative cells of B. subtilis declined rapidly, R. meliloti grew, and the other species maintained significant numbers with little or a slow decline. The populations of S. aureus, K. pneumoniae, A. tumefaciens, B. subtilis, and S. typhimurium declined in soil, but the first four species grew in sterile soil. It is suggested that some species persist in environments in which they are not indigenous because they tolerate abiotic stresses, do not lose viability readily when starved, and coexist with antagonists. The species that fails to survive need only be affected by one of these factors.     

Survival of cells and DNA of Aeromonas salmonicida released into aquatic microcosms

D. DEERE, J. PORTER, R.W. PICKUP AND C. EDWARDS. 1996. The survival of the bacterial fish pathogen Aeromonas salmonicida , and persistence of its DNA, were monitored in aquatic microcosms using selective culture and most probable number PCR. Bacterial cells and naked DNA were released into natural non-sterile microcosms consisting of lake sediment overlayered with lake water. Two different types of surface sediment were used. One was sandy in character, taken from the shoreline whilst the other was a littoral loamy surface mud. Inoculated cells and naked DNA became undetectable from water overlayers within 4 weeks of release. Colony counts of Aer. salmonicida declined below detectable limits after 4 weeks in loamy sediment or 7 weeks in sandy sediment; however, naked DNA and DNA from released cells remained detectable for more than 13 weeks.     

Whole cell fatty acid patterns of Xenorhabdus species

Thirty-three strains of the nematode-associated bacterium Xenorhabdus were characterized by traditional biochemical tests and whole cell fatty acid analysis. In traditional tests 26 strains were found to belong to X. luminescens and 7 to X. nematophilus (sensu latu). No further subdivision could be made. In fatty acid analysis, however, X. luminescens strains could be divided into three subgroups. The amount of distinction in fatty acids is similar to that at subspecies or species level found in other bacteria. Xenorhabdus nematophilus could be clearly differentiated from X. luminescens , key acids are 12: 0, 15: 0 iso, 16: 0, 17: 0 iso, 17: 0 cyclo, 18: 1 cis 11 and 19: 0 cyclo. Separation is almost at genus level. The presence of branched and hydroxy acids in Xenorhabdus and its aberrant morphology make the placement of this genus in the Enterobacteriaceae questionable. This is the first report on fatty acid profiles of Xenorhabdus species.     

Pathogenicity of Bacteria Symbiotically Associated with Insect Pathogenic Nematodes against the Greater Wax Moth,Galleria Mellonella (L.)

The bacterial symbionts isolated from the entomopathogenic nematodes were compared for their pathogenicity to last instar larvae of G. mellonella at both Phases I and II. Most bacterial symbionts at Phase I cause 100% mortality within 2-3 days post-injection with 1 times; 10 3 cells/larva. The pathogenicity of Phase I decreased in the following order: Xenorhabdus nematopbilus, Flavimonas oryzihabitans, Photorhabdus luminescens and Xenorhabdus bovienii with LD 50 values of 40, 55, 70 and 170 cells/larva. The injection of Phase II of the bacterial symbionts did not give 100% mortality even after 4 days post-injection. The time mortality response of G. mellonella larvae to both phases of the bacterial symbiont was significantly different usually at the two highest concentrations tested. The significancy in case of Phase I was in the following order from lowest to highest, F . oryzihabitans , X . nematophilus , P. luminescens and X. bovienii . It was 20.57, 23.96, 23.99 and 53.76 h, respectively. Also, F. oryzihabitans gave the lowest LT 50 value for its Phase II form. It was 36.85 h, and this is followed by X. bovienii , X. nematophilus , and P. luminescens , the LT 50 values of which were 69.29, 74.08 and 74.49 h, respectively. The results suggest that there is a direct correlation between toxin concentration and rate of killing the larvae. On the other hand, there is an inverse correlation between the LT 50 values and the injected concentration.     

Cultural and Environmental Factors Affecting the Longevity of Escherichia coli in Histosols

The survival of Escherichia coli in organic soils (Histosols) was examined. The death rate of this organism in Pahokee muck was less than that observed in Pompano fine sand. The number of viable E. coli cells found in the muck was approximately threefold greater than that found in the sand following 8 days of incubation. The initial population of the coliform affected the death rate. The rate of loss of viability varied 100-fold when the population size decreased from 2.5 × 10⁷ to 3.4 × 10⁴. Other factors affecting the viability of E. coli in muck were aerobic versus anaerobic growth of the organism and moist versus flooded conditions in the soil. The greatest survival of the coliform was noted with anaerobically grown cells amended to flooded soil. That the observed decrease in E. coli viability in soil was the result of biotic factors was demonstrated with amendment of sterile soil with E. coli. When 1.1 × 10⁵ bacteria per g of soil were added to sterile muck, a population of 3.0 × 10⁷ organisms per g of soil developed over a 10-day period. The role of the protozoa in eradication of the coliform from the muck was indicated by a sixfold increase in the protozoan population in natural soil amended with E. coli. Higher organic matter content in a Histosol compared with a mineral soil resulted in an increased survival of the fecal coliforms. Biotic factors are instrumental in the decline in coliform populations, but the potential for growth of the coliform in the organic soil could extend the survival of the organism.     

Survival of Yersinia enterocolitica in the environment

When Yersinia enterocolitica was introduced into soils (or physiological saline), very little decrease in the population was observed throughout the test period. If the soil was allowed to air dry slowly, only 0.1% (2.8 x 10(3) colony forming units/g of soil) of the original population added still remained viable by day 10. On the other hand, the introduced organisms disappeared rapidly in river water but their longevities could be extended significantly if a eucaryote inhibitor was added to the river water or the river water was passed through a 0.8-micron membrane filter to remove eucaryotic predators. Furthermore, the rapid decrease of the Yersinia population coincided with an increase in numbers of protozoans. However, when Yersinia was added to filter-sterilized river water or when small numbers of the organism, below the threshold level believed necessary for active predation to occur, were added to the river water, no response in predators was observed; nevertheless, the population of Yersinia still showed a continued decline. When the organism was introduced into sephadex-treated river water or groundwater, its survival improved significantly compared with its survival in nontreated water samples. Low ambient temperature dramatically increased its ability to survive in the aquatic environment. It is concluded that, in addition to the temperature factor, the longevity of Y. enterocolitica in river water is chiefly regulated by predators and toxin producers.     

Cultural and environmental factors affecting the longevity of Escherichia coli in Histosols.

The survival of Escherichia coli in organic soils (Histosols) was examined. The death rate of this organism in Pahokee muck was less than that observed in Pompano fine sand. The number of viable E. coli cells found in the muck was approximately threefold greater than that found in the sand following 8 days of incubation. The initial population of the coliform affected the death rate. The rate of loss of viability varied 100-fold when the population size decreased from 2.5 x 10(7) to 3.4 x 10(4). Other factors affecting the viability of E. coli in muck were aerobic versus anaerobic growth of the organism and moist versus flooded conditions in the soil. The greatest survival of the coliform was noted with anaerobically grown cells amended to flooded soil. That the observed decrease in E. coli viability in soil was the result of biotic factors was demonstrated with amendment of sterile soil with E. coli. When 1.1 x 10(5) bacteria per g of soil were added to sterile muck, a population of 3.0 x 10(7) organisms per g of soil developed over a 10-day period. The role of the protozoa in eradication of the coliform from the muck was indicated by a sixfold increase in the protozoan population in natural soil amended with E. coli. Higher organic matter content in a Histosol compared with a mineral soil resulted in an increased survival of the fecal coliforms. Biotic factors are instrumental in the decline in coliform populations, but the potential for growth of the coliform in the organic soil could extend the survival of the organism.     

Survival of Azospirillum brasilense in the Bulk Soil and Rhizosphere of 23 Soil Types

The survival of Azospirillum brasilense Cd and Sp-245 in the rhizosphere of wheat and tomato plants and in 23 types of plant-free sterilized soils obtained from a wide range of environments in Israel and Mexico was evaluated. Large numbers of A. brasilense cells were detected in all the rhizospheres tested, regardless of soil type, bacterial strain, the origin of the soil, or the amount of rainfall each soil type received prior to sampling. Survival of A. brasilense in soils without plants differed from that in the rhizosphere and was mainly related to the geographical origin of the soil. In Israeli soils from arid, semiarid, or mountain regions, viability of A. brasilense rapidly declined or populations completely disappeared below detectable levels within 35 days after inoculation. In contrast, populations in the arid soils of Baja California Sur, Mexico, remained stable or even increased during the 45-day period after inoculation. In soils from Central Mexico, viability slowly decreased with time. In all soils, percentages of clay, nitrogen, organic matter, and water-holding capacity were positively correlated with bacterial viability. High percentages of CaCO(inf3) and fine or rough sand had a highly negative effect on viability. The percentage of silt, pH, the percentage of phosphorus or potassium, electrical conductivity, and C/N ratio had no apparent effect on bacterial viability in the soil. Fifteen days after removal of inoculated plants, the remaining bacterial population in the three soil types tested began to decline sharply, reaching undetectable levels 90 days after inoculation. After plant removal, percolating the soils with water almost eliminated the A. brasilense population. Viability of A. brasilense in two artificial soils containing the same major soil components as the natural soils from Israel did was almost identical to that in the natural soils. We conclude that A. brasilense is a rhizosphere colonizer which survives poorly in most soils for prolonged periods of time; that outside the rhizosphere, seven abiotic parameters control the survival of this bacterium in the soil; and that disturbance of the soil (percolation with water or plant removal) directly and rapidly affects the population levels.     

Aerobic growth and survival of Campylobacter jejuni in food and stream water

When 40 Campylobacter jejuni isolates from human clinical cases, raw chicken and water were tested, 29 (72·5%) could be adapted to grow on nutrient agar under aerobic conditions. Once adapted, these isolates could grow on repeated aerobic subculture. An aerobically-grown Camp. jejuni isolate survived almost as well as the same isolate grown microaerophilically in sterile chicken mince at 5 °C, and survival of a cocktail of Camp. jejuni isolates under both atmospheres was comparable at 25 °C. However, at 37 °C, the decline in numbers of the aerobically-grown cells was greater. Survival of cells on chicken nuggets was poorer than in chicken mince. In filter-sterilized stream water incubated aerobically at 5 °C, survival of inocula grown under different atmospheres was again similar, but slightly better with the microaerophilically-grown cells. Adaptation to aerobic growth was not found to enhance survival under aerobic conditions.     

Effect of poly(3-hydroxybutyrate) (PHB) content on the starvation-survival of bacteria in natural waters

Abstract The effect of poly(3-hydroxybutyrate) (PHB) content on the survival of wild-type strains and PHB negative mutants of Bacillus megaterium and Alcaligenes eutrophus in natural waters was studied. The survival strategy of B. megaterium was dominated by the development of resistant forms, but the number of the wild-type vegetative cells was higher than that of PHB mutant strain. In some environmental conditions the mutant spores needed a heat shock for germination, a fact that suggests, for the first time, that PHB plays a role in this phenomenon. Survival of A. eutrophus wild-type strain in all experiments was higher compared to the PHB mutant, and differences were significant. In raw river water, survival of both species was lower than in sterile river water.     

Photorhabdus toxins: novel biological insecticides.

Following concerns over the potential for insect resistance to insecticidal Bacillus thuringiensis toxins expressed in transgenic plants, there has been recent interest in novel biological insecticides. Over the past year there has been considerable progress in the cloning of several alternative toxin genes from the bacteria Photorhabdus luminescens and Xenorhabdus nematophilus. These genes encode large insecticidal toxin complexes with little homology to other known toxins.