Inés Arana

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.

The Effect of Simulated Solar Radiation on Escherichia coli: The Relative Roles of UV-B, UV-A, and Photosynthetically Active Radiation

A bstractThe relative role of components of solar radiation (UV-B, UV-A, and photosynthetically active radiation) as well as the effect of simulated sunlight upon the physiological state of Escherichia coli in fresh water were evaluated. Simulated solar radiation had a sublethal effect on E. coli populations in a short-time exposure by provoking loss of culturability and the formation of viable but nonculturable cells. Prolonged exposure increased the damage to cells but cellular integrity was never affected. However, important differences between the way the sunlight components acted were detected. After photosynthetically active radiation (PAR) exposure, cells remained metabolically active but only 10% of the cells were culturable. When cells were exposed to UV-A, the culturable fraction was similar to the one obtained after PAR irradiation, although formation of viable but nonculturable cells was not observed. For UV-B radiation short-time exposures (6 h) were enough to provoke loss of culturability and a reduction in activity similar to that of simulated sunlight exposed cells. The effect of simulated solar radiation on E. coli cells was mainly attributable to shorter wavelengths, but a synergistic interaction of the UV-B, UV-A and PAR components was detected.

Direct viable count of Gram-positive and Gram-negative bacteria using ciprofloxacin as inhibitor of cellular division

Abstract Following the method described by Kogure et al. (Can. J. Microbiol. 25 (1979) 415–420), we used ciprofloxacin, a quinolone which inhibits cell division of Gram-positive and Gram-negative bacteria, to quantify the number of viable bacteria in a series of cultures. As test bacteria we used three Escherichia coli strains, two of them sensitive to nalidixic acid and a third resistant to this inhibitor, and three Gram-positive bacteria: one rod ( Lactobacillus plantarum ) and two cocci ( Enterococcus faecalis and Micrococcus varians ). Ciprofloxacin is an efficient inhibitor of cell division in all six strains, although the effect on the morphology of the rods and cocci was clearly different. The percentage of viable bacteria was determined in the case of the rods by the increase in either length or biovolume and, for the cocci, by the increase in cell biovolume. Image analysis was required to detect variations in cell biovolume in the case of cocci and rods. For nalidixic acid-sensitive strains, it was possible to use both, as similar percentages of viable bacteria were detected with both inhibitors. The differences between the colony forming units (CFU) and direct viable (DVC) counts were much higher in the strains showing typical arrangements than in those strains without them. Finally, we found no significant differences in the number of viable bacteria in the natural population of the Butron river when we used either ciprofloxacin or nalidixic acid as inhibitors of cell division.

Chlorination and ozonation of waste‐water: comparative analysis of efficacy through the effect on Escherichia coli membranes

The effect of chlorine and ozone on Escherichia coli cells resuspended in waste‐water was compared. Selected chlorination and ozonation conditions produced a similar decrease in culturability (2–2·5 log). Under these conditions, differences in membrane permeability and cell surface hydrophobicity, depending on the disinfectant tested, were detected. After ozonation, while no changes in cell surface hydrophobicity were observed, approximately 95·5% of cells showed altered membrane permeability. The effect of chlorine was not linked to changes in membrane permeability. After chlorination, E. coli cells showed a tendancy to aggregate. The possibility that aggregation of cells could interfere with conventional colony counts is discussed. The degree of toxicity (MicrotoxTM assay) was unrelated to the effect on cellular activity.

Factors affecting the survival of E. coli in a river

The relationships between physical, chemical and microbial characteristics of an aquatic ecosystem and the survival of E. coli have been studied. Two conditions of the ecosystem (warm and cold) are considered. T90 (time necessary for 90% of a bacterial population to die) in the warm situation shows an inverse exponential relationship with water temperature. Besides the direct relationship temperature-T90, there is an indirect effect of temperature upon T90 through the natural microflora of the water. The relationships between temperature and the heterotrophic population, and between the heterotrophic population and the bacterial consumers (P.F.U.), are exponential and linear, respectively.

Influence of light and natural microbiota of the Butrón river on E. coli survival

The survival of an E. coli strain in water samples from the Butrón river has been studied. The input of E. coli cells in the aquatic system breaks down the established balance among the components of the natural microbiota: E. coli becomes the object of the active protozoal predation whereas the autochtonous heterotrophic community become alternative preys. As a result of this new situation, the natural microbiota increases but returns to the initial values once the E. coli cells have been removed from the system. The effect of the temperature of incubation on the survival is exerted through the effect of this parameter on the predatory activity of the protozoa. Light has a lethal and direct action on the E. coli cells, the effect of this parameter is even superior to that of predation.

Relationships between Escherichia coli cells and the surrounding medium during survival processes

In Escherichia coli, during survival under adverse conditions, namely starvation and luminous radiation, two things occur. On the one hand organic substances are released into the surrounding medium and on the other there is a transition from the culturable state to viable but non-culturable (VBNC). An analysis of organic molecules released into the surrounding medium showed the presence of proteins, dissolved free amino acids, and dissolved monomeric carbohydrates. The concentration of these substances in the medium changed with exposure time, type of stress and type of molecule. The proteins accumulated in the medium and in some cases their identification revealed the presence of components of the outer membrane. Variations in the concentration of amino acids and carbohydrates point to a twofold process of excretion and uptake. Indeed, cell free supernatants supported the growth of several generations of a population of 104 cells ml–1. The survival of E. coli in supernatants previously colonized by cells in the VBNC state was greater than that observed in the control experiments, with a short delay in the loss of culturability. It was thus clear that organic molecules released into the medium play a role in the transition from culturable to VBNC state.

gfp-Tagged cells as a useful tool to study the survival of Escherichia coli in the presence of the river microbial community

We have used an Escherichia coli strain DH5a containing pGreenTIR to study the survival of this bacterium in river water. As green fluorescence was maintained throughout survival both in dark and illuminated conditions, gfp-tagged E. coli cells were clearly distinguished from the microbial community of the river Butrón. gfp-tagged E. coli cells were monitored to estimate total density as well as the density of the culturable and viable (active electron transport system, CTC+) cells. Our results indicate that autochthonous bacteria and introduced E. coli are predated by flagellates. The autochthonous bacterial community behaves as predation-escaping prey, showing a tendency to cellular miniaturization and so maintaining the density of the population. In contrast, introduced E. coli behaves as predation-non-escaping prey, so E. coli was eliminated from the system. When comparing the elimination by predation of heat-treated and non-heated gfp-tagged E. coli cells we deduce that the flagellates do not discriminate between live and heat-treated cells. Finally, in the presence of the river microbial community, the E. coli cells appeared to be ingested before cellular deterioration could occur. Thus predation reduces the quantitative importance of the viable but nonculturable (VBNC) population of E. coli in the aquatic systems.

Influence of a Survival Process in a Freshwater System upon Plasmid Transfer Between Escherichia coli Strains

A bstractSurvival and potential ability to act as recipient or donor during the survival process for one plasmid-free and four plasmid-bearing Escherichia coli strains under nonilluminated and illuminated conditions in freshwater systems were studied. The five E. coli strains showed the same behavior with respect to the microbial parameters used to characterize the survival process (culturability and viability). Under nonilluminated conditions, recipient cells did not show variation in the ability to receive and express plasmid material, while the culturability of the recipient strain remained stable. Under the same conditions, donor cells lost their ability for plasmid transfer during the survival process, in all cases more than a 90% decrease of the number of transconjugants was found after 4 days of experimentation, although viable and culturable cells of donor strains maintained the capacity to express some plasmidic genes. Under illuminated conditions, transconjugants were not detected after 2 days of experimentation. The number of transconjugants formed was dependent not only on the time donor strains remained in the water but also on the temperature (20 or 37°C) at which the mating assays were conducted.

Effect of temperature and starvation upon survival strategies of Pseudomonas fluorescens CHA0: comparison with Escherichia coli

Microorganisms in aquatic systems are exposed to continuous modifications in their environmental conditions. In these systems, both autochthonous and allochthonous bacteria respond to adverse conditions by expressing viable but nonculturable phenotype. On the basis of this common response, the behaviour of a few species is extrapolated to others. We compared the survival strategies of Escherichia coli (allochthonous, mesophile bacterium) and Pseudomonas fluorescens CHA0 (ubiquitous, psychrotrophic bacteria) under nonoptimal temperature and nutrient deprivation. In the absence of nutrients, the effect of temperature on the loss of culturability did not show a common pattern. Whereas the survival of E. coli had an inverse relationship with temperature, whereas for P. fluorescens a direct relationship between temperature and T₉₀ values was only established in the range 5-15°C, with an inverse relationship at higher temperatures. When the subproteome of the outer membrane of P. fluorescens was comparatively analysed, starvation was not the main source of change. The most relevant modifications were due to variations in temperature. OprF, the major surface protein of the genus Pseudomonas, showed a high expression in nonculturable as well as culturable populations under all the adverse situations analysed. We therefore propose OprF as a suitable marker for Pseudomonas detection in the environment.