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Previous work demonstrated that physiological, morphological, and gene expression changes as well as the time-dependent entry into the viable but not culturable (VBNC) state are used by Vibrio species to survive and cope with diverse stress conditions including seasonal temperature downshifts and starvation. To learn more about the nature and specific contribution of membrane proteins to cell adaptation and survival, we analyzed variations in the protein composition of cell envelope and related them to morphological and physiological changes that were taking place during the long-term permanence of Vibrio harveyi in seawater microcosm at 4 °C. We found that after 21 days of permanence, nearly all population (ca. 99 %) of V. harveyi acquired the VBNC phenotype. Although the size of V. harveyi cells gradually decreased during the incubation time, we found that this morphological change was not directly related to their entry into the VBNC state. Our proteomic study revealed that the level of membrane proteins playing key roles in cellular transport, maintenance of cell structure, and in bioenergetics processes remained unchanged along starvation at low temperature, thus suggesting that V. harveyi might need these proteins for the long-term survival and/or for the resuscitation process. On a contrary, the level of two proteins, elongation factor Tu (EF-TU) and bacterioferritin, greatly increased reaching the maximal values by the end of the incubation period. We further discuss the above data with respect to the putative roles likely exerted by membrane proteins during transition to and maintaining of the VBNC state.

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.

In nature, prokaryotes must face alternating periods of prosperity and adversity. Differentiating bacteria confront situations of adversity by developing resistant structures. When there is a plentiful period, they adopt a vegetative state and when the period is adverse, a resistant structure, thereby completing a cycle. Non-differentiating bacteria do not develop such morphological distinct resistant structures. It has been proposed that many of these bacteria withstand periods of adversity by adopting the viable but nonculturable phenotype (VBNC). Bacteria of this phenotype conserve detectable metabolic function but become unculturable. Is it possible that the VBNC phenotype can revert to a culturable state, and vice versa, thus establishing a life-cycle? This review presents and evaluates different hypotheses regarding this question. Moreover, it attempts to analyse and proffer answers to other questions related to this phenotype. Is this a successful phenotype which prolongs survival? Is this a strategy for the survival of individual cells, or is it a strategy for the survival of a population? Finally, is it possible that this phenotype is, in fact, an example of altruistic death?

The relationship between the microbial methylation of mercury and the microbial activities in sediments and water collected from the estuary of Bilbao (North of Spain) was studied in three different sampling points and in two different seasons. Three different cultures were prepared with a sediment slurry to distinguish between biotic and abiotic methylation pathways and the variations of the methylmercury concentration and the variations of the population of total number of bacteria (TDC), anaerobic heterotrophic bacteria (AHB), sulphate-reducing bacteria (SRB) and Desulfovibrio were measured. From this work, it can be concluded that the variation of MeHg concentrations is a result of the methylation/demethylation processes in the sediments, and that the abiotic processes have a negligible contribution to those processes. According to the statistical analysis of the results (partial least squares analysis) a significant statistical correlation was established between methylmercury and the SRB counts.

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.

After induction of the viable but nonculturable (VBNC) state in Escherichia coli populations, we analysed abiotic and biotic factors suggested to promote the resuscitation process. The response to the stressing conditions implied the formation of three subpopulations, culturable, VBNC and nonviable. In most adverse situations studied, the VBNC subpopulation did not represent the dominant fraction, decreasing with time. This suggests that, in most cases, the VBNC is not a successful phenotype. Combining methods of dilution and inhibition of remaining culturable cells, we designed a working protocol in order to distinguish unequivocally between regrowth and resuscitation. Reversion of abiotic factors inducing nonculturability as well as prevention of additional oxidative stress did not provoke resuscitation. Participation of biotic factors was studied by addition of supernatants from different origin without positive results. These results indicate that the E. coli strain used is not able to resuscitate from the VBNC state. VBNC cells release into the surrounding medium, and could thus aid in the survival of persisting culturable cells. The formation of a VBNC subpopulation could thus be considered as an adaptive process, designed for the benefit of the population as a whole.

The ability of Urografin or Percoll density gradient centrifugations to separate nonculturable subpopulations from heterogeneous Escherichia coli populations was analysed. Bacterial counts (total, active and culturable cells) and flow cytometric analyses were carried out in all recovered bands. After Urografin centrifugation, and despite the different origin of E. coli populations, a common pattern was obtained. High-density bands were formed mainly by nonculturable cells. However, the increase in cell density would not be common to all nonculturable cells, since part of this subpopulations banded in low-density zones, mixed with culturable cells. Bands obtained after Percoll centrifugation were heterogeneous and culturable and nonculturable cells were recovered along the gradient. Thus, fractionation in Urografin cannot be only attributed to changes in buoyant densities during the transition from culturable to nonculturable state. Urografin density gradients allow us to obtain enriched fractions in nonculturable subpopulations from a heterogeneous population, but working conditions should be carefully chosen to avoid Urografin toxicity.

Acinetobacter baumannii possesses a tremendous potential to thrive under hostile conditions. To learn more about its survival strategy and capacity to persist in the environment, we studied the effect of temperature, nutrient deprivation and dryness on the long-term survival of two A. baumannii strains (ATCC 19606(T) and a clinical isolate). Our results revealed that both strains show a great persistence under stress that appears to involve a bust-and-boom strategy. Bacterial survival was differentially affected by temperature and physical environment: Desiccation favored cell resistance to stress at 20 and 37 °C, while survival in aqueous environments was temperature dependent and led to changes in several cellular characteristics. In addition, we tested the ability of the A. baumannii ATCC 19606(T) strain to form biofilms by monitoring the expression of adhesion-/biofilm-related genes (ompA, bfmR and csuAB). The observed downregulation of these genes suggests that the potential difficulties to adhere to solid surfaces and form biofilms likely limit the capacity of starved cells to spread and colonize abiotic surfaces.

The survival of allochthonous bacteria in aquatic systems is affected by biotic and abiotic environmental factors. Grazing by protozoa is one of the main biological processes that control allochthonous bacterial density. Its extent depends on the concentration of bacteria and the digestion capacity of the grazer. The physiological state of bacteria is affected by multiple physicochemical stresses, to which they respond by entering a dormant, viable but non-culturable state. Starved bacteria show a tendency to shrink, and a generally enhanced resistance to heat, oxidative and osmotic shock is observed. Nutrient scarcity, temperature, osmotic stress and visible light seem to be the abiotic factors that most negatively influence survival. The negative effect of light upon the culturability of enterobacteria in aquatic systems has long been recognized. In relation to the influence of plasmids on bacterial survival, heterogeneous and contradictory results have been reported. Some authors reported that plasmid-bearing strains can survive as well as their wild-type counterparts or even better, whereas in other reports the effects of various plasmids on the survival of their hosts were very variable. Plasmid transfer could be affected by the physiological status of donors and recipients during survival. Flow cytometry is a recent approach with great potential, especially for assessing the heterogeneity of cell size, metabolic state and molecular content in the population.

Owing to their ubiquitous presence and ability to act as primary or opportunistic pathogens, Vibrio species greatly contribute to the diversity and evolution of marine ecosystems. This study was aimed at unveiling the cellular strategies enabling the marine gammaproteobacterium Vibrio harveyi to adapt and persist in natural aquatic systems. We found that, although V. harveyi incubation in seawater microcosm at 20 °C for 2 weeks did not change cell viability and culturability, it led to a progressive reduction in the average cell size. Microarray analysis revealed that this morphological change was accompanied by a profound decrease in gene expression affecting the central carbon metabolism, major biosynthetic pathways, and energy production. In contrast, V. harveyi elevated expression of genes closely linked to the composition and function of cell envelope. In addition to triggering lipid degradation via the β-oxidation pathway and apparently promoting the use of endogenous fatty acids as a major energy and carbon source, V. harveyi upregulated genes involved in ancillary mechanisms important for sustaining iron homeostasis, cell resistance to the toxic effect of reactive oxygen species, and recycling of amino acids. The above adaptation mechanisms and morphological changes appear to represent the major hallmarks of the initial V. harveyi response to starvation.

No differences were observed in the survival of plasmid-bearingand plasmid-free Escherichia coli strains in the course of a long-term survival process in wastewater, under both illuminated and non-illuminated conditions. While the CFU counts and the number of active cells decreased, the numberof nucleoid-containing cells remained constant throughout the 30 days of experimentation. Visible light efficiently contributed to the reduction in culturability, and T90 values were very different under illuminated and nonilluminatedconditions. In the latter case the time necessary to reduce theculturability of a bacterial population by 90% was 27 days, while in the former it was only 1 day. Plasmid transfer was abundant, while the survival of donor and recipient cells was extensive. After 24 hr of survival in wastewater, transfer frequency values ranged from 5.92 × 10-5 to 1.12 × 10-2, depending on mating conditions. In the absence of illumination, the potential transfer abilitiesremained for survival periods of at least 20 days. Transfer assays between free and adhered cells were carried out by means of dialysis bags and submerged membrane diffusion chambers. Transfer frequency for adhered cells was greater than for free cells (2.80 × 10-2 as opposed to 2.39 × 10-3).

The behavior of Escherichia coli immersed in aqueous systems amended with humic acids, under PAR, UV-A, UV-B, and simulated solar radiation was examined. Culturability, ability to elongate, functioning of the electron transport systems, and glucose uptake were assessed. Humic substances in the range from 1 to 50 mg L-1 protected cells from photoinactivation. Decrease in culturability and cellular activities was significantly (p0.05) less in the presence of humic material. However, humic acids were not used as nutrients. Neither irradiated nor nonirradiated humic solutions (50 mg L-1) supported the growth of 105 cells ml-1. However, humic acids dissolved in 0.9% NaC1 efficiently absorbed light over wavelengths from 270 to 500 nm. Also, a photoprotective effect against simulated sunlight was observed when humic acids were not in contact with but rather enveloped the cellular suspensions in double-wall microcosms. The protection afforded by humic acids against luminous radiation likely derives from their ability to absorb these radiations and hence reduces the amount of energy reaching the cells.

Aromatic extracts of Roncal cheese were obtained by supercritical fluid extraction (SFE) in different solvent conditions. An adequate support for sensory perception of the extracts has been selected from a group containing four commercial products: sweetened cream, quark, cottage cheese and ewes’ milk whey cheese (ricotta type). Ewes’ milk whey cheese was found to be an adequate support for assessing sensory qualities in the Roncal cheese. To assess the representativeness of the aromatic extracts, three different sensory methods were applied : ranking, similarity and matching tests. The reconstitution solvent mixture which best represented Roncal aromatic qualities was n -hexane : acetone (2 : 1).

Various sample preparation techniques and collection procedures for a supercritical fluid extraction (SFE) system were tested and compared. Special attention was paid to parameters which influence the extraction results, but are not part of the extraction process per se. The application of SFE to the extraction of the aroma components from unsmoked ewe’s milk Idiazabal cheese was undertaken for the first time. It was, therefore, necessary to optimize and validate the extraction conditions. The parameters which were optimized included sample preparation (lyophilization and sonication); sample to adsorbent ratio; reconstitution solvent; trap type and trapping temperature. The best extraction results were achieved for a comminuted cheese sample containing added alumina (1:1 ratio with cheese) as the adsorbent and n-hexane:acetone (2:1) as the reconstitution solvent, and a polar octadecylsilica (ODS) trap held at a temperature of −5°C to retain volatile compounds.

Secondary microflora in three batches of Idiazabal cheese were studied. In one of them the milk was cold-stored for 3 d and showed lowcr counts, except for psychrotrophs, Enterobacte- riaceae, Clostridium tyrobutyricum and moulds. Significant differences in NaCI, dry matter and pH in raw milk were observed. Aerobic mesophilic, psychrotrophic !lora and Enterococcus increased dur- ing coagulation, pressing and brining, while the rest of the secondary microflora was inhibited, Dur- ing ripening, Enterobacteriaceae and Micrococcaceae declincd in the first 2 ripening months; the Ente- rococcus were stable, except in the cheese from milk cold-stored for 3 d, for which they presented low counts and Clostridium tyrobutyricum, yeast and mou Ids showed an irregular evolution. © Inra/Elsevier, Paris. ewe's cheese 1physicochemicaI characteristic 1secondary microflora

The discharge of disinfected effluents affects the bacterivorous ability of protozoa and the effect depends on the disinfectant applied. Chlorine provokes a decrease in the number of protozoa and a delay in the bacterivorous ability. The discharge of ozonated and peracetic acid-treated wastewater provokes only an initial slight decrease in bacterivorous ability. No correlation was found between toxicity values detected using the Microtox™ assay and the effect of disinfected effluents on freshwater protozoa populations. After the disinfection processes, recipient systems (fresh and marine water) have different effects on the survival of Escherichia coli populations discharged to them. The effect of the freshwater recipient system is less negative than the effect provoked by sea-water, and the differences detected depend on the disinfection treatment applied. The wastewater bacterial population as a whole is able to grow after discharge of disinfected wastewater to receiving waters. However, in the absence of predation or competition, the recipient systems exert selection, with rod-shaped bacteria predominating.

Three batches of Idiazabal cheese were studied at different production stages. Counts obtained on three solid media (M17, MRS and MSE agar) showed growth during coagulation, pressing and brining operations and a decrease during the two first months of ripening. While the pH and water activity decreased, dry matter and NaCl increased during ripening. Physiological and biochemical characteristics of lactic acid bacteria were studied for their identification. Of the Lactococcus strains, 61% were identified as Lc. lactis subsp. lactis biovar diacetylactis , 30% as Lc. lactis subsp. lactis , 7% as Lc. lactis subsp. cremoris and 2% Lc. raffinolactis . All Lactobacillus isolated from milk and curd and 19% of those isolated from cheese were identified as Lb. plantarum . The predominant Lactobacillus in cheese was Lb. casei subsp. casei (64%). Leuconostoc lactis was identified in cheese.

The life and survival of the marine bacterium Vibrio harveyi during its adaptation in natural aquatic systems is highly influenced by the availability of nutrients and temperature. To learn about adaptation strategies evolved by this bacterium to cope with drastic temperature downshifts and nutrients depletion, we have studied the phenotypical and gene expression changes occurring in V. harveyi during its adaptation to cold seawater. We found that incubation in cold seawater up to 12 h did not cause any significant morphological changes in V. harveyi and had no effect on the number of viable and culturable cells. Microarray analysis revealed that the V. harveyi response to cold seawater leads to up- and downregulation of numerous genes controlling the central carbon metabolism, nucleotide and amino acid biosynthesis as well as DNA repair. In addition, expression of some genes controlling biosynthesis of lipids, molecular transport, and energy production was altered to likely affect the composition and properties of the V. harveyi cell envelope, thus implying the putative role of this compartment in adaptation to stress. Here, we discuss these results with regard to the putative adaptive responses likely triggered by V. harveyi to cope with environmental challenges in natural aquatic systems.

Escherichia coli O25:H-42 was selected to study the effect of pre-treatments on the enumeration of direct viable cells from milk samples. Before and after inducing cell elongation by cellular division inhibitors, three pre-treatments for milk-filtration were used. One involved a pre-treatment with trypsin (1.5 min at 50 degrees C), addition of hot Triton X-100 after heating and filter rinses with phosphate saline buffer. The other two involved pre-treatment with trypsin and Triton X-100 (10 min at 50 degrees C), filter rinses with hot Triton X-100 and organic solvents. Pre-treatments applied after inducing cell elongation had an effect on cell recovery from milk samples depending on the pre-treatment used. The most suitable, on the basis of the number and percentage of enlarged cells obtained was the first described. The others selectively affected recovery of elongated cells. Pre-treatments applied before inducing the cell elongation, negatively affected viability with enumeration in milk samples being significantly (P < 0.001) lower than those found in controls. However, the negative effects of first pre-treatment on viability was lower than that produced by the pre-treatments involving organic solvents.

A description is given of a pre-treatment for milk samples which allows it to be filtered through 0.2 μm pore-size membrane filters without reducing the initial number of bacteria. The efficiency of the method was demonstrated in milk samples inoculated with Escherichia coli O25:H-42, a food-borne pathogen, by comparison with a control in tryptone soya broth (TSB). No significant differences were found between direct microscopic counts in milk samples and controls in TSB.

SUMMARY 1. The influences of bacterial density and water temperature on the grazing activity of the ciliates Uronema sp. and Colpoda inflata were studied. The conditions assayed were two prey densities (106 and 4 × 107 bacteria ml−1) and three water temperatures (10, 15 and 22 °C). 2. The response of the ciliates was measured from changes in protistan biovolumes and specific clearance rates. At high prey density, both ciliates showed lower biovolumes as water temperature increased, while at low prey density this tendency was minimized. 3. At the intermediate temperature of 15 °C both ciliates filtered ten times more body volume when bacteria were scarce; however, the ingested bacteria were fewer than at high prey density. At low prey density, a decrease from 15 to 10 °C evidenced different strategies of the two ciliates, which led to a similar ingestion of bacteria: C. inflata reduced its specific clearance rates and increased its biovolume, while Uronema sp. did not show changes. At high prey density, an increase from 15 to 22 °C caused lower biovolumes and a noticeable increase in specific clearance rates in both ciliates, indicating opportunist behaviour.

Size fractionation of aminopeptidase (Amp) activity and incorporation of dissolved substrates such as glucose and thymidine were analyzed in three aquatic ecosystems: the Salvaje Beach (Spain) with low levels of nutrients, and two sampling stations of the Butron River (Spain) with higher levels of nutrients. Amp activity in the 3.0 μm size fraction represented a high percentage of the total Amp activity with mean values of 41% in the Salvaje Beach and 35% and 34% in the Butron River. The activity in this fraction could be attributed to particle attached bacteria. However, the attached bacteria represented a low percentage of the total abundance and moreover, Amp activity in the > 3.0 μm size fraction was not significantly correlated with the attached bacterial abundance, biomass, and incorporation of glucose and thymidine in this fraction, in any of the ecosystems studied. These results indicate that bacteria should not be considered the only microorganisms responsible for the Amp activity in these aquatic systems. Amp activity in the 0.2–3.0 μm size fraction correlated with bacterial abundance, biomass, and glucose and thymidine incorporation in this fraction, but only in the Salvaje Beach with low concentration of nutrients.

A study was made of the mechanisms by which visible light produces cell dormancy in Escherichia coli, resulting in loss of culturability. Visible light may act directly on the cells or generate photoproducts with a negative effect on the cells. In nonilluminated microcosms the addition of increasing concentrations of hydrogen peroxide, one of the photoproducts formed in natural aquatic systems, gave rise to the formation of nonculturable cells and injured culturable cells, and this negative effect depended on the concentration of peroxide. On the other hand, in illuminated microcosms the addition of compounds which eliminate hydrogen peroxide (i.e., catalase, sodium pyruvate, and thioglycolate) had a protective effect on the E. coli cells, as the CFU counts on minimal medium and on recuperation medium were significantly higher (P < 0.05) than those detected in the absence of these compounds. Furthermore, when hydrogen peroxide was eliminated, the CFU counts on recuperation medium did not fall significantly, indicating that nonculturable cells did not form. These results rule out the direct effect of visible light on the cells and show that hydrogen peroxide, generated photochemically, may be the cause of the loss of culturability of E. coli in illuminated systems.

A comparative study, in illuminated and non-illuminated systems, was made to determine the survival strategies of plasmid-carrier and plasmidless bacteria in sterile river water. Two strains of Escherichia coli from river water were selected: one plasmidless, EC1, and one antibiotic-resistant strain, EC7, which showed plasmid bands. By matings with EC7 as donor and E. coli K12 strain J62 as recipient, transconjugants were generated, the J62(7) strain, which showed both antibiotic resistance and plasmid bands. Ethidium bromide curing of the EC7 strain generated the EC7(2) strain which showed a partial loss of resistance and a reorganization of plasmid bands. Under non-illuminated conditions the total number of cells detected by direct count and the number of culturable cells (injured and non-injured cells) remained practically constant throughout the period of incubation. In the illuminated systems, however, the number of cfu decreased in four of the five strains studied. The greatest decreases are those of the J62 strain, followed by those of the J62(7), EC1, EC7(2) and EC7 strains. Differences in survival strategies as a consequence of the presence or absence of plasmids are discussed.

10 páginas.-- 2 figuras.-- 3 tablas.-- 39 referencias.-- El autor González Grau, Juan Miguel pertenece actualmente al Instituto de Recursos Naturales y Agrobiología de Sevilla, 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 sig- nificantly 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

Trophonema arenarium (Raski, 1956) Raski, 1957 has been found in the roots and rhizosphere of ash tree (Fraxinus angustifolia L.) at Los Molinos (Madrid), Spain. Two species of the genus (T. asoense Minagawa, 1983 and T. okamotoi Minagawa, 1983) are proposed as junior synonyms.

The disappearance of bacteria through grazing was studied. Five allochthonous and eight autochthonous bacterial strains, and the bacterioplankton of the Butron River were tested. There were differences in the susceptibility of different strains. These differences seemed to be related to intrinsic characteristics of the bacteria rather than to their origin.

The 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./hea

Physicochemical and microbiological parameters were compared for three brining times (12, 24 and 36 h) for fresh, young, semihard and hard Idiazabal cheese. Longer brining time produced higher salt, dry matter and salt–moisture ratio and lower water activity values for all types of cheese according to ripening time, while non-significant changes were observed for pH. In fresh cheese (1–15 days ripening), non-significant differences for microbiological counts in relation to brining time were observed, except for moulds. In young and hard cheeses, Lactobacillus and Leuconostoc showed lower counts with longer brining times. In contrast, Micrococcaceae, yeast and moulds were stimulated by higher salt content in matured cheeses. In addition, this work has proved that there are lower water activity values and lower microbiological counts in longer-matured Idiazabal cheeses. For the different brining and ripening times, positive correlations were observed among most of the microbial groups studied, but a different behavior was established for Enterococcus, Clostridium tyrobutyricum, yeast and moulds.

The percentage of dividing biomass was calculated for attached and free-living bacteria, in a coastal marine and a freshwater system. In the marine system with low concentrations of total and dissolved organic carbon (TOC and DOC) the percentage of dividing biomass was higher for attached (41.4 ± 13.9) than for the free-living bacteria (22.0 ± 11.7). However, in the freshwater system, which had a higher concentration of TOC and DOC, the percentage of dividing biomass was similar for both communities-attached (53.4 ± 26.5) and free-living (78.4 ± 21.9). Thus the attachment to particulate material is not necessarily an advantage in waters where dissolved organic nutrients are readily available.

Advanced disinfection processes (peracetic acid, UV irradiation and ozonation) have been tested and evaluated through bench and pilot scale studies. 3 log removals of total coliforms, faecal coliforms and faecal streptococci were achieved by 10mg/L peracetic acid at a 10min contact time, by UV radiation at 35mW.s/cm2 and by ozone at 5mg/L for 10min contact time. Higher doses are required for virus removal by UV and PAA and especially for highly resistant viruses such as F-specific bacteriophage MS2. Ozonation has the advantage of having a strong effect on all types of bacteriophages and protozoa cysts even when low treatment doses and short contact times are applied. The results of this study demonstrated that evaluation of disinfection efficiency of ozone, UV and PAA depends on the criteria and methods employed. Standard method (plate count) results showed an important disinfection effect on culturability, while results from non-standard methods (respiratory activity and β-galactosidase activity assay) indicated less reduction of viable cells. Moreover, the results confirm that disinfectants act on bacteria in different ways. It has been clearly demonstrated that b-galactosidase activity is affected by PAA while UV treatment has no or very limited effect on the enzyme activity. Even without sunlight reactivation, bacterial regrowth in seawater was observed after disinfection of sewage effluents. This study also shows that the biodegradability of sewage effluent for an E coli strain was affected differently by the oxidative disinfectants ozone and PAA. Biodegradability should therefore be considered when evaluating the total disinfection efficiency.

Viable but non-culturable transconjugant cells were detected by a modification of the direct viable count (DVC) method. This modification involved the addition of parental antimicrobial markers (kanamycin and streptomycin) to the elongation medium in order to promote selective elongation of the transconjugant cells. Presence of viable, other than culturable, transconjugants was demonstrated in matings with parental cells from TSB culture as well as with recipient cells from survival in river water (under illuminated and non-illuminated systems). In matings with a recipient strain from illuminated systems, culturable transconjugants were not detected after the third day of recipient cell survival. In spite of this, viable transconjugants were detected in numbers that exceeded 10 5 cells ml -1 . These results clearly show that a fraction of non-culturable recipient cells is able to receive and express plasmids by conjugation processes and form viable but non-culturable transconjugant cells.

Roncal and Idiazabal cheeses are manufactured from ewe's milk in the Autonomous Region of Navarre in Spain. Levels of enterococci are high in these cheeses and in other types of ewe's-milk cheeses. The present study has identified enterococci present in the milk used and in the Roncal and Idiazabal cheeses after 120 days of ripening. A total of 282 strains were isolated and identified, and the cytoplasmic and extracellular enzymatic activities of some of the strains were assessed. The dominating species were Enterococcus faecalis, which accounted for 85% of the total both in the milk as well as in the two types of cheese, and Enterococcus faecium, Enterococcus durans, and Enterococcus avium which were present in lower proportions. Aminopeptidase and proteinase activity levels in enterococci were low, and considerable variation between strains of the same species was recorded, highlighting the need to study individual strains when selecting the most suitable bacteria as a starter culture. Aminopeptidase activity levels for the enterococci were appreciably higher at pH 7 than at pH 5.5, hence aminopeptidase activity by enterococci would appear to be less significant in the normal manufacturing conditions of the two cheeses.

La flore lactique du roncal et de l'idiazabal, deux fromages AOC pur brebis fabriques dans la region de la Navarre (Espagne), a ete identifiee et comparee ; 263 isolats de lactocoques, 494 de lactobacilles et 464 de leuconostocs provenant des deux fromages au cours de l'affinage ont ete identifies d'apres leur morphologie et leur phenotype. La distribution des especes de lactocoques ne differait pas significativement pour les deux fromages, Lactococcus lactis subsp lactis etant l'espece la plus representee. Lactobacillus casei et Lactobacillus plantarum constituaient les deux especes de lactobacilles majoritaires dans les deux types de fromages. Dans le roncal, la proportion de L casei (53%) etait superieure a celle de L plantaruni (30%), mais la tendance inverse etait observee dans l'idiazabal: 39% de L casei contre 44% de L plantarum. Leuconostoc mesenteroides subsp dextranicum et subsp mesenteroides constituait l'espece majoritaire pour le roncal et l'idiazabal, mais les proportions pour les deux sous-especes etaient d'environ 3:1 pour le roncal et 1:1 pour l'idiazabal. Les resultats experimentaux de ce travail sont consideres comme une premiere etape pour concevoir des levains pour les deux fromages a partir de souches representatives isolees de chacun des fromages.

Surveys of 63 fields in the chickpea growing area of the Guadalquivir Valley of Andalucía, southern Spain, in 1990 and 1991, indicated that Pratylenchus thornei was the predominant plantparasitic nematode, being recovered from 92% of soil and 84% of root samples, with population densities ranging from 2-48/100 cm3 and 6-70 per g of fresh root. Substantial populations of the ectoparasitic nematodes Amplimerlinius magnistylus, Aorolaimus perscitus, Helicotylenchus tunisiensis, Merlinius brevidens, Mesocriconema curvatum and Paratrophurus loofi were also found. Three populations of P. thornei were tested in a glasshouse at 15, 20 and 25°C at an inoculum density of 2500 nematodes per plant to determine the effect of temperature on nematode reproduction on chickpea cv. UG 27. Reproduction increased with increase of soil temperature. A population from Jerez had a reproduction rate significantly higher than the other two populations at each of the temperatures. Shoot wight of chickpea was not affected by nematode population or soil temperature. At 25°C root weight was significantly lower with a P. thornei population from Cañete.

Pratylenchus thornei-chickpea interactions were investigated under controlled and fluctuating environmental conditions in the growth chamber, greenhouse and shadehouse. Under controlled conditions. P. thornei infected chickpea hnes 12071/10054 and P2245 and cultivars Andoum 1, JG62 and UC 27. Line P 2245 and cv. JG 62 were the most susceptible genotypes on the basis of root damage and nematode reproduction, but nematode infection did not significantly reduce root and shoot weights. Cultivars Andoum 1 and UC27 and line 12071/10054 showed the least root damage and nematode reproduction. Inoculation of cv. Andoum 1 with 2500, 5000 or lOOOO nematodes per plant in pots did not affect shoot weight, regardless ofthe conditions of water stress ofthe plants. However, root weight was significantly reduced by nematode infection in plants grown under water stress and fluctuating temperature conditions in the greenhouse, but was not affected by any other treatment. The nematode reproduction index was not affected by soil water content under shadehouse conditions, but was greater on plants watered to soil water-holding capacity than in water-stressed plants under greenhouse conditions. For both environments, the nematode reproduction index decreased when inoculum density was greater than 5000 nematodes per plant., This research was supported by grant AGF92- 0910-CO2-01 from Comision Interministerial de Ciencia y Technologia (CICYT), We thank H, M, Halila, INRA, Tunisia for providing us with seeds of cultivar Andoum 1, and F, Orgaz, Instituto de Agricultura Sostenible, CSIC, Spain for measuring water potential.

We evaluated the transfer to and from Escherichia coli of endogenously isolated plasmid material from the River Butrón during the growth of three donor strains and two recipient strains as well as after the survival of these parental cells in river water. Transfer frequency varied greatly during the growth of donor cells, with minimum values in the exponential phase; frequency remained constant, however, during the growth of recipient strains. After survival in river water, donor cells lost their ability for plasmid transfer before any other physiological variations in the cells caused by environmental stress were detected. Under the same conditions and during equal periods, however, no variation in the ability of recipient cells to receive and express plasmid material was observed.

J. IRIBERRI, I. AZUA, A. LABIRUA-ITURBURU, I. ARTOLOZAGA AND I. BARCINA. 1994. The short-term (1 h) and long-term (3 d) elimination of low and high densities of five enteric bacteria, Klebsiella pneumoniae, Aeromonas hydrophila, Escherichia coli, Enterococcus faecalis and Staphylococcus epidermidis, by flagellate and ciliate protists were measured in a freshwater system. In addition, the two processes, ingestion and digestion, which cause the disappearance of those enteric bacteria as time passes, were quantified. The results showed that the elimination of these enteric bacteria by protists depends on their initial density, which confirms that the lower the bacterial density the more difficult is their elimination. On the other hand, the short-term and long-term elimination rates of each enteric bacteria were different, and moreover, the order of priority for elimination in the two cases was not the same. Escherichia coli showed the highest elimination rate in short-term experiments, while Aer. hydrophila disappeared at highest rates in long-term experiments. This different order of priority in the elimination rates and the different digestion rates on the five enteric bacteria by phagotrophic protists indicated that the elimination in time is very much influenced by the digestive capacity on each enteric bacteria of those protists. Thus, the low digestion rates of Ent. faecalis and Staph. epidermidis by flagellates and ciliates as well as their low disappearance percentages in the long-term experiments confirm that enteric Gram-positive bacteria are eliminated from the aquatic systems at lower rates, because their digestion is difficult.

The influence of biotic and abiotic factors on plasmid transfer between Escherichia coli strains in terms of the variation in the number of transconjugants formed and the variation in transfer frequency was investigated. The density of parent cells affected the number of transconjugants, reaching a maximum when the cell density was on the order of 10(8) CFU ml-1. As the donor-to-recipient ratios varied from 10(-4) to 10(4), the number of transconjugants varied significantly (P less than 0.001), reaching a maximum with donor-to-recipient ratios between 1 and 10. The concentration of total organic carbon in the mating medium affects both the number of transconjugants and the transfer frequency, being significantly higher (P less than 0.001) when the total organic carbon concentration was higher than 1,139 mg of C liter-1. However, the transconjugants were detected even with less than 1 mg of C liter-1. Linear regression of log10 transconjugants versus mating temperature showed a highly significant regression line (P less than 0.001). Neither the transfer frequency nor the transconjugant number varied significantly in the range of pHs assayed. We can conclude that plasmid transfer by conjugation can take place within a wide range of conditions, even in such adverse conditions as the absence of nutrients and low temperatures.

Production and specific growth rates of attached and free-living bacteria were estimated in an oligotrophic marine system, La Salvaje Beach, Vizcaya, Spain, and in a freshwater system having a higher nutrient concentration, Butron River, Vizcaya, Spain. Production was calculated from [ methyl - 3 H]thymidine incorporation by estimating specific conversion factors (cells or micrograms of C produced per mole of thymidine incorporated) for attached and free-living bacteria, respectively, in each system. Conversion factors were not statistically different between attached and free-living bacteria: 6.812 � 10 11 and 8.678 � 10 11 μg of C mol −1 for free-living and attached bacteria in the freshwater system, and 1.276 � 10 11 and 1.354 � 10 11 μg of C mol −1 for free-living and attached bacteria in the marine system. Therefore, use of a unique conversion factor for the mixed bacterial population is well founded. However, conversion factors were higher in the freshwater system than in the marine system. This could be due to the different trophic conditions of the two systems. Free-living bacteria contributed the most to production in the two systems (85% in the marine system and 67% in the freshwater system) because of their greater contribution to total biomass. Specific growth rates calculated from production data and biomass data were similar for attached and free-living bacteria.

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 (Microtox assay) was unrelated to the effect on cellular activity.

Anew species of Rotylenchus Filip'ev, 1936 from Sierra de Cazorla, Spain, with males and females is decribed as R. cazorlaensis sp. n. Females are characterized by a truncate cephalic region continuous with body, large size L = 1. 79 mm (1.44-2.28) and well developed stylet. Close to R. magnus Zancada, 1986 in large size and lateral field areolated only anteriorly, it differs in stylet length 53 !J.m (46.5-56.5) compared to 64 !J.m (59-70), lip region and position of phasmids. It resembles R. robustus (de Man, 1876) Filip'ev, 1936, but differs in areolations of lateral field. R. fallorobustus Sher, 1965 is recorded and described for the first time in Spain. Keywords: taxonomy, morphology, soil nematodes, Spain, spiral nematodes.

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.

Summary Nine selective culture media were used for counting actinomycetes in marine water and sediments. The presence of glycerol and/or asparagine in the media favoured the growth of these microorganims. Thermic treatment of sediment samples resulted in a selective reduction of the non-actinomycetal heterotrophic microflora. However, the same treatment of water samples had a negative effect on the actinomycetal flora. Actinomycetes in water and sediment represented 3–4% and 5–6% of the total flora, respectively. 72.7% of the isolated strains were Streptomyces, 20.5% Micromonospora and 7% Nocardia. The isolated actynomycetes strains, especially Streptomyces, showed a high hydrolytic activity for macromolecules and non-viable Gram-negative cells. Antimicrobial activity increased when sea water was present in the culture medium.

Until now different uses of computers are known in nematology, e.g. Boag (1981) described an electronic system for measuring nematodes and Fortuner (1983) designed a computer program (NEMAID) for identifying Helicotylenchus species, including a database with measurements.

The abundance and heterotrophic activity of attached and free-living bacteria were examined seasonally in coastal water. Heterotrophic activity was determined by the uptake of [ 14 C]glucose. The density of attached bacteria was always minor, not showing a seasonal variation, whereas the free-living bacteria were more numerous and showed a marked seasonal variation, their density being higher under warmer conditions. The contribution of the attached bacteria to the total assimilation of [ 14 C]glucose (from 10 to 38%) was lower than that of the free-living bacteria, neither of them showing a seasonal variation. On a cellular basis, attached bacteria were more active, since they assimilated more [ 14 C]glucose and showed, under warmer conditions, a higher cellular volume (0.102 versus 0.047 μm 3 ). We consider that the factors responsible for these observations were the amount and quality of the particulate material, the different availability of organic matter for the two types of bacteria, and in a fundamental way, the variation in water temperature.

Protozoan predation on bacteria and bacterioplankton secondary production were simultaneously determined in La Salvaje Beach water during 1990. Protozoan grazing on bacterioplankton was measured from fluorescently labeled bacterium uptake rates; estimates of bacterial secondary production were obtained from [ 3 H]thymidine incorporation rates. Two different conversion factors were used to transform thymidine incorporation rates into bacterial production rates; both of them were specific for La Salvaje Beach and were calculated by using empirical and semitheoretical approaches. The average flagellate predation rate was 14.0 bacteria flagellate -1 h -1 ; the average population predation rate was 7.35 x 10 6 bacteria liter -1 h -1 . The estimates of bacterial production differed greatly depending on the conversion factor used, and so did the percentages of bacterial production consumed by flagellated protozoa (4.6% when the empirical conversion factor for La Salvaje Beach was used and 113% when the semitheoretical conversion factor specific for this system was used). The ecological implications of each of these values are discussed.

7 páginas.-- 4 figuras.-- 3 tablas.-- 49 referencias.-- El autor González Grau, Juan Miguel pertenece actualmente al Instituto de Recursos Naturales y Agrobiología de Sevilla, Differential decreases over time of two bacterial species, Escherichia coli and Enterococcus faecalis, in a freshwater and a marine ecosystem were observed and explained by a differential rate of digestion of these bacteria by phagotrophic flagellates and ciliates. For this purpose, fluorescence-labeled bacteria (FLB) were used and prepared from the two species cited above. The number of FLB was observed for 5 days in fresh and marine waters in the presence or absence (0.2-,unm-pore-size-filtered water) of natural microbiota. These experiments showed a longer persistence of Enterococcusfaecalis FLB is opposed to Escherichia coli FLB in the presence of natural microbiota. Removal of FLB was due to protozoan grazing because no decrease of FLB number was observed in the absence of natural microbiota. In short-term (about 40 min) ingestion experiments, we found similar clearance rates of Escherichia coli and Enterococcus faecalis FLB by assemblages of flagelates from the freshwater and the marine ecosystem and by cultured assemblages of ciliates from the marine ecosystem. Clearance rates of Enterococcus faecalis FLB were greater than those of Escherichia coli FLB for assemblages of ciliates from the freshwater ecosystem. Comparison of rates of ingestion and digestion of FLB by protozoa showed that Escherichia coli FLB were digested and ingested at similar rates. However, Enterococcusfaecalis FLB were digested slower than they were ingested. These results suggest that a longer persistence of Enterococcus faecalis as opposed to Escherichia coli can be explained by a differential digestion by flagellates and ciliates in aquatic ecosystems. Moreover, rates of ingestion and digestion were strongly correlated for both FLB types.

The objective of this study was to analyze the flux of biomass through the communities of bacteria and phagotrophic protists in the cold and warm conditions occurring seasonally in Butr6n River. Bacterial and hetero- trophic protistan (flagellate and ciliate) abundance was determined by epifluo- rescence direct counts; protistan grazing on planktonic bacteria was measured from fluorescently labeled bacteria uptake rates; and the estimate of bacterial secondary production was obtained from (3H)thymidine incorporation rates. The abundance of bacterial, flagellate, and ciliate communities was similar during cold and warm situations. However, we observed that estimates of dynamic parameters, i.e., secondary bacterial production and protistan graz- ing, in both situations were noticeably different. In the warm situation, grazing rates of flagellates and ciliates (bacteria per protist per hour) were, respec- tively, 7 times and 18 times higher than those determined in the cold situation, and the grazing rates of the protistan communities (bacteria per protists present in 1 ml of water per hour) increased up to 5 times in the case of flagellates and 42 times in the case of ciliates. Estimates of bacterial secondary production were also higher during the warm situation, showing a ninefold increase. The percentage of bacterial production preyed upon by flagellates or ciliates was not significantly different between the two conditions. These results showed that in the different conditions of a system, the flux of biomass between the trophic levels may be quite different although this process may not be reflected in the abundance of each community of bacteria, flagellates, and ciliates.