The effects of temperature, water activity and pH on the growth of Aeromonas hydrophila and on its subsequent survival in microcosm water.

AIMS: The influence of temperature, water activity and pH on the growth of Aeromonas hydrophila, and on its survival after transfer in nutrient-poor water were assessed. METHODS AND RESULTS: Experiments were carried out according to a Box-Behnken matrix at 10-30 degrees C, 0.95-0.99 water activity (aw) and pH 5-9. The effect of each factor on the kinetic parameters of growth (i.e. the maximal specific growth rate, mumax, and the lag time, lambda) and on the decline of the bacteria in microcosm water (time to obtain a reduction of 5 log, T5 log) were studied by applying central composite design. CONCLUSIONS: The major effect of temperature and water activity on the growth of A. hydrophila was highlighted, whereas the effect of pH in these experimental conditions was not significant. Models describing the effect of environmental parameters on the growth of A. hydrophila were proposed. The effect of the growth environment, and particularly the incubation temperature, have an influence on the survival ability of the bacteria in nutrient-poor water. SIGNIFICANCE AND IMPACT OF THE STUDY: The Box-Behnken design was well suited to determine the influence of environmental factors on the growth of A. hydrophila and to investigate the effect of previous growth conditions on its survival in microcosm water.

Starvation survival and viable but nonculturable states in Aeromonas hydrophila.

The behavior of Aeromonas hydrophila stored at 4 degrees C and 25 degrees C in nutrient-poor filtered sterilized distilled water was investigated. At 4 degrees C, the A. hydrophila population declined below the detection level (0.1 cell mL(-1)) after 7 weeks, whereas the number of cells with intact membrane as determined by the LIVE/DEAD method decreased only by 1 log unit. Although, this response is reminiscent of the so-called VBNC state, the cells could not be resuscitated by an upshift to 25 degrees C. A mixture of rods with normal size and elongated cells was observed in this state. At 25 degrees C, viable cells and cells with intact membrane declined only by 0.8 log unit over the 10-week storage period, and thus A. hydrophila entered the classical starvation survival state. During this state, a mixture of rods and cocci was observed. Prestarvation at 25 degrees C for 24 h and especially 49 days delayed significantly the rate of entry into the VBNC state. However, stationary phase cells were not significantly more tolerant than exponential phase cells. No significant improvements in recovery yield were obtained on LB agar plates amended with catalase or sodium pyruvate. During cold incubation, high variability in responses was observed. Intermittent cryptic regrowth might be responsible for this variability in responses.

Growth and survival of clinical vs. environmental species of Aeromonas in tap water.

The ability of four species of Aeromonas (two of clinical and two of environmental origin) to survive and/or grow in tap water microcosms supplemented with sodium thiosulphate was tested. After bottling, the autochthonous microflora reached 6 x 10(5) cfu ml(-1) after a 5-day incubation period in tap water unfiltered and which was non-autoclaved. In filtered tap water, "ultramicrocells" were detected and final populations of ca. 10(6) cfu ml(-1) after 7 days were obtained. Aeromonas was inoculated at an initial cell concentration of ca. 10(4) cfu ml(-1). All strains were able to grow in tap water samples, which were filtered and autoclaved, and a final concentration of 10(5)-10(6) cfu ml(-1) was observed. Any inherent capability of Aeromonas to grow in tap water was eliminated by the presence of autochthonous microflora and "ultramicrocells" bacteria. Survival rates were strain- and microcosm-dependent. In unfiltered-non-autoclaved water, viable counts declined to below the detection limit (i.e. 1 log cfu ml(-1)) in 1.5 to 20 days. The declines in viable counts were even more pronounced in the filtered microcosm. Although inoculation ratios (100/1 in unfiltered-non-autoclaved and 1,000/1 in filtered microcosms) were favourable for aeromonads, at least for I to 3 days, the organisms disappeared in these microcosms. Thus, competition for nutrients was an unlikely cause of the limitation of aeromonads. The bacteriolytic effect of enzymes released by membrane vesicles from the autochthonous microflora and of "tail phage-like particles" bacteriocins were suggested as an in situ control of aeromonad populations. The present study showed that environmental strains of Aeromonas had no ecological advantage over clinical isolates. Thus, waterborne infections and contaminations of foods by pathogenic Aeromonas species could not be discounted.

Changes in fatty acid composition and degree of unsaturation of (brady)rhizobia as a response to phases of growth, reduced water activities and mild desiccation.

The effects of growth phase, reductions in the water activity (a(w)) of the growth medium and mild desiccation on the composition and the degree of unsaturation of cellular fatty acids (CFA) of Sinorhizobium meliloti, Bradyrhizobium elkanii and Bradyrhizobium japonicum were studied. During the course of growth, an interchange of cis-vaccenic with lactobacillic acid and a slight increase in palmitic acid were observed while other fatty acids remained constant. The degree of unsaturation was significantly higher in the exponential phase of growth. Reductions in the a(w) of the medium led to an increase in lag phase, a reduction in growth rate and maximal optical densities (OD) in stationary phase cells. A decrease in the degree of unsaturation of CFA was also observed as the a(w) was reduced from 0.999 to 0.969 and after desiccation to 83.5% relative humidity (R.H.). The changes in the degree of unsaturation of CFA observed after growth at reduced a(w) may be one of the pre-adaptation steps to endure more severe desiccation.

Impact of thermal variations on biochemical and physiological traits in Pectinatus sp.

The influence of temperature on cellular fatty acid composition and on heat stress tolerance was studied in the two species of Pectinatus, an anaerobic gram-negative bacterium. Cellular fatty acid (FA) patterns were determined for Pectinatus species cultivated in MRS medium at various defined conditions of temperature and pH. Our study shows that fluctuations of growth temperature and pH induced important changes in the ratio of unsaturated FAs (UFAs) to saturated FAs (SFAs). The major differences in the FA composition as a function of growth temperature concerned C15:0 and C17:0 for the SFAs and C15:1 and C17:1 for the UFAs. The most significant adaptation of lipid composition to lower growth temperatures was the strong increase of UFAs, particularly for C15:1 and C17:1 concomitantly with a decrease of SFAs (C15:0 and C17:0). When the pH of the culture medium was lowered from 6.2 to 4.0, a notable drop in the synthesis of the UFAs C15:1 and C17:1 was observed together with an important increase of C18-cyclopropane (C18-cyc) and high carbon number SFAs. Thermal modifications also provoked changes in Pectinatus behaviour. We observed that P. cerevisiiphilus was more heat sensitive than P. frisingensis. Mild exponential phase cells were treated for 1 h, at 40 degrees C for P. cerevisiiphilus or at 41 degrees C for P. frisingensis. This thermal adaptation induced tolerance against heat challenge (49 and 50 degrees C for P. cerevisiiphilus and P. frisingensis, respectively). Survival of P. cerevisiiphilus and P. frisingensis adapted cells was, respectively, 3400- and 790-fold higher than control. Interestingly, adapted cells of P. cerevisiiphilus were more thermotolerant than P. frisingensis pretreated cells.

Total counts, culturable and viable, and non-culturable microflora of a French mineral water: a case study.

The changes in bacterial counts during the storage of a natural mineral water from a French spring were studied. Samples were taken from the spring and the bottling line. Viable cultivable (VC) bacteria were counted on R2A medium. Total counts, viable and dead bacteria were counted using the LIVE/DEAD Bac Light VIABILITY kit and epifluorescence microscopy. Viable but non-cultivable (VNC) bacteria were estimated by difference between viable and VC counts. Isolates were clustered by phenotype. The microflora in the spring water increased from < 10-3 x 10(5) bacteria ml-1 after 6 d in storage and then stabilized. Mechanical bottling increased the allochthonous bacteria in the water that stabilized at 10(5) bacteria ml-1. Maximal growth is controlled by the low concentration of nutrients in the mineral water and the lysis of dead cells. The allochthonous bacteria came from the aquifer and colonized the filling line. The changes in the VC and VNC populations showed that the bacteria used starvation-survival and entry into the VNC state to adapt to the bottling stress and the enclosed oligotrophic environment.

Effects of a recombinant gene product and growth conditions on plasmid stability in pectinolytic Escherichia coli cells.

The genes encoding pectin methylesterase (pme) and pectate lyase (pel) from Bacteroides thetaiotaomicron were previously cloned in Escherichia coli. In the absence of selective pressure the recombinant vectors harbouring a functional pel gene were rapidly lost. This instability was due to a toxic effect of the pel gene product when overproduced and was closely related (1) to a decrease of the growth rate, and (2) to the impossibility of transforming different strains of E. coli with the recombinant plasmids harbouring a functional pel gene. When the expression level of the pel gene was reduced and the tet gene partially deleted, the stability was greatly improved. The export of pectate lyase in the extracellular medium was significantly enhanced in the presence of glycine with a positive effect on plasmid stability for low concentrations. Furthermore, using a factorial design at two levels, the effects of tetracycline, ampicillin, glucose and magnesium on pBT4 stability were quantified.

Enumeration and characterization of bacteria in mineral water by improved direct viable count method.

Fifteen strains from two emergent mineral waters were isolated and tentatively identified with API 20NE and BIOLOG GN systems. These strains were screened for their sensitivities to seven replication-inhibiting antibiotics of the (fluoro)quinolone group (nalidixic and pipemidic acid, flumequine, norfloxacin, ofloxacin, pefloxacin and ciprofloxacin). It was shown that the direct viable count (DVC) procedure could be improved by using certain antibiotic cocktails, which were active against the isolates. Geometric bacterial features were successfully determined with image analysis and adapted software (ICONIX, Perfect Image). Elongations were significant and allowed rapid discrimination of antibiotic inhibited and non-inhibited strains. Particular isolates in a mixed culture were characterized and enumerated after only 14 h exposure with the appropriate antibiotic cocktail. This method can also be applied to other communities, such as mixed cultures in bio-fermentors or in food with known microflora.

Modeling growth for predicting the contamination level of guava nectar by Candida pelliculosa under different conditions of pH and storage temperature.

The combined effects of temperature (2-46 degrees C) and pH (1.55-6.25) on the growth of Candida pelliculosa isolated from guava nectar produced in Cameroon were studied using a turbidity method, ie measurement of optical density at 630 nm. A quadratic polynomial model was constructed to predict the effects and interactions of these two environmental conditions on the maximal optical density obtained (i2 = 0.97). The relation between optical density and population density of C. pelliculosa (CFU ml-1) was also established using an exponential regression (2 = 0.99). According to the model, maximal growth conditions were 37 degrees C and pH 6.25 for obtaining the maximal optical density of 1.25 corresponding to about 60 x 10(6) CFU ml-1. A good agreement of the model was found between the predicted values and the observed values of maximal optical density. The model was validated by the experimental values of maximal optical density obtained in the growth of C. pelliculosa in commercial guava nectar (pH 3.15).

Application of factorial and Doehlert designs for optimization of pectate lyase production by a recombinant Escherichia coli.

The expression of a recombinant pectate lyase from Bacteroides thetaiotaomicron strain 217 was studied in Escherichia coli strain HB101(pBT4). First, two sets of complete 2(4) factorial designs were used to evaluate the influences of casamino acids, glucose, magnesium, calcium, tetracycline, ampicillin, tryptophan and MOPS buffer on pectate lyase production in a basal medium. While casamino acids, glucose and magnesium were found to be the prevalent factors, the presence of tetracycline, ampicillin and MOPS buffer were necessary for the reproducibility of the process, probably by increasing the plasmid stability. Secondly, application of the Doehlert design, a response-surface methodology, allowed a good prediction of pectate lyase production according to the variation in glucose and magnesium concentrations. This optimization strategy allowed the production of biomass and recombinant pectate lyase respectively to be increased from 0.2 gl-1 to 1.9 gl-1 (dry weight) and from 10 units ml-1 to 210 units ml-1 within 24 h at 30 degrees C in shake flasks.

Response Surface Models To Describe the Effects of Temperature, pH, and Ethanol Concentration on Growth Kinetics and Fermentation End Products of a Pectinatus sp.

Growth curve data which had been fitted by use of the Gompertz and logistic functions have permitted the development of mathematical models to describe the growth of a Pectinatus sp. by several variables, namely, temperature, pH, and ethanol concentration. The activation energy of this microorganism was lower at 26 to 35(deg)C than at 15 to 22(deg)C. On the basis of the Arrhenius law, growth rate, maximum population density, and cell yield models have been developed by introducing the different activation energy (E(infa)) values. According to the model, optimal conditions were 35(deg)C, pH 6.5, and 0% (vol/vol) ethanol for the growth rate. For cell density and cell yield, optimal conditions were 32(deg)C, pH 6.0, and 1% (vol/vol) ethanol. No growth was observed for ethanol concentrations above 8% and pH values below 4.0. Other equations have also been made to describe the major end products fermented during fermentation by a Pectinatus sp. The synthesis of propionate and acetate is maximal at 28(deg)C at pHs of 5.5 and 6.25, respectively. This model completes the model suggested by Membre and Tholozan (J. Appl. Bacteriol. 77:456-460, 1994), which includes only one variable, i.e., the glucose concentration.

Variability in survival of Pectinatus cerevisiiphilus, strictly anaerobic bacteria, under different oxygen conditions.

Survival of Pectinatus cerevisiiphilus DSM 20466 in pure culture at variable temperatures under different oxygen concentrations was measured. Survival of P. cerevisiiphilus in co-culture with Saccharomyces cerevisiae under both saturated oxygen and brewing conditions was also studied. The survival of strictly anaerobic bacteria to oxygen seems to follow the classical laws of heat resistance. The D(oxy) values of P. cerevisiiphilus , calculated as a function of oxygen level, shows that the oxygen level is important for the survival duration of the bacteria. The temperature greatly influences the oxygen resistance of P. cerevisiiphilus, which increases when the temperature decreases. P. cerevisiiphilus resists better in co-culture than in pure culture under saturated oxygen conditions. Therefore, the oxygenation of the wort does not totally eliminate the risk of beer contamination by this bacterium. Under brewing conditions in co-culture at 8 degrees C, P. cerevisiiphilus grows slowly to reach a final cell concentration up to 10(6) cells/mL in beer, which is undrinkable. Pectinatus is a strictly anaerobic bacterium; however, it is resistant under certain oxygen conditions of incubation. This resistance is considerably higher in the presence of Saccharomyces cerevisiae .

Evidence of an active glucose uptake in Rhizobium meliloti.

Rhizobium meliloti M5N1 showed immediate uptake of glucose. Glucose accumulation was a saturable function of the glucose concentration, and km and Vmax values for uptake were determined. The glucose uptake system was found to be proteic with a turnover of about 6 h. This system was observed to be an active ATP-dependent process, since it was severely inhibited by uncouplers. Glucose analogues and others hexoses had some inhibitory effects on glucose uptake, but not polyols and lactose.

[Comparative evaluation of the use of glucose and fructose by the oxidative pathway in a strain of Rhizobium meliloti]. / Bilan comparé de l'utilisation par voie oxydative du glucose et du fructose par une souche de Rhizobium meliloti.

The oxidation rate in resting cells of Rhizobium meliloti was investigated with either glucose or fructose as substrate. Fructose uptake was found to be complete, whereas glucose uptake did not fully occur, yielding 2-kitogluconic acid in the medium. At the end of fructose oxidation, significant levels of this substrate and its derivatives had accumulated in the bacteria.

[Demonstration of a metabolic property of Rhibozium meliloti usable for its classification]. / Mise en évidence d'une properiété métabolique de Rhizobium meliloti utilisable pour sa classification.

Fast-growing Rhizobium and Agrobacterium strans were studied for their behaviour towards two carbohydrates (glucose and fructose) according to the following criteria: acidification and soluble exopolysaccharide production by resting cells. R. meliloti and Agrobacterium spp. showed a similar behaviour: in glucose-containing medium, acidification occurred and few exopolysaccharides were produced, whereas in medium supplemented with fructose, a neutral pH was maintained and large amounts of exopolysaccharides were synthesized. Other fast-growing Rhizobium spp. did not acidify these media and produced variable amounts of exopolysacchrides.

[Effect of 2-ketogluconic acid synthesis on the exopolysaccharide production in a Rhizobium meliloti strain]. / Effet de la synthèse d'acide 2 céto-gluconique sur la production d'exopolysaccharides par une souche de Rhizobium meliloti.

Two categories of carbon substrates are defined for Rhizobium meliloti: the first favours the synthesis of exopolysaccharides (fructose belongs to this category) while the other is not suitable (glucose belongs to this category). With fructose, resting cells synthesize polysaccharides during more than 100 h and this synthesis is at its best in aerobic conditions at 30 degrees C. With glucose, 2-ketogluconic acid accumulates and rapidly stops the synthesis. The method used to stop this acidification allows with glucose a synthesis which can be compared to the one obtained with fructose.

[Demonstration of different metabolic pathways starting with glucose or fructose in Rhizobium meliloti]. / Mise en évidence de voies de métabolisme différentes à partir du glucose ou du fructose chez Rhizobium meliloti

Rhizobium meliloti can produce many polysaccharides on D-fructose and D-mannitol. In glucose-grown cultures, few polysaccharides are observed and 2 keto-gluconate is accumulated.

[A study of polysaccharides in fast growing "Rhizobium" (author's transl)]. / Etude des polyosides de Rhizobium à croissance rapide

This work is an attempt to use the composition of exopolysaccharides in the Rhizobium genus as a criterium of taxonomic importance. The organisms were first cultured in media and under conditions which led to the production of as many polysaccharides as possible. The medium chosen for proliferation conditions was Wright's medium; for conditions of non-proliferation conditions was Wright's medium; for conditions of non-proliferation, a minimal medium without nitrogen was used. The polysaccharides synthesised in Wright's medium consists in neutral sugars such as glucose, galactose, mannose and sometimes rhamnose and in uronic acid in various quantities. If no mannose is found in the polysaccharides when they are synthesised in non-proliferation conditions and if mannans are found in yeast broth, it may be thought, as suggested by Humphrey, that mannose would come from those mannans. The study of the composition of these polysaccharides enables us to sort out two groups of fast growing Rhizobium. The first one, relatively uniform, corresponds to the strains of R. leguminosarum, trifolii and phaseoli. In these strains, the polysaccharides are made of about 70% of neutral sugars and 20% of glucuronic acid.