Habituation to acid in Escherichia coli: conditions for habituation and its effects on plasmid transfer

Induction of acid resistance (habituation) in Escherichia coli at pH 5·0 took ca 5 min in broth at 37°C and 30–60 min in minimal medium. Induction occurred at a range of pH values from 4·0 to 6·0; it was dependent on continuing protein and RNA synthesis but substantial acid resistance appeared in the presence of nalidixic acid. Acid resistance was long-lasting; organisms grown at pH 5·0 retained most of their resistance after 2 h growth at pH 7·0. Organisms grown at pH 5·0 showed increased synthesis of a number of cytoplasmic proteins compared with the level in cells grown at pH 7·0. DNA repair-deficient strains carrying recA, uvrA or polAl mutations were more acid-sensitive than the repair-proficient parents but were able to habituate at pH 5·0. Organisms grown at pH 5·0 transferred the ColV plasmid much more effectively at acid pH than did those grown at pH 7·0 and habituated recipients appeared better able to repair incoming acid-damaged plasmid DNA than did those that were non-habituated. Induction of acid resistance at pH 5·0 may be significant for the survival of organisms exposed to periodic discharges of acid effluent in the aquatic environment and habituation may also allow plasmid transfer and repair of acid-damaged plasmid DNA during or after such exposure.     

Habituation to normally lethal acidity by prior growth of Escherichia coli at a sub-lethal acid pH value

Both Col^- and ColV, I-K94⁺ strains of Escherichia coli , grown at pH 7–0, failed to grow after relatively short periods of exposure to pH 3·0 or 3·5. After growth in exposure medium initially at pH 5·0, both strains were almost unaffected by exposure to such acid pH values. Addition of catalase to nutrient agar only slightly increased plating efficiency after acid treatment and very slightly reduced the difference in survival, after acid treatment, between organisms grown from pH 5·0 and those grown from pH 7·0. Accordingly, acid resistance of organisms grown from pH 5·0 is not chiefly due to greater resistance to hydrogen peroxide already present in nutrient media.     

Habituation to alkali in Escherichia coli

Escherichia coli grown at pH 9·0 was much more resistant to extremes of alkaline pH (10·5–11·5) than when grown at pH 7·0. This is termed habituation to alkali. It was not due to ability to reduce the pH of the medium during exposure but was due to a phenotypic change during growth at pH 9·0. Habituation occurred within 60 min at pH 9·0.     

Induction of acid tolerance at neutral pH in log-phase Escherichia coli by medium filtrates from organisms grown at acidic pH

Escherichia coli grown at pH 5·0 became acid-tolerant (acid-habituated) but, in addition, neutralized medium filtrates from cultures of E. coli grown to log-phase or stationary-phase at pH 5·0 (pH 5·0 filtrates) induced acid tolerance when added to log-phase E. coli growing at pH 7·0. In contrast, filtrates from pH 7·0-grown cultures were ineffective. The pH 5·0 filtrates were inactivated by heating in a boiling water-bath but there was less activity loss at 75 °C. Protease also inactivated such filtrates, which suggested that a heat-resistant protein (or proteins) in the filtrates was essential for the induction of acid tolerance. Filtrates from cells grown at pH 5·0 plus phosphate or adenosine 3':5'-cyclic monophosphate (cAMP) were much less effective in inducing acid tolerance, while the conversion of pH 7·0-grown log-phase cells to acid tolerance by pH 5·0 filtrates was inhibited by cAMP and bicarbonate. It seems likely that the acid tolerance response (acid habituation) involved the functioning of the extracellular protein(s) as protease reduces tolerance induction if added during acid habituation. Most inducible responses are believed to involve the functioning of only intracellular reactions and components ; the present results suggest that this is not the case for acid habituation, as an extracellular protein (or proteins) is needed for induction.     

The characteristics of a new non-spore-forming cellulolytic mesophilic anaerobe strain CM126 isolated from municipal sewage sludge

A new mesophilic anaerobic cellulolytic bacterium, CM126, was isolated from an anaerobic sewage sludge digester. The organism was non-spore-forming, rod-shaped, Gram-negative and motile with peritrichous flagella. It fermented microcrystalline Avicel cellulose, xylan, Solka floc cellulose, filter paper, L-arabinose, D-xylose, β-methyl xyloside, D-glucose, cellobiose and xylitol and produced indole. The % G + C content was 36. Acetic acid, ethanol, lactic acid, pyruvic acid, carbon dioxide and hydrogen were produced as metabolic products. This strain could grow at 20–44·5°C and at pH values 5·2–7·4 with optimal growth at 37–41·5°C and pH 7. Both endoglucanase and xylanase were detected in the supernatant fluid of a culture grown on medium containing Avicel cellulose and cellobiose. Exoglucanase could not be found in either supernatant fluid or the cell lysate. When cellulose and cellobiose fermentation were compared, the enzyme production rate in cellobiose fermentation was higher than in cellulose fermentation. The optimum pH for both enzyme activities was 5·0, the optimum temperature was 40°C for the endoglucanase and 50°C for the xylanase. Both enzyme activities were inhibited at 70°C. Co-culture of this organism with a Methanosarcina sp. (A145) had no effect on cellulose degradation and both endoglucanase and xylanase were stable in the co-culture.     

Factors affecting production and activity of phospholipase C by Yersinia enterocotttica

The production of phospholipase C by Yersinia enterocolitica strain SG was optimum at 37†C at pH 6·5. No enzyme activity could be detected when the organism was grown at extreme pH values (pH > 8·5 or <5·0). The enzyme production was maximum when the organism was grown under static conditions in TSB medium. All solvents and salts inhibited the enzyme activity, whereas loss of activity was 95% in presence of methanol (20%) and 99% in presence of sodium azide (0·2 mol/l). The enzyme activity was increased twofold in the presence of cyste-ine and decreased by 98% in the presence of sodium perchlorate (0·2 mol/1).     

Survival of Listeria monocytogenes in yogurt during storage at 4°C

Cows' milk was inoculated with ca 10³and 10⁷cfu/ml Listeria monocytogenes. After fermentation at 42°C for 0–5 h, the yogurt was stored at 4°C. Low and high inocula survived for 48 h and 7 d, respectively; L. monocytogenes cells were not detectable by direct plating or cold-enrichment after 5 and 15 d, respectively. In low inoculum samples, initial pH at the time of refrigeration was 4·9; the final pH at the time of last sampling was 4·2. In the samples with high inoculum the pH decreased from 5·0 to 4·2.     

Bactericidal activity of carvacrol towards the food-borne pathogen Bacillus cereus

Carvacrol, a natural plant constituent occurring in oregano and thyme, was investigated for its bactericidal effect towards the food-borne pathogen Bacillus cereus . Carvacrol showed a dose-related growth inhibition of B. cereus . At concentrations of 0·75 mmol l⁻¹ and above, total inhibition of the growth was observed. Below this concentration, carvacrol extended the lag-phase, reduced the specific growth rate and reduced the maximum population density. Incubation for 40 min in the presence of 0·75–3 mmol l⁻¹ carvacrol decreased the number of viable cells of B. cereus exponentially. Spores were found to be approximately 2·3-fold less sensitive to carvacrol than vegetative cells. Bacillus cereus cells showed reduced susceptibility towards carvacrol at pH 7·0 compared with different values between pH 4·5 and 8·5. The culture and exposure temperatures had a significant influence on the survival of vegetative cells. The highest death rate of cells was observed at an exposure temperature of 30 °C. Membrane fluidity was found to be an important factor influencing the bactericidal activity of carvacrol.     

The role of regulatory gene products in alkali sensitization by extracellular medium components in Escherichia coli

Organisms of Escherichia coli 1829 become alkali sensitized on transfer from pH 7·0 to pH 5·5 but they also secrete extracellular agents which induce alkali sensitivity when added (in neutralized filtrates) to organisms growing at pH 7·0. In contrast, filtrates from cultures grown at pH 7·0 have no effect. Filtrates were inactivated by protease but not by heat treatment in a boiling water-bath, suggesting that a very heat-stable protein is involved in alkali sensitivity induction. A heatstable low molecular weight component (or components) may also be needed for induction, or the induction protein itself may be of low molecular weight. Strains with lesions in hns, fur or himA produced almost inactive filtrates and it therefore appears that H-NS, Fur and IHF are involved in synthesis of the induction components. As the presence of protease during incubation at pH 5·5 totally abolished alkali sensitization of strain 1829 while inhibition of sensitization induction occurred if the induction components were removed by filtration or dialysis during pH 5·5 incubation, it is proposed that the extracellular induction components (EICs) are essential for the original sensitization response. These results suggest that sensitization induction occurs by a different mechanism to that which is believed to occur for most bacterial inducible response systems; these are claimed to involve exclusively intracellular reactions and components whereas the present response involves functioning of extracellular components.     

The effect of pH, salt concentration and temperature on the survival and growth of Listeria monocytogenes

Factorially designed experiments have been used to study the growth and survival of Listeria monocytogenes in different combinations of pH and salt concentrations at ambient and chill temperatures. Survival at low pH and high salt concentration was strongly temperature dependent. The minimum pH values that allowed survival after 4 weeks from an initial 10⁴ cells were 4·66 at 30†C, 4·36 at 10†C and 4·19 at 5†C. These limits were salt dependent, low (4–6%) salt concentrations improved and higher concentrations reduced survival at limiting pH values. The lowest pH that allowed a 100-fold increase in cell numbers within 60 d was 4·66 at 30†C but this was increased to 4·83 at 10†C. At 5†C growth occurred at pH 7·0 but not at pH 5·13. Simple predictive models describing the effect of hydrogen-ion and salt concentration on the time for at least a 100-fold increase in numbers at 10†C and 30†C were constructed after analysis of the results for a least squares fit to a quadratic model. The interactions between salt and hydrogen-ion concentration on growth were found to be purely additive.     

The effect of various acidulants on the growth of Listeria monocytogenes

The ability of four Listeria monocytogenes strains to initiate growth in brain heart infusion broth adjusted to various pH values with either acetic, lactic, citric or hydrochloric acid was investigated. Acetic acid was the most effective inhibitor tested, since in broth adjusted with this acid a higher minimum pH was required for growth of the various strains at both 4 and 30°C, as compared with broth adjusted with the other acidulants. The minimum pH value required for the initiation of growth of L. monocytogenes ranged from 5·0 to 5·7 at 4°C, and from 4·3 to 5·2 at 30°C, depending upon the acidulant used.     

Effect of pH and NaCl on growth from spores of non-proteolytic Clostridium botulinum at chill temperature

The effect of combinations of temperature (2°, 3°, 4°, 5°, 8° and 10°C), pH (5·0–7·2) and NaCl (0·1–5·0% w/w) on growth from spores of non-proteolytic Clostridium botulinum types B, E and F was determined using a strictly anaerobic medium. Inoculated media were observed weekly for turbidity, and tests were made for the presence of toxin in conditions that approached the limits of growth. Growth and toxin production were detected at 3°C in 5 weeks, at 4°C in 3/4 weeks and at 5°C in 2/3 weeks. The resulting data define growth/no growth boundaries with respect to low temperature, pH, NaCl and incubation time. This is important in assessment of the risk of growth and toxin production by non-proteolytic Cl. botulinum in minimally processed chilled foods.     

The Development of Aerobic Spoilage Flora on Meat Stored at Chill Temperatures

In meat juice medium, aerobic spoilage bacteria utilized the following substrates in the order shown: Pseudomonos , glucose, amino acids, lactic acid; Acinetobacter , amino acids, lactic acid: Enterobacter , glucose, glucose-6-phosphate, amino acids; Microbacterium thermosphactum , glucose, glutamate. All the bacteria grew at their maximum rate utilizing the first and second substrates, but the growth rates declined when these were exhausted. The growth rate of Acinetobacter was reduced at pH 5·7 and below. All other species grew at their maximum rate within the pH range 5·5–7·0. On meat pseudomonads grew faster than the other species at all temperatures between 2° and 15°C. Interactions between any two species were observed only when one organism had attained its maximum cell density. Substrate exhaustion at the meat surface did not limit bacterial growth and it is suggested that the maximum cell density of aerobic spoilage cultures is determined by oxygen limitation of growth.     

Survival and reproductive performance of the desert pupfish, Cyprinodon n. nevadensis (Eigenmann and Eigenmann), in acid waters

Desert pupfish, Cyprinodon n. nevadensis , were exposed to pH levels of 8·3 (control), 7·0, 6·5, 6·0, 5·5 and 5·0 to determine the effects of acidity on reproduction. Egg production was significantly reduced at every pH level tested below the control. Egg-laying virtually ceased at pH 5·0, while egg viability was reduced to less than 50% of the control value at pH 6·5, when eggs were tested at the same pH at which they were laid. The 96 h LC₅₀ for this species was pH 4·56, considerably below that for successful reproduction. Larvae were less tolerant to acid stress than were adults.
Acclimation of reproductive performance to increased acid levels did not occur. Reproductive performance did not fully recover to control levels when the fish were placed in more favourable conditions after prolonged exposure to low pH.
Reduction in the number and development of oocytes was observed in the ovaries of acid stressed fish resulting in the decreased reproductive potential.     

Anti-Helicobacter pylori activity of Lactobacillus delbrueckii subsp. bulgaricus strains: preliminary report

Aims:  To evaluate the activities of six Lactobacillus delbrueckii subsp. bulgaricus (LB) strains against 30 Helicobacter pylori strains by agar-well diffusion method.
Methods and Results:  LB cultures [4 × 10⁸–4 × 10⁹ CFU ml⁻¹) either were prepared in milk at their native pH, 3·8–5·0, or were adjusted to pH 6·4–7·7. At low and neutralized pH, LB strains inhibited the growth by 40–86·7% and 16·7–66·7% of H. pylori strains, respectively. LB activity was strain-dependent. At low and neutralized pH, one and five H. pylori strains, respectively, were not inhibited by any LB strain. LB2 and LB3, taken together, were active against most metronidazole and clarithromycin resistant strains.
Conclusions:  All LB strains inhibited a number of H. pylori strains, including also antibiotic resistant strains. LB activity was strain-dependent and better at low pH. At low pH values, the most active LB strains were LB1, LB2 and LB3, inhibiting 86·7% of H. pylori strains, while at neutralized pH values, the most active LB strains were LB2 and LB3, inhibiting 53·3 and 66·7% of H. pylori strains, respectively.
Significance and Impact of the Study:  LB could be utilized in the treatment or prophylaxis of H. pylori infection and warrants clinical investigations.     

Factors affecting the survival of Salmonella and Escherichia coli in anaerobically fermented pig waste

The survival of Salmonella typhimurium was investigated in acidogenic, anaerobically fermented pig wastes and in synthetic media, each containing volatile fatty acids (VFA). Salm. typhimurium survived at pH 6·8, but not at pH 4·0, when incubated at 37°C for 24 h in either fermented or synthetic medium containing VFA. The minimum inhibiting concentration of VFA for Salm. typhimurium after 48 h incubation at 30°C at pH 4·0 was 0·03 mol/l and for Escherichia coli it was 0·09 mol/l. Fermented pig wastes in a digester, maintained at pH 5·9, were inoculated with Salm. typhimurium and then incubated at 37°C for 24 h. The pH was adjusted to either 4·0 or 5·0 and after a further 48 h at 30°C, Salm. typhimurium survived at pH 5·0 but not at pH 4·0. It was concluded that pH is critical in determining the survival of this organism in acidogenic anaerobically fermented pig waste.     

The effects of environmental factors on rainbow smelt Osmerus mordax embryos and larvae

Experiments were conducted to identify environmental factors that influence the survival of rainbow smelt Osmerus mordax during their early life stages. Developing rainbow smelt embryos and yolk-sac larvae were cultured under controlled conditions with different dissolved oxygen (DO; 1·09, 2·18, 4·37 and 6·55 mg l⁻¹, pH (4·0, 4·5, 5·0, 5·5, 6·0 and 7·0), nitrate ( 0·7, 3·6, 7·3, 14·6 and 29·2 mg l⁻¹), phosphate (0·04, 0·21, 0·42, 2·08 and 4·17 mg l⁻¹) and salinity (0, 5, 10, 15, 20 and 30) levels. Rainbow smelt embryos were also incubated with simulated tidal salinity fluctuations (2–28), ultraviolet radiation (irradiances of 2·8, 6·2 and 5·1 W m⁻²) and under natural conditions in two rainbow smelt spawning rivers. In the laboratory, hatch was only impaired under the lowest DO and pH conditions (0 and 13% hatch, respectively) and at highest constant salinity levels (0% hatch). Larval survival was only affected by pH levels ≤5·0. The experiment that compared hatch under natural conditions was terminated when embryos became covered with silt and fungus. These results suggest that water acidification, sediment and fungal growth may affect rainbow smelt survival during their early life stages.     

Antimicrobial activity of food-related Penicillium sp. against pathogenic bacteria in laboratory media and a cheese model system

Food-related Penicillium species ( n ep fy1 = rs 34) and Geotrichum candidum ( n = 11) grown on Czapek Dox and brie agar were tested for their ability to suppress growth of pathogenic bacteria. Ten out of 13 P. camemberti showed antagonistic activity while the other species did not interact significantly with the bacterial growth. The order of inhibition was: Gram-negative bacteria and Bacillus cereus > Listeria monocytogenes , Lactococcus sp. > Micrococcus sp. whereas Lactobacillus sp., Staphylococcus aureus and some Micrococcus sp. were unaffected. When Salmonella typhimurium was inoculated together with P. camemberti P25 in brie agar, bacterial growth was inhibited during the first 6 d of incubation whereafter growth started. The inhibition of L. monocytogenes was similar but less pronounced. The antimicrobial activity produced by P. camemberti P25 and L84 was enhanced with increasing amount of sucrose in the medium. The activity was increased at low pH and destroyed at pH above 8. It was detectable at 15°C but not at 37°C indicating that volatile metabolites might be involved. No significant accumulation of organic acids and no secondary metabolites such as mycotoxins were detected. HSGC-MS analysis indicated that acetaldehyde, benzaldehyde, 3-methylbutanal and 1-octen-3-ol were produced by P. camemberti during the period when inhibitory activity was observed. Pure acetaldehyde and benzaldehyde were shown to be inhibitory to L. monocytogenes and Salm. typhimurium when grown at 15°C and pH 5·5 and 7·0.     

Effect of the fermentation pH on the storage stability of Lactobacillus rhamnosus preparations and suitability of in vitro analyses of cell physiological functions to predict it

Aims:  To investigate how cell physiological functions can predict the stability of freeze-dried probiotics. In addition, the effect of the fermentation pH on the stability of probiotics was investigated.
Methods and Results:  Fermenter-grown (pH 5·8 or 5·0) Lactobacillus rhamnosus cells were freeze-dried and their survival was evaluated during storage at 37°C, in apple juice and during acid [hydrochloric acid (HCl) and malic acid] and bile exposure. Cells grown at pH 5·0 were generally coping better with acid-stress than cells grown at pH 5·8. Cells were more sensitive to malic acid compared with HCl. Short-term stability results of Lact. rhamnosus cells in malic acid correlated well with the long-term stability results in apple juice, whereas the results of cell membrane integrity studies were in accordance with bile exposure results.
Conclusions:  Malic acid exposure can prove useful in evaluating the long-term stability of probiotic preparations in apple juice. Fermentation at reduced pH may ensure a better performance of Lact. rhamnosus cells during the subsequent acid-stress.
Significance and Impact of the Study:  The beneficial effect of lowered fermentation pH to Lact. rhamnosus stability during storage in apple juice and the usefulness of malic acid test in predicting the stability were shown.     

Differential effects of low pH and aluminium on the caudal neurosecretory system of the brook trout, Salvelinus fontinalis

Brook trout were subjected to soft water at pH 6·5, 5·5 or 5·0 without aluminium added, or to water at pH 5·5 with 200,300 or 500 μg Al I^-1 added. The response of the caudal neurosecretory system to low pH or aluminium was evaluated after one week by measuring the urotensin I and urotensin II concentrations in the urophysis by radioimmunoassay, and by morphometric analysis of the caudal neurosecretory cells. A positive correlation was found between urotensin I concentrations and acidity, and a negative correlation was found between urotensin II concentrations and total aluminium in the water. Morphometric indices (cell size and proportion of lobed nuclei in the caudal neurosecretory cells) suggested increased synthetic activity in the caudal neurosecretory cells of fish at pH 5·5 compared to pH 6·5.