Effects of fatty acids on lysis of Streptococcus faecalis.

Palmitic, stearic, oleic, and linoleic acids at concentrations of 200 nmol/ml all inhibited autolysin activity 80% or more in whole cells or cell-free extracts. This concentration of the saturated fatty acids palmitic acid and stearic acid had little or no effect on the growth of whole cells or protoplasts. However, the unsaturated fatty acids oleic acid and linoleic acid induced lysis in both situations. This lytic effect is apparently not related to any uncoupling activity or inhibition of energy catabolism by unsaturated fatty acids. It is concluded that unsaturated fatty acids induce cell and protoplast lysis by acting as more potent membrane destabilizers than saturated fatty acids.     

Effect of Long Chain Fatty Acids on Bacterial Respiration and Amino Acid Uptake

S ummary . Long chain fatty acids stimulated oxygen uptake by Gram positive bacteria at bactericidal and protoplast lytic concentrations and produced inhibition at higher levels. The order of activity between individual acids and effects of reversal agents on respiratory activity corresponded to those which produced bactericidal activity. Protoplasts were more susceptible to inhibition than whole cells. Gram negative bacteria were inhibited to a limited extent at high fatty acid concentrations, but spheroplasts were highly sensitive. Fatty acids inhibited amino acid uptake both aerobically and anaerobically at sub-bactericidal levels. The effects were reversed by metal cations, and reflected the activity of dinitrophenol and sodium azide. The susceptibility of organisms to inhibition was of the same order as the sensitivity to other antibacterial effects. The probable mode of action of the fatty acids is discussed in terms of the interference with energy metabolism within the bacterial cell.     

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.     

Influence of pH on antimicrobial activity of organic acids against rabbit enteropathogenic strain of<Emphasis Type="Italic">Escherichia coli</Emphasis>

Susceptibility of the rabbit enteropathogenic strain Escherichia coli C6 (O128 serogroup) to C6-C14 fatty acids, oleic, citric, lactic and fumaric acid at 5 mg/mL was determined by the plating technique in the near-neutral pH region (pH approximately 6.5), and in a weakly acid and acid environment (pH 5.4 +/- 0.1 and 2.2-2.5, respectively). In the near-neutral pH region caproic and caprylic acid reduced the concentration of viable cells by 3 and 6 orders, respectively. At lower pH the bactericidal effect of caproic acid remained similar, but caprylic acid decreased the concentration of viable cells to < 100/mL. The bactericidal activity of capric acid was low at pH 6.5 but increased at pH 5.3. High environmental acidity was intrinsically bactericidal and at very low pH the effects of fatty acids were thus less pronounced. Citric acid reduced the counts of viable cells to 1/10. Antimicrobial activity of other acids examined was marginal or absent. Medium-chain fatty acids, caprylic and, to a lesser extent, also caproic and capric acid were better antimicrobials than other organic acids examined; the antimicrobial activity of fatty acids toward the C6 strain was pH-dependent. Beneficial effects of citric, lactic and fumaric acid reported by animal nutritionists are thus probably related to factors other than their direct antimicrobial action.     

Antilisterial activity of enterocin 81, a bacteriocin produced by Enterococcus faecium WHE 81 isolated from cheese

Enterocin 81, a bacteriocin produced by Enterococcus faecium WHE 81 previously isolated from cheese, exhibited a very narrow spectrum of activity, which is mainly directed against enterococci and Listeria spp. including Listeria monocytogenes. Enterocin 81 activity, which was extremely rapid with maximal effect achieved within 30 min, could not be detected after treatment with various proteolytic enzymes. This activity was bactericidal in nature and induced an important efflux of intracellular material, which was visualized under electron microscopy as filaments coming out of L. monocytogenes cells. However, enterocin 81 did not display bacterial lysis on sensitive cells, as no changes in cell morphology were detected following the bactericidal action. Furthermore, this bacteriocin was shown to be equally active at pH values ranging from 4·0 to 8·0, which, along with the narrow activity spectrum, are two factors of paramount interest with regards to possible use of this bacteriocin in fermented foods.     

Studies on the Mode of Action of the Phenolic Antibacterial Agent Fentichlor against Staphylococcus aureus and Escherichia coli II. The Effects of Fentichlor on the Bacterial Membrane and the Cytoplasmic Constituents of the Cell

Fentichlor produces rapid and marked damage to cell membranes of Staphylococcus aureus and Escherichia coli causing leakage of 260 nm material and pentose from whole cells, and lysis of spheroplasts of E. coli. Investigations indicate that Fentichlor-induced membrane damage is related to bactericidal activity. Bacteriostatic concentrations of 10 μg/ml or less do not stimulate leakage while at higher concentrations there is a marked relationship between loss of viability and of metabolic pool material released. Drug concentrations producing 99·99% kill within 1 h correspond closely to minimum concentrations required to produce total loss of 260 nm material from the pool. Membrane damage caused by bactericidal concentrations of drug is irreversible and under conditions optimal for enzyme activity additional leakage of 260 nm material resulting from autolytic breakdown of insoluble nucleic acids also occurs. There is no evidence that Fentichlor produces precipitation of cell cytoplasmic constituents.     

THE ASSESSMENT OF RELATIVE BACTERICIDAL ACTIVITY AMONG CERTAIN PHENOLS BY A PHYSICAL METHOD

SUMMARY: The addition of phenol, various alkyl phenols, or CTAB to a suspension of E. coli in distilled water caused the optical density to increase, the change being more marked as the concentration of a given compound was raised. Families of curves of similar shape were obtained with all the compounds when optical density was plotted against time for different concentrations, and figures expressing the activity relative to that of 1·2% phenol were obtained by dividing the concentration which produced the same curve as that concentration of phenol, into 1·2. These figures were very similar to those obtained for the bactericidal effects of the compounds relative to 1·2% phenol, using an 'end-point' method.
The presence of the chlorides of mono- and divalent metals at 10⁻⁴ M, or of the sulphates of trivalent metals at 10⁻⁵ M, did not affect the turbidity reaction, but higher concentrations of monovalent metal salts, e.g. 2 × 10⁻² M phosphate buffer (potassium) at pH 6·7, markedly retarded the change due to phenol. That concentration of the buffer also greatly reduced the bactericidal activity of 1·2% phenol. Much of the turbidity increase, however, occurred after all the cells of the suspensions had been killed, with phenol concentrations over 1·0%.     

Evaluation of the antimicrobial activity of citral

Citral showed appreciable antimicrobial activity against Gram-positive and Gram-negative bacteria as well as fungi. Media composition and inoculum size had no observable effect on activity but alkaline pH increased citral activity. The growth rates of Escherichia coli cultures were reduced at concentrations of citral ≥0·01% v/v while concentrations ≥0·03% v/v produced rapid reduction in viable cells followed by limited regrowth. In a non-growth medium, 0·08% and 0·1% v/v showed rapid bactericidal effects. Citral may therefore be of preservative use in addition to its other uses in the food, soap and cosmetic industries.     

Effects of culture conditions on Pectinatus cerevisiiphilus and Pectinatus frisingensis metabolism: a physiological and statistical approach

The genus Pectinatus has been often reported in beer spoilage with off-flavours. The bacteria are strictly anaerobic, Gram-negative rods. Propionate and acetate are the main fermentation products from glucose in the two species belonging to the genus, P. cerevisiiphilus and P. frisingensis. Amino acids routinely present at a high level in beer were not growth substrates for both species, and a significant accumulation of succinate was observed with lactate as growth substrate. Both Pectinatus ssp. showed almost identical fermentation balances on glucose. Growth kinetics of both glucose-grown species were unchanged under a N₂, H₂ or 20% CO₂-containing atmosphere. Combinations of culture medium pH values from pH 3·9 to pH 7·2, of glucose levels between 5 and 55 mmol l^-1, and of lactate concentrations varied from 4 to 40 mmol l^-1 demonstrated that biomass and volatile fatty acids production were proportional to glucose concentration for both Pectinatus species. A significant increase of volatile fatty acid production was measured for both species at the lowest pH values with a lactate or a glucose concentration increase. The maximum biomass production was observed at pH 6·2 for P. cerevisiiphilus , and between pH 4·5 and pH 4·9 for P. frisingensis. Glucose and lactate or pH value were dependent with regard to propionate and acetate production in P. frisingensis. On the other hand, the variations of these three parameters were independent with regard to biomass production for both strains, and to volatile fatty acids production for P. cerevisiiphilus. Addition of ethanol to glucose-grown cultures completely inhibited growth at 1·3 mol l^-1 ethanol for P. cerevisiiphilus , and at 1·8 mol l^-1 for P. frisingensis.     

Effect of Metal Cations and pH on the Antibacterial Activity and Uptake of Long Chain Fatty Acids

S ummary . The bactericidal activity of long chain saturated fatty acids was antagonized by alkaline earth metals. The activity of linoleic acid was less effectively antagonized but was more sensitive to reversal by ferric and stannous ions. With increasing pH value the bactericidal activity of lauric acid decreased but that of the longer chain saturated acids increased. Both Gram positive and Gram negative bacteria reversibly adsorbed fatty acids. Uptake increased with decreasing pH value and increasing chain length. Although adsorbed to a lesser extent, the intrinsic activity of linoleic acid was greater than lauric acid. The uptake appeared to be non-specific and governed by the physicochemical properties of both the acids and the bacterial cell surfaces. Sensitivity to the fatty acids increased with decreasing pH value. Protoplasts of Bacillus megaterium adsorbed fatty acids to a greater extent than whole cells. Resistance of the Gram negative Pseudomonas phaseolicola was not due to non-adsorption of the fatty acids.     

The antimicrobial effects of hops (Humulus lupulus L.) on ruminal hyper ammonia-producing bacteria

Aim:  To assess the antimicrobial effects of hops ( Humulus lupulus L.) on hyper ammonia producing-bacteria (HAB), which catabolize amino acids and peptides in the bovine rumen.
Methods and Results:  When media were amended with dried hops or hops extract (30·7% lupulone), ammonia production by mixed rumen bacteria was inhibited. The growth and ammonia production of pure cultures ( Peptostreptococcus anaerobius, Clostridium aminophilum, or Clostridium sticklandii ) was inhibited by 30 ppm lupulone at pH 6·7, and bactericidal activity was observed at pH 5·6. When hops extract was added to energized cell suspensions, the intracellular pH rapidly decreased and intracellular potassium was lost.
Conclusions:  The three HAB species were sensitive to the antimicrobial components in hops, and the inhibition of ammonia production by mixed rumen bacteria indicates that similar effects could be expected in the rumen.
Significance and Impact of the Study:  As much as half of the amino acids consumed by ruminants can be lost due to microbial degradation in the rumen. This study supports the idea that biologically active plant metabolites can be used to mitigate this wasteful process.     

The plasma membrane of yeast protoplasts exposed to hypotonicity becomes porous but does not disintegrate in the presence of protons or polyvalent cations

Protoplasts of Saccharomyces cerevisiae swelled, lysed and disintegrated when exposed to hypotonic solutions at neutral pH. At pH 4.5 or lower the hypotonically treated protoplasts did not disintegrate and they retained their intracellular proteins, nucleic acids and nucleotides. However, they became leaky for K+ and Ca2+, indicating that pores had been created in the surface membrane, relaxing the osmotic stress. Upon readjustment of pH to neutral, the hypotonically treated protoplasts released the intracellular content and disintegrated. Also, at low pH, protoplasts did not swell in isotonic ammonium acetate and were refractory to the permeabilizing effect of nystatin and to lysis with low concentrations of detergents. Protoplasts were similarly protected against lysis and disintegration by hypotonic treatment or by detergents, even at neutral pH, if the incubation media contained polyvalent cations, especially Zn2+, La3+, spermine, and Ca2+ chelated with EDTA. The protoplasts exposed to hypotonic stress at low pH did not respire and could not regenerate into viable cells. Effects of H+ and polyvalent cations on intramembrane forces acting between molecules of membrane phospholipids are considered along with possible changes in interactions between membrane proteins.     

Effect of divalent cations on permeabilizer-induced lysozyme lysis of Pseudomonas aeruginosa

The presence of divalent (Mg²⁺) ions greatly reduced the lysis of Pseudomonas aeruginosa strain G48 in a system at pH 7·8 or 9·0 consisting of ethylenediamine tetraacetic acid (EDTA), lysozyme and tris. Similar reductions in lysis occurred when EDTA was replaced by nitrilotriacetic acid, sodium citrate or sodium polyphosphate. The effect depended on the cation concentration. Mg²⁺ may replace cations removed from the outer membrane, or may effectively remove the permeabilizer from the system. The results suggest that the permeabilizing activity associated with these agents against this organism has a common basis in affecting the outer membrane.     

Bactericidal effect of fatty acids on mycobacteria, with particular reference to the suggested mechanism of intracellular killing

The effect of fatty acids on Mycobacterium smegmatis was examined in vitro at pH 5.0 to 7.0 to determine the role of fatty acids in the intracellular killing of mycobacteria. Unsaturated fatty acids showed strong bactericidal activity in low concentrations (0.005 to 0.02 mM), whereas saturated fatty acids, except for lauric and myristic acids, were not very effective even at a concentration of 0.2 mM. Addition of a saturated fatty acid (palmitic or stearic acid) to an unsaturated fatty acid (oleic or linoleic acid) did not strongly interfere with the bactericidal effect of the unsaturated fatty acid at pH 5.0 and 6.0. Ca2+ (3.0 mM), Mg2+ (1.0 mM), and gamma-globulin (0.4%) showed weak reversal effects on the bactericidal activity of unsaturated fatty acids at pH 5.0 and 6.0. Serum albumin and serum showed strong reversal effects. The concentrations of each fatty acid in a mixture (molar ratio, 1:1:1:1) of oleic, linoleic, palmitic, and stearic acids required for the killing of M. smegmatis in the presence of 2% serum (bovine, rabbit, or human) were 0.05 to 0.10 mM at pH 5.0 and 6.0 and 0.05 to 0.20 mM at pH 7.0, depending on the serum used. The susceptibilities of M. kansasii, M. bovis strain BCG, and M. tuberculosis to the mixture of the four fatty acids in the presence of 2% bovine serum were similar to that of M. smegmatis, although M. fortuitum was more resistant.     

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.     

Methanogenesis and ATP synthesis in a protoplast system of Methanobacterium thermoautotrophicum.

When Methanobacterium thermoautotrophicum cells were incubated in 50 mM potassium phosphate buffer (pH 7.0) containing 1 M sucrose and autolysate from Methanobacterium wolfei, they were transformed into protoplasts. The protoplasts, which possessed no cell wall, lysed in buffer without sucrose. Unlike whole cells, the protoplasts did not show convoluted internal membrane structures. The protoplasts produced methane from H2-CO2 (approximately 1 mumol min-1 mg of protein-1) at about 50% the rate obtained for whole cells, and methanogenesis was coupled with ATP synthesis. Addition of the protonophore 3,5-di-tert-butyl-4-hydroxybenzylidenemalononitrile (SF-6847) to protoplast suspensions resulted in a dissipation of the membrane potential (delta psi), and this was accompanied by a parallel decrease in the rates of ATP synthesis and methanogenesis. In this respect protoplasts differed from whole cells in which ATP synthesis and methanogenesis were virtually unaffected by the addition of the protonophore. It is concluded that the insensitivity of whole cells to protonophores could be due to internal membrane structures. Membrane preparations produced from lysis of protoplasts or by sonication of whole cells gave comparatively low rates of methanogenesis (methylcoenzyme M methylreductase activity, less than or equal to 100 nmol of CH4 min-1 mg of protein-1), and no coupling with ATP synthesis could be demonstrated.     

Natural milk fatty acids affect survival and invasiveness of Listeria monocytogenes

The effects of 12 fatty acids, naturally occurring in milk from several mammalian species, on the survival and invasion ability of Listeria monocytogenes , a food-borne pathogen, were determined. The survival was tested in the presence of 200 μg ml⁻¹ fatty acids suspended in brain hearth infusion broth or in storage conditioning solution (NaCl 1%) of Mozzarella cheese, an Italian soft unripened cheese, at pH 7·0 or 5·0. Lauric (C12:0), linoleic (C18:2) and linolenic (C18:3) acids exerted the strongest bactericidal activity. The invasive efficiency of L. monocytogenes , determined in the Caco-2 enterocyte-like cell line, was strongly decreased in the presence of the fatty acids tested (from about 20 to 500-fold). This research suggests that naturally occurring fatty acids of milk, supplemented in milk derivatives, could affect both bacterial growth and invasiveness and consequently, could serve as barriers towards L. monocytogenes infection.     

Involvement of autolysin in cellular lysis of Bacillus subtilis induced by short- and medium-chain fatty acids.

The addition of saturated C6, C8, C10, and C12 fatty acids appeared to lyse actively growing cells of Bacillus subtilis 168, as judged by a decrease in the optical density of the culture. Of these fatty acids, dodecanoic acid was the most effective, with 50% lysis occurring in about 30 min at a concentration of 0.5 mM. These conditions also decreased the amount of peptidoglycan estimated by the incorporated radioactivity of N-acetyl-D-[1-14C]glucosamine. At concentrations above 1 mM, however, bacterial lysis was not extensive. Dodecanoic acid did not affect autolysis of the cell wall. The lytic action of dodecanoic acid was greatly diminished in cells in which protein synthesis was inhibited and in an autolytic enzyme-deficient mutant. The results suggest that fatty acid-induced lysis of B. subtilis 168 is due to the induction of autolysis by an autolytic enzyme rather than massive solubilization of the cell membrane by the detergent-like action of the fatty acids.     

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.     

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.