Factors affecting the adsorption of buchnericin LB, a bacteriocin produced by Lactocobacillus buchneri

Buchnericin-LB adsorbs to gram-positive but not to gram-negative bacteria. The tested gram-positive bacteria were species of Lactobacillus, Pediococcus, Leuconostoc, Enterococcus, Lactococcus, Listeria, Bacillus, Staphylococcus; gram-negative bacteria belonged to the genera Salmonella, Escherichia, Yersinia and Pseudomonas. Buchnericin-LB adsorption depended on pH but not on time and temperature. Also some anions of salts and lipoteichoic acid reduced or inhibited its adsorption. Treatment of cells and cell walls of sensitive bacteria with detergents, organic solvents or enzymes did not affect subsequent binding of buchnericin-LB. Treatment with buchnericin-LB caused sensitive cells to lose high amounts of intracellular K⁺ ions and UV-absorbing materials and became more permeable to o-nitrophenol-β-D-galactopyranoside. Buchnericin-LB (640-2560 AU/ml) decreased the colony forming units (99%) and absorbance values of Listeria monocytogenes and Bacillus cereus. These results indicate that the mode of action of buchnericin-LB is bactericidal and its lethal effect is very rapid.     

Production of bacteriocin inhibitory to Listeria species by Enterococcus hirae.

A bovine intestinal bacterial isolate, identified as Enterococcus hirae, was found to produce a bacteriocin (designated hiraecin S) inhibitory to Listeria monocytogenes and other Listeria spp. Identification to species level was determined by comprehensive biochemical and morphological tests which were verified by DNA-DNA homology assays. The antimicrobial agent was inactivated by pronase and papain and was insensitive to catalase. The antimicrobial activity was not due to hydrogen peroxide or acid formation, nor was lysozyme or muramidase activity observed in cell-free bacteriocin preparations. Inhibition of selected gram-negative bacteria was not observed. Other enterococci were sensitive to the bacteriocin, and except for Listeria spp., no other gram-positive bacteria tested were inhibited.     

Production of bacteriocin inhibitory to Listeria species by Enterococcus hirae.

A bovine intestinal bacterial isolate, identified as Enterococcus hirae, was found to produce a bacteriocin (designated hiraecin S) inhibitory to Listeria monocytogenes and other Listeria spp. Identification to species level was determined by comprehensive biochemical and morphological tests which were verified by DNA-DNA homology assays. The antimicrobial agent was inactivated by pronase and papain and was insensitive to catalase. The antimicrobial activity was not due to hydrogen peroxide or acid formation, nor was lysozyme or muramidase activity observed in cell-free bacteriocin preparations. Inhibition of selected gram-negative bacteria was not observed. Other enterococci were sensitive to the bacteriocin, and except for Listeria spp., no other gram-positive bacteria tested were inhibited.     

Study of ectoparasitism of ultramicrobacteria of the genus Kaistia, strains NF1 and NF3 by electron and fluorescence microscopy

Transmission electron and fluorescence microscopy was used to study the character of the interaction of free-living ultramicrobacterial (UMB) strains NF1 and NF3, affiliated with the genus Kaistia, and seven species of gram-positive and gram-negative heterotrophic bacteria. Strains NF1 and NF3 were found to exhibit parasitic activity against gram-positive Bacillus subtilis and gram-negative Acidovorax delafildii. UMB cells are tightly attached to the envelopes of the victim cells and induce their lysis, thus demonstrating the features of typical ectoparasitism. The selectivity of parasitism of the studied UMB to the victim bacteria has been shown: only two soil microorganisms of the seven test objects, B. subtilis ATCC 6633 and an aerobic gram-negative bacterium A. delafildii 39, were found to be sensitive to UMB attack. Other bacteria (Micrococcus luteus VKM Ac-2230, Staphylococcus aureus 209-P, Pseudomonas putida BS394, Escherichia coli C 600, and Pantoea agglomerans ATCC 27155) were not attacked by UMB. It was established for the first time that free-living UMB may be facultative parasites not only of phototrophic bacteria, as we have previously demonstrated, but of heterotrophic bacteria as well. The UMB under study seem to play an important role in the regulation of the quantity of microorganisms and in the functioning of microbial communities in some natural ecotopes.     

Growth and Bacteriolytic Activity of a Soil Amoeba, Hartmannella glebae

A soil amoeba, Hartmannella glebae, could grow on a variety of gram-positive and gram-negative bacteria, although the rate of growth was faster in the presence of gram-negative bacteria. The amoeba, however, could not use yeasts, molds, or a green alga as a nutritional source. The extract prepared from amoebae grown in the presence of Aerobacter aerogenes and Alcaligenes faecalis could lyse intact cells and cell walls of many gram-positive bacteria at different rates. The spectrum of lytic activity was similar to that of egg-white lysozyme, with the exception that several species and strains of Bacillus, Micrococcus, and Staphylococcus were resistant to lysozyme and susceptible to the extract. The gram-negative bacteria tested were resistant.     

Mode of action of piscicocin CS526 produced by Carnobacterium piscicola CS526

AIMS: Piscicocin CS526 is a unique class IIa bacteriocin produced by Carnobacterium piscicola CS526. The mode of action against the sensitive strain Listeria monocytogenes IID581 was evaluated. METHODS AND RESULTS: Piscicocin CS526 was adsorbed on both sensitive and insensitive gram-positive and gram-negative bacterial cells. Treatment of L. monocytogenes cells with trypsin, lipase and Triton X-100 did not reduce subsequent adsorption of piscicocin CS526. The activity of piscicocin CS526 against L. monocytogenes cells was bactericidal rather than bacteriostatic, but did not cause bacteriolysis. Piscicocin CS526 induced the efflux of K+ ions from the target cells which cause dissipation of the transmembrane potential (DeltaPsi) of the cell membrane. Moreover, after exposure to piscicocin CS526, intracellular adenosine 5'-triphosphate (ATP) level of the target cells rapidly reduced without leakage of ATP from the cells, indicating that ATP depletion occurred in the cells. CONCLUSIONS: Pore formation by piscicocin CS526 caused a rapid efflux of small molecules such as K+ from the indicator cells and dissipation of proton motive force (PMF), which lead to the cell death. SIGNIFICANCE AND IMPACT OF THE STUDY: Molecular mechanism of action of piscicocin CS526 is very similar to that of other pediocin-like bacteriocins, although piscicocin CS526 possesses a unique N-terminal sequence in which Val is substituted for by Leu in the amino acid at position 7.     

Efficacy of ozonated water against various food-related microorganisms.

The antimicrobial effects of ozonated water in a recirculating concurrent reactor were evaluated against four gram-positive and four gram-negative bacteria, two yeasts, and spores of Aspergillus niger. More than 5 log units each of Salmonella typhimurium and Escherichia coli cells were killed instantaneously in ozonated water with or without addition of 20 ppm of soluble starch (SS). In ozonated water, death rates among the gram-negative bacteria--S. typhimurium, E. coli, Pseudomonas aeruginosa, and Yersinia enterocolitica--were not significantly different (P > 0.05). Among gram-positive bacteria, Listeria monocytogenes was significantly P < 0.05) more sensitive than either Staphylococcus aureus or Enterococcus faecalis. In the presence of organic material, death rates of S. aureus compared with L. monocytogenes and E. coli compared with S. typhimurium in ozonated water were not significantly (P > 0.05) affected by SS addition but were significantly reduced (P < 0.05) by addition of 20 ppm of bovine serum albumin (BSA). More than 4.5 log units each of Candida albicans and Zygosaccharomyces bailii cells were killed instantaneously in ozonated water, whereas less than 1 log unit of Aspergillus niger spores was killed after a 5-min exposure. The average ozone output levels in the deionized water (0.188 mg/ml) or water with SS (0.198 mg/ml) did not differ significantly (P < 0.05) but were significantly lower in water containing BSA (0.149 mg/ml).     

Heterogenetic antigens of gram-positive bacteria

Chorpenning, Frank W. (The Ohio State University, Columbus), and Matthew C. Dodd. Heterogenetic antigens of gram-positive bacteria. J. Bacteriol. 91:1440-1445. 1966.-Soluble antigens obtained by various methods from gram-positive bacteria were used to modify erythrocytes whose hemagglutinating reactions with immune rabbit sera and normal human sera were then studied. Antigens from all gram-positive organisms studied except corynbacteria altered red cells, causing them to react with specific bacterial antisera and with normal human sera; however, cross-absorption and inhibition tests indicated that at least three different specificites were involved. One of these antigens seemed to be similar to Rantz's streptococcal NSS, which is shared with Staphylococcus aureus and Bacillus spp., and is therefore heterogenetic. Another was found in streptococci but was apparently not present in S. aureus and Bacillus spp. A third antigen, also heterogenetic, appeared to be shared by several species of Bacillus and by S. aureus, but not by streptococci or any gram-negative bacteria. The third antigen was heat-stable at pH 8.0, and appeared to be essentially polysaccharide in nature. Normal human sera varied in their content of antibodies which reacted with erythrocytes modified by extracts from gram-positive bacteria. Whereas some sera reacted very broadly with red cells modified by extracts of practically any gram-positive organism, other sera agglutinated only cells which had been modified by streptococcal antigen.     

Lipopolysaccharide Layer Protection of Gram-Negative Bacteria Against Inhibition by Long-Chain Fatty Acids

Growth, amino acid transport, and oxygen consumption of Escherichia coli and Salmonella typhimurium are inhibited by short-chain (C(2)-C(6)) but not by medium or long-chain fatty acids (C(10)-C(18)) at concentrations at which these processes are completely inhibited in Bacillus subtilis. The resistance of gram-negative organisms is not correlated with their ability to metabolize fatty acids, since an E. coli mutant unable to transport oleic acid is still resistant. However, mutants of both E. coli and S. typhimurium in which the lipopolysaccharide layer does not contain the residues beyond the 2-keto-3-deoxyoctonate core are inhibited by medium (C(10)) but not by long-chain (C(18)) fatty acids. Furthermore, removal of a portion of the lipopolysaccharide layer by ethylenediaminetetraacetate treatment renders the organisms sensitive to medium and partially sensitive to long-chain fatty acids. The intact lipopolysaccharide layer of gram-negative organisms apparently screens the cells against medium and long-chain fatty acids and prevents their accumulation on the inner cell membrane (site of amino acid transport) at inhibitory concentrations. These results are relevant to the use of antimicrobial food additives, and they allow the characterization of gram-positive versus gram-negative bacteria and their lipopolysaccharide mutants.     

Effects of nisin on growth of bacteria attached to meat

Nisin had an inhibitory effect on gram-positive bacteria (Listeria monocytogenes, Staphylococcus aureus, and Streptococcus lactis) but did not have an inhibitory effect on gram-negative bacteria (Serratia marcescens, Salmonella typhimurium, and Pseudomonas aeruginosa) attached to meat. Nisin delayed bacterial growth on meats which were artificially inoculated with L. monocytogenes or Staphylococcus aureus for at least 1 day at room temperature. If the incubation temperature was 5 degrees C, growth of L. monocytogenes was delayed for more than 2 weeks, and growth of Staphylococcus aureus did not occur. We also found that the extractable activity of nisin decreased rapidly when the meats were incubated at ambient temperatures and that this decrease was inversely related to the observed inhibitory effect. These findings disclosed that nisin delays the growth of some gram-positive bacteria attached to meat. However, nisin alone may not be sufficient to prevent meat spoilage because of the presence of gram-negative and other nisin-resistant gram-positive bacteria.     

Effects of nisin on growth of bacteria attached to meat.

Nisin had an inhibitory effect on gram-positive bacteria (Listeria monocytogenes, Staphylococcus aureus, and Streptococcus lactis) but did not have an inhibitory effect on gram-negative bacteria (Serratia marcescens, Salmonella typhimurium, and Pseudomonas aeruginosa) attached to meat. Nisin delayed bacterial growth on meats which were artificially inoculated with L. monocytogenes or Staphylococcus aureus for at least 1 day at room temperature. If the incubation temperature was 5 degrees C, growth of L. monocytogenes was delayed for more than 2 weeks, and growth of Staphylococcus aureus did not occur. We also found that the extractable activity of nisin decreased rapidly when the meats were incubated at ambient temperatures and that this decrease was inversely related to the observed inhibitory effect. These findings disclosed that nisin delays the growth of some gram-positive bacteria attached to meat. However, nisin alone may not be sufficient to prevent meat spoilage because of the presence of gram-negative and other nisin-resistant gram-positive bacteria.     

Expanding the bactericidal action of the food color additive phloxine B to gram-negative bacteria

Phloxine B (D&C red no. 28) is a color additive for food, drugs, and cosmetics. It has been previously shown to have anti-Staphylococcus aureus activities. In this work, the effect of Phloxine B on various gram-negative bacteria and other gram-positive bacteria including Bacillus cereus, Bacillus thuringiensis, Bacillus mycoides, Bacillus subtilis, Bacillus aureus, Salmonella, Escherichia coli and Shigella was studied, along with the mechanism of anti-microbial activity. In the presence of fluorescent light, the viable count for gram-positive bacteria, (Bacillus spp. and S. aureus) decreased in a dose and time dependent manner when incubated with Phloxine B. The viability of gram-positive bacteria was reduced by 99.99% in 40 min, while there was no effect on gram-negative bacteria (Salmonella choleraesuis, E. coli and Shigella flexneri). However, the use of ethylenediaminetetraacetic acid (EDTA) expands the spectrum of activity for Phloxine B to include gram-negative bacteria. EDTA increased membrane-permeability by releasing lipopolysaccharide. Overall, in an Agar diffusion test the light-dependent bactericidal activity of 1 microg of Phloxine B had a potency of 0.64 units of chloramphenicol and 0.5 units of tetracycline when tested on B. cereus, and had a potency of 0.7 units of chloramphenicol and 0.2 units of tetracycline when tested on S. aureus. The data suggest that the dye may have some potential anti-microbial applications.     

Bactericidal effect of gentamicin-induced membrane vesicles derived from Pseudomonas aeruginosa PAO1 on gram-positive bacteria

Previous studies have shown that gentamicin-induced membrane vesicles (g-MVs) from Pseudomonas aeruginosa PAO1 possess both the antibiotic (gentamicin) and a potent peptidoglycan hydrolase (PGase; autolysin) that is effective in killing gram-negative pathogens. This present study evaluated the therapeutic potential of g-MVs against four gram-positive bacteria. Bactericidal assays and electron microscopy of thin sections revealed that Bacillus subtilis 168 and Staphylococcus aureus D2C were susceptible to killing mediated by g-MVs, Listeria monocytogenes ATCC 19113 was slightly susceptible, whereas Enterococcus hirae ATCC 9790 was unaffected. g-MVs were generally more effective against the bacteria than was soluble gentamicin, suggesting they could have more killing power than natural membrane vesicles containing no antibiotic. Electron microscopy and hydrophobic interaction chromatography showed that more membrane vesicles (MVs) initially attached to B. subtilis (hydrophilic) than to predominantly hydrophobic E. hirae, L. monocytogenes, and S. aureus. Zymograms containing murein sacculi as an enzyme substrate illustrated that all organisms except E. hirae were sensitive to the 26-kDa autolysin to varying degrees. Peptidoglycan O-acetylation did not influence susceptibility to MV-mediated lysis. Though not universally effective, the g-MV delivery system remains a promising therapeutic alternative for specific gram-positive infections.     

Lipid Requirement for the Lipase Production by Geotrichum candidum Link

The injection of live or heat-killed bacteria into larvae of the silkworm, Bombyx mori, induced antibacterial activity in the hemolymph. A wide variety of gram-negative and gram-positive bacteria were effective as inducing agents, but saline alone, yeast cells and fungal spores were not effective. The antibacterial activities were separated into six bands on native polyacrylamide gel electrophoresis, which were sensitive to trypsin. Some of these antibacterial proteins were partially purified by CM-cellulose column chromatography. The proteins were heat-stable and showed no lysozyme activity. The proteins repressed the growth of various gram-positive and gram-negative bacteria.     

Physiological responses of bacteria to cytochalasin A: effects on growth, transport, and enzyme induction.

Cytochalasin A at 5 to 25 microgram/ml (1.0 x 10(-5) to 5.2 x 10(-5) M) inhibited the growth of three gram-positive bacteria, Arthrobacter sialophilus, Staphyloccus aureus, and Bacillus amyloliquifaciens, but had little or no effect on the growth of three gram-negative bacteria, Excherichia coli, Pseudomonas maltophilia, and Aeromonas proteolytica. A. sialophilus and S. aureus recovered spontaneously from cytochalasin A-mediated growth inhibition after a considerable lag period, which was dependent on the drug dose. It was demonstrated that this long-term recovery did not involve selection of resistant variants. Cytochalasin A had no detrimental effect on cell viability in A. sialophilus or S. aureus, but caused lysis of B. amyloliquifaciens. The drug prevented enzyme inductions and inhibited transport of valine, uridine, and glucose in the gram-positive organisms. It had little or no effect on these processes in the gram-negative organisms. In studies with A. sialophilus, the drug inhbitied respiration of exogenous substrates, but did not depress endogenous respiration. These results constitute the first unequivocal evidence for the bacteriostatic properties of this class of compounds and indicate that cytochalasin A halts various physiological processes in gram-positive bacteria primarily by inhibiting solute transport.     

Isolation and identification of a Paenibacillus polymyxa strain that coproduces a novel lantibiotic and polymyxin

A new bacterial strain, displaying potent antimicrobial properties against gram-negative and gram-positive pathogenic bacteria, was isolated from food. Based on its phenotypical and biochemical properties as well as its 16S rRNA gene sequence, the bacterium was identified as Paenibacillus polymyxa and it was designated as strain OSY-DF. The antimicrobials produced by this strain were isolated from the fermentation broth and subsequently analyzed by liquid chromatography-mass spectrometry. Two antimicrobials were found: a known antibiotic, polymyxin E1, which is active against gram-negative bacteria, and an unknown 2,983-Da compound showing activity against gram-positive bacteria. The latter was purified to homogeneity, and its antimicrobial potency and proteinaceous nature were confirmed. The antimicrobial peptide, designated paenibacillin, is active against a broad range of food-borne pathogenic and spoilage bacteria, including Bacillus spp., Clostridium sporogenes, Lactobacillus spp., Lactococcus lactis, Leuconostoc mesenteroides, Listeria spp., Pediococcus cerevisiae, Staphylococcus aureus, and Streptococcus agalactiae. Furthermore, it possesses the physico-chemical properties of an ideal antimicrobial agent in terms of water solubility, thermal resistance, and stability against acid/alkali (pH 2.0 to 9.0) treatment. Edman degradation, mass spectroscopy, and nuclear magnetic resonance were used to sequence native and chemically modified paenibacillin. While details of the tentative sequence need to be elucidated in future work, the peptide was unequivocally characterized as a novel lantibiotic, with a high degree of posttranslational modifications. The coproduction of polymyxin E1 and a lantibiotic is a finding that has not been reported earlier. The new strain and associated peptide are potentially useful in food and medical applications.     

Membrane damage and microbial inactivation by chlorine in the absence and presence of a chlorine-demanding substrate

The relationship between cell inactivation and membrane damage was studied in two gram-positive organisms, Listeria monocytogenes and Bacillus subtilis, and two gram-negative organisms, Yersinia enterocolitica and Escherichia coli, exposed to chlorine in the absence and presence of 150 ppm of organic matter (Trypticase soy broth). L. monocytogenes and B. subtilis were more resistant to chlorine in distilled water. The addition of small amounts of organic matter to the chlorination medium drastically increased the resistance of both types of microorganisms, but this effect was more marked in Y. enterocolitica and E. coli. In addition, the survival curves for these microorganisms in the presence of organic matter had a prolonged shoulder. Sublethal injury was not detected under most experimental conditions, and only gram-positive cells treated in distilled water showed a relevant degree of injury. The exposure of bacterial cells to chlorine in distilled water caused extensive permeabilization of the cytoplasmic membrane, but the concentrations required were much higher than those needed to inactivate cells. Therefore, there was no relationship between the occurrence of membrane permeabilization and cell death. The addition of organic matter to the treatment medium stabilized the cytoplasmic membrane against permeabilization in both the gram-positive and gram-negative bacteria investigated. Exposure of E. coli cells to the outer membrane-permeabilizing agent EDTA increased their sensitivity to chlorine and caused the shoulders in the survival curves to disappear. Based on these observations, we propose that bacterial envelopes could play a role in cell inactivation by modulating the access of chlorine to the key targets within the cell.     

Rapid method for distinction of gram-negative from gram-positive bacteria

Summary A rapid method for distinction between gram-negative and grampositive bacteria by means of a 3% solution of potassium hydroxide is tested on 71 gram-positive and 55 gram-negative bacterial strains. The method proved reliable with one exception only, a Bacillus macerans strain. That strain was definately gram-negative on staining. Other Bacillus strains were proved gram-positive by the test, even those being gram-negative on staining.     

Photodynamic antimicrobial activity of avian eggshell pigments

Pigmentation in avian eggshells appears to be associated with shell strength, temperature regulation, and camouflage. The pigments found in eggshells are mainly porphyrins, which have been utilized therapeutically as photosensitizers. Here, we examined the photoinactivation of gram-positive (Staphylococcus aureus, Bacillus cereus) and gram-negative bacteria (Escherichia coli, Salmonella enteritidis) by hen eggshells and their pigments. The results indicated that eggshells have a light-dependent antimicrobial activity against gram-positive, but not gram-negative, bacteria. Our results indicate the possibility that the natural pigments used therapeutically have evolved in nature as a defence system.     

Phosphatidylinositol-specific phospholipase C of Bacillus anthracis down-modulates the immune response

Phosphatidylinositol-specific phospholipases (PI-PLCs) are virulence factors produced by many pathogenic bacteria, including Bacillus anthracis and Listeria monocytogenes. Bacillus PI-PLC differs from Listeria PI-PLC in that it has strong activity for cleaving GPI-anchored proteins. Treatment of murine DCs with Bacillus, but not Listeria, PI-PLC inhibited dendritic cell (DC) activation by TLR ligands. Infection of mice with Listeria expressing B. anthracis PI-PLC resulted in a reduced Ag-specific CD4 T cell response. These data indicate that B. anthracis PI-PLC down-modulates DC function and T cell responses, possibly by cleaving GPI-anchored proteins important for TLR-mediated DC activation.