Hirokazu Ogihara

Inactivation of Geobacillus stearothermophilus Spores by High-Pressure Carbon Dioxide Treatment

ABSTRACT High-pressure CO2 treatment has been studied as a promising method for inactivating bacterial spores. In the present study, we compared this method with other sterilization techniques, including heat and pressure treatment. Spores of Bacillus coagulans, Bacillus subtilis, Bacillus cereus, Bacillus licheniformis, and Geobacillus stearothermophilus were subjected to CO2 treatment at 30 MPa and 35°C, to high-hydrostatic-pressure treatment at 200 MPa and 65°C, or to heat treatment at 0.1 MPa and 85°C. All of the bacterial spores except the G. stearothermophilus spores were easily inactivated by the heat treatment. The highly heat- and pressure-resistant spores of G. stearothermophilus were not the most resistant to CO2 treatment. We also investigated the influence of temperature on CO2 inactivation of G. stearothermophilus. Treatment with CO2 and 30 MPa of pressure at 95°C for 120 min resulted in 5-log-order spore inactivation, whereas heat treatment at 95°C for 120 min and high-hydrostatic-pressure treatment at 30 MPa and 95°C for 120 min had little effect. The activation energy required for CO2 treatment of G. stearothermophilus spores was lower than the activation energy for heat or pressure treatment. Although heat was not necessary for inactivationby CO2 treatment of G. stearothermophilus spores, CO2 treatment at 95°C was more effective than treatment at 95°C alone.

Fosmidomycin Resistance in Adenylate Cyclase Deficient (cya) Mutants of Escherichia coli

Adenylate cyclase deficient (cya) mutants of E. coli K-12 were found to be resistant to fosmidomycin, a specific inhibitor of the non-mevalonate pathway, just like to fosfomycin. E. coli glpT mutants were resistant to fosfomycin and also to fosmidomycin. This fact shows that fosmidomycin was transported inside via the glycerol-3-phosphate transporter, GlpT. DNA micro-array analysis showed that the transcription of glpT and other genes concerning glycerol utilization were highly dependent on the presence of cAMP.

Sugar fatty acid esters inhibit biofilm formation by food-borne pathogenic bacteria.

Effects of food additives on biofilm formation by food-borne pathogenic bacteria were investigated. Thirty-three potential food additives and 3 related compounds were added to the culture medium at concentrations from 0.001 to 0.1% (w/w), followed by inoculation and cultivation of five biofilm-forming bacterial strains for the evaluation of biofilm formation. Among the tested food additives, 21 showed inhibitory effects of biofilm formation by Staphylococcus aureus and Escherichia coli, and in particular, sugar fatty acid esters showed significant anti-biofilm activity. Sugar fatty acid esters with long chain fatty acid residues (C14-16) exerted their inhibitory effect at the concentration of 0.001% (w/w), but bacterial growth was not affected at this low concentration. Activities of the sugar fatty acid esters positively correlated with the increase of the chain length of the fatty acid residues. Sugar fatty acid esters inhibited the initial attachment of the S. aureus cells to the abiotic surface. Sugar fatty acid esters with long chain fatty acid residues (C14-16) also inhibited biofilm formation by Streptococcus mutans and Listeria monocytogenes at 0.01% (w/w), while the inhibition of biofilm formation by Pseudomonas aeruginosa required the addition of a far higher concentration (0.1% (w/w)) of the sugar fatty acid esters.

Inhibition of Streptococcus mutans Biofilm Formation by Streptococcus salivarius FruA

The oral microbial flora consists of many beneficial species of bacteria that are associated with the healthy condition and control the progression of oral disease. Cooperative interactions between oral streptococci and the pathogens play important roles in the development of dental biofilms in the oral cavity. To determine the roles of oral streptococci in multi-species biofilm development and the effects of the streptococci in biofilm formation, the active substances inhibiting S. mutans biofilm formation were purified from Streptococcus salivarius ATCC 9759 and HT9R culture supernatants using ion-exchange and gel filtration chromatography. MALDI-TOF mass spectrometry analysis was performed and the results were compared to data bases. The S. salivarius HT9R genome sequence was determined; and used to indentify candidate proteins for inhibition. The candidates inhibiting biofilms were identified as S. salivarius fructosyltransferase (FTF) and exo-beta-D-fructosidase (FruA). The activity of the inhibitors was elevated in the presence of sucrose; and the inhibitory effects were dependent on the sucrose concentration in the biofilm formation assay medium. Purified and commercial FruA from Aspergillus niger (31.6% identity and 59.6% similarity to the amino acid sequence of FruA from S. salivarius HT9R) completely inhibited S. mutans GS-5 biofilm formation on saliva-coated polystyrene and hydroxyapatite surfaces. The inhibition was induced by decreasing polysaccharide production dependent on sucrose digestion rather than fructan digestion. The data indicate S. salivarius produces large quantities of FruA; and FruA alone may play an important role in multi-species microbial interactions for sucrose-dependent biofilm formation in the oral cavity.ABSTRACT The oral microbial flora consists of many beneficial species of bacteria that are associated with a healthy condition and control the progression of oral disease. Cooperative interactions between oral streptococci and the pathogens play important roles in the development of dental biofilms in the oral cavity. To determine the roles of oral streptococci in multispecies biofilm development and the effects of the streptococci in biofilm formation, the active substances inhibiting Streptococcus mutans biofilm formation were purified from Streptococcus salivarius ATCC 9759 and HT9R culture supernatants using ion exchange and gel filtration chromatography. Matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry analysis was performed, and the results were compared to databases. The S. salivarius HT9R genome sequence was determined and used to indentify candidate proteins for inhibition. The candidates inhibiting biofilms were identified as S. salivarius fructosyltransferase (FTF) and exo-beta-d-fructosidase (FruA). The activity of the inhibitors was elevated in the presence of sucrose, and the inhibitory effects were dependent on the sucrose concentration in the biofilm formation assay medium. Purified and commercial FruA from Aspergillus niger (31.6% identity and 59.6% similarity to the amino acid sequence of FruA from S. salivarius HT9R) completely inhibited S. mutans GS-5 biofilm formation on saliva-coated polystyrene and hydroxyapatite surfaces. Inhibition was induced by decreasing polysaccharide production, which is dependent on sucrose digestion rather than fructan digestion. The data indicate that S. salivarius produces large quantities of FruA and that FruA alone may play an important role in multispecies microbial interactions for sucrose-dependent biofilm formation in the oral cavity.

Effect of high pressure gaseous carbon dioxide on the germination of bacterial spores

Effect of high pressure gaseous carbon dioxide treatment (HGCT) at 6.5 MPa, 35 degrees C on the germination of bacterial spores was investigated. Germination of bacterial spores was estimated by the decrease of heat tolerance. Approximately, 40% of Bacillus coagulans and 70% of Bacillus licheniformis were germinated by HGCT for 120 min at 35 degrees C, respectively. Germination was confirmed by phase contrast microscopy. The effect of hydrostatic pressure treatment (HPT) at 6.5 MPa, 35 degrees C on the germination of B. coagulans and B. licheniformis spores were also investigated. Spores did not germinate by HPT alone at 6.5 MPa for 120 min.

The Importance of Inter-Species Cell-Cell Co-Aggregation between Lactobacillus plantarum ML11-11 and Saccharomyces cerevisiae BY4741 in Mixed-Species Biofilm Formation

Cells of Lactobacillus plantarum ML11-11, an isolate from Fukuyama pot vinegar, and the yeast Saccharomyces cerevisiae formed significant mixed-species biofilms with concurrent inter-species co-aggregation. The co-aggregation did not occur with heated or proteinase K-treated ML11-11 cells, or in the presence of D-mannose, suggesting that surface proteins of ML11-11 and mannose-containing surface substance(s) of yeast were the predominant contributing factors. Sugar fatty acid ester inhibited mixed-species biofilm formation, but did not inhibit co-aggregation, suggesting that the cell-cell adhesion and cell-polystylene adhesion are controlled by different mechanisms. Microscopic observation and microflora analysis revealed that inter-species co-aggregation plays an important role in the formation of the mixed-species biofilm.

Biofilm formation by Escherichia coli in hypertonic sucrose media

High osmotic environments produced by NaCl or sucrose have been used as reliable and traditional methods of food preservation. We tested, Escherichia coli as an indicator of food-contaminating bacterium, to determine if it can form biofilm in a hyperosmotic environment. E. coli K-12 IAM1264 did not form biofilm in LB broth that contained 1 M NaCl. However, the bacterium formed biofilm in LB broth that contained 1 M sucrose, although the planktonic growth was greatly suppressed. The biofilm, formed on solid surfaces, such as titer-plate well walls and glass slides, solely around the air-liquid interface. Both biofilm forming cells and planktonic cells in the hypertonic medium adopted a characteristic, fat and filamentous morphology with no FtsZ rings, which are a prerequisite for septum formation. Biofilm forming cells were found to be alive based on propidium iodide staining. The presence of 1 M sucrose in the food environment is not sufficient to prevent biofilm formation by E. coli.

Effect of skimmed milk and its fractions on the inactivation of Escherichia coli K12 by high hydrostatic pressure treatment

We investigated the effects of skimmed milk and its protein fractions (casein, whey, globulin, and albumin) on the injury and inactivation of Escherichia coli K-12 by high hydrostatic pressure (HHP) treatment. The protective effect of skimmed milk on HHP-mediated inactivation and injury of E. coli increased with increases in the skimmed milk concentration. However, protein fractions derived from skimmed milk did not exhibit this protective effect. Microscopy analysis by DAPI/PI staining indicated that some cells were localized in the solid portion of skimmed milk, and some of these cells were alive. The coagulated fraction derived from the autoclaved whey fraction also showed a significant protective effect. We speculate that the solid portion in skimmed milk could provide the protective effect to bacterial cells.

Mixed-Species Biofilm Formation by Direct Cell-Cell Contact between Brewing Yeasts and Lactic Acid Bacteria

Mixed-species biofilm was remarkably formed in a static co-culture of Lactobacillus plantarum ML11-11 and Saccharomyces cerevisiae Y11-43 isolated from brewing samples of Fukuyama pot vinegar. Mixed-species biofilm is probably formed by direct cell-cell contact between ML11-11 and S. cerevisiae including Y11-43 and laboratory yeast strains. Scanning electron microscopic observation suggested that the mixed-species biofilm had a thick, bi-layer structure.

L-Tryptophan prevents Escherichia coli biofilm formation and triggers biofilm degradation.

The effect of deletion of trp operon and tna operon on the Escherichia coli biofilm formation was investigated in order to elucidate the role of L-tryptophan metabolism in biofilm formation. trp operon deletion mutants ΔtrpC, ΔtrpD and ΔtrpE deficient in L-tryptophan biosynthesis showed higher biofilm formation. In addition, ΔtnaC with increased L-tryptophan degradation activity showed higher biofilm formation. On the contrary, ΔtnaA deletion mutant which lost L-tryptophan degradation activity showed low biofilm formation. From these results, it was suggested that decrease of intracellular L-tryptophan level induced biofilm formation and increase of L-tryptophan repressed biofilm formation. So the effect of the addition of L-tryptophan to the medium on the E. coli biofilm formation was investigated. L-Tryptophan addition at starting culture decreased biofilm formation and furthermore L-tryptophan addition after 16 h culture induced the degradation of preformed biofilm. From the above results, it was suggested that maintenance of high intracellular L-tryptophan concentration prevents E. coli biofilm formation and elevation of intracellular L-tryptophan concentration triggers degradation of matured biofilm.