Steric microstructure of mixed-species biofilm formed by interaction between Lactobacillus plantarum ML11-11 and Saccharomyces cerevisiae.

The mixed-species biofilm of Lactobacillus plantarum ML11-11 (LAB) and yeast had a double-layered structure with the ground layer composed of LAB cells, and the upper layer composed of coaggregates of LAB and yeast cells. The ability of LAB to adhere to both, the solid surface and the yeast cells, enabled the formation and maintenance of the biofilm as an ecosystem for LAB and yeast.

Screening of lactic acid bacteria that can form mixed-species biofilm with Saccharomyces cerevisiae.

The abilities of lactic acid bacteria (LAB) to form mixed-species biofilm with Saccharomyces cerevisiae in a static co-culture were investigated out of 168 LAB stock cultures, and two Lactobacillus plantarum strains (D71 and E31) and one Leuconostoc mesenteroides strain K01 were found to form mixed-species biofilm with S. cerevisiae BY4741. SEM observation showed that there was no significant difference in morphological properties among these three mixed-species biofilms and they resembled that formed by S. cerevisiae with L. plantarum ML11-11 previously isolated from a brewing sample of Fukuyama pot vinegar. The co-aggregation assays showed that L. plantarum D71 and L. plantarum E31 could co-aggregate with S. cerevisiae similarly to L. plantarum ML11-11, while L. mesenteroides K01 had no ability to co-aggregate with yeast. The above results indicate that aggregation followed by direct cell-to-cell contact is required for mixed-species biofilm formation between these L. plantarum strains and S. cerevisiae, though some different mechanism may be involved in biofilm formation between L. mesenteroides strain and S. cerevisiae.

Acid resistance contributes to the high-pressure carbon dioxide resistance of Escherichia coli K-12.

Effect of deletion of acid resistant genes of E. coli on the high-pressure carbon dioxide (HPC) resistance was investigated. Genes coding amino acid decarboxylases, such as lysine, arginine, and glutamate decarboxylase, were found to contribute to HPC resistance. Protonophore-treated cells showed hypersensitivity to HPC, confirming that HPC induced cytoplasm acidification and exerted severe damage on cells by intrusion of gaseous carbon dioxide into cytoplasm.

Fimbriae and lipopolysaccharides are necessary for co-aggregation between Lactobacilli and Escherichia coli.

Cells of Lactobacilli co-aggregated with Escherichia coli K-12 cells to form co-aggregates under mixed-culture conditions at 37 °C for 24 h. Co-aggregation was inhibited by sodium dodecyl sulfate but not by protease. E. coli deletion mutants of fimbriae formation and lipopolysaccharide (LPS) formation did not co-aggregate with Lactobacilli. These results showed that fimbriae and LPS are necessary for co-aggregation between Lactobacilli and E. coli.

Sublethal high hydrostatic pressure treatment reveals the importance of genes coding cytoskeletal protein in Escherichia coli morphogenesis.

We studied morphologic changes after sublethal high hydrostatic pressure treatment (HPT) of Escherichia coli K-12 strains in which genes related to the cytoskeleton, cell wall, and cell division had been deleted. Some long filamentous and swelling cells were observed in wild-type bacteria, while some spherical, branched, or collapsed cells were observed in deletion mutants. In particular, ΔzapA and ΔrodZ showed distinguished morphologies. ZapA supports FtsZ, a cytoskeletal protein, forming ring with ZapB. RodZ, a cytoskeletal protein, interacts with MreB, also a cytoskeletal protein, and both factors are necessary for maintaining the rod shape of the cell. These results showed that insufficient formation of FtsZ rings induced cell elongation and that insufficient formation of MreB induced a branched and collapsed cell shape. Therefore, the correct formation of the bacteria cytoskeleton by FtsZ rings and MreB is important for keeping normal cell shape during growth after HPT, and the polymerization of cytoskeletal proteins was a critical target of sublethal HPT. These results indicate that sublethal HPT induces bacterial cell morphologic change and provide important information on the role of genes involved in morphogenesis. Therefore, sublethal HPT may be a good tool for studying the morphogenesis of bacterial cells.

Antibacterial Activity of Hexanol Vapor In Vitro and on the Surface of Vegetables.

Hexanol is a volatile alcohol and a major component of plant essential oils (EOs). However, the antibacterial activity of hexanol vapor has not been well studied. This study aimed to evaluate the antibacterial activity of hexanol. In this study, seven food-related bacteria were exposed to 1-, 2- or 3-hexanol vapor on agar media to evaluate their growth. Additionally, the total viable counts in three vegetables when exposed to 1-hexanol vapor were measured. The results showed that 1-hexanol exhibited antibacterial effects against Gram-negative bacteria but did not affect Gram-positive bacteria. However, compounds 2- and 3-hexanol did not show antimicrobial activity against any bacteria. For the vegetables, exposure to 1-hexanol vapor decreased the total viable bacterial counts in cabbage and carrot and inhibited bacterial growth in eggplants. In cabbage, 1-hexanol vapor at concentrations over 50 ppm decreased the total viable count within 72 h, and 25 ppm of vapor showed bacteriostatic activity for 168 h. However, 1-hexanol vapor also caused discoloration in cabbage. In summary, 1-hexanol has the potential to act as an antibacterial agent, but further studies are required for practical use. Moreover, the study results may help determine the antimicrobial activity of various EOs in the future.

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.

Yeast mannan structure necessary for co-aggregation with Lactobacillus plantarum ML11-11.

Lactic acid bacteria (LAB) Lactobacillus plantarum ML11-11, an isolate from Fukuyama pot vinegar, and yeast Saccharomyces cerevisiae form significant mixed-species biofilm with direct cell-cell contact. Co-aggregation of L. plantarum ML11-11 and S. cerevisiae cells, mediated by the interaction between surface protein(s) on L. plantarum ML11-11 cells and surface mannan of S. cerevisiae cells, contributes significantly to mixed-species biofilm formation. In this study, co-aggregation activities of yeast mutants that were deleted of genes related to mannan biosynthesis were investigated to clarify the mannan structures essential for interaction with L. plantarum ML11-11. Among the 12 deletion mutants which had various incomplete mannan structures, only the mnn2 mutant lost the co-aggregation activity. In the mnn2 mutant, the gene coding the activity of attaching first branching mannose residue to mannan main chain is deleted and therefore the mnn2 mutant has unbranched mannan. From this result, it is clarified that the specific structure, consisted of mannan main chain to which are attached side chains containing one or more mannose residues, is critical for co-aggregation with L. plantarum ML11-11.

Mutants of Lactobacillus plantarum ML11-11 deficient in co-aggregation with yeast exhibited reduced activities of mixed-species biofilm formation.

Lactic acid bacteria (LAB) mutants deficient in inter-species co-aggregation with yeast were spontaneously derived from Lactobacillus plantarum ML11-11, a significant mixed-species biofilm former in static co-cultures with budding yeasts. These non-co-aggregative mutants also showed significant decreases in mixed-species biofilm formation. These results suggest the important role of co-aggregation between LAB and yeast in mixed-species biofilm formation. Cell surface proteins obtained by 5 M LiCl extraction from the wild-type cells and non-co-aggregative mutant cells were analyzed by SDS-PAGE. There was an obvious difference in protein profiles. The protein band at 30 kDa was present abundantly in the wild-type cell surface fraction but was significantly decreased in the mutant cells. This band assuredly corresponded to the LAB surface factors that contribute to co-aggregation with yeasts.

Identification of changes in the microflora composition of Japanese horse mackerel (Trachurus japonicus) during storage to identify specific spoilageorganisms.

Japanese horse mackerel (Trachurus japonicus) is an important marine resource, and its loss and waste should be reduced. This study aimed to identify the changes in the microflora composition during storage and specific spoilage organisms (SSOs) in Japanese horse mackerel, for spoilage prevention. They were stored at either 20 °C or 4 °C aerobically, and the bacterial viable counts, concentration of total volatile basic nitrogen (TVB-N), and microflora composition for each group were analyzed. Samples stored at 20 °C for 48 h showed similar viable counts to those stored at 4 °C for 168 h; however, the TVB-N concentrations increased at 20 °C, but not at 4 °C. 16S rRNA metagenome analysis showed that Shewanella became dominant genus in the microflora regardless of the storage temperature. However, dominant amplicon sequence variants (ASVs), which are a more detailed classification level than the genus, differed depending on the storage temperatures; therefore, dominant ASVs at 20 °C were assumed to be potential SSOs. Shewanella sp. Strain NFH-SH190041, which was genetically closely related to the dominant ASVs at 20 °C, was isolated, and its spoilage ability was verified. The strain NFH-SH190041 may be considered a novel SSO of Japanese horse mackerel because its 16S rRNA sequence is clearly different from those of known species.

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 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.

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.

Roles of multidrug efflux pumps on the biofilm formation of Escherichia coli K-12.

We genetically analyzed the roles of multidrug efflux pumps on the biofilm formation of Escherichia coli K-12 BW25113. We used 22 mutants missing various genes related to the multidrug efflux pumps and found that the biofilm formation of emrD, emrE, emrK, acrD, acre and mdtE-deleted mutants was extremely decreased. These results indicate that some multidrug efflux pumps significantly contribute to the biofilm formation of E. coli.

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.

Microbiological and biochemical survey on the transition of fermentative processes in Fukuyama pot vinegar brewing.

Traditional brewing of Fukuyama pot vinegar is a process that has been continued in Fukuyama, Kagoshima, Japan, for almost 200 years. The entire process proceeds from raw materials, including steamed rice, rice koji (steamed rice grown with a fungus, Aspergillus oryzae) and water, to produce vinegar in roughly capped large pots laid in the open air. No special fermentative manipulation is required, except for scattering dried rice koji (called furi-koji) on the surface of the mash to form a cap-like mat on the surface at the start of brewing. As the biochemical mechanism of the natural transition of the fermentative processes during brewing has not been fully explained, we conducted a microbiological and biochemical study on the transition. First, a distinct biochemical change was observed in the brewing of spring preparation; that is, a sharp decline in pH from 6.5 to 3.5 within the first 5 days of brewing was observed due to lactic acid fermentation. Alcoholic fermentation also proceeded with a sharp increase to 4.5% ethanol within the first 5 days under the acidic conditions, suggesting that saccharification and both fermentations proceed in parallel. Acidic conditions and ethanol accumulation restricted the growth of most microorganisms in the mash, and in turn provided a favorable growth condition for acetic acid bacteria which are acid resistant and "ethanol-philic." Acetic acid was detected from day 16 and gradually increased in concentration, reaching a maximum of 7% at day 70 that was maintained thereafter. Empirically furi-koji naturally sinks into the mash after around day 40 by an unknown mechanism, allowing acetic acid bacteria to easily form pellicles on the mash surface and promoting efficient acetic acid fermentation. Dominant microbial species involved in the three fermentations were identified by denaturing gradient gel electrophoresis analysis using PCR-amplified defined-regions of small rDNA from microorganisms in the brewing mash or colony direct PCR applied to isolated microorganisms from the mash.

Cell-cycle independent chromosome condensation in Schizosaccharomyces pombe induced by high hydrostatic pressure treatment.

We exposed Schizosaccharomyces pombe to high hydrostatic pressure treatment (HPT) of 75 MPa at 28 degrees Celsius for 30 min and then observed that the DAPI-stained chromosomal DNA had shrunk compactly. We termed this phenomenon HPT-induced chromosome condensation (HPT-CC). HPT did not significantly decrease viability. The condensed state was released when HPT cells were cultured at 28 degrees Celsius for 30 min. The condensation was not caused by shrinking of the nuclear envelope, which was visualized by YFP-tagged importin alpha. HPT-CC was cell cycle independent, because it was observed in almost all randomly cultured cells. The condensin complex (Cut3, Cut14, and three other proteins) is responsible for cell cycle dependent CC. Studies with Cut3-YFP and ts mutants of Cut3 and Cut14 confirmed that HPT-CC was independent of condensin molecules. HPT-CC was also observed in Saccharomyces cerevisiae. HPT-CC appears likely to be a temporal stress response to high hydrostatic pressure found at least in yeasts.

Comparative Evaluation of Selective Media for the Detection of Bacillus cereus.

The commercially available 3 types of selective media in Japan were compared for the detection of Bacillus cereus. When assessed inclusivity using 25 B. cereus strains, MYP agar, NGKG agar, and chromogenic X-BC agar demonstrated excellent inclusivity. For exclusivity study using 50 non-B. cereus strains, MYP, NGKG, and X-BC allowed to grow 11, 7, and 3 strains, respectively. Of the grown bacteria on each strains tested, only 2 strains of B. thuringiensis formed typical B. cereus colonies on all selective media tested. The NGKG and X-BC were compared with MYP as a reference using artificially contaminated food (fried rice, plain rice, fried noodle, and potato salad ), since MYP is recommended in ISO 7932: 2004. The both correlation coefficients between NGKG and MYP, and X-BC and MYP were 0.999. Therefore, we demonstrated that NGKG and X-BC can be adapted to ISO 7932: 2004 method for selected food as well as MYP.

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.

Cessation of cytokinesis in Schizosaccharomyces pombe during growth after release from high hydrostatic pressure treatment.

On the basis of our previous study concerning the effect of high hydrostatic pressure treatment (HPT) on Escherichia coli FtsZ ring (bacterial cytoskeleton) formation, we aimed to determine the effect of HPT on the growth properties of a representative eukaryotic microbe, Schizosaccharomyces pombe, in relation to the behavior of genuine cytoskeletons. Microtubules were visualized with GFP-linked alpha-tubulin. Actin-related cytoskeletons were fluorescently stained with rhodamine-phalloidin. We observed growth retardation of about 10 h in post growth after HPT (75 MPa, 30 min, 28 degrees C), which caused only a little loss of viable cells. In accordance with the period of growth retardation, cessation of cytokinesis and disappearance of the contractile ring (composed of actin, myosin II, and other proteins), directly participates in cytokinesis, continued for 18 h after HPT. On the other hand, the microtubules disappeared only for 6 h after HPT. Based on these observations, the contractile ring was the site most sensitive to HPT resulting in the cessation of cytokinesis.

Comparison of Chromogenic Selective Media for the Detection of Cronobacter spp. (Enterobacter sakazakii).

　The four types of chromogenic selective media that are commercially available in Japan were compared for establishing a Japanese standard method for detecting Cronobacter spp. based on ISO/TS 22964:2006. When assessed using 9 standard Cronobacter spp. strains and 29 non-Cronobacter strains, Enterobacter sakazakii isolation agar, ChromocultTM Enterobacter sakazakii agar, CHROMagarTM E. sakazakii, and XM-sakazakii agar demonstrated excellent inclusivity and exclusivity. Using the ISO/TS 22964:2006 method, the recovered numbers of 38 Cronobacter spp. strains, including 29 C. sakazakii isolates obtained from each medium, were equivalent, indicating that there was no significant difference (p > 0.05) among the four types of chromogenic selective media. Thus, we demonstrated that these four chromogenic selective media are suitable alternatives when using the standard method for detecting Cronobacter spp. in Japan, based on the ISO/TS 22964:2006.