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.

Inhibitory effect of the combination of xylitol and funoran on Streptococcus mutans biofilm formation on the uncoated surface.

We investigated the effect of xylitol or/and funoran on biofilm formation by Streptococcus mutans, one of cariogenic bacteria, on the surfaces coated and non-coated with saliva. Effects of xylitol and/or funoran were observed on biofilm formation of S. mutans in non-coated and salivary components-coated polystyrene microtiter 96-well plates (s-plate) and flow cell system. Xylitol did not strongly affect biofilm formation of S. mutans UA159 on non-coated and s-plates and, however, changed the quality of the biofilm on the cells in a flow cell system. Funoran had effects on biofilm formation, and the combination of xylitol and funoran strongly inhibited S. mutans biofilm formation on non-coated plates. In particular, funoran had inactivation effects on membrane vesicles (MVs) and inhibited MV-dependent biofilm formation of S. mutans on non-coated plate surfaces but not on the s-plate. These findings suggest that the combination of xylitol and funoran might be useful to remove the oral biofilm formation in elderly individuals with decreased saliva production. This result suggests that the synergistic effect of funoran and xylitol might be useful for the prevention of biofilm-associated diseases such as dental caries in saliva-decreased patients such as elderly patients.

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.

Complete Genome Sequence of Lactobacillus curvatus NFH-Km12, Isolated from the Japanese Traditional Fish Fermented Food Kabura-zushi.

Kabura-zushi is a traditional Japanese fermented food made from yellowtail, rice, salt, and koji. In this study, the complete genomic sequence of Lactobacillus curvatus NFH-Km12, isolated from this unique food, is reported. NFH-Km12 has a 1.9-Mbp chromosome and contains 5 plasmids.

Enzymatic kinetics of the quinol peroxidase of an aggressive periodontopathic bacterium.

Aggregatibacter actinomycetemcomitans is an oral pathogen for aggressive periodontitis, and encodes a triheme c-containing membrane-bound enzyme, quinol peroxidase (QPO) that catalyzes peroxidase activity using quinol in the respiratory chain. In the previous work, we have characterized recombinant QPO purified from the membrane fraction of Escherichia coli harboring a plasmid containing QPO gene. Irreversible inactivation of QPO by high concentration of H2O2 exhibited pseudo-first order kinetics. Analysis of initial-rate kinetics of QPO may suggest that enzyme catalytic mechanism is explained by a Ping Pong Bi Bi system rather than sequential systems. In addition, the redox reactions of cytochrome c in the presence of several values of [Q1H2]/[Q1] were at equilibrium, and only about 2/3 of the cytochrome c of QPO is reduced at high ratios of [Q1H2]/[Q1]. These results indicated that one of the three heme c moieties of QPO is maintained in an oxidized form even at increased ratios of [Q1H2]/[Q1], suggesting that QPO is reduced in the absence of H2O2 and only two of the three heme c moieties are reduced in the presence of high concentration of the Q1H2. Product inhibition of QPO accorded with our theoretical model for the reaction mechanism. Considered together, the enzymatic kinetics data for QPO confirm the Ping Pong Bi Bi system.

Contribution of Streptococcus gordonii Hsa Adhesin to Biofilm Formation.

Adhesion of oral mitis group streptococci, such as Streptococcus gordonii, to acquired pellicle on the tooth surface is the first step in oral biofilm formation. S. gordonii strain DL1 possesses an Hsa adhesin, which recognizes the terminal sialic acid of host sialoglycoconjugates. The aim of the present study was to investigate the role of the Hsa adhesin in biofilm formation. The biofilm-forming ability of a S. gordonii hsa mutant on microtiter plates pre-coated with saliva, fetuin, or mucin was significantly lower than that of wild-type strain DL1. In contrast, no significant difference in biofilm-forming ability was observed in plates pre-coated with bovine serum albumin, which does not contain sialic acid. The biofilm-forming ability of strain DL1 in saliva-coated microtiter plates was also significantly reduced when the plate was pre-treated with neuraminidase. The sialic acid-dependent biofilm-forming ability of different wild-type S. gordonii strains varied. However, Southern and western blot analyses showed that all the tested wild-type strains possessed and expressed hsa homologs, respectively. These results indicate that the binding of Hsa adhesin to sialoglycoconjugates is associated with biofilm formation of S. gordonii DL1, and imply variation in the contribution of Hsa and its homologs to S. gordonii biofilm formation.

Inhibition of Streptococcus mutans biofilm formation using extracts from Assam tea compared to green tea.

OBJECTIVE: Streptococcus mutans, a gram-positive oral bacterium, has been identified as one of the principal etiological agents of human dental caries. To clarify the nature of the difference anti-biofilm effect against S. mutans between Assam tea from Camellia sinensis var. assamica, partially fermented, and green tea from Camellia sinensis, non-fermented, active agents from the teas were purified. METHODS: Effects of Assam tea and green tea samples on biofilm were assessed by using the conventional titer plate method and the human saliva-coated hydroxyapatite discs. The purification and identification of inhibitors were performed by using ultrafiltration with centrifugal filter devices and high performance liquid chromatography. RESULTS: Assam tea has stronger biofilm inhibition activity against S. mutans than green tea. A substance of <10kDa in mass in Assam tea had a high concentration of galloylated catechins and a stronger biofilm inhibiting activity than green tea. In contrast, substances >10kDa in mass from green tea included higher concentrations of polysaccharides composed of galacturonic acid, such as pectin, that enhance biofilm formation. CONCLUSIONS: The higher concentrations of galloylated catechins in Assam tea may assist in prevention of dental caries, whereas in green tea, this mode of inhibition was likely offset by the presence of pectin. Purification of catechins in partially fermented Assam tea with lower-molecular-weight polysaccharide than pectin may be useful for developing oral care products such as toothpaste and oral care gel pastes.

CD56(dim)CD16(high) and CD56(bright)CD16(-) cell percentages associated with maximum knee extensor strength and incidence of death in elderly.

Physical fitness is an indicator of systemic well-being in humans. Little is known about the role of physical fitness for maintaining systemic health in the elderly. Here, we study elderly subjects to determine the relationships between physical fitness and CD56 and CD16 surface NK cell markers on peripheral blood lymphocytes, as well as to analyze the relationship between the surface markers and incidence of death. We selected 253 independent elderly subjects (122 female; 131 male) who were 79-80 years old. Subjects having a higher proportion of CD56(dim)CD16(high) within CD56(+)CD16(+) cells, or ration of CD56(dim)CD16(high) and CD56(dim)CD16(-) cells had a significant positive correlation with maximum bilateral knee extensor strength/weight (kg) (r = 0.425; P < 0.0001 or r = 0.323; P < 0.0001). In contrast, an increased proportion of CD56(bright)CD16(-) cells within lymphocyte significantly negatively correlated with the maximum bilateral knee extensor strength/weight (kg) (r = -0.290; P = 0.0004); and these subjects had a significantly lower mortality during the 5 years following measurement of death. Therefore, we found that a synergistic effect of the right and left leg muscle strength was associated with proportion of matured NK and NKT cells and induced a low proportion of CD56(bright)CD16(-) cells within lymphocyte. Moreover, the low proportion of CD56(bright)CD16(-) cells was associated with incidence of death. In conclusion, measurements of physical fitness, the proportion of CD56(dim)CD16(high) within CD56(+)CD16(+) cells, the ratio of CD56(dim)CD56(high) and CD56(dim)CD16(-) cells, and the proportion of CD56(bright)C16(-) cells in lymphocytes are important indicators to check elderly health.

Competence-dependent endogenous DNA rearrangement and uptake of extracellular DNA give a natural variant of Streptococcus mutans without biofilm formation.

The production of water-insoluble glucan (WIG) enables Streptococcus mutans to survive and persist in the oral niche. WIG is produced from sucrose by glucosyltransferase encoded tandemly by the highly homologous gtfB and gtfC genes. Conversely, a single hybrid gene from the endogenous recombination of gtfB and gtfC is easily generated using RecA, resulting in S. mutans UA159 WIG- (rate of ∼1.0×10(-3)). The pneumococcus recA gene is regulated as a late competence gene. comX gene mutations did not lead to the appearance of WIG- cells. The biofilm collected from the flow cell had more WIG- cells than among the planktonic cells. Among the planktonic cells, WIG- cells appeared after 16 h and increased ∼10-fold after 32 h of cultivation, suggesting an increase in planktonic WIG- cells after longer culture. The strain may be derived from the biofilm environment. In coculture with donor WIG+ and recipient WIG- cells, the recipient cells reverted to WIG+ and acquired an intact gtfBC region from the environment, indicating that the uptake of extracellular DNA resulted in the phenotypic change. Here we demonstrate that endogenous DNA rearrangement and uptake of extracellular DNA generate WIG- cells and that both are induced by the same signal transducer, the com system. Our findings may help in understanding how S. mutans can adapt to the oral environment and may explain the evolution of S. mutans.

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.

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.

E2F-1-deficient NOD/SCID mice developed showing decreased saliva production.

The non-obese diabetic mouse (NOD) is the most characterized model used to study insulin-dependent type 1 diabetes mellitus (IDDM) and Sjoögren's syndrome (SS). In a previous report, we found NOD.E2f1(-/-) mice show a greater progressive development to IDDM and SS compared to NOD mice. Our previous data indicated a progressive decrease in regulatory T cells (CD4(+)CD25(+)) and a decrease in the systemic secretion systems for insulin, and saliva was associated with the progression of IDDM and SS. Therefore, to define the mechanism of early-onset IDDM SS in E2F-1 deficient NOD mice required further investigation by producing E2F-1 deficient NOD/SCID mice in which the T and B cells do not develop. The purpose here was to analyze the essential function of the E2F-1 molecule in the development of IDDM and SS; and the dysfunction of the pancreas islet and salivary gland in the NOD background using NOD/SCID mice. We produced NOD/SCID.E2f1(-/-) mice using homologous recombination; determined diabetes development; measured saliva and insulin production; and performed a histological analysis. The deficient mice showed a decreasing volume of saliva; no infiltration of lymphocytes into salivary glands; no development of diabetes; and no protein localization of FGFR-2b in the ducts of the salivary gland that regulates submandibular gland proliferation and morphogenesis. Therefore, we considered a deficiency in E2F-1 induces a decrease in regulatory T cells and an increase in auto-reactive T cells; however, the E2F-1 deficiency is not associated with T and B cells-independent dysfunction of pancreatic beta cell in insulin secretion. Further, the E2F-1 deficiency is associated with T and B cells-independent dysfunction of the salivary gland exhibits a decrease in saliva production volume. We suggest E2F-1 may be also associated with the differentiation of exocrine cells in the duct where FGFR-2b is expressed in the salivary gland. The E2F-1 deficient NOD/SCID mouse model is useful for showing the development of the salivary gland; and is also useful for various experiments in humanized mice.

Effects of IgY against Candida albicans and Candida spp. Adherence and Biofilm Formation.

The fungal pathogen Candida albicans is an opportunistic fungal pathogen that causes oral and vaginal mucosal infections as well as systemic disease. The ability of C. albicans to adhere to host surfaces is positively correlated with its pathogenicity. We prepared a polyclonal anti-Candida albicans antibody in chicken egg yolk (anti-C. albicans IgY) and investigated its in vitro effectiveness in preventing C. albicans adherence and biofilm formation. Anti-C. albicans IgY significantly reduced the adherence of C. albicans SC5314 to human oral epithelial cells in a dose-dependent manner. The same effect was also observed in other Candida spp. including C. albicans serotype A and B. Further, the IgY inhibited biofilm formation of C. albicans in medium without serum, but the inhibition was slightly restored in medium conditioned with 10% serum. The data indicate that anti-C. albicans IgY cross-reacted with various Candida spp. and may have a protective effect against oral candidiasis and reduce the dissemination of Candida spp. This effect may be due to the blocking of the binding of Candida spp. to the host cells. However, the blocking did not play a role when Candida formed a germ tube in the presence of serum. Therefore, anti-C. albicans IgY may be considered as a prophylactic immunotherapy or possibly an adjunctive antifungal therapy under limited conditions.

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.

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.

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.

Cytoplasmic acidification may occur in high-pressure carbon dioxide-treated Escherichia coli K12.

While studying the mechanism by which high-pressure carbon dioxide treatment (HCT) inactivates bacteria, we found that the efficiency of DNA recovery via phenol extraction was extraordinarily low from E. coli K12 cells that had been subjected to HCT. DAPI staining of the treated cells, however, revealed that nuclear DNA was present. Most DNA from the cells subjected to HCT was probably caught in the denatured protein layer during phenol extraction. The efficiency of DNA recovery from proteinase-treated crude extracts from cells subjected to HCT was high. Crude extracts of E. coli K12 cells that had not undergone HCT were intentionally acidified with acetic acid to pH 5.2 to cause acidic coagulation of cytoplasmic proteins. The efficiency of DNA recovery from the acidified extracts was low. These results suggest that in cells subjected to HCT, cytoplasmic pH is reduced to around pH 5.2, and that nuclear DNA becomes entangled in coagulated cytoplasmic proteins. Acidification of the cytoplasm might be the primary mechanism by which HCT inactivates bacteria.

Mixed-species biofilm formation by lactic acid bacteria and rice wine yeasts.

We found that species combinations such as Lactobacillus casei subsp. rhamnosus IFO3831 and Saccharomyces cerevisiae Kyokai-10 can form a mixed-species biofilm in coculture. Moreover, the Kyokai-10 yeast strain can form a biofilm in monoculture in the presence of conditioned medium (CM) from L. casei IFO3831. The active substance(s) in bacterial CM is heat sensitive and has a molecular mass of between 3 and 5 kDa. In biofilms from cocultures or CM monocultures, yeast cells had a distinct morphology, with many hill-like protrusions on the cell surface.

High-hydrostatic-pressure treatment impairs actin cables and budding in Saccharomyces cerevisiae.

We previously reported that the postgrowth of Escherichia coli K-12 after high-hydrostatic-pressure treatment (HPT) as moderate as 75 MPa for 30 min at 37 degrees C induced the formation of elongated cells due to an HPT-induced disorder in FtsZ ring formation, which is essential for cell division. Because an FtsZ ring is known as a bacterial cytoskeleton, we examined the effect of HPT on a eukaryotic cytoskeleton, such as actin cables (long bundles of actin filaments), of Saccharomyces cerevisiae. We found that actin cables disappeared after HPT (100 MPa) and were not reorganized until 3.5 h of growth after HPT. As long as actin cables disappeared, budding did not start. We also demonstrated that the in vitro polymerization of actin monomers was highly sensitive to HPT.