Effects of subchronic and mild social defeat stress on the intestinal microbiota and fecal bile acid composition in mice.

The gut microbiota plays a crucial role in both the pathogenesis and alleviation of host depression by modulating the brain-gut axis. We have developed a murine model of human depression called the subchronic and mild social defeat stress (sCSDS) model, which impacts not only behavior but also the host gut microbiota and gut metabolites, including bile acids. In this study, we utilized liquid chromatography/mass spectrometry (LC/MS) to explore the effects of sCSDS on the mouse fecal bile acid profile. sCSDS mice exhibited significantly elevated levels of deoxycholic acid (DCA) and lithocholic acid (LCA) in fecal extracts, leading to a notable increase in total bile acids and 7α-dehydroxylated secondary bile acids. Consequently, a noteworthy negative correlation was identified between the abundances of DCA and LCA and the social interaction score, an indicator of susceptibility in stressed mice. Furthermore, analysis of the colonic microbiome unveiled a negative correlation between the abundance of CDCA and Turicibacter. Additionally, DCA and LCA exhibited positive correlations with Oscillospiraceae and Lachnospiraceae but negative correlations with the Eubacterium coprostanoligenes group. These findings suggest that sCSDS impacts the bidirectional interaction between the gut microbiota and bile acids and is associated with reduced social interaction, a behavioral indicator of susceptibility in stressed mice.

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.

Cell-surface protein YwfG of Lactococcus lactis binds to α-1,2-linked mannose.

Lactococcus lactis strains are used as starter cultures in the production of fermented dairy and vegetable foods, but the species also occurs in other niches such as plant material. Lactococcus lactis subsp. lactis G50 (G50) is a plant-derived strain and potential candidate probiotics. Western blotting of cell-wall proteins using antibodies generated against whole G50 cells detected a 120-kDa protein. MALDI-TOF MS analysis identified it as YwfG, a Leu-Pro-any-Thr-Gly cell-wall-anchor-domain-containing protein. Based on a predicted domain structure, a recombinant YwfG variant covering the N-terminal half (aa 28-511) of YwfG (YwfG28-511) was crystallized and the crystal structure was determined. The structure consisted of an L-type lectin domain, a mucin-binding protein domain, and a mucus-binding protein repeat. Recombinant YwfG variants containing combinations of these domains (YwfG28-270, YwfG28-336, YwfG28-511, MubR4) were prepared and their interactions with monosaccharides were examined by isothermal titration calorimetry; the only interaction observed was between YwfG28-270, which contained the L-type lectin domain, and d-mannose. Among four mannobioses, α-1,2-mannobiose had the highest affinity for YwfG28-270 (dissociation constant = 34 µM). YwfG28-270 also interacted with yeast mannoproteins and yeast mannan. Soaking of the crystals of YwfG28-511 with mannose or α-1,2-mannobiose revealed that both sugars bound to the L-type lectin domain in a similar manner, although the presence of the mucin-binding protein domain and the mucus-binding protein repeat within the recombinant protein inhibited the interaction between the L-type lectin domain and mannose residues. Three of the YwfG variants (except MubR4) induced aggregation of yeast cells. Strain G50 also induced aggregation of yeast cells, which was abolished by deletion of ywfG from G50, suggesting that surface YwfG contributes to the interaction with yeast cells. These findings provide new structural and functional insights into the interaction between L. lactis and its ecological niche via binding of the cell-surface protein YwfG with mannose.

Complete Genome Sequence of Lactococcus cremoris Strain 7-1, a Lactic Acid Bacterium Isolated from a Traditional Mongolian Milk Product Possessing Mucin-Adhesive Ability.

We report the complete genome sequence of Lactococcus cremoris strain 7-1, which was isolated from urum, a traditional Mongolian milk product. Strain 7-1 adhered to porcine gastric mucin in a carbon source-dependent manner. The genome consists of a circular chromosome (2,557,589 bp; GC content, 35.7%) and two circular plasmids.

Complete Genome Sequence of Carotenoid-Producing Enterococcus gilvus CR1, Isolated from Raw Cow's Milk.

Enterococcus gilvus CR1, isolated from raw cow's milk, can produce carotenoids. The complete genome sequence of this strain was determined using the PacBio RS II platform. The assembly was found to contain a circular chromosome, including carotenoid biosynthesis genes, and comprises 2,863,043 bp, with a G+C content of 41.86% and three plasmids.

Indole-3-Pyruvic Acid, an Aryl Hydrocarbon Receptor Activator, Suppresses Experimental Colitis in Mice.

Aryl hydrocarbon receptor (AHR) agonists are promising immunomodulators that potentially maintain immune tolerance. In this study, we examined the ability of indole-3-pyruvic acid (IPA), a major precursor of microbiota-derived AHR agonists and a proagonist of AHR, to activate AHR. The anti-inflammatory effects of IPA were also evaluated in a mouse model of colitis in comparison with other aromatic pyruvic acids (phenylpyruvic acid and 4-hydroxyphenylpyruvic acid). Among them, IPA showed the strongest ability to activate AHR in vitro and in vivo, and only IPA improved chronic inflammation in an experimental colitis model. IPA attenuated the expression of genes encoding Th1 cytokines and enhanced Il-10 gene expression in the colon. Oral administration of IPA decreased the frequency of IFN-γ+ IL-10- CD4+ T cells and increased that of IFN-γ- IL-10+ CD4+ T cells in the colon lamina propria in a T cell-mediated colitis model. IPA directly promoted the differentiation of type 1 regulatory T cells in vitro. Furthermore, IPA administration attenuated the ability of dendritic cells (DCs) in the mesenteric lymph nodes (MLN) to induce IFN-γ-producing T cells, increased the frequency of CD103+ CD11b- DCs, and decreased the frequency of CD103- CD11b+ DCs in the MLN. Adoptive transfer of MLN CD103+ CD11b- DCs significantly improved the severity of colon inflammation. Treatment with an AHR antagonist inhibited IPA-induced differentiation of type 1 regulatory T cells and the IPA-induced increase in CD103+ CD11b- DCs and attenuated the anti-inflammatory effect of IPA. These findings suggest that IPA potently prevents chronic inflammation in the colon by activating AHR.

Reduced fucosylation in the distal intestinal epithelium of mice subjected to chronic social defeat stress.

Psychological stress can cause dysfunction of the gastrointestinal tract by regulating its interaction with central nervous system (brain-gut axis). Chronic social defeat stress (CSDS) is widely used to produce a rodent model of stress-induced human mood disorders and depression. We previously showed that CSDS significantly affects the intestinal ecosystem including cecal and fecal microbiota, intestinal gene expression profiles and cecal metabolite profiles. Here, we investigated whether the glycosylation pattern in the intestinal epithelium was affected in C57BL/6 mice exposed to CSDS (hereinafter referred to as CSDS mice). A lectin microarray analysis revealed that CSDS significantly reduced the reactivity of fucose-specific lectins (rAOL, TJA-II, rAAL, rGC2, AOL, AAL, rPAIIL and rRSIIL) with distal intestinal mucosa, but not with mucosa from proximal intestine and colon. Flow cytometric analysis confirmed the reduced TJA-II reactivity with intestinal epithelial cells in CSDS mice. In addition, distal intestine expression levels of the genes encoding fucosyltransferase 1 and 2 (Fut1 and Fut2) were downregulated in CSDS mice. These findings suggest that CSDS alters the fucosylation pattern in the distal intestinal epithelium, which could be used as a sensitive marker for CSDS exposure.

Complete Genome Sequence of Lactobacillus paracasei EG9, a Strain Accelerating Free Amino Acid Production during Cheese Ripening.

Lactobacillus paracasei EG9 is a strain isolated from well-ripened cheese and accelerates free amino acid production during cheese ripening. Its complete genome sequence was determined using the PacBio RS II platform, revealing a single circular chromosome of 2,927,257 bp, a G+C content of 46.59%, and three plasmids.

Complete Genome Sequence of Lactobacillus plantarum Strain LQ80, Selected for Preparation of Fermented Liquid Feed for Pigs.

Lactobacillus plantarum LQ80 is a strain isolated from liquid feed for pigs. We determined the complete genome sequence of this strain using the PacBio RS II platform. LQ80 contained a single circular chromosome of 3,230,192 bp, with 44.66% G+C content and seven plasmids.

Complete Genome Sequence of Lactococcus lactis subsp. lactis G50 with Immunostimulating Activity, Isolated from Napier Grass.

Lactococcus lactis subsp. lactis G50 is a strain with immunostimulating activity, isolated from Napier grass (Pennisetum purpureum). We determined the complete genome sequence of this strain using the PacBio RS II platform. The single circular chromosome consists of 2,346,663 bp, with 35.03% G+C content and no plasmids.

The distinct effects of orally administered Lactobacillus rhamnosus GG and Lactococcus lactis subsp. lactis C59 on gene expression in the murine small intestine.

The molecular mechanisms of strain-specific probiotic effects and the impact of the oral administration of probiotic strains on the host's gene expression are not yet well understood. The aim of this study was to investigate the strain-specific effects of probiotic strain intake on gene expression in the murine small intestine. Two distinct strains of lactic acid bacteria, Lactobacillus rhamnosus GG (GG) and Lactococcus lactis subsp. lactis C59 (C59), were orally administered to BALB/c mice, daily for 2 weeks. The total RNA was isolated from the upper (including the duodenum) and lower (the terminal ileum) small intestine, and gene expression was assessed by microarray analysis. The data revealed (1) oral administration of C59 and GG markedly down-regulated the expression of genes encoding fibrinogen subunits and plasminogen in the upper small intestine; (2) administration of more than 1 × 107 CFU/day of GG changed the gene expression of the host ileum. (3) strain- and dose-related effects on various GO biological processes; and (4) enrichment for B cell-related Gene Ontology terms among up-regulated genes in the terminal ileum of mice administered the 1 × 109 CFU/day of GG. The distinct effects of GG and C59 on gene expression in the intact small intestine provide clues to understand how the health beneficial effects of specific strains of probiotic bacteria are mediated by interactions with intestinal cells.

First Complete Genome Sequence of the Skin-Improving Lactobacillus curvatus Strain FBA2, Isolated from Fermented Vegetables, Determined by PacBio Single-Molecule Real-Time Technology.

The first complete genome sequence of Lactobacillus curvatus was determined by PacBio RS II. The single circular chromosome (1,848,756 bp, G+C content of 42.1%) of L. curvatus FBA2, isolated from fermented vegetables, contained low G+C regions (26.9% minimum) and 43 sets of >1,000-bp identical sequence pairs. No plasmids were detected.

Omics Studies of the Murine Intestinal Ecosystem Exposed to Subchronic and Mild Social Defeat Stress.

The microbiota-gut-brain axis plays an important role in the development of stress-induced mental disorders. We previously established the subchronic and mild social defeat stress (sCSDS) model, a murine experimental model of depression, and investigated the metabolomic profiles of plasma and liver. Here we used omics approaches to identify stress-induced changes in the gastrointestinal tract. Mice exposed to sCSDS for 10 days showed the following changes: (1) elevation of cholic acid and reduction of 5-aminovaleric acid among cecal metabolites; (2) downregulation of genes involved in the immune response in the terminal ileum; (3) a shift in the diversity of the microbiota in cecal contents and feces; and (4) fluctuations in the concentrations of cecal metabolites produced by gut microbiota reflected in plasma and hepatic metabolites. Operational taxonomic units within the family Lachnospiraceae showed an inverse correlation with certain metabolites. The social interaction score correlated with cecal metabolites, IgA, and cecal and fecal microbiota, suggesting that sCSDS suppressed the ileal immune response, altering the balance of microbiota, which together with host cells and host enzymes resulted in a pattern of accumulated metabolites in the intestinal ecosystem distinct from that of control mice.

Dietary intake of heat-killed Lactococcus lactis H61 delays age-related hearing loss in C57BL/6J mice.

Age-related hearing loss (AHL) is a common disorder associated with aging. In this study, we investigated the effect of the intake of heat-killed Lactococcus lactis subsp. cremoris H61 (strain H61) on AHL in C57BL/6J mice. Measurement of the auditory brainstem response (ABR) demonstrated that female mice at 9 months of age fed a diet containing 0.05% strain H61 for 6 months maintained a significantly lower ABR threshold than control mice. The age-related loss of neurons and hair cells in the cochlea was suppressed by the intake of strain H61. Faecal analysis of bacterial flora revealed that the intake of strain H61 increased the prevalence of Lactobacillales, which is positively correlated with hearing ability in mice. Furthermore, plasma fatty acid levels were negatively correlated with hearing ability. Overall, the results supported that the intake of heat-killed strain H61 for 6 months altered the intestinal flora, affected plasma metabolite levels, including fatty acid levels, and retarded AHL in mice.

Effect of sodium acetate on the adhesion to porcine gastric mucin in a Lactococcus lactis strain grown on fructose.

The association of lactic acid bacteria with mucosal surfaces plays important roles in the beneficial effects of these bacteria on human health, such as colonization of the gastrointestinal tract for pathogen antagonism. Previously, we found that the adhesion of Lactococcus lactis 7-1 to porcine gastric mucin was higher with fructose than with lactose, galactose or xylose as the carbon source. In this study, we examined the effect of growth conditions on the adhesion of strain 7-1 grown on fructose. Medium components affect the adhesion: the adhesion of strain 7-1 grown with sodium acetate was higher than that without it. The enhancement of adhesion by sodium acetate was not observed under aerobic conditions. Cellular properties grown with or without sodium acetate were characterized: strain 7-1 grown with sodium acetate had similar sugar contents, and different fatty acid composition to those grown without it. Strain 7-1 grown with sodium acetate showed significantly lower cell yield and significantly higher hydrophobicity than those grown without it, which is associated with higher adhesion. Fructose and sodium acetate are frequently used in the food industry; this study may reveal a simple way to enhance the adhesion of lactic acid bacteria by growing them with these substances.

Growth characteristics of Lactobacillus brevis KB290 in the presence of bile.

Live Lactobacillus brevis KB290 have several probiotic activities, including immune stimulation and modulation of intestinal microbial balance. We investigated the adaptation of L. brevis KB290 to bile as a mechanism of intestinal survival. Strain KB290 was grown for 5 days at 37 °C in tryptone-yeast extract-glucose (TYG) broth supplemented with 0.5% sodium acetate (TYGA) containing 0.15%, 0.3%, or 0.5% bile. Growth was determined by absorbance at 620 nm or by dry weight. Growth was enhanced as the broth's bile concentration increased. Bile-enhanced growth was not observed in TYG broth or with xylose or fructose as the carbon source, although strain KB290 could assimilate these sugars. Compared with cells grown without bile, cells grown with bile had twice the cell yield (dry weight) and higher hydrophobicity, which may improve epithelial adhesion. Metabolite analysis revealed that bile induced more lactate production by glycolysis, thus enhancing growth efficiency. Scanning electron microscopy revealed that cells cultured without bile for 5 days in TYGA broth had a shortened rod shape and showed lysis and aggregation, unlike cells cultured for 1 day; cells grown with bile for 5 days had an intact rod shape and rarely appeared damaged. Cellular material leakage through autolysis was lower in the presence of bile than in its absence. Thus lysis of strain KB290 cells cultured for extended periods was suppressed in the presence of bile. This study provides new role of bile and sodium acetate for retaining an intact cell shape and enhancing cell yield, which are beneficial for intestinal survival.

New lactic acid bacterial strains from traditional Mongolian fermented milk products have altered adhesion to porcine gastric mucin depending on the carbon source.

Attachment of lactic acid bacteria to the mucosal surface of the gastrointestinal tract is a major property of probiotics. Here, we examined the ability of 21 lactic acid bacterial strains isolated from traditional fermented milk products in Mongolia to adhere to porcine gastric mucin in vitro. Higher attachment was observed with Lactobacillus delbrueckii subsp. bulgaricus strains 6-8 and 8-1 than with Lactobacillus rhamnosus GG (positive control). Lactococcus lactis subsp. cremoris strain 7-1 adhered to mucin as effectively as did strain GG. Heat inactivation decreased the adhesive ability of strains 6-8 and 8-1 but did not affect strain 7-1. The adhesion of strains 6-8, 7-1 and 8-1 was significantly inhibited when the cells were pretreated with periodate and trypsin, indicating that proteinaceous and carbohydrate-like cell surface compounds are involved in the adhesion of these strains. The adhesion of strain 7-1 was affected by the type of carbohydrate present in the growth medium, being higher with fructose than with lactose, galactose or xylose as the carbon source. The sugar content of 7-1 cells grown on various carbohydrates was negatively correlated with its adhesive ability. We provide new probiotic candidate strains and new information regarding carbohydrate preference that influences lactic acid bacterial adhesion to mucin.

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.

Protective effect of indole-3-pyruvate against ultraviolet b-induced damage to cultured HaCaT keratinocytes and the skin of hairless mice.

Previous investigations demonstrated that pyruvate protects human keratinocytes against cell damage stemming from exposure to ultraviolet B (UVB) radiation. This study endeavoured to elucidate the protective capacity of aromatic pyruvates (e.g., phenylpyruvate (PPyr), 4-hydroxyphenylpyruvate (HPPyr), and indole-3-pyruvate (IPyr)) against UVB-induced injury to skin cells, both in vitro and in vivo. Cultured human HaCaT keratinocytes were irradiated with UVB light (60 mJ/cm2) and maintained with or without test compounds (1-25 mM).In addition, the dorsal skin of hairless mice (HR-1) was treated with test compounds (10 µmol) and exposed to UVB light (1 J/cm2) twice [corrected]. The ability of the test compounds to ameliorate UVB-induced cytotoxicity and inflammation was then assessed. Aromatic pyruvates reduced cytotoxicity in UVB-irradiated HaCaT keratinocytes, and also diminished the expression of interleukin 1ß (IL-1ß) and interleukin 6 (IL-6). IPyr was more efficacious than either PPyr or HPPyr. Furthermore, only IPyr inhibited cyclooxygenase-2 (Cox-2) expression at both the mRNA and the protein level in UVB-treated keratinocytes. Topical application of IPyr to the dorsal skin of hairless mice reduced the severity of UVB-induced skin lesions, the augmentation of dermal thickness, and transepithelial water loss. Overproduction of IL-1ß and IL-6 in response to UVB radiation was also suppressed in vivo by the topical administration of IPyr. These data strongly suggest that IPyr might find utility as a UVB-blocking reagent in therapeutic strategies to lessen UVB-induced inflammatory skin damage.