Keita Nishiyama

Immunomodulatory lactic acid bacteria (immunobiotics) have been proposed to prevent bovine mastitis through the enhancement of host immune responses. Furthermore, accumulating evidence suggests that non-viable immunobiotics (postimmunobiotics) can be as effective as viable bacteria in modulating immunity in the bovine host. However, the molecular mechanisms underlying the immunomodulatory effects of heat-inactivated lactic acid bacteria in bovine mammary epithelial cells remain incompletely understood. In the present study, we evaluated the effects of heat-killed Ligilactobacillus salivarius FFIG56 (hk-FFIG56) on the Toll-like receptor (TLR)-4-mediated innate immune response in bovine mammary epithelial cells (BMECs) in vitro. BMECs were pre-stimulated with hk-FFIG56 and subsequently challenged with Escherichia coli-derived lipopolysaccharide (LPS). Transcriptomic analyses revealed that, compared with cells stimulated with LPS alone, hk-FFIG56-pretreated BMECs exhibited 743 upregulated and 881 downregulated genes, indicating substantial changes in the LPS-induced transcriptional response. Among the inflammatory mediators evaluated, hk-FFIG56 pretreatment significantly reduced LPS-induced interleukin-6 (IL-6) and interleukin-8 (IL-8) expression at 12 h, as well as CCL2 expression at 6 and 12 h, compared with cells challenged with LPS alone at the corresponding time points. Differential regulation of the negative regulators of TLR signaling, IRAK-M, Tollip, and Mitogen-activated protein kinase phosphatase 1 (MKP-1), was also detected in BMECs pretreated with hk-FFIG56. In addition, hk-FFIG56 pretreatment prevented LPS-induced reduction in IκBα protein levels, whereas no significant changes were detected in the p-ERK/ERK ratio at the evaluated time points. Furthermore, hk-FFIG56 pretreatment attenuated the LPS-induced reduction in CSN3 (κ-casein) mRNA expression in BMECs. Overall, these results suggest that hk-FFIG56 modulates LPS-induced inflammatory responses and CSN3 mRNA expression in BMECs, supporting its potential as a postimmunobiotic candidate for further investigation in the context of bovine mastitis prevention.

Flavonoids are plant-derived polyphenols with diverse biological activities; however, the mechanisms by which individual compounds inhibit intestinal pathogens remain unclear. In this study, we found that taxifolin (dihydroquercetin) showed pronounced inhibitory activity against Clostridium perfringens , a major gram-positive enteropathogen, whereas most other bacterial species tested were less affected. Notably, at concentrations below those causing complete growth inhibition, taxifolin significantly impaired mucin adhesion and biofilm formation, indicating suppression of colonization-associated phenotypes rather than bactericidal activity. In an antibiotic-pretreated mouse infection model, oral administration of taxifolin significantly reduced C. perfringens intestinal colonization during the early phase of infection. Mechanistically, proteomic analysis of C. perfringens revealed marked alterations in surface-associated and extracellular proteins, including stress response factors (MreB, LytR, and ClpB), and enzymes linked to iron- and redox-dependent metabolism, consistent with an iron-limiting stress response. Scanning electron microscopy further revealed pronounced cell elongation in taxifolin-treated cells, consistent with impaired cell division under stress conditions. Importantly, Fe ²⁺ supplementation partially restored bacterial growth, normal morphology, mucin adhesion, and biofilm formation, whereas supplementation with other divalent ions failed to restore growth. Similar phenotypes were observed following treatment with the iron-specific chelator 2,2′-bipyridyl. Collectively, our findings support a working model in which iron limitation-associated physiological stress contributes to the inhibitory effects of taxifolin on C. perfringens growth and colonization-related phenotypes. This study provides insights into the interaction between dietary flavonoids and clostridial pathogens, highlighting the potential of taxifolin as a plant-derived compound for limiting C. perfringens intestinal colonization. IMPORTANCE: Clostridium perfringens , a clinically important pathogen in both humans and animals, causes various histotoxic and enteric diseases, including gas gangrene and foodborne or non-foodborne diarrhea. Its control remains challenging due to increasing antimicrobial resistance and the need to preserve beneficial gut microbiota. Here, we identified taxifolin, a naturally occurring flavonoid, as a potential inhibitor of C. perfringens that suppressed its growth and colonization-associated phenotypes while exerting comparatively limited effects on most other tested bacterial species, including commensal bacteria. Proteomic and physiological analyses revealed stress responses associated with iron metabolism, redox balance, and cell division, which were accompanied by reduced mucin adhesion, biofilm formation, and bacterial proliferation. Our findings suggest that taxifolin can reduce the colonization capacity of C. perfringens and support its potential use as a plant-derived approach for limiting C. perfringens intestinal colonization in humans and animals.

The gut–skin axis is a bidirectional communication network linking the gastrointestinal tract, skin, microbiota, and immune system. As major barrier organs, the gut and skin harbor complex microbial communities that contribute to tissue homeostasis, immune regulation, and protection from environmental insults. Increasing evidence indicates that dysbiosis of the gut and skin microbiota is associated with inflammatory diseases through interconnected microbial, metabolic, and immune pathways. Clinical observations further reveal strong associations between gastrointestinal disorders, including inflammatory bowel disease (IBD) and celiac disease, and cutaneous manifestations. Atopic dermatitis (AD) is characterized by epithelial barrier dysfunction, immune dysregulation, microbial imbalance, and environmental influences. Patients with AD frequently exhibit reduced gut microbial diversity, depletion of beneficial short-chain fatty acid (SCFA)-producing bacteria, and enrichment of potentially pathogenic microorganisms. Cutaneous dysbiosis, particularly expansion of Staphylococcus aureus, can further impair the skin barrier and sustain inflammation. Microbial metabolites, including SCFAs, tryptophan-derived aryl hydrocarbon receptor ligands, and bile acid metabolites, may mediate gut–skin communication by regulating epithelial integrity, immune tolerance, and inflammatory signaling. IBD is also increasingly recognized as a systemic disorder involving alterations in skin microbiota and cutaneous immunity. Intestinal inflammation may disrupt immune tolerance to skin commensals and promote cutaneous inflammation through cytokine signaling and immune-cell trafficking, while emerging evidence suggests reciprocal skin-to-gut effects. Epidemiologic, genetic, and clinical studies indicate an association between AD and IBD, potentially reflecting shared genetic susceptibility, barrier dysfunction, dysbiosis, and immune pathways. This review summarizes current evidence linking the gut–skin axis to AD and IBD.

This protocol describes a microfluidic co-culture system to investigate interactions between live Lactiplantibacillus plantarum JCM 1149T and swine intestinal epithelial cells (SIECs). We optimize the bacterial and epithelial cell culture conditions, perform co-culture under continuous microfluidic flow, and collect samples suitable for downstream transcriptomic and metabolomic analyses following co-culture. This protocol provides a reproducible platform for investigating live bacteria–epithelial interactions while limiting bacterial overgrowth and maintaining epithelial barrier integrity.

DNA methylation plays a fundamental role in maintaining intestinal homeostasis, immune tolerance, and inflammatory balance. Active DNA demethylation, mediated by the ten-eleven translocation family of dioxygenases (TET1, TET2, and TET3), has emerged as an important epigenetic mechanism linking environmental and metabolic cues to gene regulatory programs in the gut. In the intestinal epithelium, TET-dependent DNA hydroxymethylation contributes to intestinal stem cell maintenance, epithelial differentiation, regeneration, and barrier integrity. Perturbations in TET activity are associated with epithelial dysfunction, chronic inflammation, and increased susceptibility to colorectal tumorigenesis. Within the immune compartment, TET-mediated demethylation is required for the epigenetic stabilization of gut-associated immune cells. Altered TET function has been implicated in immune imbalance in inflammatory bowel disease, Hirschsprung’s disease, and colitis-associated colorectal cancer. Emerging evidence further indicates that intestinal microbiota-derived metabolites, including short-chain fatty acids and aryl hydrocarbon receptor ligands, modulate TET activity, positioning TET enzymes as epigenetic sensors of microbial and metabolic signals. In turn, TET-dependent programs shape immune responses to commensal microbes and pathogens, establishing a bidirectional microbiota–epigenetic axis that influences both intestinal and systemic immunity. In this review, we summarize and critically evaluate current evidence on the roles of TET enzymes in intestinal epithelial biology, immune cell regulation, and host–microbiota interactions in colorectal inflammation and disease.

Lactic acid bacteria (LAB), particularly members of the genus Lactobacillus, have emerged as promising biological agents with antimicrobial and anti-biofilm properties. While numerous individual studies have reported their inhibitory effects against pathogenic microorganisms, a systematic understanding that integrates their functional components, molecular mechanisms, and material-based applications remains lacking. In this review, we provide a comprehensive and component-oriented overview of LAB-mediated antimicrobial strategies. We first summarize secreted factors, including organic acids, bacteriocins, hydrogen peroxide, and extracellular vesicles, which collectively contribute to direct pathogen inhibition and environmental modulation. We then discuss cell-associated components such as surface-layer proteins and exopolysaccharides, highlighting their roles in adhesion interference and competitive exclusion. In addition, we examine whole-cell effects, including niche competition, quorum sensing disruption, and host immune modulation. Importantly, we place particular emphasis on the anti-biofilm activity of lactobacilli, detailing mechanisms involved in the prevention of the pathogen initial adhesion, disruption of extracellular polymeric substance matrices, and destabilization of mature biofilms. Finally, we explore emerging strategies that integrate lactobacilli with biomaterials, particularly hydrogel-based systems, to achieve controlled delivery, enhanced stability, and sustained antimicrobial activity. These biohybrid approaches represent a promising direction for the development of next-generation antimicrobial materials. These findings support the concept of LAB-based living antimicrobial materials as a next-generation strategy to combat biofilm-associated infections. Overall, this review aims to bridge the gap between molecular functions and translational applications of lactobacilli, providing new insights into its potential as a versatile platform for antimicrobial and anti-biofilm interventions.

Aims: Our aim was to evaluate calcium alginate beads as a physical cultivation platform for probiotic bacteria and investigate their effects on bacterial growth localization, release dynamics, and viability under different environmental conditions. Methods and Results: Calcium alginate beads were fabricated using sodium alginate at different concentrations (1.0–2.0%, w/v), and their structural stability, permeability, and pH resistance were assessed. Lacticaseibacillus paracasei was used as the primary model organism. Among the tested formulations, 2.0% alginate exhibited the lowest permeability (~40%) compared with 1.0% and 1.5% alginate (~60%) and maintained structural integrity under alkaline conditions (pH 11–12). Encapsulated L. paracasei proliferated predominantly within the bead matrix, while less than 1% of total bacteria were released into the surrounding medium during the first 12 h and approximately 8% released after 24 h. Encapsulation also maintained higher bacterial viability over 24–48 h compared with conventional liquid culture conditions. Encapsulated cells could be efficiently recovered using sodium citrate without marked loss of viability. In addition, encapsulation enabled the growth of Bifidobacterium animalis subsp. lactis under aerobic incubation conditions, suggesting the formation of locally protective microenvironments within the gel matrix. Conclusions: Calcium alginate beads function as controllable microenvironmental regulators that influence bacterial growth, release behavior, and long-term viability. These findings suggest that alginate-based encapsulation systems may provide a simple and scalable platform for controlled microbial cultivation and probiotic-related applications.

Enterotoxigenic Escherichia coli (ETEC) is a leading cause of post-weaning diarrhea in piglets, and the rise of antimicrobial resistance underscores the urgent need for antibiotic alternatives in swine production. Bacillus species are promising candidates owing to their ability to produce antimicrobial secondary metabolites, particularly cyclic lipopeptides such as surfactins, iturins, and fengycins. Although Bacillus lipopeptides are known to exhibit antibacterial activity against E. coli , the specific compounds responsible for the inhibition of ETEC in probiotic-relevant Bacillus strains remain undefined. Here, we sought to identify extracellular metabolite(s) associated with the anti-ETEC activity of Bacillus subtilis DB9011. DB9011 culture supernatant strongly inhibited the growth of ETEC and other enteric pathogens, including Salmonella Typhimurium. Bioactivity-guided fractionation of the culture supernatant, followed by liquid chromatography–quadrupole time-of-flight mass spectrometry, identified C17 fengycin B as a major active metabolite enriched in inhibitory fractions, but absent from the non-inhibitory type strain NBRC13719ᵀ. A fengycin-containing fraction enriched in C17 fengycin B exhibited concentration-dependent antibacterial activity against ETEC and enhanced growth inhibition when combined with colistin, whereas incomplete inhibition at native supernatant-equivalent concentrations indicated that additional metabolites contribute to the overall antibacterial phenotype. While fengycins have been primarily recognized for antifungal activity and, more recently, for effects on gram-negative bacteria, our findings identify C17 fengycin B as a major contributor to the ETEC-inhibitory phenotype of B. subtilis DB9011. These findings provide mechanistic insights into how probiotic-relevant Bacillus strains inhibit enteric pathogens; nonetheless, further studies are required to evaluate in vivo relevance and functional performance under intestinal conditions. IMPORTANCE: Antimicrobial resistance poses a major threat to livestock production, where antibiotics remain widely used to control post-weaning diarrhea caused by enterotoxigenic Escherichia coli (ETEC). Supplementation with Bacillus subtilis DB9011 is known to reduce pathogenic E. coli colonization in piglets; however, the metabolites responsible for this protective effect remain unidentified. Here, using a bioactivity-guided fractionation approach, we identify C17 fengycin B as a major contributing metabolite associated with the anti-ETEC activity of DB9011 and show enhanced growth inhibition during co-treatment with colistin. These findings clarify the functional contribution of Bacillus -derived metabolites to pathogen inhibition and establish a mechanistic basis for strain-specific antimicrobial activity.

Sialidase facilitates the utilization of host mucin glycans and milk oligosaccharides by intestinal bacteria. We identified Bifidobacterium canis SY61 as a sialidase-producing canine gut bacterium and sequenced its genome, providing the first gap-free genomic resource for this species and advancing our understanding of carbohydrate metabolism in the canine intestinal microbiota.