Mechanism of Cooperative Degradation of Gum Arabic Arabinogalactan Protein by Bifidobacterium longum Surface Enzymes.

Gum arabic is an arabinogalactan protein (AGP) that is effective as a prebiotic for the growth of bifidobacteria in the human intestine. We recently identified a key enzyme in the glycoside hydrolase (GH) family 39, 3-O-α-d-galactosyl-α-l-arabinofuranosidase (GAfase), for the assimilation of gum arabic AGP in Bifidobacterium longum subsp. longum. The enzyme released α-d-Galp-(1→3)-l-Ara and ß-l-Arap-(1→3)-l-Ara from gum arabic AGP and facilitated the action of other enzymes for degrading the AGP backbone and modified sugar. In this study, we identified an α-l-arabinofuranosidase (BlArafE; encoded by BLLJ_1850), a multidomain enzyme with both GH43_22 and GH43_34 catalytic domains, as a critical enzyme for the degradation of modified α-l-arabinofuranosides in gum arabic AGP. Site-directed mutagenesis approaches revealed that the α1,3/α1,4-Araf double-substituted gum arabic AGP side chain was initially degraded by the GH43_22 domain and subsequently cleaved by the GH43_34 domain to release α1,3-Araf and α1,4-Araf residues, respectively. Furthermore, we revealed that a tetrasaccharide, α-l-Rhap-(1→4)-ß-d-GlcpA-(1→6)-ß-d-Galp-(1→6)-d-Gal, was a limited degradative oligosaccharide in the gum arabic AGP fermentation of B. longum subsp. longum JCM7052. The oligosaccharide was produced from gum arabic AGP by the cooperative action of the three cell surface-anchoring enzymes, GAfase, exo-ß1,3-galactanase (Bl1,3Gal), and BlArafE, on B. longum subsp. longum JCM7052. Furthermore, the tetrasaccharide was utilized by the commensal bacteria. IMPORTANCE Terminal galactose residues of the side chain of gum arabic arabinogalactan protein (AGP) are mainly substituted by α1,3/α1,4-linked Araf and ß1,6-linked α-l-Rhap-(1→4)-ß-d-GlcpA residues. This study found a multidomain BlArafE with GH43_22 and GH43_34 catalytic domains showing cooperative action for degrading α1,3/α1,4-linked Araf of the side chain of gum arabic AGP. In particular, the GH43_34 domain of BlArafE was a novel α-l-arabinofuranosidase for cleaving the α1,4-Araf linkage of terminal galactose. α-l-Rhap-(1→4)-ß-d-GlcpA-(1→6)-ß-d-Galp-(1→6)-d-Gal tetrasaccharide was released from gum arabic AGP by the cooperative action of GAfase, GH43_24 exo-ß-1,3-galactanase (Bl1,3Gal), and BlArafE and remained after B. longum subsp. longum JCM7052 culture. Furthermore, in vitro assimilation test of the remaining oligosaccharide using Bacteroides species revealed that cross-feeding may occur from bifidobacteria to other taxonomic groups in the gut.

Novel 3-O-α-d-Galactosyl-α-l-Arabinofuranosidase for the Assimilation of Gum Arabic Arabinogalactan Protein in Bifidobacterium longum subsp. longum.

Gum arabic arabinogalactan (AG) protein (AGP) is a unique dietary fiber that is degraded and assimilated by only specific strains of Bifidobacterium longum subsp. longum Here, we identified a novel 3-O-α-d-galactosyl-α-l-arabinofuranosidase (GAfase) from B. longum JCM7052 and classified it into glycoside hydrolase family 39 (GH39). GAfase released α-d-Galp-(1→3)-l-Ara and ß-l-Arap-(1→3)-l-Ara from gum arabic AGP and ß-l-Arap-(1→3)-l-Ara from larch AGP, and the α-d-Galp-(1→3)-l-Ara release activity was found to be 594-fold higher than that of ß-l-Arap-(1→3)-l-Ara. The GAfase gene was part of a gene cluster that included genes encoding a GH36 α-galactosidase candidate and ABC transporters for the assimilation of the released α-d-Galp-(1→3)-l-Ara in B. longum Notably, when α-d-Galp-(1→3)-l-Ara was removed from gum arabic AGP, it was assimilated by both B. longum JCM7052 and the nonassimilative B. longum JCM1217, suggesting that the removal of α-d-Galp-(1→3)-l-Ara from gum arabic AGP by GAfase permitted the cooperative action with type II AG degradative enzymes in B. longum The present study provides new insight into the mechanism of gum arabic AGP degradation in B. longumIMPORTANCE Bifidobacteria harbor numerous carbohydrate-active enzymes that degrade several dietary fibers in the gastrointestinal tract. B. longum JCM7052 is known to exhibit the ability to assimilate gum arabic AGP, but the key enzyme involved in the degradation of gum arabic AGP remains unidentified. Here, we cloned and characterized a GH39 3-O-α-d-galactosyl-α-l-arabinofuranosidase (GAfase) from B. longum JCM7052. The enzyme was responsible for the release of α-d-Galp-(1→3)-l-Ara and ß-l-Arap-(1→3)-l-Ara from gum arabic AGP. The presence of a gene cluster including the GAfase gene is specifically observed in gum arabic AGP assimilative strains. However, GAfase carrier strains may affect GAfase noncarrier strains that express other type II AG degradative enzymes. These findings provide insights into the bifidogenic effect of gum arabic AGP.

Structure of a Core Promoter in Bifidobacterium longum NCC2705.

Bacterial promoters consist of core sequence motifs termed -35 and -10 boxes. The consensus motifs are TTGACA and TATAAT, respectively, which were identified from leading investigations on Escherichia coli However, the consensus sequences are not likely to fit genetically divergent bacteria. The sigma factor of the genus Bifidobacterium has a characteristic polar domain in the N terminus, suggesting the possibility of specific promoter recognition. We reevaluated the structure of Bifidobacterium longum NCC2705 promoters and compared them to other bacteria. Transcriptional start sites (TSSs) of the B. longum NCC2705 strain were identified using transcriptome sequencing (RNA-Seq) analysis to extract promoter regions. Conserved motifs of a bifidobacterial promoter were determined using regions upstream of TSSs and a hidden Markov model. As a result, consensus motifs of the -35 and -10 boxes were TTGTGC and TACAAT, respectively. To assess each base of both motifs, we constructed 37 plasmids based on pKO403-TPCTcon, including the hup promoter connected with a chloramphenicol acetyltransferase as a reporter gene. This reporter assay showed two optimal motifs of the -35 and -10 boxes, namely, TTGNNN and TANNNT, respectively. We further analyzed spacer lengths between the -35 and -10 boxes via a bioinformatics approach. The spacer lengths predominant in bacteria have been generally reported to be approximately 17 bp. In contrast, the predominant spacer lengths in the genus Bifidobacterium and related species were 11 bp, in addition to 17 bp. A reporter assay to assess the spacer lengths indicated that the 11-bp spacer length produced unusually high activity.IMPORTANCE The structures of sigma factors vary among bacterial strains, indicating that recognition rules may also vary. Therefore, we investigated the promoter structure of Bifidobacterium longum NCC2705 using a bioinformatics approach and wet analyses. The most frequent and optimal motifs were similar to other bacterial consensus motifs. The optimal spacer length between the two boxes was reported to be 17 bp. It is widely applied to a bioinformatics approach for other bacteria. Unexpectedly, conserved spacer lengths were 11 bp as well as 17 bp in the genus Bifidobacterium Moreover, the sigma factor of the genus Bifidobacterium has a characteristic domain in the N terminus which may contribute to the additional functions. Hence, it would be valuable to reevaluate the promoter in other organisms.

Human gut-associated Bifidobacterium species salvage exogenous indole, a uremic toxin precursor, to synthesize indole-3-lactic acid via tryptophan.

Indole in the gut is formed from dietary tryptophan by a bacterial tryptophan-indole lyase. Indole not only triggers biofilm formation and antibiotic resistance in gut microbes but also contributes to the progression of kidney dysfunction after absorption by the intestine and sulfation in the liver. As tryptophan is an essential amino acid for humans, these events seem inevitable. Despite this, we show in a proof-of-concept study that exogenous indole can be converted to an immunomodulatory tryptophan metabolite, indole-3-lactic acid (ILA), by a previously unknown microbial metabolic pathway that involves tryptophan synthase ß subunit and aromatic lactate dehydrogenase. Selected bifidobacterial strains converted exogenous indole to ILA via tryptophan (Trp), which was demonstrated by incubating the bacterial cells in the presence of (2-13C)-labeled indole and l-serine. Disruption of the responsible genes variedly affected the efficiency of indole bioconversion to Trp and ILA, depending on the strains. Database searches against 11,943 bacterial genomes representing 960 human-associated species revealed that the co-occurrence of tryptophan synthase ß subunit and aromatic lactate dehydrogenase is a specific feature of human gut-associated Bifidobacterium species, thus unveiling a new facet of bifidobacteria as probiotics. Indole, which has been assumed to be an end-product of tryptophan metabolism, may thus act as a precursor for the synthesis of a host-interacting metabolite with possible beneficial activities in the complex gut microbial ecosystem.

Synbiotics of Bifidobacterium breve MCC1274 and lactulose enhances production of tryptophan metabolites in fermented human fecal communities.

Probiotics and prebiotics have beneficial effects on host physiology via metabolites from the gut microbiota in addition to their own. Here, we used a pH-controlled single-batch fermenter as a human gut microbiota model. We conducted fecal fermentation with Bifidobacterium breve MCC1274 (probiotic), lactulose (prebiotic), or a combination of both (synbiotic) to evaluate their influence on the gut environment. Fecal inoculum without the probiotic and prebiotic was used as the control. Principal coordinate analysis (PCoA), based on the composition of gut microbiota, showed a significant difference among the groups. The relative abundance of Bifidobacterium was significantly higher in the synbiotic group, compared to that in the other three treatment groups. The relative abundance of Blautia was the highest in the control group among the four groups. CE-TOFMS and LC-TOFMS showed that the number of metabolites detected in the synbiotic group was the highest (352 in total); 29 of the 310 hydrophilic metabolites and 17 of the 107 lipophilic metabolites were significantly different among the four groups in the Kruskal-Wallis test. A clustering based on 46 metabolites indicated that tryptophan-metabolites such as indole-3-lactic acid (ILA), indole-3-ethanol, and indole-3-carboxaldehyde, were included in a sub cluster composed of metabolites enriched in the synbiotic group. Spermidine, a major polyamine, was enriched in the two groups supplemented with the probiotic whereas spermine was enriched only in the synbiotic group. Not all metabolites enriched in the probiotic and/or synbiotic groups were found in the monocultures of the probiotic strain with or without the prebiotics. This implies that some of the metabolites were produced through the interaction of the fecal microbiota with the inoculated probiotic strain. Co-abundance networking analysis indicated the differences in the correlations between the relative abundance of the fecal microbiota genus and the tryptophan metabolites in each group. There was a strong correlation between ldh4 gene abundance and ILA concentration in the fecal fermentation. The copy number of ldh4 gene was significantly higher in the groups with the probiotic than that in the control group. In conclusion, synbiotics could enhance the production of signaling molecules in the gut environment. Our results provide an insight into more effective administration of probiotics at the molecular level.

Impact of Bifidobacterium adolescentis in patients with abdominal aortic aneurysm: a cross-sectional study.

The relationships between various diseases and the human gut microbiota (GM) have been revealed. However, the relationships between the human abdominal aortic aneurysm (AAA) and GM remains unknown. The aim of this cross-sectional study was to clarify the association between the human AAA and GM. Stool samples from 30 consecutive patients with AAA before aneurysm repair and those of 30 controls without vascular diseases were analyzed by 16S rRNA gene (V3-4) sequencing using an Illumina MiSeq system and QIIME 2. There was no significant difference in age (75 vs. 75 years) or gender (80% vs. 87% males) between the groups. No significant difference in GM composition was observed in principal coordinate analysis between the two groups, whereas the AAA group showed a significantly lower abundance of Bifidobacterium adolescentis (p<0.01) at the species level than the controls. This study demonstrated that the abundance of B. adolescentis decreased in patients with AAA. This is the first study to show the characteristics of the GM in patients with AAA. Studies are needed to reveal if causal relationships exists between the human AAA and GM.

Assimilation of arabinogalactan side chains with novel 3-O-ß-L-arabinopyranosyl-α-L-arabinofuranosidase in Bifidobacterium pseudocatenulatum.

Aim: Dietary plant fibers affect gut microbiota composition; however, the underlying microbial degradation pathways are not fully understood. We previously discovered 3-O-α-D-galactosyl-α-L-arabinofuranosidase (GAfase), a glycoside hydrolase family 39 enzyme involved in the assimilation of side chains of arabinogalactan protein (AGP), from Bifidobacterium longum subsp. longum (B. longum) JCM7052. Although GAfase homologs are not highly prevalent in the Bifidobacterium genus, several Bifidobacterium strains possess the homologs. To explore the differences in substrate specificity among the homologs, a homolog of B. longum GAfase in Bifidobacterium pseudocatenulatum MCC10289 (MCC10289_0425) was characterized. Methods: Gum arabic, larch, wheat AGP, and sugar beet arabinan were used to determine the substrate specificity of the MCC10289_0425 protein. An amino acid replacement was introduced into GAfase to identify a critical residue that governs the differentiation of substrate specificity. The growth of several Bifidobacterium strains on ß-L-arabinopyranosyl disaccharide and larch AGP was examined. Results: MCC10289_0425 was identified to be an unprecedented 3-O-ß-L-arabinopyranosyl-α-L-arabinofuranosidase (AAfase) with low GAfase activity. A single amino acid replacement (Asn119 to Tyr) at the catalytic site converted GAfase into AAfase. AAfase releases sugar source from AGP, thereby allowing B. pseudocatenulatum growth. Conclusion: Bifidobacteria have evolved several homologous enzymes with overlapping but distinct substrate specificities depending on the species. They have acquired different fitness abilities to respond to diverse plant polysaccharide structures.

2'-Fucosyllactose Increases the Abundance of Blautia in the Presence of Extracellular Fucosidase-Possessing Bacteria.

Blautia is a genus of anaerobic bacteria that is widely distributed in the mammalian gut. Recently, an increasing body of research has demonstrated a link between this genus and human health, suggesting applications as a novel probiotic strain. Moreover, we have previously shown that 2'-fucosyllactose (2'-FL), a major component of human milk oligosaccharides, increases the relative abundance of Blautia sp., particularly Blautia wexlerae, in the cultured fecal microbiota of healthy adults using a pH-controlled single-batch fermenter. However, the effects of 2'-FL on Blautia proliferation vary among individuals. In this study, we assessed the impact of the intrinsic gut microbiota on the prebiotic effects of 2'-FL. Metagenomic analysis of feces collected from all donors showed that the homolog of the intracellular GH95 α-l-fucosidase gene was considerably enriched in two non-responders (individuals who showed no increase in Blautia proliferation), whereas the homologous genes encoding extracellular α-l-fucosidase were more abundant in responders, suggesting that lactose and fucose released into the environment could be substrates mediating the growth of Blautia. In vitro assays confirmed the ability of B. wexlerae to utilize the two carbohydrates but not 2'-FL. We also observed that B. wexlerae utilized fucose released from 2'-FL by Bifidobacterium bifidum, which possessed extracellular GH95 α-l-fucosidase, in co-cultures of these two organisms. Finally, increasing the proportion of extracellular GH95 by the addition of a B. bifidum strain led to Blautia proliferation by 2'-FL in fecal cultures of the two non-responders. These findings provided valuable perspectives on individualized nutritional approaches to properly control the gut microbiota. Future clinical trials are needed to obtain further insights into the characteristics of responders vs. non-responders.

Production of Hydroxycarboxylic Acid Receptor 3 (HCA3) Ligands by Bifidobacterium.

Hydroxycarboxylic acid receptor 3 (HCA3) was recently identified in the genomes of humans and other hominids but not in other mammals. We examined the production of HCA3 ligands by Bifidobacterium spp. In addition to 4-hydroxyphenyllactic acid, phenyllactic acid (PLA), and indole-3-lactic acid (ILA), we found that LeuA was produced by Bifidobacterium as an HCA3 ligand. The four ligands produced were the mixtures of enantiomers, and D-ILA, D-PLA, and D-LeuA showed stronger activity of the HCA3 ligand than their respective L-isomers. However, there was no difference in AhR activity between the two ILA enantiomers. These results provide new insights into the HCA3 ligands produced by Bifidobacterium and suggest the importance of investigating the absolute stereo structures of these metabolites.

Colonization of Supplemented Bifidobacterium breve M-16V in Low Birth Weight Infants and Its Effects on Their Gut Microbiota Weeks Post-administration.

The colonization and persistence of probiotics introduced into the adult human gut appears to be limited. It is uncertain, however, whether probiotics can successfully colonize the intestinal tracts of full-term and premature infants. In this study, we investigated the colonization and the effect of oral supplementation with Bifidobacterium breve M-16V on the gut microbiota of low birth weight (LBW) infants. A total of 22 LBW infants (12 infants in the M-16V group and 10 infants in the control group) were enrolled. B. breve M-16V was administrated to LBW infants in the M-16V group from birth until hospital discharge. Fecal samples were collected from each subject at weeks (3.7-9.3 weeks in the M-16V group and 2.1-6.1 weeks in the control group) after discharge. qPCR analysis showed that the administrated strain was detected in 83.3% of fecal samples in the M-16V group (at log10 8.33 ± 0.99 cell numbers per gram of wet feces), suggesting that this strain colonized most of the infants beyond several weeks post-administration. Fecal microbiota analysis by 16S rRNA gene sequencing showed that the abundance of Actinobacteria was significantly higher (P < 0.01), whereas that of Proteobacteria was significantly lower (P < 0.001) in the M-16V group as compared with the control group. Notably, the levels of the administrated strain and indigenous Bifidobacterium bacteria were both significantly higher in the M-16V group than in the control group. Our findings suggest that oral administration of B. breve M-16V led to engraftment for at least several weeks post-administration and we observed a potential overall improvement in microbiota formation in the LBW infants' guts.

Rice open beak is a negative regulator of class 1 knox genes and a positive regulator of class B floral homeotic gene.

Numerous genes are involved in the regulation of plant development, including those that regulate floral homeotic genes, We identified two recessive allelic rice mutants, open beak-1 (opb-1) and opb-2, which exhibited pleiotropic defects in leaf morphogenesis, inflorescence architecture, and floral organ identity. Abnormal cell proliferation was observed in the leaves and spikelets, and ectopic or overexpression of several class 1 knox genes was detected; thus, the abnormal cell proliferation in opb mutants is probably caused by ectopic class 1 knox gene expression. The opb mutants also had defects in floral organ identity, resulting in the development of mosaic organs, including gluminous lodicules, staminoid lodicules, and pistiloid stamens. These results, together with the reduced expression of a class B gene, indicate that OPB positively regulates the expression of class B genes. Map-based cloning revealed that OPB encodes a transcription factor that is orthologous to the Arabidopsis JAGGED gene and is expressed in leaf primordia, inflorescence meristem, rachis branch meristems, floral meristem, and floral organ primordia. Taken together, our data suggest that the OPB gene affects cellular proliferation and floral organ identity through the regulation of class 1 knox genes and floral homeotic genes.

Relationship between oral and gut microbiota in elderly people.

AIM: Recent studies have suggested that oral bacteria induce systemic inflammation through the alteration of gut microbiota. We examined the relationship between oral and gut microbiota to evaluate the transition of oral bacteria to the gastrointestinal tract. METHODS: Oral samples from subgingival plaque and tongue-coating and fecal samples were collected from 29 elderly subjects (age, 80.2 ± 9.1 years) and 30 adults (age, 35.9 ± 5.0 years). Genomic DNA was extracted from all samples, and DNA sequencing of bacterial 16S rRNA genes was performed for microbiota analysis. UniFrac distances were calculated to evaluate the similarity between microbial communities. RESULTS: Unweighted UniFrac distance indicated that the elderly group had a higher similarity between fecal and subgingival plaque microbiota than the adult group. Indeed, some bacterial taxa found in oral samples had a significantly higher prevalence in the feces of the elderly group than in that of the adult group. CONCLUSIONS: The prevalence of oral bacterial transition to gut may be higher in the elderly than in adults, expecting that oral health care in the elderly will affect their gut microbiota composition and consequently promote human health.

Effects of orally administered bovine lactoperoxidase on dextran sulfate sodium-induced colitis in mice.

The effect of lactoperoxidase (LPO) on dextran sulfate sodium-induced colitis was examined in mice. After 9 d of colitis induction, weight loss, colon shortening, and the histological score were significantly suppressed in mice orally administered LPO (62.5 mg/body/d) as compared to a group administered bovine serum albumin. These results suggest that LPO exhibits anti-inflammatory effects in the gastrointestinal tract.

Genomic diversity and distribution of Bifidobacterium longum subsp. longum across the human lifespan.

Bifidobacterium longum subsp. longum represents one of the most prevalent bifidobacterial species in the infant, adult and elderly (human) gut. In the current study, we performed a comparative genome analysis involving 145 B. longum representatives, including 113 B. longum subsp. longum strains obtained from healthy Japanese subjects aged between 0 and 98 years. Although MCL clustering did not reveal any correlation between isolated strains and subject age, certain characteristics appear to be more prevalent among strains corresponding to specific host ages, such as genes involved in carbohydrate metabolism and environmental response. Remarkably, a substantial number of strains appeared to have been transmitted across family members, a phenomenon that was shown not to be confined to mother-infant pairs. This suggests that the ubiquitous distribution of B. longum subsp. longum across the human lifespan is at least partly due to extensive transmission between relatives. Our findings form a foundation for future research aimed at unraveling the mechanisms that allow B. longum strains to successfully transfer between human hosts, where they then colonize and persist in the gut environment throughout the host's lifespan.

superwoman1-cleistogamy, a hopeful allele for gene containment in GM rice.

Cleistogamy is an efficient strategy for preventing gene flow from genetically modified (GM) crops. We identified a cleistogamous mutant of rice harbouring a missense mutation (the 45th residue isoleucine to threonine; I45T) in the class-B MADS-box gene SUPERWOMAN1 (SPW1), which specifies the identities of lodicules (equivalent to petals) and stamens. In the mutant, spw1-cls, the stamens are normal, but the lodicules are transformed homeotically to lodicule-glume mosaic organs, thereby engendering cleistogamy. Since this mutation does not affect other agronomic traits, it can be used in crosses to produce transgenic lines that do not cause environmental perturbation. Molecular analysis revealed that the reduced heterodimerization ability of SPW1(I45T) with its counterpart class-B proteins OsMADS2 and OsMADS4 caused altered lodicule identity. spw1-cls is the first useful mutant for practical gene containment in GM rice. Cleistogamy is possible in many cereals by engineering class-B floral homeotic genes and thereby inducing lodicule identity changes.

Fecal microbiome in dogs with inflammatory bowel disease and intestinal lymphoma.

Although alteration of commensal microbiota is associated with chronic gastrointestinal (GI) diseases such as inflammatory bowel disease (IBD) in dogs, the microbiota composition in intestinal lymphoma, an important differential diagnosis of canine IBD, has not been investigated. The objective of this study was to compare the fecal microbiota in dogs with IBD, dogs with intestinal lymphoma, and healthy dogs. Eight dogs with IBD, eight dogs with intestinal lymphoma, and fifteen healthy dogs were included in the study. Fecal samples were analyzed by 16S rRNA gene next-generation sequencing. Rarefaction analysis failed to reveal any difference in bacterial diversity among healthy dogs and diseased dogs. Based on PCoA plots of unweighted UniFrac distances, the bacterial composition in dogs with intestinal lymphoma was different from those observed in dogs with IBD and healthy dogs. When compared with healthy dogs, intestinal lymphoma subjects showed significant increases in organisms belonging to the Eubacteriaceae family. The proportion of the family Paraprevotellaceae and the genus Porphyromonas was significantly higher in dogs with IBD compared to healthy dogs. These observations suggest that dysbiosis is associated with intestinal lymphoma as well as IBD in dogs.

Fecal dysbiosis in miniature dachshunds with inflammatory colorectal polyps.

Chronic gastrointestinal disease is associated with the alteration of gastrointestinal microbiota. Inflammatory colorectal polyps (ICRPs) are commonly observed in miniature dachshunds (MDs) in Japan and are characterized by multiple polyps that are restricted in the colorectal mucosa with severe neutrophil infiltration. This study was aimed to compare the fecal microbiota of ICRP-affected MDs with that of healthy MDs. High-throughput sequencing of amplicons derived from the V3-V4 region of the 16S rRNA gene was applied using the Illumina MiSeq system. Principal coordinates analysis revealed that fecal microbiota of ICRP-affected MDs was significantly altered compared with that of healthy MDs. Proportions of Fusobacteriaceae, Helicobacteraceae, Porphyromonadaceae, and Turicibacteraceae were significantly more abundant in ICRP-affected MDs, while those of Lachnospiraceae were significantly less abundant in ICRP-affected MDs compared with healthy MDs. These results suggest that the dysbiosis is associated with ICRPs and is a potential therapeutic target, though further investigations are needed.

Comparative Genomics Revealed Genetic Diversity and Species/Strain-Level Differences in Carbohydrate Metabolism of Three Probiotic Bifidobacterial Species.

Strains of Bifidobacterium longum, Bifidobacterium breve, and Bifidobacterium animalis are widely used as probiotics in the food industry. Although numerous studies have revealed the properties and functionality of these strains, it is uncertain whether these characteristics are species common or strain specific. To address this issue, we performed a comparative genomic analysis of 49 strains belonging to these three bifidobacterial species to describe their genetic diversity and to evaluate species-level differences. There were 166 common clusters between strains of B. breve and B. longum, whereas there were nine common clusters between strains of B. animalis and B. longum and four common clusters between strains of B. animalis and B. breve. Further analysis focused on carbohydrate metabolism revealed the existence of certain strain-dependent genes, such as those encoding enzymes for host glycan utilisation or certain membrane transporters, and many genes commonly distributed at the species level, as was previously reported in studies with limited strains. As B. longum and B. breve are human-residential bifidobacteria (HRB), whereas B. animalis is a non-HRB species, several of the differences in these species' gene distributions might be the result of their adaptations to the nutrient environment. This information may aid both in selecting probiotic candidates and in understanding their potential function as probiotics.

Effect of oral administration of metronidazole or prednisolone on fecal microbiota in dogs.

Gastrointestinal microbiota have been implicated in the pathogenesis of various gastrointestinal disorders in dogs, including acute diarrhea and chronic enteropathy. Metronidazole and prednisolone are commonly prescribed for the treatment of these diseases; however, their effects on gastrointestinal microbiota have not been investigated. The objective of this study was to evaluate the effects of these drugs on the gastrointestinal microbiota of dogs. Metronidazole was administered twice daily at 12.5 mg/kg to a group of five healthy dogs, and prednisolone at 1.0 mg/kg daily to a second group of five healthy dogs for 14 days. Fecal samples were collected before and after administration (day 0 and 14), and 14 and 28 days after cessation (day 28 and 42). DNA was extracted, and the bacterial diversity and composition of each sample were determined based on 16S ribosomal RNA (rRNA) gene sequences using next-generation sequencing (Illumina MiSeq). In the group administered metronidazole, bacterial diversity indices significantly decreased at day 14, and recovered after the cessation. Principal coordinates analysis and hierarchical dendrogram construction based on unweighted and weighted UniFrac distance matrices revealed that bacterial composition was also significantly altered by metronidazole at day 14 compared with the other time points. The proportions of Bacteroidaceae, Clostridiaceae, Fusobacteriaceae, Lachnospiraceae, Ruminococcaceae, Turicibacteraceae, and Veillonellaceae decreased, while Bifidobacteriaceae, Enterobacteriaceae, Enterococcaceae, and Streptococcaceae increased at day 14 and returned to their initial proportions by day 42. Conversely, no effect of prednisolone was observed on either the bacterial diversity or composition. Reducing pathogenic bacteria such as Fusobacteria and increasing beneficial bacteria such as Bifidobacterium through the administration of metronidazole may be beneficial for promoting gastrointestinal health; however, further investigations into the effects on diseased dogs are needed.

Orally administered lactoperoxidase ameliorates dextran sulfate sodium-induced colitis in mice by up-regulating colonic interleukin-10 and maintaining peripheral regulatory T cells.

We previously demonstrated orally administered bovine lactoperoxidase (LPO) ameliorated dextran sulfate sodium-induced colitis in mice. Here, we examine the mechanism of action of LPO. Three days after colitis induction, expression of interferon-gamma mRNA in colonic tissue was significantly decreased in mice administered LPO; while mRNA expression of interleukin (IL)-10 and regulatory T cell (Treg) marker, Foxp3, were significantly increased. The proportion of CD4+CD25+ Tregs in peripheral CD4+ T cells was also significantly elevated when LPO was administered. Nine days after colitis induction, the severity of colitis symptoms, including body weight loss and colon shortening, was reduced and expression of IL-10 mRNA was increased in mice administered LPO. The proportion of CD4+CD25+ Tregs in peripheral leukocytes was also significantly elevated when LPO was administered. These results suggest LPO ameliorates colitis by up-regulating colonic anti-inflammatory cytokines and maintaining peripheral regulatory T cells.