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.

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.

Growth-promoting effect of alginate on Faecalibacterium prausnitzii through cross-feeding with Bacteroides.

Faecalibacterium prausnitzii is a commensal gut bacterium that is thought to provide protection against inflammatory diseases. However, this bacterium is extremely oxygen sensitive, which limits its industrial application as a probiotic. The use of prebiotics to increase the abundance of this bacterium in the gut is an alternative strategy to achieve its possible health-promoting effect. We evaluated nine substances as candidate prebiotics for F. prausnitzii using a pH-controlled single-batch fermenter as a human gut microbiota model. Of them, alginate markedly increased the relative abundance of F. prausnitzii, as determined by the significant increase in the number of 16S rRNA sequences corresponding to this bacterial taxon in the fecal fermentation samples detected by real-time PCR. However, F. prausnitzii strains were incapable of utilizing alginate in monoculture, implying that an interaction with another gut microbe was required. There was a positive correlation between the relative abundance of F. prausnitzii and that of Bacteroides when cultured in medium containing alginate as the sole carbon source, indicative of cross-feeding between these bacteria. Interestingly, the ratio of acetic acid, a known substrate for F. prausnitzii, produced by Bacteroides was significantly higher in the alginate-containing medium than in media containing other prebiotic candidates. Bacterially degraded alginate oligosaccharides (AOS) remained in the medium after Bacteroides monoculture, and an isolate of F. prausnitzii was able to utilize a portion of them. Genomic sequencing revealed that the strain that consumed the AOS contained an ATP-binding cassette transporter, an alginate lyase, and AlgQ1/2 homologs encoding solute-binding proteins. Furthermore, in real-time PCR analyses, AlgQ1/2 homologs were detected in fecal samples collected from 309 of 452 (68.4%) Japanese subjects. Thus, the products of alginate assimilation by Bacteroides may promote the growth of F. prausnitzii.

Tolerance mechanisms of human-residential bifidobacteria against lysozyme.

We previously reported that lysozyme present in breast milk is a selection factor for bifidobacterial colonization in infant human intestines. This study is aimed at examining their underlying mechanisms. Human-residential bifidobacteria (HRB) generally exhibited higher tolerance than non-HRB to lysozymes, except B. bifidum subspecies. To assess the involvement of enzymatic activity of lysozyme, peptidoglycan (PG) was isolated and the degree of O-acetylation (O-Ac) in 19 strains, including both HRB and non-HRB, was determined. Variety in the degree of O-Ac was observed among each of the Bifidobacterium species; however, all purified PGs were found to be tolerant to lysozyme, independent of their O-Ac degree. In addition, De-O-Ac of PGs affected the sensitivity to lysozyme of only B. longum-derived PG. To examine the non-enzymatic antibacterial activity of lysozyme on bifidobacteria, lysozyme was heat-denatured. The HRB and non-HRB strains exhibited similar patterns of susceptibility to intact lysozyme as they did to heat-denatured lysozyme. In addition, strains of B. bifidum (30 strains), which showed various tolerance of lysozyme, also exhibited similar patterns of susceptibility to intact lysozyme as they did to heat-denatured lysozyme. These results suggest that bifidobacteria are resistant to the peptidoglycan-degrading property of lysozyme, and the tolerance to lysozyme among some HRB strains is due to resistance to the non-enzymatic antibacterial activity of lysozyme.

Age-related changes in gut microbiota composition from newborn to centenarian: a cross-sectional study.

BACKGROUND: It has been reported that the composition of human gut microbiota changes with age; however, few studies have used molecular techniques to investigate the long-term, sequential changes in gut microbiota composition. In this study, we investigated the sequential changes in gut microbiota composition in newborn to centenarian Japanese subjects. RESULTS: Fecal samples from 367 healthy Japanese subjects between the ages of 0 and 104 years were analyzed by high-throughput sequencing of amplicons derived from the V3-V4 region of the 16S rRNA gene. Analysis based on bacterial co-abundance groups (CAGs) defined by Kendall correlations between genera revealed that certain transition types of microbiota were enriched in infants, adults, elderly individuals and both infant and elderly subjects. More positive correlations between the relative abundances of genera were observed in the elderly-associated CAGs compared with the infant- and adult-associated CAGs. Hierarchical Ward's linkage clustering based on the abundance of genera indicated five clusters, with median (interquartile range) ages of 3 (0-35), 33 (24-45), 42 (32-62), 77 (36-84) and 94 (86-98) years. Subjects were predominantly clustered with their matched age; however, some of them fell into mismatched age clusters. Furthermore, clustering based on the proportion of transporters predicted by phylogenetic investigation of communities by reconstruction of unobserved states (PICRUSt) showed that subjects were divided into two age-related groups, the adult-enriched and infant/elderly-enriched clusters. Notably, all the drug transporters based on Kyoto Encyclopedia of Genes and Genomes (KEGG) Orthology groups were found in the infant/elderly-enriched cluster. CONCLUSION: Our results indicate some patterns and transition points in the compositional changes in gut microbiota with age. In addition, the transporter property prediction results suggest that nutrients in the gut might play an important role in changing the gut microbiota composition with age.

Differences in folate production by bifidobacteria of different origins.

Bifidobacteria are known to produce folate, a vital nutrient for humans. Previous studies have suggested that the ability to produce folate is strain dependent, but further adequate evaluation is needed. In this study, a total of 44 strains, including 12 species and 7 subspecies, of bifidobacteria were investigated for the production of folate during cultivation in medium containing essential levels of folate for growth of the tested strains. An in vitro assay showed that all strains of human-residential bifidobacteria (HRB) were able to produce folate, whereas most strains of non-HRB were not, with the exception of the B. thermophilum and B. longum ssp. suis strains. The differences in the in vivo production of folate by HRB and non-HRB were confirmed using mono-associated mice. The fecal folate concentrations, blood levels of hemoglobin and mean corpuscular volumes were significantly higher in the mice colonized with a folate producer, B. longum subsp. longum, compared with mice colonized with a nonproducer, B. animalis subsp. lactis. Our results confirmed the differences in folate production between HRB and non-HRB strains and suggested the benefit of HRB to hosts from the perspective of potential folate delivery.

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.