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Bifidobacteria are among the first colonizers of the infant gut, and human milk oligosaccharides (HMOs) in breastmilk are instrumental for the formation of a bifidobacteria-rich microbiota. However, little is known about the assembly of bifidobacterial communities. Here, by applying assembly theory to a community of four representative infant-gut associated Bifidobacterium species that employ varied strategies for HMO consumption, we show that arrival order and sugar consumption phenotypes significantly affected community formation. Bifidobacterium bifidum and Bifidobacterium longum subsp. infantis, two avid HMO consumers, dominate through inhibitory priority effects. On the other hand, Bifidobacterium breve, a species with limited HMO-utilization ability, can benefit from facilitative priority effects and dominates by utilizing fucose, an HMO degradant not utilized by the other bifidobacterial species. Analysis of publicly available breastfed infant faecal metagenome data showed that the observed trends for B. breve were consistent with our in vitro data, suggesting that priority effects may have contributed to its dominance. Our study highlights the importance and history dependency of initial community assembly and its implications for the maturation trajectory of the infant gut microbiota., 母乳栄養児の腸内におけるビフィズス菌コミュニティー形成には先住効果が大きな影響を及ぼす --ヒトミルクオリゴ糖利用能力の低いビフィズス菌B. breveが優勢となる仕組み--. 京都大学プレスリリース. 2022-07-27.

Bifidobacteria are widely used as a probiotic for their health-promoting effects. To promote their growth, bifidogenic prebiotics, including human milk oligosaccharides (HMOs), have been added to supplements and infant formula. However, the efficacy of both probiotic and prebiotic interventions is often debated, as clinical responses vary significantly by case. Here, we review clinical studies that aimed to proliferate human-residential Bifidobacterium (HRB) strains in the gut, and we highlight the difference between responders and non-responders to such interventions through an ecological, niche-based perspective and an examination of the prevalence of genes responsible for prebiotic assimilation in HRB genomes. We discuss the criteria necessary to better evaluate the efficacy of probiotic and prebiotic interventions and the recent therapeutic potential shown by synbiotics.

Mucinolytic bacteria modulate host–microbiota symbiosis and dysbiosis through their ability to degrade mucin O-glycans. However, how and to what extent bacterial enzymes are involved in the breakdown process remains poorly understood. Here we focus on a glycoside hydrolase family 20 sulfoglycosidase (BbhII) from Bifidobacterium bifidum, which releases N-acetylglucosamine-6-sulfate from sulfated mucins. Glycomic analysis showed that, in addition to sulfatases, sulfoglycosidases are involved in mucin O-glycan breakdown in vivo and that the released N-acetylglucosamine-6-sulfate potentially affects gut microbial metabolism, both of which were also supported by a metagenomic data mining analysis. Enzymatic and structural analysis of BbhII reveals the architecture underlying its specificity and the presence of a GlcNAc-6S-specific carbohydrate-binding module (CBM) 32 with a distinct sugar recognition mode that B. bifidum takes advantage of to degrade mucin O-glycans. Comparative analysis of the genomes of prominent mucinolytic bacteria also highlights a CBM-dependent O-glycan breakdown strategy used by B. bifidum. [Figure not available: see fulltext.].

Bifidobacterium longum subsp. infantis (B. infantis) is a prevalent beneficial bacterium that colonizes the human neonatal gut and is uniquely adapted to efficiently use human milk oligosaccharides (HMOs) as a carbon and energy source. Multiple studies have focused on characterizing the elements of HMO utilization machinery in B. infantis; however, the regulatory mechanisms governing the expression of these catabolic pathways remain poorly understood. A bioinformatic regulon reconstruction approach used in this study implicated NagR, a transcription factor from the ROK family, as a negative global regulator of genomic loci encoding lacto-N-biose/galacto-N-biose (LNB/GNB), lacto-N-tetraose (LNT), and lacto-N-neotetraose (LNnT) utilization pathways in B. infantis. This conjecture was corroborated by transcriptome profiling upon nagR genetic inactivation and experimental assessment of binding of recombinant NagR to predicted DNA operators. The latter approach also implicated N-acetylglucosamine (GlcNAc), a universal intermediate of LNT and LNnT catabolism, and its phosphorylated derivatives as plausible NagR transcriptional effectors. Reconstruction of NagR regulons in various Bifidobacterium lineages revealed multiple regulon expansion events, suggesting evolution from a local regulator of GlcNAc catabolism in ancestral bifidobacteria to a global regulator controlling foraging of mixtures of GlcNAc-containing host-derived glycans in mammalian gut-colonizing B. infantis and Bifidobacterium bifidum.ImportanceThe predominance of bifidobacteria in the gut of breastfed infants is attributed to the ability of these bacteria to utilize human milk oligosaccharides (HMOs). Thus, individual HMOs such as lacto-N-tetraose (LNT) and lacto-N-neotetraose (LNnT) are considered promising prebiotics that would stimulate the growth of bifidobacteria and confer multiple health benefits to preterm and malnourished children suffering from impaired (stunted) gut microbiota development. However, the rational selection of HMO-based prebiotics is hampered by the incomplete knowledge of regulatory mechanisms governing HMO utilization in target bifidobacteria. This study describes NagR-mediated transcriptional regulation of LNT and LNnT utilization in Bifidobacterium longum subsp. infantis. The elucidated regulatory network appears optimally adapted to simultaneous utilization of multiple HMOs, providing a rationale to add HMO mixtures (rather than individual components) into infant formulas. The study also provides insights into the evolutionary trajectories of complex regulatory networks controlling carbohydrate metabolism in bifidobacteria.

Human milk oligosaccharides (HMOs), which are natural bifidogenic prebiotics, were recently commercialized to fortify formula milk. However, HMO-assimilation phenotypes of bifidobacteria vary by species and strain, which has not been fully linked to strain genotype. We have recently shown that specialized uptake systems, particularly for the internalization of major HMOs (fucosyllactose (FL)), are associated with the formation of a bifidobacteria-rich gut microbial community. Phylogenetic analysis has revealed that FL transporters have diversified into two clades harboring four clusters within the Bifidobacterium genus, but the underpinning functional diversity associated with this divergence remains underexplored. In this study, we examined the HMO-consumption phenotypes of two bifidobacterial species, Bifidobacterium catenulatum subspecies kashiwanohense and Bifidobacterium pseudocatenulatum, which both possess FL binding proteins that belong to phylogenetic clusters with unknown specificities. Growth assays, heterologous gene expression experiments, and HMO-consumption analysis showed that the FL transporter type from B. catenulatum subspecies kashiwanohense JCM 15439T conferred a novel HMO-uptake pattern that includes the complex fucosylated HMOs (lacto-N-fucopentaose II and lacto-N-difucohexaose I/II). Further genomic landscape analyses of FL transporter-positive bifidobacterial strains revealed that H-antigen or Lewis antigen-specific fucosidase gene(s) and FL transporter specificities were largely aligned. These results suggest that bifidobacteria have acquired FL transporters along with the corresponding gene sets necessary to utilize the imported HMOs. Our results provide insight into the species- and strain-dependent adaptation strategies of bifidobacteria to HMO-rich environments. Importance The gut of breastfed infants is generally dominated by health-promoting bifidobacteria. Human milk oligosaccharides (HMOs) from breastmilk selectively promote the growth of specific taxa such as bifidobacteria, thus forming an HMO-mediated, host-microbe symbiosis. While the co-evolution of humans and bifidobacteria has been proposed, the underpinning adaptive strategies employed by bifidobacteria require further research. Here, we analyzed the divergence of the critical fucosyllactose (FL) HMO transporter within Bifidobacterium. We have shown that the diversification of the solute-binding proteins of the FL-transporter led to uptake specificities of fucosylated sugars ranging from simple trisaccharides to complex hexasaccharides. This transporter and the congruent acquisition of the necessary intracellular enzymes allows for bifidobacteria to import different types of HMOs in a predictable and strain-dependent manner. These findings explain the adaptation and proliferation of bifidobacteria in the competitive and HMO-rich infant gut environment and enable accurate specificity annotation of transporters from metagenomic data.

The gut microbiome is a dynamic community that significantly affects host health; it is frequently disturbed by medications such as antibiotics. Recently, probiotics have been proposed as a remedy for antibiotic-induced dysbiosis, but the efficacy of such treatments remains uncertain. Thus, the effect of specific antibiotic-probiotic combinations on the gut microbiome and host health warrants further research. We tested the effect vancomycin, amoxicillin, and ciprofloxacin on mice. Antibiotic administration was followed by one of the following recovery treatments: Bifidobacterium bifidum JCM 1254 as a probiotic (PR); fecal transplant (FT); or natural recovery (NR). Each antibiotic administration and recovery treatment was repeated three times over 9 weeks. We used the Shannon Index and Chao1 Index to determine gut microbiome diversity and assessed recovery by quantifying the magnitude of microbial shift using the Bray-Curtis Index of Dissimilarity. We determined the community composition by sequencing the V3–V4 regions of the 16S ribosomal RNA gene. To assess host health, we measured body weight and cecum weight, as well as mRNA expression of inflammation-related genes by reverse-transcription quantitative PCR. Our results show that community response varied by the type of antibiotic used, with vancomycin having the most significant effects. As a result, the effect of probiotics and fecal transplants also varied by antibiotic type. For vancomycin, the first antibiotic disturbance substantially increased the relative abundance of inflammatory species in the phylum Proteobacteria, such as Proteus, but the effect of subsequent disturbances was less pronounced, suggesting that the gut microbiome is affected by past disturbance events. Furthermore, although gut microbiome diversity did not recover, probiotic supplementation was effective in limiting cecum size enlargement and colonic inflammation caused by vancomycin. However, for amoxicillin and ciprofloxacin, the relative abundances of proinflammatory species were not greatly affected, and consequently, the effect of probiotic supplementation on community structure, cecum weight, and expression of inflammation-related genes was comparatively negligible. These results indicate that probiotic supplementation is effective, but only when antibiotics cause proinflammatory species-induced gut inflammation, suggesting that the necessity of probiotic supplementation is strongly influenced by the type of disturbance introduced to the community.

Aim: 16S rRNA gene-based microbiota analyses (16S metagenomes) using next-generation sequencing (NGS) technologies are widely used to examine the microbial community composition in environmental samples. However, the sequencing capacity of NGS is sometimes insufficient to cover the whole microbial community, especially when analyzing soil and fecal microbiotas. This limitation may have hampered the detection of minority species that potentially affect microbiota formation and structure. Methods: We developed a simple method, termed 16S metagenome-DRIP (Deeper Resolution using an Inhibitory Primer), that not only enhances minority species detection but also increases the accuracy of their abundance estimation. The method relies on the inhibition of normal amplicon formation of the 16S rRNA gene of a target major (abundant) species during the first PCR step. The addition of a biotinylated primer that is complementary to the variable sequence of the V3-V4 region of the target species inhibits a normal amplification process to form an aberrant short amplicon. The fragment is then captured by streptavidin beads for removal from the reaction mixture, and the resulting mixture is utilized for the second PCR with barcode-tag primers. Thus, this method only requires two additional experimental procedures to the conventional 16S metagenome analysis. A proof-of-concept experiment was first conducted using a mock sample consisting of the genomes of 14 bacterial species. Then, the method was applied to infant fecal samples using a Bifidobacterium-specific inhibitory primer (n = 11). Results: As a result, the reads assigned to the family Bifidobacteriaceae decreased on average from 16,657 to 1718 per sample without affecting the total read counts (36,073 and 34,778 per sample for the conventional and DRIP methods, respectively). Furthermore, the minority species detection rate increased with neither affecting Bray-Curtis dissimilarity calculated by omitting the target Bifidobacterium species (median: 0.049) nor changing the relative abundances of the non-target species. While 115 amplicon sequence variants (ASVs) were unique to the conventional method, 208 ASVs were uniquely detected for the DRIP method. Moreover, the abundance estimation for minority species became more accurate, as revealed thorough comparison with the results of quantitative PCR analysis. Conclusion: The 16S metagenome-DRIP method serves as a useful technique to grasp a deeper and more accurate microbiota composition when combined with conventional 16S metagenome analysis methods.

Summary The human body houses a variety of microbial ecosystems, such as the microbiotas on the skin, in the oral cavity and in the digestive tract. The gut microbiota is one such ecosystem that contains trillions of bacteria, and it is well established that it can significantly influence host health and diseases. With the advancement in bioinformatics tools, numerous comparative studies based on 16S ribosomal RNA (rRNA) gene sequences, metabolomics, pathological and epidemical analyses have revealed the correlative relationship between the abundance of certain taxa and disease states or amount of certain causative bioactive compounds. However, the 16S rRNA‐based taxonomic analyses using next‐generation sequencing (NGS) technology essentially detect only the majority species. Although the entire gut microbiome consists of 1013 microbial cells, NGS read counts are given in multiples of 106, making it difficult to determine the diversity of the entire microbiota. Some recent studies have reported instances where certain minority species play a critical role in creating locally stable conditions for other species by stabilizing the fundamental microbiota, despite their low abundance. These minority species act as ‘keystone species’, which is a species whose effect on the community is disproportionately large compared to its relative abundance. One of the attributes of keystone species within the gut microbiota is its extensive enzymatic capacity for substrates that are rare or difficult to degrade for other species, such as dietary fibres or host‐derived complex glycans, like human milk oligosaccharides (HMOs). In this paper, we propose that more emphasis should be placed on minority taxa and their possible role as keystone species in gut microbiota studies by referring to our recent studies on HMO‐mediated microbiota formation in the infant gut.

The traditional debate on alternative community states has been over whether or not they exist. Studies of community assembly have examined the role of assembly history in driving community divergence, but the context in which assembly history becomes important is a continued topic of interest. In this study, we created communities of bacterivorous ciliated protists in laboratory microcosms and manipulated assembly history, disturbance frequency, and the presence of dispersal among local communities to investigate the mechanisms behind community divergence. Specifically, we sought to understand how the role of assembly history changed in response to disturbance, dispersal, and the combination of the two. Assembly history influenced the identity of the dominant species through priority effects, and dispersal and disturbance showed interactive effects on both alpha and beta diversity. Dispersal substantially increased alpha diversity, but only in the absence of disturbance, and it reduced beta diversity, but not in the presence of low or mixed disturbance. These results demonstrate that the role of assembly history and the strength of priority effects depend on community context, suggesting that understanding the interactions between various factors shaping community assembly is important for understanding how ecological communities are structured.

Certain species of the genus Bifidobacterium represent human symbionts. Many studies have shown that the establishment of symbiosis with such bifidobacterial species confers various beneficial effects on human health. Among the more than ten (sub)species of human gut-associated Bifidobacterium that have significantly varied genetic characteristics at the species level, Bifidobacterium bifidum is unique in that it is found in the intestines of a wide age group, ranging from infants to adults. This species is likely to have adapted to efficiently degrade host-derived carbohydrate chains, such as human milk oligosaccharides (HMOs) and mucin O-glycans, which enabled the longitudinal colonization of intestines. The ability of this species to assimilate various host glycans can be attributed to the possession of an adequate set of extracellular glycoside hydrolases (GHs). Importantly, the polypeptides of those glycosidases frequently contain carbohydrate-binding modules (CBMs) with deduced affinities to the target glycans, which is also a distinct characteristic of this species among members of human gut-associated bifidobacteria. This review firstly describes the prevalence and distribution of B. bifidum in the human gut and then explains the enzymatic machinery that B. bifidum has developed for host glycan degradation by referring to the functions of GHs and CBMs. Finally, we show the data of co-culture experiments using host-derived glycans as carbon sources, which underpin the interesting altruistic behavior of this species as a cross-feeder.