Multi-omics Approaches To Decipher the Impact of Diet and Host Physiology on the Mammalian Gut Microbiome.

In recent years, various studies have demonstrated that the gut microbiota influences host metabolism. However, these studies were focused primarily on a single or a limited range of host species, thus preventing a full exploration of possible taxonomic and functional adaptations by gut microbiota members as a result of host-microbe coevolution events. In the current study, the microbial taxonomic profiles of 250 fecal samples, corresponding to 77 host species that cover the mammalian branch of the tree of life, were reconstructed by 16S rRNA gene-based sequence analysis. Moreover, shotgun metagenomics was employed to investigate the metabolic potential of the fecal microbiomes of 24 mammals, and subsequent statistical analyses were performed to assess the impact of host diet and corresponding physiology of the digestive system on gut microbiota composition and functionality. Functional data were confirmed and extended through metatranscriptome assessment of gut microbial populations of eight animals, thus providing insights into the transcriptional response of gut microbiota to specific dietary lifestyles. Therefore, the analyses performed in this study support the notion that the metabolic features of the mammalian gut microbiota have adapted to maximize energy extraction from the host's diet.IMPORTANCE Diet and host physiology have been recognized as main factors affecting both taxonomic composition and functional features of the mammalian gut microbiota. However, very few studies have investigated the bacterial biodiversity of mammals by using large sample numbers that correspond to multiple mammalian species, thus resulting in an incomplete understanding of the functional aspects of their microbiome. Therefore, we investigated the bacterial taxonomic composition of 250 fecal samples belonging to 77 host species distributed along the tree of life in order to assess how diet and host physiology impact the intestinal microbial community by selecting specific microbial players. Conversely, the application of shotgun metagenomics and metatranscriptomics approaches to a group of selected fecal samples allowed us to shed light on both metabolic features and transcriptional responses of the intestinal bacterial community based on different diets.

Metagenomic dissection of the canine gut microbiota: insights into taxonomic, metabolic and nutritional features.

Domestication of dogs from wolves is the oldest known example of ongoing animal selection, responsible for generating more than 300 dog breeds worldwide. In order to investigate the taxonomic and functional evolution of the canine gut microbiota, a multi-omics approach was applied to six wild wolves and 169 dog faecal samples, the latter encompassing 51 breeds, which fully covers currently known canine genetic biodiversity. Specifically, 16S rRNA gene and bifidobacterial Internally Transcribed Spacer (ITS) profiling were employed to reconstruct and then compare the canine core gut microbiota to those of wolves and humans, revealing that artificial selection and subsequent cohabitation of dogs with their owners influenced the microbial population of canine gut through loss and acquisition of specific bacterial taxa. Moreover, comparative analysis of the intestinal bacterial population of dogs fed on Bones and Raw Food (BARF) or commercial food (CF) diet, coupled with shotgun metagenomics, highlighted that both bacterial composition and metabolic repertoire of the canine gut microbiota have evolved to adapt to high-protein or high-carbohydrates intake. Altogether, these data indicate that artificial selection and domestication not only affected the canine genome, but also shaped extensively the bacterial population harboured by the canine gut.

Bifidobacterium bifidum and the infant gut microbiota: an intriguing case of microbe-host co-evolution.

Bifidobacterium bifidum is reported to be among the first colonizers of the newborn's gastrointestinal tract due to its ability to metabolize human milk oligosaccharides (HMOs). In order to investigate biological features that allow this bifidobacterial species to colonize a newborn, bifidobacterial internally transcribed spacer profiling of stool samples of 50 mother-infant dyads, as well as corresponding breastmilk samples, was performed. Hierarchical clustering based on bifidobacterial population profiles found in infant faecal samples revealed the presence of four bifidobacterial clusters or the so-called bifidotypes. Bifidobacterium bifidum was shown to be a key member among bifidotypes, in which its presence correlate with several different bifidobacterial species retrieved in infant faecal samples. For this reason, we investigated cross-feeding behaviour facilitated by B. bifidum on a bioreactor model using human milk as growth substrate. Transcriptional profiles of this strain were evaluated when grown on nine specific glycans typically constituting HMOs. Remarkably, these analyses suggest extensive co-evolution with the host and other bifidobacterial species in terms of resource provision and sharing, respectively, activities that appear to support a bifidobacteria-dominant microbiome.

Dissecting the Evolutionary Development of the Species Bifidobacterium animalis through Comparative Genomics Analyses.

Bifidobacteria are members of the gut microbiota of animals, including mammals, birds, and social insects. In this study, we analyzed and determined the pangenome of Bifidobacterium animalis species, encompassing B. animalis subsp. animalis and the B. animalis subsp. lactis taxon, which is one of the most intensely exploited probiotic bifidobacterial species. In order to reveal differences within the B. animalis species, detailed comparative genomics and phylogenomics analyses were performed, indicating that these two subspecies recently arose through divergent evolutionary events. A subspecies-specific core genome was identified for both B. animalis subspecies, revealing the existence of subspecies-defining genes involved in carbohydrate metabolism. Notably, these in silico analyses coupled with carbohydrate profiling assays suggest genetic adaptations toward a distinct glycan milieu for each member of the B. animalis subspecies, resulting in a divergent evolutionary development of the two subspecies.IMPORTANCE The majority of characterized B. animalis strains have been isolated from human fecal samples. In order to explore genome variability within this species, we isolated 15 novel strains from the gastrointestinal tracts of different animals, including mammals and birds. The present study allowed us to reconstruct the pangenome of this taxon, including the genome contents of 56 B. animalis strains. Through careful assessment of subspecies-specific core genes of the B. animalis subsp. animalis/lactis taxon, we identified genes encoding enzymes involved in carbohydrate transport and metabolism, while unveiling specific gene acquisition and loss events that caused the evolutionary emergence of these two subspecies.

Understanding the gut-kidney axis in nephrolithiasis: an analysis of the gut microbiota composition and functionality of stone formers.

OBJECTIVES: The involvement of the gut microbiota in the pathogenesis of calcium nephrolithiasis has been hypothesised since the discovery of the oxalate-degrading activity of Oxalobacter formigenes, but never comprehensively studied with metagenomics. The aim of this case-control study was to compare the faecal microbiota composition and functionality between recurrent idiopathic calcium stone formers (SFs) and controls. DESIGN: Faecal samples were collected from 52 SFs and 48 controls (mean age 48±11). The microbiota composition was analysed through 16S rRNA microbial profiling approach. Ten samples (five SFs, five controls) were also analysed with deep shotgun metagenomics sequencing, with focus on oxalate-degrading microbial metabolic pathways. Dietary habits, assessed through a food-frequency questionnaire, and 24-hour urinary excretion of prolithogenic and antilithogenic factors, including calcium and oxalate, were compared between SFs and controls, and considered as covariates in the comparison of microbiota profiles. RESULTS: SFs exhibited lower faecal microbial diversity than controls (Chao1 index 1460±363vs 1658±297, fully adjusted p=0.02 with stepwise backward regression analysis). At multivariate analyses, three taxa (Faecalibacterium, Enterobacter, Dorea) were significantly less represented in faecal samples of SFs. The Oxalobacter abundance was not different between groups. Faecal samples from SFs exhibited a significantly lower bacterial representation of genes involved in oxalate degradation, with inverse correlation with 24-hour oxalate excretion (r=-0.87, p=0.002). The oxalate-degrading genes were represented in several bacterial species, whose cumulative abundance was inversely correlated with oxaluria (r=-0.85, p=0.02). CONCLUSIONS: Idiopathic calcium SFs exhibited altered gut microbiota composition and functionality that could contribute to nephrolithiasis physiopathology.

Tracking the Taxonomy of the Genus Bifidobacterium Based on a Phylogenomic Approach.

For decades, bacterial taxonomy has been based on in vitro molecular biology techniques and comparison of molecular marker sequences to measure the degree of genetic similarity and deduce phylogenetic relatedness of novel bacterial species to reference microbial taxa. Due to the advent of the genomic era, access to complete bacterial genome contents has become easier, thereby presenting the opportunity to precisely investigate the overall genetic diversity of microorganisms. Here, we describe a high-accuracy phylogenomic approach to assess the taxonomy of members of the genus Bifidobacterium and identify apparent misclassifications in current bifidobacterial taxonomy. The developed method was validated by the classification of seven novel taxa belonging to the genus Bifidobacterium by employing their overall genetic content. The results of this study demonstrate the potential of this whole-genome approach to become the gold standard for phylogenomics-based taxonomic classification of bacteria.IMPORTANCE Nowadays, next-generation sequencing has given access to genome sequences of the currently known bacterial taxa. The public databases constructed by means of these new technologies allowed comparison of genome sequences between microorganisms, providing information to perform genomic, phylogenomic, and evolutionary analyses. In order to avoid misclassifications in the taxonomy of novel bacterial isolates, new (bifido)bacterial taxons should be validated with a phylogenomic assessment like the approach presented here.

Phylotype-Level Profiling of Lactobacilli in Highly Complex Environments by Means of an Internal Transcribed Spacer-Based Metagenomic Approach.

The genus Lactobacillus is a widespread taxon, members of which are highly relevant to functional and fermented foods, while they are also commonly present in host-associated gut and vaginal microbiota. Substantial efforts have been undertaken to disclose the genetic repertoire of all members of the genus Lactobacillus, and yet their species-level profiling in complex matrices is still undeveloped due to the poor phylotype resolution of profiling approaches based on the 16S rRNA gene. To overcome this limitation, an internal transcribed spacer (ITS)-based profiling method was developed to accurately profile lactobacilli at the species level. This approach encompasses a genus-specific primer pair combined with a database of ITS sequences retrieved from all available Lactobacillus genomes and a script for the QIIME software suite that performs all required steps to reconstruct a species-level profile. This methodology was applied to several environments, i.e., human gut and vagina and the ceca of free-range chickens, as well as whey and fresh cheese. Interestingly, the data collected confirmed a relevant role of lactobacilli present in functional and fermented foods in defining the population harbored by the human gut, while, unsurprisingly perhaps, the ceca of free-range chickens were observed to be dominated by lactobacilli characterized in birds living in natural environments. Moreover, vaginal swabs confirmed the existence of previously hypothesized community state types, while analysis of whey and fresh cheese revealed a dominant presence of single Lactobacillus species used as starters for cheese production. Furthermore, application of this ITS profiling method to a mock Lactobacillus community allowed a minimal resolution level of <0.006 ng/µl.IMPORTANCE The genus Lactobacillus is a large and ubiquitous taxon of high scientific and commercial relevance. Despite the fact that the genetic repertoire of Lactobacillus species has been extensively characterized, the ecology of this genus has been explored by metataxonomic techniques that are accurate down to the genus or phylogenetic group level only. Thus, the distribution of lactobacilli in environmental or processed food samples is relatively unexplored. The profiling protocol described here relies on the use of the internal transcribed spacer to perform an accurate classification in a target population of lactobacilli with a <0.006-ng/µl sensitivity. This approach was used to analyze five sample types collected from both human and animal host-associated microbiota, as well as from the cheese production chain. The availability of a tool for species-level profiling of lactobacilli may be highly useful for both academic research and a wide range of industrial applications.

Comparative genomic and phylogenomic analyses of the Bifidobacteriaceae family.

BACKGROUND: Members of the Bifidobacteriaceae family represent both dominant microbial groups that colonize the gut of various animals, especially during the suckling stage of their life, while they also occur as pathogenic bacteria of the urogenital tract. The pan-genome of the genus Bifidobacterium has been explored in detail in recent years, though genomics of the Bifidobacteriaceae family has not yet received much attention. Here, a comparative genomic analyses of 67 Bifidobacteriaceae (sub) species including all currently recognized genera of this family, i.e., Aeriscardovia, Alloscardovia, Bifidobacterium, Bombiscardovia, Gardnerella, Neoscardovia, Parascardovia, Pseudoscardovia and Scardovia, was performed. Furthermore, in order to include a representative of each of the 67 (currently recognized) (sub) species belonging to the Bifidobacteriaceae family, we sequenced the genomes of an additional 11 species from this family, accomplishing the most extensive comparative genomic analysis performed within this family so far. RESULTS: Phylogenomics-based analyses revealed the deduced evolutionary pathway followed by each member of the Bifidobacteriaceae family, highlighting Aeriscardovia aeriphila LMG 21773 as the deepest branch in the evolutionary tree of this family. Furthermore, functional analyses based on genome content unveil connections between a given member of the family, its carbohydrate utilization abilities and its corresponding host. In this context, bifidobacterial (sub) species isolated from humans and monkeys possess the highest relative number of acquired glycosyl hydrolase-encoding genes, probably in order to enhance their metabolic ability to utilize different carbon sources consumed by the host. CONCLUSIONS: Within the Bifidobacteriaceae family, genomics of the genus Bifidobacterium has been extensively investigated. In contrast, very little is known about the genomics of members of the other eight genera of this family. In this study, we decoded the genome sequences of each member of the Bifidobacteriaceae family. Thanks to subsequent comparative genomic and phylogenetic analyses, the deduced pan-genome of this family, as well as the predicted evolutionary development of each taxon belonging to this family was assessed.

Untangling the cecal microbiota of feral chickens by culturomic and metagenomic analyses.

Different factors may modulate the gut microbiota of animals. In any particular environment, diet, genetic factors and human influences can shape the bacterial communities residing in the gastrointestinal tract. Metagenomic approaches have significantly expanded our knowledge on microbiota dynamics inside hosts, yet cultivation and isolation of bacterial members of these complex ecosystems may still be necessary to fully understand interactions between bacterial communities and their host. A dual approach, involving culture-independent and -dependent techniques, was used here to decipher the microbiota communities that inhabit the gastro intestinal tract of free-range, broiler and feral chickens. In silico analysis revealed the presence of a core microbiota that is typical of those animals that live in different geographical areas and that have limited contact with humans. Anthropic influences guide the metabolic potential and the presence of antibiotic resistance genes of these different bacterial communities. Culturomics attempts, based on different cultivation conditions, were applied to reconstruct in vitro the microbiota of feral chickens. A unique strain collection representing members of the four major phyla of the poultry microbiota was assembled, including bacterial strains that are not typically retrieved from the chicken gut.

The Sortase-Dependent Fimbriome of the Genus Bifidobacterium: Extracellular Structures with Potential To Modulate Microbe-Host Dialogue.

Bifidobacteria are important gut commensals of mammals, including humans, of any age. However, the molecular mechanisms by which these microorganisms establish themselves in the mammalian gut and persist in this environment are largely unknown. Here, we analyzed the genetic diversity of the predicted arsenal of sortase-dependent pili of known and sequenced members of the Bifidobacterium genus and constructed a bifidobacterial sortase-dependent fimbriome database. Our analyses revealed considerable genetic variability of the sortase-dependent fimbriome among bifidobacterial (sub)species, which appears to have been due to horizontal gene transfer events and for which we were able to perform evolutionary mapping. Functional assessment by transcriptome analysis and binding assays involving different substrates demonstrates how bifidobacterial pili are pivotal in promoting various abilities for adhesion to glycans and extracellular matrix proteins, thereby supporting the ecological success of bifidobacteria in the mammalian gut.IMPORTANCE Adhesion of bifidobacterial cells to the mucosa of the large intestine is considered a hallmark for the persistence and colonization of these bacteria in the human gut. In this context, we analyzed the genetic diversity of the predicted arsenal of sortase-dependent pili of known and sequenced members of the Bifidobacterium genus, and constructed a bifidobacterial sortase-dependent fimbriome database. Our analyses revealed considerable genetic variability of the sortase-dependent fimbriome among bifidobacterial (sub)species, which appears to have been due to horizontal gene transfer events. In addition, functional assessment by transcriptome analysis and binding assays involving different substrates demonstrates how bifidobacterial pili are crucial in promoting various abilities for adhesion to glycans and extracellular matrix proteins, thereby supporting the ecological success of bifidobacteria in the mammalian gut. This study represents a complete genomic study regarding the presence of fimbriae in the genus Bifidobacterium.

Prevalence of Antibiotic Resistance Genes among Human Gut-Derived Bifidobacteria.

The microbiota of the human gastrointestinal tract (GIT) may regularly be exposed to antibiotics, which are used to prevent and treat infectious diseases caused by bacteria and fungi. Bacterial communities of the gut retain a reservoir of antibiotic resistance (AR) genes, and antibiotic therapy thus positively selects for those microorganisms that harbor such genetic features, causing microbiota modulation. During the first months following birth, bifidobacteria represent some of the most dominant components of the human gut microbiota, although little is known about their AR gene complement (or resistome). In the current study, we assessed the resistome of the Bifidobacterium genus based on phenotypic and genotypic data of members that represent all currently recognized bifidobacterial (sub)species. Moreover, a comparison between the bifidobacterial resistome and gut metagenome data sets from adults and infants shows that the bifidobacterial community present at the first week following birth possesses a reduced AR arsenal compared to that present in the infant bifidobacterial population in subsequent weeks of the first year of life. Our findings reinforce the concept that the early infant gut microbiota is more susceptible to disturbances by antibiotic treatment than the gut microbiota developed at a later life stage. IMPORTANCE: The spread of resistance to antibiotics among bacterial communities has represented a major concern since their discovery in the last century. The risk of genetic transfer of resistance genes between microorganisms has been extensively investigated due to its relevance to human health. In contrast, there is only limited information available on antibiotic resistance among human gut commensal microorganisms such as bifidobacteria, which are widely exploited by the food industry as health-promoting microorganisms or probiotic ingredients. In the current study, we explored the occurrence of antibiotic resistance genes in the genomes of bifidobacteria and evaluated their genetic mobility to other human gut commensal microorganisms.

Bifidobacterium vansinderenii sp. nov., isolated from faeces of emperor tamarin (Saguinus imperator).

A novel Bifidobacterium strain, Tam10BT, i.e. LMG 30126T, was isolated from emperor tamarin (Saguinus imperator). Cells were Gram-positive, non-motile, non-sporulating, non-haemolytic, facultative anaerobic and fructose 6-phosphate phosphoketolase-positive. Phylogenetic analyses based on 16S rRNA genes as well as multilocus sequences (representing hsp60, rpoB, dnaJ, dnaG and clpC genes) and the core genome revealed that Bifidobacterium Tam10BT exhibited close phylogenetic relatedness to Bifidobacterium tissieri DSM 100201T. Comparative analysis of 16S rRNA gene sequences confirmed the phylogenetic results showing the highest gene sequence identity with strain B. tissieri DSM 100201T (96.5 %). Furthermore, genotyping based on the genome sequence of Tam 10B, in combination with phenotypic analyses, clearly showed that strain Tam10BT is distinct from each of the type strains of the so far recognized Bifidobacterium species. The type strain Tam10BT (=LMG 30126T=CCUG 70655T) represents a novel species, for which the name Bifidobacteriumvansinderenii sp. nov is proposed.

Insights into the biodiversity of the gut microbiota of broiler chickens.

The gastrointestinal tract of poultry is densely populated with microorganisms, which are presumed to interact with the host and ingested feed. Comparison of the gut microbiota of chickens used for large-scale commercial production (Broiler Chicken, BC) and those grown in semi-wild conditions (Free-Range Chicken, FRC) revealed that at phylum level Firmicutes was the dominant phylum of the gut community in BC, while the gut microbiota of FRC contained higher levels of Bacteroidetes and Proteobacteria. Such differences may be due to the diet and/or the intensive use of antibiotics in BC. Indeed, analysis of the resistome of the cecal microbiomes showed a marked richness in BC datasets, with a modulation of the cecal microbiota toward antibiotic resistant bacteria. Functional characterization of the microbiome of FRC samples revealed an increase in gene pathways involved in degradation of complex carbohydrates. Furthermore, in silico analyses of the microbiomes of FRC and BC revealed a higher presence in genes involved in formate production in BC samples. Notably, compared to the BC microbiomes the FRC microbiomes were shown to contain a higher abundance of genes involved in the pathway for acetate production.

Role of sortase-dependent pili of Bifidobacterium bifidum PRL2010 in modulating bacterium-host interactions.

Bifidobacteria represent one of the dominant groups of microorganisms colonizing the human infant intestine. Commensal bacteria that interact with a eukaryotic host are believed to express adhesive molecules on their cell surface that bind to specific host cell receptors or soluble macromolecules. Whole-genome transcription profiling of Bifidobacterium bifidum PRL2010, a strain isolated from infant stool, revealed a small number of commonly expressed extracellular proteins, among which were genes that specify sortase-dependent pili. Expression of the coding sequences of these B. bifidum PRL2010 appendages in nonpiliated Lactococcus lactis enhanced adherence to human enterocytes through extracellular matrix protein and bacterial aggregation. Furthermore, such piliated L. lactis cells evoked a higher TNF-α response during murine colonization compared with their nonpiliated parent, suggesting that bifidobacterial sortase-dependent pili not only contribute to adherence but also display immunomodulatory activity.

Insights from genomes of representatives of the human gut commensal Bifidobacterium bifidum.

Bifidobacteria are bacterial gut commensals of mammals, birds and social insects that are perceived to influence the metabolism/physiology of their host. In this context, members of the Bifidobacterium bifidum species are believed to significantly contribute to the overall microbiota of the human gut at infant stage. However, the molecular reasons for their adaptation to this environment are poorly understood. In this study, we analysed the pan-genome of B. bifidum species by decoding genomes of 15 B. bifidum strains, which highlighted the existence of a conserved gene uniquely present in this bifidobacterial taxon, underscoring a nutrient acquisition strategy that targets host-derived glycans, such as those present in mucin. Growth experiments and corresponding transcriptomic analyses confirmed the in silico data and supported these intriguing and unique host glycan-specific saccharolytic features. The ubiquity of the genetic features of B. bifidum for the breakdown of host glycans was confirmed by interrogating metagenomic datasets, thereby supporting the notion that metabolic access to host-derived glycans is a potent evolutionary force that has shaped B. bifidum genomes and consequently the ecology of the infant intestinal microbiota.

Exploring Vertical Transmission of Bifidobacteria from Mother to Child.

Passage through the birth canal and consequent exposure to the mother's microbiota is considered to represent the initiating event for microbial colonization of the gastrointestinal tract of the newborn. However, a precise evaluation of such suspected vertical microbiota transmission has yet to be performed. Here, we evaluated the microbiomes of four sample sets, each consisting of a mother's fecal and milk samples and the corresponding infant's fecal sample, by means of amplicon-based profiling supported by shotgun metagenomics data for two key samples. Notably, targeted genome reconstruction from microbiome data revealed vertical transmission of a Bifidobacterium breve strain and a Bifidobacterium longum subsp. longum strain from mother to infant, a notion confirmed by strain isolation and genome sequencing. Furthermore, PCR analyses targeting unique genes from these two strains highlighted their persistence in the infant gut at 6 months. Thus, this study demonstrates the existence of specific bifidobacterial strains that are common to mother and child and thus indicative of vertical transmission and that are maintained in the infant for at least relatively short time spans.

Genomic characterization and transcriptional studies of the starch-utilizing strain Bifidobacterium adolescentis 22L.

Bifidobacteria are members of the gut microbiota, but the genetic basis for their adaptation to the human gut is poorly understood. The analysis of the 2,203,222-bp genome of Bifidobacterium adolescentis 22L revealed a nutrient acquisition strategy that targets diet/plant-derived glycans, in particular starch and starch-like carbohydrates. Starch-like carbohydrates were shown to support the growth of B. adolescentis 22L. Transcriptome profiling of 22L cultures grown under in vitro conditions or during colonization of the murine gut by RNA sequencing and quantitative real-time PCR assays revealed the expression of a set of chromosomal loci responsible for starch metabolism as well as for pilus production. Such extracellular structures include so-called sortase-dependent and type IVb pili, which may be involved in gut colonization of 22L through adhesion to extracellular matrix proteins.

Investigation of the evolutionary development of the genus Bifidobacterium by comparative genomics.

The Bifidobacterium genus currently encompasses 48 recognized taxa, which have been isolated from different ecosystems. However, the current phylogeny of bifidobacteria is hampered by the relative paucity of genotypic data. Here, we reassessed the taxonomy of this bacterial genus using genome-based approaches, which demonstrated that the previous taxonomic view of bifidobacteria contained several inconsistencies. In particular, high levels of genetic relatedness were shown to exist between particular Bifidobacterium taxa which would not justify their status as separate species. The results presented are here based on average nucleotide identity analysis involving the genome sequences for each type strain of the 48 bifidobacterial taxa, as well as phylogenetic comparative analysis of the predicted core genome of the Bifidobacterium genus. The results of this study demonstrate that the availability of complete genome sequences allows the reconstruction of a more robust bifidobacterial phylogeny than that obtained from a single gene-based sequence comparison, thus discouraging the assignment of a new or separate bifidobacterial taxon without such a genome-based validation.

Genomic encyclopedia of type strains of the genus Bifidobacterium.

Bifidobacteria represent one of the dominant microbial groups that are present in the gut of various animals, being particularly prevalent during the suckling stage of life of humans and other mammals. However, the overall genome structure of this group of microorganisms remains largely unexplored. Here, we sequenced the genomes of 42 representative (sub)species across the Bifidobacterium genus and used this information to explore the overall genetic picture of this bacterial group. Furthermore, the genomic data described here were used to reconstruct the evolutionary development of the Bifidobacterium genus. This reconstruction suggests that its evolution was substantially influenced by genetic adaptations to obtain access to glycans, thereby representing a common and potent evolutionary force in shaping bifidobacterial genomes.

Untangling Species-Level Composition of Complex Bacterial Communities through a Novel Metagenomic Approach.

16S small-subunit (SSU) rRNA gene-based bacterial profiling is the gold standard for cost-effective taxonomic reconstruction of complex bacterial populations down to the genus level. However, it has been proven ineffective in clinical and research settings requiring higher taxonomic resolution. We therefore developed a bacterial profiling method based on the internal transcribed spacer (ITS) region employing optimized primers and a comprehensive ITS database for accurate cataloguing of bacterial communities at (sub)species resolution. Performance of the microbial ITS profiling pipeline was tested through analysis of host-associated, food, and environmental matrices, while its efficacy in clinical settings was assessed through analysis of mucosal biopsy specimens of colorectal cancer, leading to the identification of putative novel biomarkers. The data collected indicate that the proposed pipeline represents a major step forward in cost-effective identification and screening of microbial biomarkers at (sub)species level, with relevant impact in research, industrial, and clinical settings.IMPORTANCE We developed a novel method for accurate cataloguing of bacterial communities at (sub)species level involving amplification of the internal transcribed spacer (ITS) region through optimized primers, followed by next-generation sequencing and taxonomic classification of amplicons by means of a comprehensive database of bacterial ITS sequences. Host-associated, food, and environmental matrices were employed to test the performance of the microbial ITS profiling pipeline. Moreover, mucosal biopsy samples from colorectal cancer patients were analyzed to demonstrate the scientific relevance of this profiling approach in a clinical setting through identification of putative novel biomarkers. The results indicate that the ITS-based profiling pipeline proposed here represents a key metagenomic tool with major relevance for research, industrial, and clinical settings.