Exploring the Ecology of Bifidobacteria and Their Genetic Adaptation to the Mammalian Gut.

The mammalian gut is densely inhabited by microorganisms that have coevolved with their host. Amongst these latter microorganisms, bifidobacteria represent a key model to study host-microbe interaction within the mammalian gut. Remarkably, bifidobacteria naturally occur in a range of ecological niches that are either directly or indirectly connected to the animal gastrointestinal tract. They constitute one of the dominant bacterial members of the intestinal microbiota and are among the first colonizers of the mammalian gut. Notably, the presence of bifidobacteria in the gut has been associated with several health-promoting activities. In this review, we aim to provide an overview of current knowledge on the genetic diversity and ecology of bifidobacteria. Furthermore, we will discuss how this important group of gut bacteria is able to colonize and survive in the mammalian gut, so as to facilitate host interactions.

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.

Bifidobacterium adolescentis as a key member of the human gut microbiota in the production of GABA.

Gamma aminobutyric acid (GABA) is the principal inhibitory neurotransmitter playing a key role in anxiety and depression disorders in mammals. Recent studies revealed that members of the gut microbiota are able to produce GABA modulating the gut-brain axis response. Among members of the human gut microbiota, bifidobacteria are well known to establish many metabolic and physiologic interactions with the host. In this study, we performed genome analyses of more than 1,000 bifidobacterial strains publicly available revealing that Bifidobacterium adolescentis taxon might represent a model GABA producer in human gastrointestinal tract. Moreover, the in silico screening of human/animal metagenomic datasets showed an intriguing association/correlation between B. adolescentis load and mental disorders such as depression and anxiety. Interestingly, in vitro screening of 82 B. adolescentis strains allowed identifying two high GABA producers, i.e. B. adolescentis PRL2019 and B. adolescentis HD17T2H, which were employed in an in vivo trial in rats. Feeding Groningen rats with a supplementation of B. adolescentis strains, confirmed the ability of these microorganisms to stimulate the in vivo production of GABA highlighting their potential implication in gut-brain axis interactions.

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.

Ecology of Lactobacilli Present in Italian Cheeses Produced from Raw Milk.

Among the bacterial genera that are used for cheese production, Lactobacillus is a key taxon of high industrial relevance that is commonly present in commercial starter cultures for dairy fermentations. Certain lactobacilli play a defining role in the development of the organoleptic features during the ripening stages of particular cheeses. We performed an in-depth 16S rRNA gene-based microbiota analysis coupled with internally transcribed spacer-mediated Lactobacillus compositional profiling of 21 common Italian cheeses produced from raw milk in order to evaluate the ecological distribution of lactobacilli associated with this food matrix. Statistical analysis of the collected data revealed the existence of putative Lactobacillus community state types (LCSTs), which consist of clusters of Lactobacillus (sub)species. Each LCST is dominated by one or two taxa that appear to represent keystone elements of an elaborate network of positive and negative interactions with minor components of the cheese microbiota. The results obtained in this study reveal the existence of peculiar cheese microbiota assemblies that represent intriguing targets for further functional studies aimed at dissecting the species-specific role of bacteria in cheese manufacturing.IMPORTANCE The microbiota is known to play a key role in the development of the organoleptic features of dairy products. Lactobacilli have been reported to represent one of the main components of the nonstarter bacterial population, i.e., bacteria that are not deliberately added to the milk, harbored by cheese, although the species-level composition of this microbial population has never been assessed in detail. In the present study, we applied a recently developed metagenomic approach that employs an internally transcribed spacer to profile the Lactobacillus population harbored by cheese produced from raw milk at the (sub)species level. The obtained data revealed the existence of particular Lactobacillus community state types consisting of clusters of Lactobacillus (sub)species that tend to cooccur in the screened cheeses. Moreover, analysis of covariances between members of this genus indicate that these taxa form an elaborate network of positive and negative interactions that define specific clusters of covariant lactobacilli.

Investigating bifidobacteria and human milk oligosaccharide composition of lactating mothers.

Human milk is known to carry its own microbiota, of which the precise origin remains obscure. Breastfeeding allows mother-to-baby transmission of microorganisms as well as the transfer of many other milk components, such as human milk oligosaccharides (HMOs), which act as metabolizable substrates for particular bacteria, such as bifidobacteria, residing in infant intestinal tract. In the current study, we report the HMO composition of 249 human milk samples, in 163 of which we quantified the abundance of members of the Bifidobacterium genus using a combination of metagenomic and flow cytometric approaches. Metagenomic data allowed us to identify four clusters dominated by Bifidobacterium adolescentis and Bifidobacterium pseudolongum, Bifidobacterium crudilactis or Bifidobacterium dentium, as well as a cluster represented by a heterogeneous mix of bifidobacterial species such as Bifidobacterium breve and Bifidobacterium longum. Furthermore, in vitro growth assays on HMOs coupled with in silico glycobiome analyses allowed us to elucidate that members of the Bifidobacterium bifidum and B. breve species exhibit the greatest ability to degrade and grow on HMOs. Altogether, these findings indicate that the bifidobacterial component of the human milk microbiota is not strictly correlated with their ability to metabolize HMOs.

Characterization of the phylogenetic diversity of two novel species belonging to the genus Bifidobacterium: Bifidobacterium cebidarum sp. nov. and Bifidobacterium leontopitheci sp. nov.

Two Bifidobacterium strains, i.e., 2176BT and 2177BT, were isolated from Golden-Headed Lion Tamarin (Leontopithecus chrysomelas) and Goeldi's monkey (Callimico goeldii). Isolates were shown to be Gram-positive, non-motile, non-sporulating, facultative anaerobic and d-fructose 6-phosphate phosphoketolase-positive. Phylogenetic analyses based on 16S rRNA sequences, multilocus sequences (including hsp60, rpoB, dnaJ, dnaG and clpC genes) and the core genome revealed that bifidobacterial strains 2176BT and 2177BT exhibit close phylogenetic relatedness to Bifidobacterium felsineum DSM 103139T and Bifidobacterium bifidum LMG 11041T, respectively. Further genotyping based on the genome sequence of the isolated strains combined with phenotypic analyses, clearly show that these strains are distinct from each of the type strains of the so far recognized Bifidobacterium species. Thus, Bifidobacterium cebidarum sp. nov. (2176BT=LMG 31469T=CCUG 73785T) and Bifidobacterium leontopitheci sp. nov. (2177BT=LMG 31471T=CCUG 73786T are proposed as novel Bifidobacterium species.

Deciphering the Bifidobacterial Populations within the Canine and Feline Gut Microbiota.

During the course of evolution, dogs and cats have been subjected to extensive domestication, becoming the principal companion animals for humans. For this reason, their health care, including their intestinal microbiota, is considered of considerable importance. However, the canine and feline gut microbiota still represent a largely unexplored research area. In the present work, we profiled the microbiota of 23 feline fecal samples by 16S rRNA gene and bifidobacterial internally transcribed spacer (ITS) approaches and compared this information with previously reported data from 138 canine fecal samples. The obtained data allowed the reconstruction of the core gut microbiota of the above-mentioned samples coupled with their classification into distinct community state types at both genus and species levels, identifying Bacteroides, Fusobacterium, and Prevotella 9 as the main bacterial components of the canine and feline gut microbiota. At the species level, the intestinal bifidobacterial gut communities of dogs and cats differed in terms of both species number and composition, as emphasized by a covariance analysis. Together, our findings show that the intestinal populations of cats and dogs are similar in terms of genus-level taxonomical composition, while at the bifidobacterial species level, clear differences were observed, indicative of host-specific colonization behavior by particular bifidobacterial taxa.IMPORTANCE Currently, domesticated dogs and cats are the most cherished companion animals for humans, and concerns about their health and well-being are therefore important. In this context, the gut microbiota plays a crucial role in maintaining and promoting host health. However, despite the social relevance of domesticated dogs and cats, their intestinal microbial communities are still far from being completely understood. In this study, the taxonomical composition of canine and feline gut microbiota was explored at genus and bifidobacterial species levels, allowing classification of these microbial populations into distinct gut community state types at either of the two investigated taxonomic levels. Furthermore, the reconstruction of core gut microbiota coupled with covariance network analysis based on bifidobacterial internally transcribed spacer (ITS) profiling revealed differences in the bifidobacterial compositions of canine and feline gut microbiota, suggesting that particular bifidobacterial species have developed a selective ability to colonize a specific host.

The infant gut microbiome as a microbial organ influencing host well-being.

Initial establishment of the human gut microbiota is generally believed to occur immediately following birth, involving key gut commensals such as bifidobacteria that are acquired from the mother. The subsequent development of this early gut microbiota is driven and modulated by specific dietary compounds present in human milk that support selective colonization. This represents a very intriguing example of host-microbe co-evolution, where both partners are believed to benefit. In recent years, various publications have focused on dissecting microbial infant gut communities and their interaction with their human host, being a determining factor in host physiology and metabolic activities. Such studies have highlighted a reduction of microbial diversity and/or an aberrant microbiota composition, sometimes referred to as dysbiosis, which may manifest itself during the early stage of life, i.e., in infants, or later stages of life. There are growing experimental data that may explain how the early human gut microbiota affects risk factors related to adult health conditions. This concept has fueled the development of various nutritional strategies, many of which are based on probiotics and/or prebiotics, to shape the infant microbiota. In this review, we will present the current state of the art regarding the infant gut microbiota and the role of key commensal microorganisms like bifidobacteria in the establishment of the first microbial communities in the human gut.

Bifidobacterium bifidum: A Key Member of the Early Human Gut Microbiota.

Bifidobacteria typically represent the most abundant bacteria of the human gut microbiota in healthy breast-fed infants. Members of the Bifidobacterium bifidum species constitute one of the dominant taxa amongst these bifidobacterial communities and have been shown to display notable physiological and genetic features encompassing adhesion to epithelia as well as metabolism of host-derived glycans. In the current review, we discuss current knowledge concerning particular biological characteristics of the B. bifidum species that support its specific adaptation to the human gut and their implications in terms of supporting host health.

Bifidobacterial Distribution Across Italian Cheeses Produced from Raw Milk.

Cheese microbiota is of high industrial relevance due to its crucial role in defining the organoleptic features of the final product. Nevertheless, the composition of and possible microbe-microbe interactions between these bacterial populations have never been assessed down to the species-level. For this reason, 16S rRNA gene microbial profiling combined with internally transcribed spacer (ITS)-mediated bifidobacterial profiling analyses of various cheeses produced with raw milk were performed in order to achieve an in-depth view of the bifidobacterial populations present in these microbially fermented food matrices. Moreover, statistical elaboration of the data collected in this study revealed the existence of community state types characterized by the dominance of specific microbial genera that appear to shape the overall cheese microbiota through an interactive network responsible for species-specific modulatory effects on the bifidobacterial population.

Uncovering Bifidobacteria via Targeted Sequencing of the Mammalian Gut Microbiota.

Bifidobacteria are among the most prevalent gut commensals in mammals, playing crucial functional roles that start from their early colonization of the infant gastrointestinal tract and last throughout the life span of their host. Metagenomic approaches have been employed to unveil the genetic features of bifidobacteria in order to understand how they participate in the correct development of a healthy microbiome. Nevertheless, their low relative abundance in many environmental samples may represent a major limitation for metagenomics approaches. To overcome this restriction, we applied an enrichment method that allows amplification of bifidobacterial DNA obtained from human or animal fecal samples for up to 26,500-fold, resulting in the metagenomic reconstruction of genomes belonging to bifidobacterial strains, present at very low abundance in collected samples. Functional predictions of the genes from these reconstructed genomes allows us to identify unique signatures among members of the same bifidobacterial species, highlighting genes correlated with the uptake of nutrients and adhesion to the intestinal mucosa.

Bifidobacterial Transfer from Mother to Child as Examined by an Animal Model.

Bifidobacteria commonly constitute the most abundant group of microorganisms in the healthy infant gut. Their intestinal establishment is believed to be maternally driven, and their acquisition has even been postulated to occur during pregnancy. In the current study, we evaluated bifidobacterial mother-to infant transmission events in a rat model by means of quantitative PCR (qPCR), as well as by Internally Transcribed Spacer (ITS) bifidobacterial profiling. The occurrence of strains supplied by mothers during pregnancy to their corresponding newborns was observed and identified by analysis immediately following C-section delivery. These findings provide intriguing support for the existence of an unknown route to facilitate bifidobacterial transfer during the very early stages of life.

The impact of human-facilitated selection on the gut microbiota of domesticated mammals.

Domestication is the process by which anthropogenic forces shape lifestyle and behavior of wild species to accommodate human needs. The impact of domestication on animal physiology and behavior has been extensively studied, whereas its effect on the gut microbiota is still largely unexplored. For this reason, 16S rRNA gene-based and internal transcribed spacer-mediated bifidobacterial profiling, together with shotgun metagenomics, was employed to investigate the taxonomic composition and metabolic repertoire of 146 mammalian fecal samples, corresponding to 12 domesticated-feral dyads. Our results revealed that changes induced by domestication have extensively shaped the taxonomic composition of the mammalian gut microbiota. In this context, the selection of microbial taxa linked to a more efficient feed conversion into body mass and putative horizontal transmission of certain bacterial genera from humans were observed in the fecal microbiota of domesticated animals when compared to their feral relatives and to humans. In addition, profiling of the metabolic arsenal through metagenomics highlighted extensive functional adaptation of the fecal microbial community of domesticated mammals to changes induced by domestication. Remarkably, domesticated animals showed, when compared to their feral relatives, increased abundance of specific glycosyl hydrolases, possibly due to the higher intake of complex plant carbohydrates typical of commercial animal feeds.

Exploring the effects of COLOSTRONONI on the mammalian gut microbiota composition.

COLOSTRONONI is a dietary supplement consisting of bovine colostrum and Morinda citrifolia fruit (Noni). In this study, we tested the capability of COLOSTRONONI to influence gut microbiota composition using an in vivo evaluation in rats. Furthermore, we analyzed the effect of COLOSTRONONI on the systemic inflammatory responses as well as on the gut permeability of the animals. Altogether, our analyses supported the concept of COLOSTRONONI as a natural food supplement that doesn't affect (neither negatively nor positively) gut microbiota homeostasis in healthy conditions. Moreover, COLOSTRONONI highlighted a lower effect in the expression of genes coding for IL-10, Il-12 and TNF-α response allowing us to hypothesize an immunomodulatory activity of this dietary supplement.

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.

Isolation of novel gut bifidobacteria using a combination of metagenomic and cultivation approaches.

Whole metagenome shotgun (WMGS) sequencing is a method that provides insights into the genomic composition and arrangement of complex microbial consortia. Here, we report how WMGS coupled with a cultivation approach allows the isolation of novel bifidobacteria from animal fecal samples. A combination of in silico analyses based on nucleotide and protein sequences facilitate the identification of genetic material belonging to putative novel species. Consequently, the prediction of metabolic properties by in silico analyses permits the identification of specific substrates that are then employed to isolate these species through a cultivation method.

Ability of bifidobacteria to metabolize chitin-glucan and its impact on the gut microbiota.

Chitin-glucan (CG) represents a natural carbohydrate source for certain microbial inhabitants of the human gut and may act as a prebiotic for a number of bacterial taxa. However, the bifidogenic activity of this substrate is still unknown. In the current study, we evaluated the ability of chitin-glucan to influence growth of 100 bifidobacterial strains belonging to those species commonly identified within the bifidobacterial communities residing in the infant and adult human gut. Such analyses were coupled with transcriptome experiments directed to explore the transcriptional effects of CG on Bifidobacterium breve 2L, which was shown to elicit the highest growth performance on this natural polysaccharide. In addition, an in vivo trial involving a rat model revealed how the colonization efficiency of this bifidobacterial strain was enhanced when the animals were fed with a diet containing CG. Altogether our analyses indicate that CG is a valuable novel prebiotic compound that may be added to the human diet in order to re-establish/reinforce bifidobacteria colonization in the mammalian gut.

Colonization of the human gut by bovine bacteria present in Parmesan cheese.

The abilities of certain microorganisms to be transferred across the food production chain, persist in the final product and, potentially, colonize the human gut are poorly understood. Here, we provide strain-level evidence supporting that dairy cattle-associated bacteria can be transferred to the human gut via consumption of Parmesan cheese. We characterize the microbial communities in samples taken from five different locations across the Parmesan cheese production chain, confirming that the final product contains microorganisms derived from cattle gut, milk, and the nearby environment. In addition, we carry out a human pilot study showing that Bifidobacterium mongoliense strains from cheese can transiently colonize the human gut, a process that can be enhanced by cow milk consumption.

Characterization of the phylogenetic diversity of five novel species belonging to the genus Bifidobacterium: Bifidobacterium castoris sp. nov., Bifidobacterium callimiconis sp. nov., Bifidobacterium goeldii sp. nov., Bifidobacterium samirii sp. nov. and Bifidobacterium dolichotidis sp. nov.

Five Bifidobacterium strains, i.e. 2020BT, 2028BT, 2033BT, 2034BT and 2036BT, were isolated from European beaver (Castor fiber), Goeldi's marmoset (Callimicogoeldii), black-capped squirrel monkey (Saimiriboliviensissubsp. peruviensis) and Patagonian mara (Dolichotispatagonum). All of these isolates were shown to be Gram-positive, facultative anaerobic, d-fructose 6-phosphate phosphoketolase-positive, non-motile and non-sporulating. Phylogenetic analyses based on 16S rRNA gene sequences, multilocus sequences (including hsp60, rpoB, dnaJ, dnaG and clpC genes) and the core genome revealed that bifidobacterial strains 2020BT, 2028BT, 2033BT, 2034BT and 2036BT exhibit close phylogenetic relatedness to Bifidobacterium biavatii DSM 23969T, Bifidobacterium bifidum LMG 11041T, Bifidobacterium choerinum LMG 10510T, Bifidobacterium gallicum LMG 11596T, Bifidobacterium imperatoris LMG 30297T, Bifidobacterium italicum LMG 30187T and Bifidobacterium vansinderenii LMG 30126T, respectively. Further genotyping based on the genome sequence of the isolated strains combined with phenotypic analyses, clearly show that these strains are distinct from each of the type strains of the so far recognized Bifidobacterium species. Thus, Bifidobacterium castoris sp. nov. (2020BT=LMG 30937T=CCUG 72816T), Bifidobacterium callimiconis sp. nov. (2028BT=LMG 30938T=CCUG 72814T), Bifidobacterium samirii sp. nov. (2033BT=LMG 30940T=CCUG 72817T), Bifidobacterium goeldii sp. nov. (2034BT=LMG 30939T=CCUG 72815T) and Bifidobacterium dolichotidis sp. nov. (2036BT=LMG 30941T=CCUG 72818T) are proposed as novel Bifidobacterium species.