Bifidobacterial Species Research Articles

Compendium of Bifidobacterium-based probiotics: characteristics and therapeutic impact on human diseases

The human microbiota, a complex community of microorganisms residing in and on the human body, plays a crucial role in maintaining health and preventing disease. Bifidobacterium species have shown remarkable therapeutic potential across a range of health conditions, thus being considered optimal probiotic bacteria. This review provides insights into the concept of probiotics and explores the impact of bifidobacteria on human health, focusing on the gastrointestinal, respiratory, skeletal, muscular, and nervous systems. It also integrates information on the available genetic bases underlying the beneficial effects of each bifidobacterial probiotic species on different aspects of human physiology. Notably, Bifidobacterium -based probiotics have proven effective in managing gastrointestinal conditions such as constipation, antibiotic-associated diarrhea, irritable bowel syndrome (IBS), inflammatory bowel disease (IBD), and Helicobacter pylori infections. These benefits are achieved by modulating the intestinal microbiota, boosting immune responses, and strengthening the gut barrier. Moreover, Bifidobacterium species have been reported to reduce respiratory infections and asthma severity. Additionally, these probiotic bacteria offer benefits for skeletal and muscular health, as evidenced by Bifidobacterium adolescentis and Bifidobacterium breve , which have shown anti-inflammatory effects and symptom relief in arthritis models, suggesting potential in treating conditions like rheumatoid arthritis. Furthermore, probiotic therapies based on bifidobacterial species have shown promising effects in alleviating anxiety and depression, reducing stress, and enhancing cognitive function. Overall, this review integrates the extensive scientific literature now available that supports the health-promoting applications of probiotic Bifidobacterium species and underscores the need for further research to confirm their clinical efficacy across different body systems.

From raw milk cheese to the gut: investigating the colonization strategies of Bifidobacterium mongoliense.

The microbial ecology of raw milk cheeses is determined by bacteria originating from milk and milk-producing animals. Recently, it has been shown that members of the Bifidobacterium mongoliense species may become transmitted along the Parmigiano Reggiano cheese production chain and ultimately may colonize the consumer intestine. However, there is a lack of knowledge regarding the molecular mechanisms that mediate the interaction between B. mongoliense and the human gut. Based on 128 raw milk cheeses collected from different Italian regions, we isolated and characterized 10 B. mongoliense strains. Comparative genomics allowed us to unveil the presence of enzymes required for the degradation of sialylated host-glycans in B. mongoliense, corroborating the appreciable growth on de Man-Rogosa-Sharpe (MRS) medium supplemented with 3'-sialyllactose (3'-SL) or 6'-sialyllactose (6'-SL). The B. mongoliense BMONG18 was chosen, due to its superior ability to utilize 3'-SL and mucin as representative strain, to investigate its behavior when co-inoculated with other bifidobacterial species. Conversely, members of other bifidobacterial species did not appear to benefit from the presence of BMONG18, highlighting a competitive scenario for nutrient acquisition. Transcriptomic data of BMONG18 reveal no significant differences in gene expression when cultivated in a gut simulating medium (GSM), regardless of whether cheese was included or not. Furthermore, BMONG18 was shown to exhibit high adhesion capabilities to HT29-MTX human cells, in line with its colonization ability of a human host.IMPORTANCEFermented foods are nourishments produced through controlled microbial growth that play an essential role in worldwide human nutrition. Research interest in fermented foods has increased since the 80s, driven by growing awareness of their potential health benefits beyond mere nutritional content. Bifidobacterium mongoliense, previously identified throughout the production process of Parmigiano Reggiano cheese, was found to be capable of establishing itself in the intestines of its consumers. Our study underscores molecular mechanisms through which this bifidobacterial species, derived from food, interacts with the host and other gut microbiota members.

Molecular cross-talk among human intestinal bifidobacteria as explored by a human gut model.

Bifidobacteria are well known as common and abundant colonizers of the human gut and are able to exert multiple beneficial effects on their host, although the cooperative and competitive relationships that may occur among bifidobacterial strains are still poorly investigated. Therefore, to dissect possible molecular interactions among bifidobacterial species that typically colonize the human gut, three previously identified bifidobacterial prototypes, i.e., B. bifidum PRL2010, B. breve PRL2012, and B. longum PRL2022 were cultivated individually as well as in bi- and tri-association in a human gut-simulating medium. Transcriptomic analyses of these co-associations revealed up-regulation of genes predicted to be involved in the production of extracellular structures including pili (i.e., flp pilus assembly TadE protein gene), exopolysaccharides (i.e., GtrA family protein gene) and teichoic acids (i.e., ABC transporter permease), along with carbohydrate, amino acid and vitamin metabolism-related genes (i.e., exo-alpha-sialidase; beta-galactosidase and pyridoxamine kinase), suggesting that co-cultivation of bifidobacteria induces a response, in individual bifidobacterial strains, aimed at enhancing their proliferation and survival, as well as their ability to cooperate with their host to promote their persistence. Furthermore, exposure of the selected prototypes to human cell line monolayers unveiled the ability of the bifidobacterial tri-association to communicate with their host by increasing the expression of genes involved in adherence to/interaction with intestinal human cells. Lastly, bifidobacterial tri-association promoted the transcriptional upregulation of genes responsible for maintaining the integrity and homeostasis of the intestinal epithelial barrier.

Unveiling metabolic pathways of selected plant-derived glycans by Bifidobacterium pseudocatenulatum.

Bifidobacteria are commonly encountered members of the human gut microbiota that possess the enzymatic machinery necessary for the metabolism of certain plant-derived, complex carbohydrates. In the current study we describe differential growth profiles elicited by a panel of 21 newly isolated Bifidobacterium pseudocatenulatum strains on various plant-derived glycans. Using a combination of gene-trait matching and comparative genome analysis, we identified two distinct xylanases responsible for the degradation of xylan. Furthermore, three distinct extracellular α-amylases were shown to be involved in starch degradation by certain strains of B. pseudocatenulatum. Biochemical characterization showed that all three α-amylases can cleave the related substrates amylose, amylopectin, maltodextrin, glycogen and starch. The genes encoding these enzymes are variably found in the species B. pseudocatenulatum, therefore constituting a strain-specific adaptation to the gut environment as these glycans constitute common plant-derived carbohydrates present in the human diet. Overall, our study provides insights into the metabolism of these common dietary carbohydrates by a human-derived bifidobacterial species.

Integrative genomic reconstruction of carbohydrate utilization networks in bifidobacteria: global trends, local variability, and dietary adaptation.

Bifidobacteria are among the earliest colonizers of the human gut, conferring numerous health benefits. While multiple Bifidobacterium strains are used as probiotics, accumulating evidence suggests that the individual responses to probiotic supplementation may vary, likely due to a variety of factors, including strain type(s), gut community composition, dietary habits of the consumer, and other health/lifestyle conditions. Given the saccharolytic nature of bifidobacteria, the carbohydrate composition of the diet is one of the primary factors dictating the colonization efficiency of Bifidobacterium strains. Therefore, a comprehensive understanding of bifidobacterial glycan metabolism at the strain level is necessary to rationally design probiotic or synbiotic formulations that combine bacterial strains with glycans that match their nutrient preferences. In this study, we systematically reconstructed 66 pathways involved in the utilization of mono-, di-, oligo-, and polysaccharides by analyzing the representation of 565 curated metabolic functional roles (catabolic enzymes, transporters, transcriptional regulators) in 2973 non-redundant cultured Bifidobacterium isolates and metagenome-assembled genomes (MAGs). Our analysis uncovered substantial heterogeneity in the predicted glycan utilization capabilities at the species and strain level and revealed the presence of a yet undescribed phenotypically distinct subspecies-level clade within the Bifidobacterium longum species. We also identified Bangladeshi isolates harboring unique gene clusters tentatively implicated in the breakdown of xyloglucan and human milk oligosaccharides. Predicted carbohydrate utilization phenotypes were experimentally characterized and validated. Our large-scale genomic analysis considerably expands the knowledge of carbohydrate metabolism in bifidobacteria and provides a foundation for rationally designing single- or multi-strain probiotic formulations of a given bifidobacterial species as well as synbiotic combinations of bifidobacterial strains matched with their preferred carbohydrate substrates.

The role of Bifidobacterium genus in modulating the neonate microbiota: implications for antibiotic resistance acquisition in early life

ABSTRACT Resistance to antibiotics in newborns is a huge concern as their immune system is still developing, and infections and resistance acquisition in early life have short- and long-term consequences for their health. Bifidobacterium species are important commensals capable of dominating the infant gut microbiome and are known to be less prone to possess antimicrobial resistance genes than other taxa that may colonize infants. We aimed to study the association between Bifidobacterium-dominated infant gut microbiota and the antibiotic resistant gene load in neonates, and to ascertain the perinatal factors that may contribute to the antibiotic resistance acquisition. Two hundred infant fecal samples at 7 days and 1 month of age from the MAMI birth cohort were included in the study and for whom maternal-neonatal clinical records were available. Microbiota profiling was carried out by 16S rRNA amplicon sequencing, and targeted antibiotic resistance genes (ARGs) including tetM, tetW, tetO, blaTEM, blaSHV and ermB were quantified by qPCR. Infant microbiota clustered into two distinct groups according to their Bifidobacterium genus abundance: high and low. The main separation of groups or clusters at each time point was performed with an unsupervised non-linear algorithm of k-means partitioning to cluster data by time points based on Bifidobacterium genus relative abundance. Microbiota composition differed significantly between both groups, and specific bifidobacterial species were enriched in each cluster. Lower abundance of Bifidobacterium in the infant gut was associated with a higher load of antibiotic resistance genes. Our results highlight the relevance of Bifidobacterium genus in the early acquisition and establishment of antibiotic resistance in the gut. Further studies are needed to develop strategies to promote a healthy early colonization and fight against the spread of antibiotic resistances.

Evaluation of Safety and Beneficial Health Effects of the Human-Milk Strain Bifidobacterium breve DSM32583: An Infant Pilot Trial.

Human milk promotes the growth of bifidobacteria in the infant gut. Adding bifidobacterial species to infant formula may contribute to increasing their presence in the gut of formula-fed infants. Therefore, the safety and anti-infectious effects of Bifidobacterium breve DSM32583, a breast milk isolate, were assessed in a pilot trial involving 3-month-old infants. The infants were randomly assigned to either the probiotic (PG) or the control (CG) groups. All the infants consumed the same formula, although it was supplemented with the strain (1 × 107 cfu/g of formula) in the PG. Overall, 160 infants (80 per group) finished the intervention. Infants in CG gained more weight compared to PG (p < 0.05), but the weights for age Z-scores at 6 months were within the normal distribution for this age group. The rates of infections affecting the gastrointestinal and respiratory tracts and antibiotic therapy were significantly lower in the PG. The bifidobacterial population and the level of short-chain fatty acids were higher (p < 0.05) in the fecal samples of PG infants. No adverse events related to formula consumption were observed. In conclusion, the administration of an infant formula with B. breve DSM32583 was safe and exerted potential beneficial effects on gut health.

Effects of 2'-fucosyllactose on the viability of starter cultures and Bifidobacterium strains of human origin in yogurt during refrigerated storage.

2'-Fucosyllactose (2'-FL) is postulated to provide health benefits and promote the growth of probiotics. This work was undertaken to study the effects of 2'-FL on the viability of starter cultures and Bifidobacterium strains of human origin in yogurt during refrigerated storage. Yogurts were produced containing 2'-FL (0 or 2g/L) and Bifidobacterium strains of human origin (Bifidobacterium longum subsp. longum BB536 or Bifidobacterium longum subsp. infantis ATCC 15697) at a concentration of at least 109 CFU/mL. All yogurts were stored at 4°C for 5weeks. Results showed that 2'-FL was stable in yogurts for at least 5 weeks of cold storage, and the addition of 2'-FL did not significantly alter yogurt fermentation parameters, associated metabolites, and the viability of mixed yogurt starter cultures and Bifidobacterium strains (p>0.05). The addition of bifidobacteria had a negative impact (p<0.05) on the survival rate of starter cultures, Streptococcus thermophilus and Lactobacillus delbureckii subsp. bulgaricus. Meanwhile, it is difficult to maintain a high survival rate of bifidobacteria in final yogurt products, and the addition of 2'-FL could not enhance the viability of bifidobacteria. B. longum BB536 survived at a level higher than 106 CFU/g for 28 days, while B. infantis ATCC15697 maintained this level for only 7 days. In summary, this study has shown the impact of 2'-FL and bifidobacterial species on yogurt properties, and results suggest that it is promising to use 2'-FL in yogurt products as a prebiotic. PRACTICAL APPLICATION: Yogurt is known for its beneficial effects on human health and nutrition. This study reported the production of symbiotic yogurt containing bifidobacteria and 2'-fucosyllactose (2'-FL) as a functional food for specified health uses. The viability of yogurt starter cultures and probiotic bifidobacterial strains was analyzed in this study. Moreover, this research demonstrated that 2'-FL could be added to yogurt without affecting the characteristics of yogurt significantly.

Gut microbiota and inflammatory mediators differentiate IgE mediated and non-IgE mediated cases of cow's milk protein at diagnosis.

Analyze fecal and blood samples at point of diagnosis in IgE mediated cow's milk protein allergy (CMPA) and non-IgE mediated (NIM)-CMPA patients to look for potential new biomarkers. Fourteen patients with IgE mediated CMPA and 13 with NIM-CMPA were recruited in three hospitals in the north of Spain, and were compared with 25 infants from a control group of the same age range. To characterize intestinal microbiota, 16S rDNA gene and internal transcribed spaceramplicons of bifidobacteria were sequenced with Illumina technology. Fatty acids were analyzed by gas chromatography, meanwhile intestinal inflammation markers were quantified by enzyme-linked immunosorbent assayand a multiplex system. Immunological analysis of blood was performed by flow cytometry. The fecal results obtained in the NIM-CMPA group stand out. Among them, a significant reduction in the abundance of Bifidobacteriaceae and Bifidobacterium sequences with respect to controls was observed. Bifidobacterial species were also different, highlighting the lower abundance of Bifidobacterium breve sequences. Fecal calprotectin levels were found to be significantly elevated in relation to IgE mediated patients. Also, a higher excretion of IL-10 and a lower excretion of IL-1ra and platelet derived growth factor-BB was found in NIM-CMPA patients. The differential fecal parameters found in NIM-CMPA patients could be useful in the diagnosis of NIM food allergy to CM proteins.

Harnessing the endogenous Type I-C CRISPR-Cas system for genome editing in Bifidobacterium breve.

Bifidobacterium breve, one of the main bifidobacterial species colonizing the human gastrointestinal tract in early life, has received extensive attention for its purported beneficial effects on human health. However, exploration of the mode of action of such beneficial effects exerted by B. breve is cumbersome due to the lack of effective genetic tools, which limits its synthetic biology application. The widespread presence of CRISPR-Cas systems in the B. breve genome makes endogenous CRISPR-based gene editing toolkits a promising tool. This study revealed that Type I-C CRISPR-Cas systems in B. breve can be divided into two groups based on the amino acid sequences encoded by cas gene clusters. Deletion of the gene coding uracil phosphoribosyl-transferase (upp) was achieved in five B. breve strains from both groups using this system. In addition, translational termination of uracil phosphoribosyl-transferase was successfully achieved in B. breve FJSWX38M7 by single-base substitution of the upp gene and insertion of three stop codons. The gene encoding linoleic acid isomerase (bbi) in B. breve, being a characteristic trait, was deleted after plasmid curing, which rendered it unable to convert linoleic acid into conjugated linoleic acid, demonstrating the feasibility of successive editing. This study expands the toolkit for gene manipulation in B. breve and provides a new approach toward functional genome editing and analysis of B. breve strains.IMPORTANCEThe lack of effective genetic tools for Bifidobacterium breve is an obstacle to studying the molecular mechanisms of its health-promoting effects, hindering the development of next-generation probiotics. Here, we introduce a gene editing method based on the endogenous CRISPR-Cas system, which can achieve gene deletion, single-base substitution, gene insertion, and successive gene editing in B. breve. This study will facilitate discovery of functional genes and elucidation of molecular mechanisms of B. breve pertaining to health-associated benefits.

GH136-encoding gene (perB) is involved in gut colonization and persistence by Bifidobacterium bifidum PRL2010.

Bifidobacteria are commensal microorganisms that typically inhabit the mammalian gut, including that of humans. As they may be vertically transmitted, they commonly colonize the human intestine from the very first day following birth and may persist until adulthood and old age, although generally at a reduced relative abundance and prevalence compared to infancy. The ability of bifidobacteria to persist in the human intestinal environment has been attributed to genes involved in adhesion to epithelial cells and the encoding of complex carbohydrate-degrading enzymes. Recently, a putative mucin-degrading glycosyl hydrolase belonging to the GH136 family and encoded by the perB gene has been implicated in gut persistence of certain bifidobacterial strains. In the current study, to better characterize the function of this gene, a comparative genomic analysis was performed, revealing the presence of perB homologues in just eight bifidobacterial species known to colonize the human gut, including Bifidobacterium bifidum and Bifidobacterium longum subsp. longum strains, or in non-human primates. Mucin-mediated growth and adhesion to human intestinal cells, in addition to a rodent model colonization assay, were performed using B. bifidum PRL2010 as a perB prototype and its isogenic perB-insertion mutant. These results demonstrate that perB inactivation reduces the ability of B. bifidum PRL2010 to grow on and adhere to mucin, as well as to persist in the rodent gut niche. These results corroborate the notion that the perB gene is one of the genetic determinants involved in the persistence of B. bifidum PRL2010 in the human gut.

Folate-producing bifidobacteria: metabolism, genetics, and relevance.

Folate (the general term for all bioactive forms of vitamin B9) plays a crucial role in the evolutionary highly conserved one-carbon (1C) metabolism, a network including central reactions such as DNA and protein synthesis and methylation of macromolecules. Folate delivers 1C units, such as methyl and formyl, between reactants. Plants, algae, fungi, and many bacteria can naturally produce folate, whereas animals, including humans, must obtain folate from external sources. For humans, folate deficiency is, however, a widespread problem. Bifidobacteria constitute an important component of human and many animal microbiomes, providing various health advantages to the host, such as producing folate. This review focuses on bifidobacteria and folate metabolism and the current knowledge of the distribution of genes needed for complete folate biosynthesis across different bifidobacterial species. Biotechnologies based on folate-trophic probiotics aim to create fermented products enriched with folate or design probiotic supplements that can synthesize folate in the colon, improving overall health. Therefore, bifidobacteria (alone or in association with other microorganisms) may, in the future, contribute to reducing widespread folate deficiencies prevalent among vulnerable human population groups, such as older people, women at child-birth age, and people in low-income countries.

Bifidobacterium adolescentis - a beneficial microbe.

Bifidobacterium adolescentis is one of the most abundant bifidobacterial species in the human large intestine, and is prevalent in 60-80% of healthy human adults with cell densities ranging from 109-1010 cells/g of faeces. Lower abundance is found in children and in elderly individuals. The species is evolutionary adapted to fermenting plant-derived glycans and is equipped with an extensive sugar transporter and degradation enzymes repertoire. Consequently, the species is strongly affected by dietary carbohydrates and is able to utilize a wide range of prebiotic molecules. B. adolescentis is specialized in metabolizing resistant starch and is considered a primary starch degrader enabling growth of other beneficial bacteria by cross-feeding. The major metabolic output is acetate and lactate in a ratio of 3:2. Several health-beneficial properties have been demonstrated in certain strains of B. adolescentis in vitro and in rodent models, including enhancement of the intestinal barrier function, anti-inflammatory and immune-regulatory effects, and the production of neurotransmitters (GABA), and vitamins. Although causalities have not been established, reduced abundance of B. adolescentis as part of a dysbiotic colonic microbiota in human observational studies has been associated with inflammatory bowel diseases, irritable bowel syndrome, coeliac disease, cystic fibrosis, Helicobacter pylori infection, type 1 and 2 diabetes, metabolic syndrome, nonalcoholic steatohepatitis, and certain allergies. It is therefore reasonable to conceive B. adolescentis as a health-associated, or even health-promoting bacterial species in humans.

High purity galacto-oligosaccharides: Optimal process design and prebiotic effect

Prebiotic unpurified commercial galacto-oligosaccharides (GOS) often contain significant amounts of glucose, galactose and lactose. Current purification processes remove these digestible components, especially lactose with a high risk of affecting functionality.In the current study a high purity GOS (HP-GOS) was prepared by treating GOS with β-galactosidase to hydrolyze the remaining lactose into glucose and galactose. Subsequently mono sugars were removed by sequential simulated moving bed (SSMB) chromatography. To this end, a design of experiments (DOE) approach was applied to determine the dry matter content and β-galactosidase concentration that optimally support lactose hydrolysis in such a way that the detrimental effects on GOS composition are minimized to preserve its original oligosaccharide composition. The HP-GOS product obtained using the identified optimal settings for lactose hydrolysis contained more than 75% of the original DP2 GOS fraction while the fraction with a DP above 2 was unaffected by the purification. Using fecal fermentations, it was demonstrated that the GOS parts of the HP-GOS and the parent GOS have comparable effects on gut microbiota composition. Both GOS products equally increased the relative abundances of Bifidobacterium. Furthermore, there were no differences in alpha diversity and in the distribution of the different bifidobacterial species. Based on these outcomes it was concluded that the lactose hydrolysis step of the purification process did not influence gut microbiota modulating effects of GOS.

Preferential sugar utilization by bifidobacterial species.

Aim: Bifidobacteria benefit host health and homeostasis by breaking down diet- and host-derived carbohydrates to produce organic acids in the intestine. However, the sugar utilization preference of bifidobacterial species is poorly understood. Thus, this study aimed to investigate the sugar utilization preference (i.e., glucose or lactose) of various bifidobacterial species. Methods: Strains belonging to 40 bifidobacterial species/subspecies were cultured on a modified MRS medium supplemented with glucose and/or lactose, and their preferential sugar utilization was assessed using high-performance thin-layer chromatography. Comparative genomic analysis was conducted with a focus on genes involved in lactose and glucose uptake and genes encoding for carbohydrate-active enzymes. Results: Strains that preferentially utilized glucose or lactose were identified. Almost all the lactose-preferring strains harbored the lactose symporter lacS gene. However, the comparative genomic analysis could not explain all their differences in sugar utilization preference. Analysis based on isolate source revealed that all 10 strains isolated from humans preferentially utilized lactose, whereas all four strains isolated from insects preferentially utilized glucose. In addition, bifidobacterial species isolated from hosts whose milk contained higher lactose amounts preferentially utilized lactose. Lactose was also detected in the feces of human infants, suggesting that lactose serves as a carbon source not only for infants but also for gut microbes in vivo. Conclusion: The different sugar preference phenotypes of Bifidobacterium species may be ascribed to the residential environment affected by the dietary habits of their host. This study is the first to systematically evaluate the sugar uptake preference of various bifidobacterial species.

Antibiotic Susceptibility Testing and Establishment of Tentative Species-Specific Microbiological Cut-off Values for Bifidobacteria Isolated from Chinese Population.

Bifidobacteria are commonly used as probiotics in the food industry. The resistance of Bifidobacterium species to antibiotics is closely linked to food safety. However, we still lack a system for the safety evaluation of antibiotic resistance in bifidobacteria, and genus-level microbiological cut-off values remain in use for the determination of phenotypic resistance of Bifidobacterium strains to a given antibiotic. Here, we collected a total of 422 gut-derived bifidobacterial strains isolated from Chinese population and identified their phenotypic resistance profiles against ampicillin, amoxicillin, ciprofloxacin, chloramphenicol, clindamycin, erythromycin, rifampicin, tetracycline, trimethoprim, and vancomycin. Different Bifidobacterium species were found to have varying tolerances to the same antibiotic; therefore, we further established species-specific cut-off values for bifidobacterial species to ten antibiotics. Species-specific rather than genus-specific cut-off values for species belonging to the same taxon were considered more suitable to determine the phenotypic resistance of a Bifidobacterium strain. Moreover, a comprehensive scanning of antibiotic resistance genes in all Bifidobacterium strains tested revealed that the existence of the tetracycline resistance gene tet(W) and the erythromycin/clindamycin resistance gene ErmX is closely related to host phenotypes. Our findings provide guidance and reference values at both phenotype and genotype levels for the safe application of bifidobacteria in the food industry and the development of probiotic resistance evaluation standards.

Faecal Microbiota Characterisation of Potamochoerus porcus Living in a Controlled Environment.

Intestinal bacteria establish a specific relationship with the host animal, which causes the acquisition of gut microbiota with a unique composition classified as the enterotype. As the name suggests, the Red River Hog is a wild member of the pig family living in Africa, in particular through the West and Central African rainforest. To date, very few studies have analysed the gut microbiota of Red River Hogs (RRHs) both housed under controlled conditions and in wild habitats. This study analysed the intestinal microbiota and the distribution of Bifidobacterium species in five Red River Hog (RRH) individuals (four adults and one juvenile), hosted in two different modern zoological gardens (Parco Natura Viva, Verona, and Bioparco, Rome) with the aim of disentangling the possible effects of captive different lifestyle and host genetics. Faecal samples were collected and studied both for bifidobacterial counts and isolation by means of culture-dependent method and for total microbiota analysis through the high-quality sequences of the V3-V4 region of bacterial 16S rRNA. Results showed a host-specific bifidobacterial species distribution. Indeed, B. boum and B. thermoacidophilum were found only in Verona RRHs, whereas B. porcinum species were isolated only in Rome RRHs. These bifidobacterial species are also typical of pigs. Bifidobacterial counts were about 106 CFU/g in faecal samples of all the individuals, with the only exception for the juvenile subject, showing 107 CFU/g. As in human beings, in RRHs a higher count of bifidobacteria was also found in the young subject compared with adults. Furthermore, the microbiota of RRHs showed qualitative differences. Indeed, Firmicutes was found to be the dominant phylum in Verona RRHs whereas Bacteroidetes was the most represented in Roma RRHs. At order level, Oscillospirales and Spirochaetales were the most represented in Verona RRHs compared with Rome RRHs, where Bacteroidales dominated over the other taxa. Finally, at the family level, RRHs from the two sites showed the presence of the same families, but with different levels of abundance. Our results highlight that the intestinal microbiota seems to reflect the lifestyle (i.e., the diet), whereas age and host genetics are the driving factors for the bifidobacterial population.

Exploring Molecular Interactions between Human Milk Hormone Insulin and Bifidobacteria.

Multiple millennia of human evolution have shaped the chemical composition of breast milk toward an optimal human body fluid for nutrition and protection and for shaping the early gut microbiota of newborns. This biological fluid is composed of water, lipids, simple and complex carbohydrates, proteins, immunoglobulins, and hormones. Potential interactions between hormones present in mother's milk and the microbial community of the newborn are a very fascinating yet unexplored topic. In this context, insulin, in addition to being one of the most prevalent hormones in breast milk, is also involved in a metabolic disease that affects many pregnant women, i.e., gestational diabetes mellitus (GDM). Analysis of 3,620 publicly available metagenomic data sets revealed that the bifidobacterial community varies in relation to the different concentrations of this hormone in breast milk of healthy and diabetic mothers. Starting from this assumption, in this study, we explored possible molecular interactions between this hormone and bifidobacterial strains that represent bifidobacterial species commonly occurring in the infant gut using 'omics' approaches. Our findings revealed that insulin modulates the bifidobacterial community by apparently improving the persistence of the Bifidobacterium bifidum taxon in the infant gut environment compared to other typical infant-associated bifidobacterial species. IMPORTANCE Breast milk is a key factor in modulating the infant's intestinal microbiota composition. Even though the interaction between human milk sugars and bifidobacteria has been extensively studied, there are other bioactive compounds in human milk that may influence the gut microbiota, such as hormones. In this article, the molecular interaction of the human milk hormone insulin and the bifidobacterial communities colonizing the human gut in the early stages of life has been explored. This molecular cross talk was assessed using an in vitro gut microbiota model and then analyzed by various omics approaches, allowing the identification of genes associated with bacterial cell adaptation/colonization in the human intestine. Our findings provide insights into the manner by which assembly of the early gut microbiota may be regulated by host factors such as hormones carried by human milk.

SMRT sequencing and ddPCR reveal the complexity of developmental trajectories and temporal dynamics of gut bifidobacterial communities in infants

Infant intestinal microbiome is closely linked with health and risk of disease. Bifidobacterium are important components of the infant gut and are known to confer various health effects on the host. However, few studies have described the precise composition and dynamics of early infant gut bifidobacterial communities. Thus, this was a pilot study aiming to describe the developmental trajectories and temporal dynamics of bifidobacterial communities in infants before 6 months of age. A total of 28 fecal samples from 4 infants (GF, ZZ, QM, TN, respectively) were collected and analyzed after 5, 15, 30, 60, 90, 120, 150, and 180 days of birth by a bifidobacteria-target method (based on single-molecule real-time sequencing of partial bifidobacterial rpsK genes) in conjunction with droplet digital polymerase chain reaction (ddPCR). The infant fecal microbiota comprised a total of 11 bifidobacterial species, including 4 major species, i.e., B. dentium (37.35 %), B. catenulatum (32.04 %), B. breve (22.24 %), and B. animalis (8.02 %). The infant microbiota showed highly individualized developmental trajectories. The leading species for GF was B. catenulatum, with a relatively stable developmental trajectory. In ZZ, B. breve was enriched, and the developmental trajectory was rather fluctuating. The most abundant species for QM and TN was B. dentium. The developmental trajectory of B. dentium in QM showed a trend of gradual decrease, whereas an opposite trend was seen in samples of TN. The results of ddPCR confirmed large variations in quantities of bifidobacteria between infants and suggested discordances in temporal dynamics of bifidobacterial communities during the first half year of infancy. In conclusion, our results suggested that the early infant gut bifidobacterial microbiota was highly complex and temporal dynamics, with individualized developmental trajectories, which should be considered in future research of infant gut microbiota.

Arabinoxylan-based substrate preferences and predicted metabolic properties of Bifidobacterium longum subspecies as a basis to design differential media

Arabinoxylan (AX) and arabinoxylo-oligosaccharides (AXOS) derived therefrom are emergent prebiotics with promising health promoting properties, likely linked to its capacity to foster beneficial species in the human gut. Bifidobacteria appear to be one taxa that is frequently promoted following AX or AXOS consumption, and that is known to establish metabolic cross-feeding networks with other beneficial commensal species. Therefore, probiotic bifidobacteria with the capability to metabolize AX-derived prebiotics represent interesting candidates to develop novel probiotic and synbiotic combinations with AX-based prebiotics. In this work we have deepen into the metabolic capabilities of bifidobacteria related to AX and AXOS metabolization through a combination of in silico an in vitro tools. Both approaches revealed that Bifidobacterium longum and, particularly, B. longum subsp. longum, appears as the better equipped to metabolize complex AX substrates, although other related subspecies such as B. longum subsp. infantis, also hold some machinery related to AXOS metabolization. This correlates to the growth profiles exhibited by representative strains of both subspecies in AX or AXOS enriched media. Based on these results, we formulated a differential carbohydrate free medium (CFM) supplemented with a combination of AX and AXOS that enabled to recover a wide diversity of Bifidobacterium species from complex fecal samples, while allowing easy discrimination of AX metabolising strains by the appearance of a precipitation halo. This new media represent an appealing alternative to isolate novel probiotic bifidobacteria, rapidly discriminating their capacity to metabolize structurally complex AX-derived prebiotics. This can be convenient to assist formulation of novel functional foods and supplements, including bifidobacterial species with capacity to metabolize AX-derived prebiotic ingredients.