Bifidobacterial biofilm formation is a multifactorial adaptive phenomenon in response to bile exposure.

In the current study, we show that biofilm formation by various strains and species belonging to Bifidobacterium, a genus that includes gut commensals with reported health-promoting activities, is induced by high concentrations of bile (0.5% (w/v) or higher) and individual bile salts (20 mM or higher), rather than by acid or osmotic stress. The transcriptomic response of a bifidobacterial prototype Bifidobacterium breve UCC2003 to such high bile concentrations was investigated and a random transposon bank of B. breve UCC2003 was screened for mutants that affect biofilm formation in order to identify genes involved in this adaptive process. Eleven mutants affected in their ability to form a biofilm were identified, while biofilm formation capacity of an insertional mutation in luxS and an exopolysaccharide (EPS) negative B. breve UCC2003 was also studied. Reduced capacity to form biofilm also caused reduced viability when exposed to porcine bile. We propose that bifidobacterial biofilm formation is an adaptive response to high concentrations of bile in order to avoid bactericidal effects of high bile concentrations in the gastrointestinal environment. Biofilm formation appears to be a multi-factorial process involving EPS production, proteins and extracellular DNA release, representing a crucial strategy in response to bile stress in order to enhance fitness in the gut environment.

Bifidobacterium breve UCC2003 Exopolysaccharide Modulates the Early Life Microbiota by Acting as a Potential Dietary Substrate.

BACKGROUND: Bifidobacterium represents an important early life microbiota member. Specific bifidobacterial components, exopolysaccharides (EPS), positively modulate host responses, with purified EPS also suggested to impact microbe-microbe interactions by acting as a nutrient substrate. Thus, we determined the longitudinal effects of bifidobacterial EPS on microbial communities and metabolite profiles using an infant model colon system. METHODS: Differential gene expression and growth characteristics were determined for each strain; Bifidobacterium breve UCC2003 and corresponding isogenic EPS-deletion mutant (B. breve UCC2003del). Model colon vessels were inoculated with B. breve and microbiome dynamics monitored using 16S rRNA sequencing and metabolomics (NMR). RESULTS: Transcriptomics of EPS mutant vs. B. breve UCC2003 highlighted discrete differential gene expression (e.g., eps biosynthetic cluster), though overall growth dynamics between strains were unaffected. The EPS-positive vessel had significant shifts in microbiome and metabolite profiles until study end (405 h); with increases of Tyzzerella and Faecalibacterium, and short-chain fatty acids, with further correlations between taxa and metabolites which were not observed within the EPS-negative vessel. CONCLUSIONS: These data indicate that B. breve UCC2003 EPS is potentially metabolized by infant microbiota members, leading to differential microbial metabolism and altered metabolite by-products. Overall, these findings may allow development of EPS-specific strategies to promote infant health.

Linoleic acid induces metabolic stress in the intestinal microorganism Bifidobacterium breve DSM 20213.

Despite clinical and research interest in the health implications of the conjugation of linoleic acid (LA) by bifidobacteria, the detailed metabolic pathway and physiological reasons underlying the process remain unclear. This research aimed to investigate, at the molecular level, how LA affects the metabolism of Bifidobacterium breve DSM 20213 as a model for the well-known LA conjugation phenotype of this species. The mechanisms involved and the meaning of the metabolic changes caused by LA to B. breve DSM 20213 are unclear due to the lack of comprehensive information regarding the responses of B. breve DSM 20213 under different environmental conditions. Therefore, for the first time, an untargeted metabolomics-based approach was used to depict the main changes in the metabolic profiles of B. breve DSM 20213. Both supervised and unsupervised statistical methods applied to the untargeted metabolomic data allowed confirming the metabolic changes of B. breve DSM 20213 when exposed to LA. In particular, alterations to the amino-acid, carbohydrate and fatty-acid biosynthetic pathways were observed at the stationary phase of growth curve. Among others, significant up-regulation trends were detected for aromatic (such as tyrosine and tryptophan) and sulfur amino acids (i.e., methionine and cysteine). Besides confirming the conjugation of LA, metabolomics suggested a metabolic reprogramming during the whole growth curve and an imbalance in redox status following LA exposure. Such redox stress resulted in the down-accumulation of peroxide scavengers such as low-molecular-weight thiols (glutathione- and mycothiol-related compounds) and ascorbate precursors, together with the up-accumulation of oxidized (hydroxy- and epoxy-derivatives) forms of fatty acids. Consistently, growth was reduced and the levels of the oxidative stress marker malondialdehyde were higher in LA-exposed B. breve DSM 20213 than in the control.

Metabolism of biosynthetic oligosaccharides by human-derived Bifidobacterium breve UCC2003 and Bifidobacterium longum NCIMB 8809.

This work aimed to investigate the ability of two human-derived bifidobacterial strains, i.e. Bifidobacterium breve UCC2003 and Bifidobacterium longum NCIMB 8809, to utilize various oligosaccharides (i.e., 4-galactosyl-kojibiose, lactulosucrose, lactosyl-oligofructosides, raffinosyl-oligofructosides and lactulose-derived galacto-oligosaccharides) synthesized by means of microbial glycoside hydrolases. With the exception of raffinosyl-oligofructosides, these biosynthetic oligosaccharides were shown to support growth acting as a sole carbon and energy source of at least one of the two studied strains. Production of short-chain fatty acids (SCFAs) as detected by HPLC analysis corroborated the suitability of most of the studied novel oligosaccharides as fermentable growth substrates for the two bifidobacterial strains, showing that acetic acid is the main metabolic end product followed by lactic and formic acids. Transcriptomic and functional genomic approaches carried out for B. breve UCC2003 allowed the identification of key genes encoding glycoside hydrolases and carbohydrate transport systems involved in the metabolism of 4-galactosyl-kojibiose and lactulosucrose. In particular, the role of ß-galactosidases in the hydrolysis of these particular trisaccharides was demonstrated, highlighting their importance in oligosaccharide metabolism by human bifidobacterial strains.

Effects of Bifidobacterium breve A1 on the cognitive function of older adults with memory complaints: a randomised, double-blind, placebo-controlled trial.

In our previous study, we reported the therapeutic potential of Bifidobacterium breve A1 in preventing cognitive impairment in a mouse model of Alzheimer's disease and participants with mild cognitive impairment; we suggested that probiotic supplementation is an effective therapeutic strategy for managing cognitive function. Accordingly, we conducted a randomised, double-blind, placebo-controlled trial to assess whether 12-week B. breve A1 supplementation could affect the cognitive function of elderly subjects with memory complaints. We assessed cognitive function using the Japanese version of the Repeatable Battery for the Assessment of Neuropsychological Status (RBANS) and Mini-Mental State Examination (MMSE) at baseline and after 12 weeks of probiotic supplementation. A total of 121 participants were randomised and received B. breve A1 capsules or placebo daily for 12 weeks; of these, 117 participants completed the study. At 12 weeks, neuropsychological test scores significantly increased in both groups; no significant intergroup difference was observed in terms of changes in scores from the baseline scores. However, a stratified analysis revealed a significant difference between B. breve A1 and placebo groups in terms of the subscale 'immediate memory' of RBANS and MMSE total score in the subjects with low RBANS total score at baseline. No significant differences in terms of blood parameters between the groups or adverse effects caused by B. breve A1 intervention were observed. The results of the present study suggest the safety of B. breve A1 supplementation and its potential in maintaining cognitive function in elderly subjects with memory complaints. However, future large-scale studies on individuals with impaired cognitive function are required to validate the present findings.

Bifidobacterium breve MCC1274 with glycosidic activity enhances in vivo isoflavone bioavailability.

Polyphenols are plant derived compounds that exert many beneficial health effects to the human host. However, associated health benefits of dietary polyphenol are highly dependent on their intestinal metabolism, bioavailability, and absorption. Bifidobacteria, which represent the key members of gut microbiota, have been suggested to promote gut microbial homeostasis and may be involved in the metabolism of polyphenols. In this study, the capabilities of thirteen Bifidobacterium strains in hydrolysing polyphenol glycosides were evaluated. Among the tested strains, Bifidobacterium breve MCC1274 was found to possess the highest ß-glucosidase activity and strong capability to convert daidzin and trans-polydatin to their aglycones; while kinetic analysis revealed that B. breve MCC1274 hydrolysed more than 50% of daidzin and trans-polydatin at less than 3 h of incubation. Further investigation using rats with an antibiotics-disturbed microbiome revealed that following the ingestion of daidzin glycoside, oral administration of B. breve MCC1274 significantly enhanced the plasma concentration of daidzein in rats pre-treated with antibiotics as compared to antibiotics-pre-treated control and non-treated control groups. The relative abundance of Actinobacteria and the total numbers of B. breve were also significantly higher in antibiotics-pre-treated rats administered with B. breve MCC1274 than that of the control groups. These findings suggest that B. breve MCC1274 is effective in enhancing the bioavailability of daidzein in the gut under dysbiosis conditions and may potentially improve intestinal absorption of isoflavones and promote human health.

Bifidobacteriumbreve Bif195 Protects Against Small-Intestinal Damage Caused by Acetylsalicylic Acid in Healthy Volunteers.

BACKGROUND & AIMS: Enteropathy and small-intestinal ulcers are common adverse effects of nonsteroidal anti-inflammatory drugs such as acetylsalicylic acid (ASA). Safe, cytoprotective strategies are needed to reduce this risk. Specific bifidobacteria might have cytoprotective activities, but little is known about these effects in humans. We used serial video capsule endoscopy (VCE) to assess the efficacy of a specific Bifidobacterium strain in healthy volunteers exposed to ASA. METHODS: We performed a single-site, double-blind, parallel-group, proof-of-concept analysis of 75 heathy volunteers given ASA (300 mg) daily for 6 weeks, from July 31 through October 24, 2017. The participants were randomly assigned (1:1) to groups given oral capsules of Bifidobacterium breve (Bif195) (≥5 × 1010 colony-forming units) or placebo daily for 8 weeks. Small-intestinal damage was analyzed by serial VCE at 6 visits. The area under the curve (AUC) for intestinal damage (Lewis score) and the AUC value for ulcers were the primary and first-ranked secondary end points of the trial, respectively. RESULTS: Efficacy data were obtained from 35 participants given Bif195 and 31 given placebo. The AUC for Lewis score was significantly lower in the Bif195 group (3040 ± 1340 arbitrary units) than the placebo group (4351 ± 3195) (P = .0376). The AUC for ulcer number was significantly lower in the Bif195 group (50.4 ± 53.1 arbitrary units) than in the placebo group (75.2 ± 85.3 arbitrary units) (P = .0258). Twelve adverse events were reported from the Bif195 group and 20 from the placebo group. None of the events was determined to be related to Bif195 intake. CONCLUSIONS: In a randomized, double-blind trial of healthy volunteers, we found oral Bif195 to safely reduce the risk of small-intestinal enteropathy caused by ASA. ClinicalTrials.gov no: NCT03228589.

Prevention of Rotavirus Diarrhea in Suckling Rats by a Specific Fermented Milk Concentrate with Prebiotic Mixture.

Several microbial modulatory concepts, such as certain probiotics and prebiotics, confer protection against gastrointestinal infections, among which is acute diarrhea caused by the rotavirus (RV). Other microbiota modulators, such as postbiotics, produced during fermentation, might also have the potential to counteract RV infection. In light of this, a fermented milk, made by using Bifidobacterium breve C50 (BbC50) and Streptococcus thermophilus 065 (St065) with a prebiotic mixture-short chain galactooligosaccharides/long chain fructooligosaccharides (scGOS/lcFOS 9:1)-with potential to impact the intestinal microbiota composition was tested. An RV infected rat model was used to evaluate the amelioration of the infectious process and the improvement of the immune response induced by the fermented milk with prebiotic mixture. The dietary intervention caused a reduction in the clinical symptoms of diarrhea, such as severity and incidence. Furthermore, a modulation of the immune response was observed, which might enhance the reduction of the associated diarrhea. In addition, the fermented milk with prebiotic mixture was able to bind the virus and reduce its clearance. In conclusion, the postbiotic components in the fermented milk in combination with the prebiotics used here showed protective properties against RV infection.

Bifidobacterium bifidum ATCC 15696 and Bifidobacterium breve 24b Metabolic Interaction Based on 2'-O-Fucosyl-Lactose Studied in Steady-State Cultures in a Freter-Style Chemostat.

Infants fed breast milk harbor a gut microbiota in which bifidobacteria are generally predominant. The metabolic interactions of bifidobacterial species need investigation because they may offer insight into the colonization of the gut in early life. Bifidobacterium bifidum ATCC 15696 hydrolyzes 2'-O-fucosyl-lactose (2FL; a major fucosylated human milk oligosaccharide) but does not use fucose released into the culture medium. However, fucose is a growth substrate for Bifidobacterium breve 24b, and both strains utilize lactose for growth. The provision of fucose and lactose by B. bifidum (the donor) allowing the growth of B. breve (the beneficiary) conforms to the concept of syntrophy, but both strains will compete for lactose to multiply. To determine the metabolic impact of this syntrophic/competitive relationship on the donor, the transcriptomes of B. bifidum were determined and compared in steady-state monoculture and coculture using transcriptome sequencing (RNA-seq) and reverse transcription-quantitative PCR (RT-qPCR). B. bifidum genes upregulated in coculture included those encoding alpha-l-fucosidase and carbohydrate transporters and those involved in energy production and conversion. B. bifidum abundance was the same in coculture as in monoculture, but B. breve dominated the coculture numerically. Cocultures during steady-state growth in 2FL medium produced mostly acetate with little lactate (acetate:lactate molar ratio, 8:1) compared to that in monobatch cultures containing lactose (2:1), which reflected the maintenance of steady-state cells in log-phase growth. Darwinian competition is an implicit feature of bacterial communities, but syntrophy is a phenomenon putatively based on cooperation. Our results suggest that the regulation of syntrophy, in addition to competition, may shape bacterial communities.IMPORTANCE This study addresses the microbiology and function of a natural ecosystem (the infant bowel) using in vitro experimentation with bacterial cultures maintained under controlled growth and environmental conditions. We studied the growth of bifidobacteria whose nutrition centered on the hydrolysis of a human milk oligosaccharide. The results revealed responses relating to metabolism occurring in a Bifidobacterium bifidum strain when it provided nutrients that allowed the growth of Bifidobacterium breve, and so discovered biochemical features of these bifidobacteria in relation to metabolic interaction in the shared environment. These kinds of experiments are essential in developing concepts of bifidobacterial ecology that relate to the development of the gut microbiota in early life.

Arabinoxylan from Argentinian whole wheat flour promote the growth of Lactobacillus reuteri and Bifidobacterium breve.

Arabinoxylans are part of dietary fibre and have received attention given their emergent prebiotic character. Four arabinoxylans extracts were obtained from Argentinian soft and hard wheat. In vitro assays were performed to describe the extent to which the extracts from whole wheat flour support selective growth of Bifidobacterium breve and probiotic Lactobacillus reuteri ATCC23272 in a defined media. The prebiotic effect was evaluated by three quantitative scores: relative growth, prebiotic activity score and prebiotic index. For prebiotic index equation the growth of Bacteroides and Clostridium strains was compared to that of bifidobacteria and lactic acid bacteria. All the arabinoxylans extracts supported the growth of Lactobacillus and Bifidobacterium, reaching higher prebiotic activity score values than inulin (0·37 and 0·36 for Lactobacillus and Bifidobacterium respectively). AX2 from soft wheat and AX4 from hard showed similar prebiotic index value to commercial inulin (2·64, 2·52 and 2·22 respectively), and AX3 extract presented higher prebiotic index value (4·09) than the positive control and other prebiotic index reported for arabinoxylans. These extracts could be used as prebiotic, synbiotic compositions or novel food prototypes to treat dysbiosis associated with many diseases. SIGNIFICANCE AND IMPACT OF THE STUDY: The present work demonstrates that AX extracts from Argentinian soft and hard wheat promote efficiently the growth of probiotic strain L. reuteri ATCC23272 and B. breve 286, validated with three different parameters that consider the growth of representative strains of Bacteria genera found in the gut. The evaluation of AX extracts as a food supplement in a murine model could confirm their ability to modulate the microbiome. Novel food prototypes including AX and probiotics could relieve local symptoms and may act as psychobiotics with a beneficial effect on microbiome-brain axis.

Effects of lactoferrin on neonatal pathogens and Bifidobacterium breve in human breast milk.

Supplementation with probiotics in preterm infants reduces necrotizing enterocolitis and sepsis. Bovine lactoferrin is a promising supplement that may further reduce disease burden, but its effects on probiotic bacteria in human breast milk has not been evaluated. We aimed to characterise the antimicrobial activity of bovine and human lactoferrin in human breast milk against probiotics and typical neonatal sepsis pathogens. Lactoferrin levels were determined by enzyme linked immunosorbent assay in fresh and pasteurised human breast milk. The neonatal pathogens Staphylococcus epidermidis and Escherichia coli, and the probiotic Bifidobacterium breve strain M-16V were cultured in human breast milk or infant formula in the presence or absence of clinically relevant doses of bovine or human lactoferrin. Standard microbiological methods were used to determine the effects of lactoferrin on bacterial growth. Unpasteurised human breast milk contained significantly higher lactoferrin levels and resulted in superior inhibition of pathogenic bacterial growth compared to infant formula and pasteurised human breast milk. Human lactoferrin was significantly more effective at inhibiting bacterial growth, when compared to bovine lactoferrin. Supplementation with human lactoferrin or high dose bovine lactoferrin inhibited growth of the probiotic strain B. breve M-16V in pasteurised human breast milk. Although unpasteurised human breast milk and human lactoferrin had the greatest antimicrobial activity against all bacterial species tested, higher doses of bovine lactoferrin also showed activity against B. breve and. S. epidermidis. This study suggests that simultaneous administration of lactoferrins and probiotics may affect colonisation with probiotic bacteria, warranting further investigations.

Two extracellular sialidases from Bifidobacterium bifidum promote the degradation of sialyl-oligosaccharides and support the growth of Bifidobacterium breve.

We investigated the roles of extracellular sialidases (SiaBb1 and SiaBb2) in cross-feeding between sialidase-carrying Bifidobacterium bifidum and sialic acid-utilizing Bifidobacterium breve. Using 6' sialyllactose (6'SL) as a carbon source, the number of wild-type B. bifidum cells increased while that of a siabb2-inactivated strain (Δsiabb2) did not. Coculture of these two strains in the presence of 6'SL resulted in similar increase in cell numbers. Coculture of wild-type B. bifidum, but not the Δsiabb2 strain, with sialic acid-utilizing Bifidobacterium breve, which cannot release sialic acids from carbohydrates, in the presence of 6'SL increased the number of B. breve cells. Moreover, when mucin was used as a carbon source, B. breve growth was increased in cocultures with B. bifidum wild-type and Δsiabb2 strains, suggesting that SiaBb1 may be involved. Additionally, B. breve cell numbers increased during cultivation with recombinant SiaBb1-and SiaBb2-treated mucin as the sole carbon source. These results indicated that B. bifidum SiaBb2 liberated sialic acid from sialyl-human milk oligosaccharides and -mucin glycans, supporting the growth of B. breve through sialic acid cross-feeding. SiaBb1 may assist in the degradation of mucin glycan. Collectively, our results revealed that both the B. bifidum extracellular sialidases promote the utilization of sialylated carbohydrates and supply free sialic acid to other Bifidobacterium strains.

The potential synergistic behaviour of inter- and intra-genus probiotic combinations in the pattern and rate of short chain fatty acids formation during fibre fermentation.

This study compared the rate of short chain fatty acid (SCFA) production by different probiotic combinations of Lactobacillus and Bifidobacterium to determine any synergistic effects. Six different fibre fractions were fermented with nine combinations of Lactobacillus rhamnosus (LR), Lactobacillus acidophilus (LA), Bifidobacterium longum (BL) and Bifidobacterium breve (BB) for 0, 6, 24 and 48 h. SCFAs were quantified by gas chromatography. Inter-genus combinations of bacteria produced more SCFA, especially BB + BL + LR, compared to intra-genus that yielded the lowest SCFA production. Acetate was the most abundant, while propionate and butyrate were the most utilised. The SCFA formation was as acetate > propionate > butyrate and the total dietary fibre produced most of the SCFA. Most combinations utilised 60-80% of the fibre; BB + BL + LR digested the fibre completely. The quantity, pattern and the time of release of SCFA depends on the genus, but the combination of pre and probiotics is of great importance for the outcome.

The essential genomic landscape of the commensal Bifidobacterium breve UCC2003.

Bifidobacteria are common gut commensals with purported health-promoting effects. This has encouraged scientific research into bifidobacteria, though recalcitrance to genetic manipulation and scarcity of molecular tools has hampered our knowledge on the precise molecular determinants of their health-promoting attributes and gut adaptation. To overcome this problem and facilitate functional genomic analyses in bifidobacteria, we created a large Tn5 transposon mutant library of the commensal Bifidobacterium breve UCC2003 that was further characterized by means of a Transposon Directed Insertion Sequencing (TraDIS) approach. Statistical analysis of transposon insertion distribution revealed a set of 453 genes that are essential for or markedly contribute to growth of this strain under laboratory conditions. These essential genes encode functions involved in the so-called bifid-shunt, most enzymes related to nucleotide biosynthesis and a range of housekeeping functions. Comparison to the Bifidobacterium and B. breve core genomes highlights a high degree of conservation of essential genes at the species and genus level, while comparison to essential gene datasets from other gut bacteria identified essential genes that appear specific to bifidobacteria. This work establishes a useful molecular tool for scientific discovery of bifidobacteria and identifies targets for further studies aimed at characterizing essential functions not previously examined in bifidobacteria.

Prebiotic effects of bovine lactoferrin on specific probiotic bacteria.

Bovine lactoferrin (bLf) is a natural iron-binding protein and it has been suggested to be a prebiotic agent, but this finding remains inconclusive. This study explores the prebiotic potential of bLf in 14 probiotics. Initially, bLf (1-32 mg/mL) treatment showed occasional and slight prebiotic activity in several probiotics only during the late experimental period (48, 78 h) at 37 °C. We subsequently supposed that bLf exerts stronger prebiotic effects when probiotic growth has been temperately retarded. Therefore, we incubated the probiotics at different temperatures, namely 37 °C, 28 °C, room temperature (approximately 22-24 °C), and 22 °C, to retard or inhibit their growth. As expected, bLf showed more favorable prebiotic activity in several probiotics when their growth was partially retarded at room temperature. Furthermore, at 22 °C, the growth of Bifidobacterium breve, Lactobacillus coryniformis, L. delbrueckii, L. acidophilus, B. angulatum, B. catenulatum, and L. paraplantarum were completely blocked. Notably, these probiotics started regrowing in the presence of bLf (1-32 mg/mL) in a significant and dose-dependent manner. Accordingly, bLf significantly increased the growth of Pediococcus pentosaceus, L. rhamnosus, and L. paracasei (BCRC 17483; a locally isolated strain) when their growth was retarded by incubation at 22 °C. In conclusion, bLf showed inconsistent prebiotic activity in the 14 probiotics at 37 °C, but revealed strong prebiotic activity in 10 probiotic strains at 22 °C. Therefore, this study enables determining additional roles of Lf in probiotic strains, which can facilitate developing novel combinational approaches by simultaneously using Lf and specific probiotics.

Mechanistic Study of Utilization of Water-Insoluble Saccharomyces cerevisiae Glucans by Bifidobacterium breve Strain JCM1192.

Bifidobacteria exert beneficial effects on hosts and are extensively used as probiotics. However, due to the genetic inaccessibility of these bacteria, little is known about their mechanisms of carbohydrate utilization and regulation. Bifidobacterium breve strain JCM1192 can grow on water-insoluble yeast (Saccharomyces cerevisiae) cell wall glucans (YCWG), which were recently considered as potential prebiotics. According to the results of 1H nuclear magnetic resonance (NMR) spectrometry, the YCWG were composed of highly branched (1→3,1→6)-ß-glucans and (1→4,1→6)-α-glucans. Although the YCWG were composed of 78.3% ß-glucans and 21.7% α-glucans, only α-glucans were consumed by the B. breve strain. The ABC transporter (malEFG1) and pullulanase (aapA) genes were transcriptionally upregulated in the metabolism of insoluble yeast glucans, suggesting their potential involvement in the process. A nonsense mutation identified in the gene encoding an ABC transporter ATP-binding protein (MalK) led to growth failure of an ethyl methanesulfonate-generated mutant with yeast glucans. Coculture of the wild-type strain and the mutant showed that this protein was responsible for the import of yeast glucans or their breakdown products, rather than the export of α-glucan-catabolizing enzymes. Further characterization of the carbohydrate utilization of the mutant and three of its revertants indicated that this mutation was pleiotropic: the mutant could not grow with maltose, glycogen, dextrin, raffinose, cellobiose, melibiose, or turanose. We propose that insoluble yeast α-glucans are hydrolyzed by extracellular pullulanase into maltose and/or maltooligosaccharides, which are then transported into the cell by the ABC transport system composed of MalEFG1 and MalK. The mechanism elucidated here will facilitate the development of B. breve and water-insoluble yeast glucans as novel synbiotics.IMPORTANCE In general, Bifidobacterium strains are genetically intractable. Coupling classic forward genetics with next-generation sequencing, here we identified an ABC transporter ATP-binding protein (MalK) responsible for the import of insoluble yeast glucan breakdown products by B. breve JCM1192. We demonstrated the pleiotropic effects of the ABC transporter ATP-binding protein in maltose/maltooligosaccharide, raffinose, cellobiose, melibiose, and turanose transport. With the addition of transcriptional analysis, we propose that insoluble yeast glucans are broken down by extracellular pullulanase into maltose and/or maltooligosaccharides, which are then transported into the cell by the ABC transport system composed of MalEFG1 and MalK. The mechanism elucidated here will facilitate the development of B. breve and water-insoluble yeast glucans as novel synbiotics.

Bifidobacterium breve UCC2003 metabolises the human milk oligosaccharides lacto-N-tetraose and lacto-N-neo-tetraose through overlapping, yet distinct pathways.

In this study, we demonstrate that the prototype B. breve strain UCC2003 possesses specific metabolic pathways for the utilisation of lacto-N-tetraose (LNT) and lacto-N-neotetraose (LNnT), which represent the central moieties of Type I and Type II human milk oligosaccharides (HMOs), respectively. Using a combination of experimental approaches, the enzymatic machinery involved in the metabolism of LNT and LNnT was identified and characterised. Homologs of the key genetic loci involved in the utilisation of these HMO substrates were identified in B. breve, B. bifidum, B. longum subsp. infantis and B. longum subsp. longum using bioinformatic analyses, and were shown to be variably present among other members of the Bifidobacterium genus, with a distinct pattern of conservation among human-associated bifidobacterial species.

Glycosulfatase-Encoding Gene Cluster in Bifidobacterium breve UCC2003.

Bifidobacteria constitute a specific group of commensal bacteria typically found in the gastrointestinal tract (GIT) of humans and other mammals. Bifidobacterium breve strains are numerically prevalent among the gut microbiota of many healthy breastfed infants. In the present study, we investigated glycosulfatase activity in a bacterial isolate from a nursling stool sample, B. breve UCC2003. Two putative sulfatases were identified on the genome of B. breve UCC2003. The sulfated monosaccharide N-acetylglucosamine-6-sulfate (GlcNAc-6-S) was shown to support the growth of B. breve UCC2003, while N-acetylglucosamine-3-sulfate, N-acetylgalactosamine-3-sulfate, and N-acetylgalactosamine-6-sulfate did not support appreciable growth. By using a combination of transcriptomic and functional genomic approaches, a gene cluster designated ats2 was shown to be specifically required for GlcNAc-6-S metabolism. Transcription of the ats2 cluster is regulated by a repressor open reading frame kinase (ROK) family transcriptional repressor. This study represents the first description of glycosulfatase activity within the Bifidobacterium genus. IMPORTANCE: Bifidobacteria are saccharolytic organisms naturally found in the digestive tract of mammals and insects. Bifidobacterium breve strains utilize a variety of plant- and host-derived carbohydrates that allow them to be present as prominent members of the infant gut microbiota as well as being present in the gastrointestinal tract of adults. In this study, we introduce a previously unexplored area of carbohydrate metabolism in bifidobacteria, namely, the metabolism of sulfated carbohydrates. B. breve UCC2003 was shown to metabolize N-acetylglucosamine-6-sulfate (GlcNAc-6-S) through one of two sulfatase-encoding gene clusters identified on its genome. GlcNAc-6-S can be found in terminal or branched positions of mucin oligosaccharides, the glycoprotein component of the mucous layer that covers the digestive tract. The results of this study provide further evidence of the ability of this species to utilize mucin-derived sugars, a trait which may provide a competitive advantage in both the infant gut and adult gut.