Safety Evaluation of Bifidobacterium breve IDCC4401 Isolated from Infant Feces for Use as a Commercial Probiotic.

Previously, our research group isolated Bifidobacterium breve IDCC4401 from infant feces as a potential probiotic. For this study, we evaluated the safety of B. breve IDCC4401 using genomic and phenotypic analyses. Whole genome sequencing was performed to identify genomic characteristics and investigate the potential presence of genes encoding virulence, antibiotic resistance, and mobile genetic elements. Phenotypic analyses including antibiotic susceptibility, enzyme activity, production of biogenic amines (BAs), and proportion of D-/L-lactate were evaluated using E-test, API ZYM test, high-performance liquid chromatography (HPLC), and D-/L-lactic acid assay respectively. The genome of B. breve IDCC4401 consists of 2,426,499 bp with a GC content of 58.70% and 2,016 coding regions. Confirmation of the genome as B. breve was provided by its 98.93% similarity with B. breve DSM20213. Furthermore, B. breve IDCC4401 genes encoding virulence and antibiotic resistance were not identified. Although B. breve IDCC4401 showed antibiotic resistance against vancomycin, we confirmed that this was an intrinsic feature since the antibiotic resistance gene was not present. B. breve IDCC4401 showed leucine arylamidase, cystine arylamidase, α-galactosidase, ß-galactosidase, and α-glucosidase activities, whereas it did not show production of harmful enzymes such as ß-glucosidase and ß-glucuronidase. In addition, B. breve IDCC4401 did not produce any tyramine, histamine, putrescine, cadaverine, or 2-phenethylamine, which are frequently detected BAs during fermentation. B. breve IDCC4401 produced 95.08% of L-lactate and 4.92% of Dlactate. Therefore, our findings demonstrate the safety of B. breve IDCC 4401 as a potential probiotic for use in the food industry.

Impact of synbiotics on gut microbiota during early life: a randomized, double-blind study.

Human milk is considered the optimal nutrition for infants and found to contain significant numbers of viable bacteria. The aim of the study was to assess the effects of a specific synbiotic combination at doses closer to the bacterial cells present in human milk, on intestinal bifidobacteria proportions (relative abundance), reduction of potential pathogens and gut physiological conditions. A clinical study was conducted in 290 healthy infants aged from 6 to 19 weeks. Infants received either a control infant formula or one of the two investigational infant formulas (control formula with 0.8 g/100 ml scGOS/lcFOS and Bifidobacterium breve M-16V at either 1 × 104 cfu/ml or 1 × 106 cfu/ml). Exclusively breastfed infants were included as a reference. Analyses were performed on intention-to-treat groups and all-subjects-treated groups. After 6 weeks of intervention, the synbiotics at two different doses significantly increased the bifidobacteria proportions in healthy infants. The synbiotic supplementation also decreased the prevalence (infants with detectable levels) and the abundance of C. difficile. Closer to the levels in the breastfed reference group, fecal pH was significantly lower while L-lactate concentrations and acetate proportions were significantly higher in the synbiotic groups. All formulas were well tolerated and all groups showed a comparable safety profile based on the number and severity of adverse events and growth. In healthy infants, supplementation of infant-type bifidobacterial strain B. breve M-16V, at a dose close to bacterial numbers found in human milk, with scGOS/lcFOS (9:1) created a gut environment closer to the breastfed reference group. This specific synbiotic mixture may also support gut microbiota resilience during early life.Clinical Trial Registration This clinical study named Color Synbiotics Study, was registered in ClinicalTrials.gov on 18 March 2013. Registration number is NCT01813175. https://clinicaltrials.gov/ct2/show/NCT01813175 .

Human milk and mucosa-associated disaccharides impact on cultured infant fecal microbiota.

Human milk oligosaccharides (HMOs) are a mixture of structurally diverse carbohydrates that contribute to shape a healthy gut microbiota composition. The great diversity of the HMOs structures does not allow the attribution of specific prebiotic characteristics to single milk oligosaccharides. We analyze here the utilization of four disaccharides, lacto-N-biose (LNB), galacto-N-biose (GNB), fucosyl-α1,3-GlcNAc (3FN) and fucosyl-α1,6-GlcNAc (6FN), that form part of HMOs and glycoprotein structures, by the infant fecal microbiota. LNB significantly increased the total levels of bifidobacteria and the species Bifidobacterium breve and Bifidobacterium bifidum. The Lactobacillus genus levels were increased by 3FN fermentation and B. breve by GNB and 3FN. There was a significant reduction of Blautia coccoides group with LNB and 3FN. In addition, 6FN significantly reduced the levels of Enterobacteriaceae family members. Significantly higher concentrations of lactate, formate and acetate were produced in cultures containing either LNB or GNB in comparison with control cultures. Additionally, after fermentation of the oligosaccharides by the fecal microbiota, several Bifidobacterium strains were isolated and identified. The results presented here indicated that each, LNB, GNB and 3FN disaccharide, might have a specific beneficial effect in the infant gut microbiota and they are potential prebiotics for application in infant foods.

Contributions to human breast milk microbiome and enteromammary transfer of Bifidobacterium breve.

Increasing evidence supports the importance of the breast milk microbiome in seeding the infant gut. However, the origin of bacteria in milk and the process of milk microbe-mediated seeding of infant intestine need further elucidation. Presumed sources of bacteria in milk include locations of mother-infant and mother-environment interactions. We investigate the role of mother-infant interaction on breast milk microbes. Shotgun metagenomics and 16S rRNA gene sequencing identified milk microbes of mother-infant pairs in breastfed infants and in infants that have never latched. Although breast milk has low overall biomass, milk microbes play an important role in seeding the infant gut. Breast milk bacteria were largely comprised of Staphylococcus, Streptococcus, Acinetobacter, and Enterobacter primarily derived from maternal areolar skin and infant oral sites in breastfeeding pairs. This suggests that the process of breastfeeding is a potentially important mechanism for propagation of breast milk microbes through retrograde flux via infant oral and areolar skin contact. In one infant delivered via Caesarian section, a distinct strain of Bifidobacteria breve was identified in maternal rectum, breast milk and the infant's stool potentially suggesting direct transmission. This may support the existence of microbial translocation of this anaerobic bacteria via the enteromammary pathway in humans, where maternal bacteria translocate across the maternal gut and are transferred to the mammary glands. Modulating sources of human milk microbiome seeding potentially imply opportunities to ultimately influence the development of the infant microbiome and health.

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.

Carbohydrate Syntrophy enhances the establishment of Bifidobacterium breve UCC2003 in the neonatal gut.

The non-digestible oligosaccharide fraction of maternal milk represents an important of carbohydrate and energy source for saccharolytic bifidobacteria in the gastrointestinal tract during early life. However, not all neonatal bifidobacteria isolates can directly metabolise the complex sialylated, fucosylated, sulphated and/or N-acetylglucosamine-containing oligosaccharide structures present in mothers milk. For some bifidobacterial strains, efficient carbohydrate syntrophy or crossfeeding is key to their establishment in the gut. In this study, we have adopted advanced functional genomic approaches to create single and double in-frame deletions of the N-acetyl glucosamine 6-phosphate deacetylase encoding genes, nagA1 and nagA2, of B. breve UCC2003. In vitro phenotypic analysis followed by in vivo studies on co-colonisation, mother to infant transmission, and evaluation of the relative co-establishment of B. bifidum and B. breve UCC2003 or UCC2003ΔnagA1ΔnagA2 in dam-reared neonatal mice demonstrates the importance of crossfeeding on sialic acid, fucose and N-acetylglucosamine-containing oligosaccharides for the establishment of B. breve UCC2003 in the neonatal gut. Furthermore, transcriptomic analysis of in vivo gene expression shows upregulation of genes associated with the utilisation of lactose, sialic acid, GlcNAc-6-S and fucose in B. breve UCC2003, while for UCC2003ΔnagA1ΔnagA2 only genes for lactose metabolism were upregulated.

Evaluation of Potential Probiotics Isolated from Human Milk and Colostrum.

Several studies have demonstrated a diversity of bacterial species in human milk, even in aseptically collected samples. The present study evaluated potential probiotic bacteria isolated from human milk and associated maternal variables. Milk samples were collected from 47 healthy women and cultured on selective and universal agar media under aerobic and anaerobic conditions. Bacterial isolates were counted and identified by Biotyper Matrix-Assisted Laser Desorption Ionization-Time of Flight mass spectrometry and then tested for probiotic properties. Total bacteria in human milk ranged from 1.5 to 4.0 log10 CFU/mL. The higher bacterial counts were found in colostrum (mean = 3.9 log10 CFU/mL, 95% CI 3.14-4.22, p = 0.00001). The most abundant species was Staphylococcus epidermidis (n = 76). The potential probiotic candidates were Lactobacillus gasseri (n = 4), Bifidobacterium breve (n = 1), and Streptococcus salivarius (n = 4). Despite the small sample size, L. gasseri was isolated only in breast milk from mothers classified into a normal weight range and after a vaginally delivered partum. No potential probiotics showed antagonism against pathogens, but all of them agglutinated different pathogens. Nine bacterial isolates belonging to the species L. gasseri, B. breve, and S. salivarius were selected as potential probiotics. The present study confirms the presence in breast milk of a bacterial microbiota that could be the source of potential probiotic candidates to be used in the formula of simulated maternal milk.

Bacteremia induced by Bifidobacterium breve in a newborn with cloacal exstrophy.

Bifidobacterium breve is an effective probiotic agent used in the field of neonatology. Although B. breve has been considered safe, a case of B. breve bacteremia has been reported. The pathogenic mechanism underlying the bacteremia is unknown. Herein, we report a second case of B. breve bacteremia that developed in a neonate with multiple abdominal organ anomalies. Following surgical repair immediately after birth, B. breve treatment was started. After 1 week, the infant developed B. breve bacteremia following the onset of adhesive ileus. The bacteremia was thought to have been associated with an intestinal obstruction. A pediatric culture bottle is theoretically unsuitable for incubating B. breve because B. breve is an obligate anaerobic bacterium. It was, however, cultured from pediatric culture bottles in the present case, suggesting that pediatric culture bottles may be useful for procuring B. breve and for determining antimicrobial susceptibility for screening purposes in neonatal patients.