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The galacto-oligosaccharide (GOS) OLIGOMATE 55N (Yakult) is a mixture of oligosaccharides, the main component of which is 4'-galactosyllactose (4'-GL). Numerous reports have shown that GOSs are non-digestible, reach the colon and selectively stimulate the growth of bifidobacteria. The product has been used as a food ingredient and its applications have expanded rapidly. However, the bifidobacterial glycoside hydrolases and transporters responsible for utilizing GOSs have not been characterized sufficiently. In this study, we aimed to identify and characterize genes responsible for metabolizing 4'-GL in Bifidobacterium breve strain Yakult. We attempted to identify B. breve Yakult genes induced by 4'-GL using transcriptional profiling during growth in basal medium containing 4'-GL with a custom microarray. We found that BbrY_0420, which encodes solute-binding protein (SBP), and BbrY_0422, which encodes β-galactosidase, were markedly upregulated relative to that during growth in basal medium containing lactose. Investigation of the substrate specificity of recombinant BbrY_0420 protein using surface plasmon resonance showed that BbrY_0420 protein bound to 4'-GL, but not to 3'-GL and 6'-GL, structural isomers of 4'-GL. Additionally, BbrY_0420 had a strong affinity for 4-galactobiose (4-GB), suggesting that this SBP recognized the non-reducing terminal structure of 4'-GL. Incubation of purified recombinant BbrY_0422 protein with 4'-GL, 3'-GL, 6'-GL and 4-GB revealed that the protein efficiently hydrolysed 4'-GL and 4-GB, but did not digest 3'-GL, 6'-GL or lactose, suggesting that BbrY_0422 digested the bond within Gal1,4-β-Gal. Thus, BbrY_0420 (SBP) and BbrY_0422 (β-galactosidase) had identical, strict substrate specificity, suggesting that they were coupled by co-induction to facilitate the transportation and hydrolysis of 4'-GL.

The composition of the human gut microbiota is related to host health, and it is thought that dietary habits may play a role in shaping this composition. Here, we examined the population size and prevalence of six predominant bacterial genera and the species compositions of genus Bifidobacterium (g-Bifid) and Bacteroides fragilis group (g-Bfra) in 42 healthy Belgian adults by quantitative PCR (qPCR) over a period of one month. The population sizes and prevalence of these bacteria were basically stable throughout the study period. The predominant g-Bifid species were Bifidobacterium adolescentis and Bifidobacterium longum ss. longum, and the predominant g-Bfra species were Bacteroides vulgatus, Bacteroides uniformis, and Bacteroides ovatus. The Belgian gut microbiota data were then compared with gut microbiota data from 46 Japanese subjects collected according to the same protocol (Matsuki et al., Appl. Environ. Microbiol. 70, 167-173, 2004). The population size and prevalence of Bifidobacterium catenulatum group were significantly lower in the Belgian gut microbiota than in the Japanese gut microbiota (P < 0.001); however, the population size and prevalence of g-Bifid did not differ. This species-level qPCR analysis will be helpful for investigating the diversity of gut microbiota among ethnic groups., (Copyright © 2013 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.)

In an attempt to identify and characterize how symbiotic bacteria of the gut microbiota affect the molecular and cellular mechanisms of epithelial homeostasis, intestinal epithelial cells were co-cultured with either Lactobacillus or Bifidobacterium as bona fide symbionts to examine potential gene modulations. In addition to genes involved in the innate immune response, genes encoding check-point molecules controlling the cell cycle were among the most modulated in the course of these interactions. In the m-ICcl2 murine cell line, genes encoding cyclin E1 and cyclin D1 were strongly down regulated by L. casei and B. breve respectively. Cell proliferation arrest was accordingly confirmed. Short chain fatty acids (SCFA) were the effectors of this modulation, alone or in conjunction with the acidic pH they generated. These results demonstrate that the production of SCFAs, a characteristic of these symbiotic microorganisms, is potentially an essential regulatory effector of epithelial proliferation in the gut.

Purpose: Probiotics are expected to be effective in prophylaxis of infection in cancer patient, since infections in neutropenics are mainly caused by endogenous flora through the intestinal mucosa. However, the experience with the use of probiotics in immunocompromised patients is limited, and precise fecal bacteria analysis has not been reported. The aim of the study was to evaluate the effects of the enteral administration of the probiotic, Bifidobacterium breve strain Yakult, on its ability to prevent infection, fecal micro flora, and intestinal environments in cancer patients on chemotherapy., Methods: A placebo-controlled trial was performed at Juntendo University Hospital. Patients with malignancies admitted for chemotherapy (n = 42) were randomized into two groups receiving probiotic or placebo. The effects on infectious complications, natural killer cells, fecal micro flora, fecal organic acid concentrations, and fecal pH were studied., Results: The frequency of fever and the use of intravenous antibiotics were lower in the probiotic group than the placebo group. The probiotic administration enhanced the habitation of anaerobes. Disruption of the intestinal microbiota after chemotherapy such as the increase in the population levels of Enterobacteriaceae was observed at more pronounced manner in the placebo group in comparison to the probiotic group. The concentrations of total organic acids were maintained most of the time at the normal level, which constantly maintained the pH below 7.0 only in the probiotic group., Conclusion: These data, although based on a limited number of patients and samples, suggest that administration of B. breve strain Yakult could be an effective approach for achieving clinical benefits in immunocompromised hosts by improving their intestinal environments.

A highly sensitive quantitative PCR detection method has been developed and applied to the distribution analysis of human intestinal bifidobacteria by combining real-time PCR with Bifidobacterium genus- and species-specific primers. Real-time PCR detection of serially diluted DNA extracted from cultured bifidobacteria was linear for cell counts ranging from 10(6) to 10 cells per PCR assay. It was also found that the method was applicable to the detection of Bifidobacterium in feces when it was present at concentrations of >10(6) cells per g of feces. Concerning the distribution of Bifidobacterium species in intestinal flora, the Bifidobacterium adolescentis group, the Bifidobacterium catenulatum group, and Bifidobacterium longum were found to be the three predominant species by examination of DNA extracted from the feces of 46 healthy adults. We also examined changes in the population and composition of Bifidobacterium species in human intestinal flora of six healthy adults over an 8-month period. The results showed that the composition of bifidobacterial flora was basically stable throughout the test period.

16SrDNA-targeted genus- and species-specific PCR primers have been developed and used for the identification and detection of bifidobacteria. These primers cover all of the described species that inhabit the human gut, or occur in dairy products. Identification of cultured bifidobacteria using PCR primer pairs is rapid and accurate, being based on nucleic acid sequences. Detection of bifidobacteria can be achieved using DNA extracted from human faeces as template in PCR reactions. We have found that, in adult faeces, the Bifidobacterium catenulatum group was the most commonly detected species, followed by Bifidobacterium longum, Bifidobacterium adolescentis, and Bifidobacterium bifidum. In breastfed infants, Bifidobacterium breve was the most frequently detected species, followed by Bifidobacterium infantis, B. longum and B. bifidum. It was notable that the B. catenulatum group was detected with the highest frequency in adults, although it has often been reported that B. adolescentis is the most common species. Real-time, quantitative PCR using primers targeting 16S rDNA shows promise in the enumeration of bifidobacteria in faecal samples. The approach to detect the target bacteria with quantitative PCR described in this review will contribute to future studies of the composition and dynamics of the intestinal microflora.

Methods that enabled the identification, detection, and enumeration of Bifidobacterium species by PCR targeting the transaldolase gene were tested. Bifidobacterial species isolated from the feces of human adults and babies were identified by PCR amplification of a 301-bp transaldolase gene sequence and comparison of the relative migrations of the DNA fragments in denaturing gradient gel electrophoresis (DGGE). Two subtypes of Bifidobacterium longum, five subtypes of Bifidobacterium adolescentis, and two subtypes of Bifidobacterium pseudocatenulatum could be differentiated using PCR-DGGE. Bifidobacterium angulatum and B. catenulatum type cultures could not be differentiated from each other. Bifidobacterial species were also detected directly in fecal samples by this combination of PCR and DGGE. The number of species detected was less than that detected by PCR using species-specific primers targeting 16S ribosomal DNA (rDNA). Real-time quantitative PCR targeting a 110-bp transaldolase gene sequence was used to enumerate bifidobacteria in fecal samples. Real-time quantitative PCR measurements of bifidobacteria in fecal samples from adults correlated well with results obtained by culture when either a 16S rDNA sequence or the transaldolase gene sequence was targeted. In the case of samples from infants, 16S rDNA-targeted PCR was superior to PCR targeting the transaldolase gene for the quantification of bifidobacterial populations.

Abstract: Quantitative (qt) real time PCR using 16SrDNA primers is useful for determination of the bacterial composition of the gastric microbiota in Mongolian gerbils. The aim of this study was to determine the change in the gastric microbiota after long-term infection with Helicobacter pylori. One year after inoculation with H. pylori, five gerbils were determined as H. pylori-positive and 6 gerbils H. pylori-negative by culture and real time qt PCR methods. The gastric microbiota of each group of gerbils was also compared with that of 6 gerbils uninfected with H. pylori. DNA from the Atopobium cluster, Bifidobacterium spp., Clostridium coccoides group, Clostridium leptum subgroup, Enterococcus spp. and Lactobacillus spp. were detected in the gastric mucus of both infected and uninfected gerbils. In contrast, Eubacterium cylindroides group and Prevotella spp. were detected only in H. pylori-negative gerbils. The numbers of C. leptum subgroup, C. coccoides group and Bifidobacterium spp. in gastric mucus of H. pylori-negative Mongolian gerbils were significantly lower than those in non-infected gerbils. The results obtained suggest that the composition of gastric indigenous microbiota in Mongolian gerbils may be disturbed by long-term infection with H. pylori, and that these changes may in fact inhibit H. pylori infection. [Copyright &y& Elsevier]

Examines the distribution of bifidobacterial species in human intestinal microflora. Use of 16S rRNA-gene-targeted species-specific primers in the examination; B. catenulatum group as the most common among the distribution; Demonstration of a rapid and accurate detection of bifidobacterial species in human feces.