Distribution of Bifidobacterial Species in Human Intestinal Microflora Examined with 16S rRNA-Gene-Targeted Species-Specific Primers

In order to clarify the distribution of bifidobacterial species in the human intestinal tract, a 16S rRNA-gene-targeted species-specific PCR technique was developed and used with DNAs extracted from fecal samples obtained from 48 healthy adults and 27 breast-fed infants. To cover all of the bifidobacterial species that have been isolated from and identified in the human intestinal tract, species-specific primers for Bifidobacterium longum, B. infantis, B. dentium, and B. gallicum were developed and used with primers for B. adolescentis, B. angulatum, B. bifidum, B. breve, and the B. catenulatum group (B. catenulatum and B. pseudocatenulatum) that were developed in a previous study (T. Matsuki, K. Watanabe, R. Tanaka, and H. Oyaizu, FEMS Microbiol. Lett. 167:113–121, 1998). The specificity of the nine primers was confirmed by PCR, and the species-specific PCR method was found to be a useful means for identifying Bifidobacterium strains isolated from human feces. The results of an examination of bifidobacterial species distribution showed that the B. catenulatum group was the most commonly found taxon (detected in 44 of 48 samples [92%]), followed by B. longum and B. adolescentis, in the adult intestinal bifidobacterial flora and that B. breve, B. infantis, and B. longum were frequently found in the intestinal tracts of infants. The present study demonstrated that qualitative detection of the bifidobacterial species present in human feces can be accomplished rapidly and accurately.     

Prevention of <Emphasis Type="Italic">Escherichia coli</Emphasis> O157:H7 infection in gnotobiotic mice associated with <Emphasis Type="Italic">Bifidobacterium</Emphasis> strains

Previous reports have shown that Escherichia coli O157:H7 infection is strongly modified by intestinal microbes. In this paper, we examined whether bifidobacteria protect against E. coli O157:H7 infections using gnotobiotic mice di-associated with Bifidobacterium strains (6 species, 9 strains) and E. coli O157:H7. Seven days after oral administration of each Bifidobacterium strain, the mice were orally infected with E. coli O157:H7 and their mortality was examined. Bifidobacterium longum subsp. infantis 157F-4-1 (B. infantis 157F) and B. longum subsp. longum NCC2705 (B. longum NS) protected against the lethal infection, while mice associated with all other Bifidobacterium strains, including type strains of B. longum subsp. infantis and B. longum subsp. longum, died. There were no significant differences in the numbers of E. coli O157:H7 in the faeces among the Bifidobacterium-associated mouse groups. However, the Shiga toxin concentrations in the cecal contents and sera of the GB mice associated with B. infantis 157F and B. longum NS were significantly lower than those of the other groups. However, there were no significant differences in the volatile fatty acid concentrations and histopathological lesions between these two groups. These data suggest that some strains of B. longum subsp. longum/infantis can protect against the lethal infections of E. coli O157:H7 by preventing Shiga toxin production in the cecum and/or Shiga toxin transfer from the intestinal lumen to the bloodstream.     

Molecular analysis of the replication functions of the bifidobacterial conjugative megaplasmid pMP7017

pMP7017 is a conjugative megaplasmid isolated from the gut commensal Bifidobacterium breve JCM7017 and was shown to encode two putative replicases, designated here as RepA and RepB. In the current work, RepB was identified as the pMP7017 replicative initiator, as the repB gene, and its surrounding region was shown to be sufficient to allow autonomous replication in two bifidobacterial species, B. breve and Bifidobacterium longum subsp. longum. RepB was shown to bind to repeat sequence downstream of its coding sequence and this region was determined to be essential for efficient replication. Based on our results, we hypothesize that pMP7017 is an iteron-regulated plasmid (IRP) under strict auto-regulatory control. Recombinantly produced and purified RepB was determined to exist as a dimer in solution, differing from replicases of other IRPs, which exist as a mix of dimers and monomers. Furthermore, a stable low-copy Bifidobacterium-E. coli shuttle vector, pRD1.3, was created which can be employed for cloning and expression of large genes, as was demonstrated by the cloning and heterologous expression of the 5.1 kb apuB gene encoding the extracellular amylopullulanase from B. breve UCC2003 into B. longum subsp. longum NCIMB8809.     

Interactions between Bifidobacterium and Bacteroides Species in Cofermentations Are Affected by Carbon Sources,Including Exopolysaccharides Produced by Bifidobacteria

Cocultures of strains from two Bifidobacterium and two Bacteroides species were performed with exopolysaccharides (EPS) previously purified from bifidobacteria, with inulin, or with glucose as the carbon source. Bifidobacterium longum NB667 and Bifidobacterium breve IPLA20004 grew in glucose but showed poor or no growth in complex carbohydrates (inulin, EPS E44, and EPS R1), whereas Bacteroides grew well in the four carbon sources tested. In the presence of glucose, the growth of Bacteroides thetaiotaomicron DSM-2079 was inhibited by B. breve, whereas it remained unaffected in the presence of B. longum. Ba. fragilis DSM-2151 contributed to a greater survival of B. longum, promoting changes in the synthesis of short-chain fatty acids (SCFA) and organic acids in coculture with respect to monocultures. In complex carbohydrates, cocultures of bifidobacterium strains with Ba. thetaiotaomicron did not modify the behavior of Bacteroides nor improve the poor growth of bifidobacteria. The metabolic activity of Ba. fragilis in coculture with bifidobacteria was not affected by EPS, but greater survival of bifidobacteria at late stages of incubation occurred in cocultures than in monocultures, leading to a higher production of acetic acid than in monocultures. Therefore, cocultures of Bifidobacterium and Bacteroides can behave differently against fermentable carbohydrates as a function of the specific characteristics of the strains from each species. These results stress the importance of considering specific species and strain interactions and not simply higher taxonomic divisions in the relationship among intestinal microbial populations and their different responses to probiotics and prebiotics.     

Differences in Composition and Mucosal Adhesion of Bifidobacteria Isolated from Healthy Adults and Healthy Seniors

Fifty-one Bifidobacterium strains were isolated from the feces of healthy adults (30–40 years old) and seniors (older than 70 years of age). B. adolescentis, B. breve, B. infantis, and B. longum were isolated from the healthy adults and B. adolescentis and B. longum from elderly subjects. The tested bacteria bound, in vitro, to intestinal mucus in a strain dependent manner. The strains isolated from healthy adults, and especially B. adolescentis, bound better to intestinal mucus than those isolated from seniors. These results indicate that the mucosal adhesive properties of the human Bifidobacterium flora were reduced with the aging of the host. This shift to a Bifidobacterium flora with reduced adhesive abilities may explain the decrease in bifidobacteria levels in the intestinal microflora of aging people. Received: 7 February 2001 / Accepted: 3 April 2001     

Variation in Consumption of Human Milk Oligosaccharides by Infant Gut-Associated Strains of Bifidobacterium breve

Human milk contains a high concentration of complex oligosaccharides that influence the composition of the intestinal microbiota in breast-fed infants. Previous studies have indicated that select species such as Bifidobacterium longum subsp. infantis and Bifidobacterium bifidum can utilize human milk oligosaccharides (HMO) in vitro as the sole carbon source, while the relatively few B. longum subsp. longum and Bifidobacterium breve isolates tested appear less adapted to these substrates. Considering the high frequency at which B. breve is isolated from breast-fed infant feces, we postulated that some B. breve strains can more vigorously consume HMO and thus are enriched in the breast-fed infant gastrointestinal tract. To examine this, a number of B. breve isolates from breast-fed infant feces were characterized for the presence of different glycosyl hydrolases that participate in HMO utilization, as well as by their ability to grow on HMO or specific HMO species such as lacto-N-tetraose (LNT) and fucosyllactose. All B. breve strains showed high levels of growth on LNT and lacto-N-neotetraose (LNnT), and, in general, growth on total HMO was moderate for most of the strains, with several strain differences. Growth and consumption of fucosylated HMO were strain dependent, mostly in isolates possessing a glycosyl hydrolase family 29 α-fucosidase. Glycoprofiling of the spent supernatant after HMO fermentation by select strains revealed that all B. breve strains can utilize sialylated HMO to a certain extent, especially sialyl-lacto-N-tetraose. Interestingly, this specific oligosaccharide was depleted before neutral LNT by strain SC95. In aggregate, this work indicates that the HMO consumption phenotype in B. breve is variable; however, some strains display specific adaptations to these substrates, enabling more vigorous consumption of fucosylated and sialylated HMO. These results provide a rationale for the predominance of this species in breast-fed infant feces and contribute to a more accurate picture of the ecology of the developing infant intestinal microbiota.     

Deoxyribonucleic acid homology among the species of the genus Bifidobacterium isolated from animals

Summary Representatives of the species of the genus Bifidobacterium isolated from bovine rumen, intestinal content of honey bees and pig feces were studied for their deoxyribonucleic acid similarities with DNA-DNA filter hybridization-competition experiments. The species were: B. asteroides, B. indicum and B. coryneforme from honey bees; B. ruminale and B. globosum from rumen; B. suis from pig feces; Mitsuoka's species B. thermophilum and B. pseudolongum. Strains of B. bifidum, B. infantis, B. longum and B. breve from human sources were included in some comparative experiments. The group of bacteria investigated is largely heterogeneous: virtually no homology exists among the species from rumen and pig feces; the species or types from bees, although unrelated with rumen and pig types, share with these a significant portion of the genome; the competitor DNA's from human strains did not at all react with any of the homologous systems. The annealing reaction was carried out under stringent conditions to ensure against mismatching of nucleotide sequences (70°C in Denhardt's reactive mixture). The separation within the genus Bifidobacterium of several specific entities is fully substantiated.This investigation was supported by a research grant of C. N. R. (Consiglio Nazionale delle Ricerche, Roma).     

Characterization of Bifidobacterium spp. strains for the treatment of enteric disorders in newborns

Several studies support the use of probiotics for the treatment of minor gastrointestinal problems in infants. Positive effects on newborn colics have been evidenced after administration of Lactobacillus strains, whereas no studies have been reported regarding the use of bifidobacteria for this purpose. This work was therefore aimed at the characterization of Bifidobacterium strains capable of inhibiting the growth of pathogens typical of the infant gastrointestinal tract and of coliforms isolated from colic newborns. Among the 46 Bifidobacterium strains considered, 16 showed high antimicrobial activity against potential pathogens; these strains were further characterized from a taxonomic point of view, for the presence and transferability of antibiotic resistances, for citotoxic effects and adhesion to nontumorigenic gut epithelium cell lines. Moreover, their ability to stimulate gut health by increasing the metabolic activity and the immune response of epithelial cells was also studied. The examination of all these features allowed to identify three Bifidobacterium breve strains and a Bifidobacterium longum subsp. longum strain as potential probiotics for the treatments of enteric disorders in newborns such as infantile colics. A validation clinical trial involving the selected strains is being planned.     

Conditions affecting cell surface properties of human intestinal bifidobacteria

The cell surface properties of human intestinal bifidobacteria have been characterized for 30 strains isolated from a fecal sample. Strain identification to the species level was obtained by restriction analysis of the amplified 16S rRNA gene and confirmed by DNA/DNA reassociation experiments. The isolates were grouped in four genetically homogeneous clusters whose members belonged to Bifidobacterium bifidum, Bifidobacterium adolescentis, Bifidobacterium longum and Bifidobacterium pseudocatenulatum species. Cell surface properties of Bifidobacterium strains were evaluated by determining the level of hydrophobicity, adhesion to hydrocarbons and contact angle measurements, and their autoaggregation ability. The results showed high and homogeneous level of hydrophobicity in all tested strains when contact angle measurements values were considered. On the contrary, autoaggregation assays and bacterial adhesion to hydrocarbons detected interesting differences in cell surface properties among the tested Bifidobacterium strains. The highest levels of autoaggregation, detected in B. bifidum and B. adolescentis strains, were strictly dependent on the pH of the medium. Moreover, protease treatment experiments suggested that proteins had a key role in the autoaggregating ability of B. bifidum and B. adolescentis strains.     

Gnotobiotic Mouse Immune Response Induced by Bifidobacterium sp. Strains Isolated from Infants

Bifidobacterium, which is a dominant genus in infants’ fecal flora and can be used as a probiotic, has shown beneficial effects in various pathologies, including allergic diseases, but its role in immunity has so far been little known. Numerous studies have shown the crucial role of the initial intestinal colonization in the development of the intestinal immune system, and bifidobacteria could play a major role in this process. For a better understanding of the effect of Bifidobacterium on the immune system, we aimed at determining the impact of Bifidobacterium on the T-helper 1 (T_H1)/T_H2 balance by using gnotobiotic mice. Germfree mice were inoculated with Bifidobacterium longum NCC2705, whose genome is sequenced, and with nine Bifidobacterium strains isolated from infants’ fecal flora. Five days after inoculation, mice were killed. Transforming growth factor β1 (TGF-β1), interleukin-4 (IL-4), IL-10, and gamma interferon (IFN-γ) gene expressions in the ileum and IFN-γ, tumor necrosis factor alpha (TNF-α), IL-10, IL-4, and IL-5 secretions by splenocytes cultivated for 48 h with concanavalin A were quantified. Two Bifidobacterium species had no effect (B. adolescentis) or little effect (B. breve) on the immune system. Bifidobacterium bifidum, Bifidobacterium dentium, and one B. longum strain induced T_H1 and T_H2 cytokines at the systemic and intestinal levels. One B. longum strain induced a T_H2 orientation with high levels of IL-4 and IL-10, both secreted by splenocytes, and of TGF-β gene expression in the ileum. The other two strains induced T_H1 orientations with high levels of IFN-γ and TNF-α splenocyte secretions. Bifidobacterium's capacity to stimulate immunity is species specific, but its influence on the orientation of the immune system is strain specific.     

Characterization of Bifidobacterium Strains Using Box Primers

Twenty-five Bifidobacterium strains isolated from infants' faeces were identified by Rep-PCR. Using BOX-PCR, characteristic bands of Bifidobacterium bifidum, Bifidobacterium breve, Bifidobacterium longum, Bifidobacterium infantis and Bifidobacterium adolescentis were found in 40 strains of bidfidobacteria. These bands were not found in lactobacilli. By computerized numerical analysis strains were grouped in two major clusters. Strains of B. bifidum fell into a well-differentiated cluster that joined the cluster of the remaining species at 0.771 of similarity. The predominant species among the isolated strains were Bifidobacterium bifidum, Bifidobacterium longum andBifidobacterium breve . In another set of experiments, DNA was extracted from bacteria harvested from fermented milks to which different concentrations of bifidobacteria had been added. In all cases characteristic bands in the agarose gel belonging to lactobacilli and streptococci were detected. Bifidobacterium was detected only when 10⁸CFU/ml were added to the fermented milks. On the basis of our results, we propose this methodology as another tool in the polyphasic taxonomy.     

Distribution of genes of toxin-antitoxin systems of MazEF and RelBE families in bifidobacteria from human intestinal microbiota

The in silico analysis of 36 sequenced genomes of bacteria of the Bifidobacterium genus determined the presence of 19 genes of toxin-antitoxin (TA) system that belong to the MazEF and RelBE families, including five mazF and two relE genes that encode toxins and 12 relB genes that encode antitoxins. A high level of gene (at the level of nucleotide changes) and genomic (presence or absence of genes in distinct genomes) polymorphism in the investigated genes was revealed. The highest level of polymorphism was observed in strains of the Bifidobacterium longum species, primarily in relB1-relB10 genes. Gene and genomic polymorphism might be used to identify the strain of B. longum species. PCR analysis of genomic DNA of 30 bifidobacteria strains belonging to the three species, B. longum, B. adolescentis, and B. bifidum, isolated from the intestinal microbiota of astronauts demonstrated the presence of mazF and relB genes. The observed polymorphism of TA genes indicates the presence of differences in bifidobacteria strains isolated from the intestinal microbiota of astronauts before and after space flight and the control group.     

Discovery of a Conjugative Megaplasmid in Bifidobacterium breve

Bifidobacterium breve is a common and sometimes very abundant inhabitant of the human gut. Genome sequencing of B. breve JCM 7017 revealed the presence of an extrachromosomal element, designated pMP7017 consisting of >190 kb, thus representing the first reported bifidobacterial megaplasmid. In silico characterization of this element revealed several genomic features supporting a stable establishment of the megaplasmid in its host, illustrated by predicted CRISPR-Cas functions that are known to protect the host against intrusion of foreign DNA. Interestingly, pMP7017 is also predicted to encode a conjugative DNA transfer apparatus and, consistent with this notion, we demonstrate here the conjugal transfer of pMP7017 to representative strains of B. breve and B. longum subsp. longum. We also demonstrate the presence of a megaplasmid with homology to pMP7017 in three B. longum subsp. longum strains.     

Antibiotic Susceptibility of Lactobacillus and Bifidobacterium Species from the Human Gastrointestinal Tract

One hundred and twenty-two strains of Bifidobacterium and Lactobacillus species have been tested against 12 antibiotics and two antibiotic mixtures by a commercial system (Sensititre Anaero3; Treck Diagnostic Systems). The upper limits of some minimum inhibitory concentrations (MICs) were completed on MRS agar plates by the NCCLS procedure. All strains were sensitive to chloramphenicol and imipenem and most of the strains were resistant to metronidazole. Bifidobacteria isolates were susceptible to cefoxitin, whereas about half of the lactobacilli were resistant. Approximately 30% of the Bifidobacterium isolates were resistant to tetracycline, as well as five Lactobacillus strains belonging to four different species. None of the tested Bifidobacterium isolates was resistant to vancomycin, whereas a species-dependent resistance was found among the lactobacilli. Single strains of Bifidobacterium longum, Bifidobacterium pseudocatenulatum, Lactobacillus acidophilus, Lactobacillus rhamnosus, and Lactobacillus brevis were resistant to erythromycin and/or clindamycin. Most of the observed resistances seemed to be intrinsic, but some others could be compatible with transmissible determinants.     

Growth-promoting Effects of N-Acetylneuraminic Acid-containing Substances on Bifidobacteria

The use of N-acetylneuraminic acid, sialyl-lactose, and glyco-macropeptide by bifidobacteria and lactobacilli, and their growth-promoting effects on B. longum, B. breve, B. bifidum, and B. infantis were investigated. The data presented here suggest that fortification with N-acetylneuraminic acid-containing substances of infant formula may provide formula-fed infants with a function that human milk possesses.     

A Bifidobacterial pilus‐associated protein promotes colonic epithelial proliferation

Development of the human gut microbiota commences at birth, with certain bifidobacterial species representing dominant and early colonisers of the newborn gastrointestinal tract. The molecular basis of Bifidobacterium colonisation, persistence and presumed communication with the host has remained obscure. We previously identified tight adherence (Tad) pili from Bifidobacterium breve UCC2003 as an essential colonisation factor. Here, we demonstrate that bifidobacterial Tad pili also promote in vivo colonic epithelial proliferation. A significant increase in cell proliferation was detectable 5 days postadministration of B. breve UCC2003. Using advanced functional genomic approaches, bacterial strains either (a) producing the Tad₂₀₀₃ pili or (b) lacking the TadE or TadF pseudopilins were created. Analysis of the ability of these mutant strains to promote epithelial cell proliferation in vivo demonstrated that the pilin subunit, TadE, is the bifidobacterial molecule responsible for this proliferation response. These findings were confirmed in vitro using purified TadE protein. Our data imply that bifidobacterial Tad pili may contribute to the maturation of the naïve gut in early life through the production of a specific scaffold of extracellular protein structures, which stimulate growth of the neonatal mucosa.     

Application of Several Molecular Techniques to Study Numerically Predominant Bifidobacterium spp. and Bacteroidales Order Strains in the Feces of Healthy Children

Bifidobacteria and Bacteroides-like bacteria are strictly anaerobic nonpathogenic members of human intestinal microflora. Here we describe an analysis of the species and subspecies composition of these bacterial populations in healthy children using a combination of culture and molecular methods at two different time points. It was found that B. bifidum and B. longum are the most common dominant taxons in infants aged between 8 and 16 months. The majority of the infants carried several dominant Bifidobacterium strains belonging to different species. Examination of the dominant bifidoflora in some of these children after a 5-year period showed major shifts in both species and strain composition, but the dominant strains remained unchanged in two children. The majority of dominant Bacteroides-like isolates belonged to species B. vulgatus and B. uniformis, but members of genera Alistipes and Barnesiella were common too. In addition, a novel approach to species identification of Bacteroidales order bacteria using amplified ribosomal DNA restriction analysis (ARDRA) is described.     

Resistance to Simulated Gastrointestinal Conditions and Adhesion to Mucus as Probiotic Criteria for Bifidobacterium longum Strains

Eight Bifidobacterium longum strains, including reported probiotic strains (commercial and noncommercial), collection strains, and laboratory isolates, were investigated for their ability to adhere to mucin as well as their ability to tolerate acid and bile. Strains could be discriminated based on their sensitivity at pH values of 2.0 to 2.5 and bile concentrations of 0.5% to 2.0%. B. longum NCC 2705, a strain known for its probiotic properties, showed the highest resistance to gastrointestinal conditions, whereas the commercial probiotic strains B. longum BB 536 and SP 07/3 were the least resistant. In parallel, the human isolate B. longum BIF 53 showed the highest adhesion to mucin, whereas the commercial probiotic strains B. longum W 11, BB 536, and SP 07/3 were the least adhesive. The bacterial adhesion to mucin of strains B. longum NCC 2705 and BIF 53 could be reduced by lysozyme, indicating that cell-wall components are involved in the adhesion process. These results showed that there is no obvious link between adhesion and resistance to gastrointestinal conditions and the probiotic status of the studied strains. This calls for a definition of conditions for in vitro tests that better predict the in vivo functionality of probiotic strains.     

A versatile and scalable strategy for glycoprofiling bifidobacterial consumption of human milk oligosaccharides

Human milk contains approximately 200 complex oligosaccharides believed to stimulate the growth and establishment of a protective microbiota in the infant gut. The lack of scalable analytical techniques has hindered the measurement of bacterial metabolism of these and other complex prebiotic oligosaccharides. An in vitro, multi‐strain, assay capable of measuring kinetics of bacterial growth and detailed oligosaccharide consumption analysis by FTICR‐MS was developed and tested simultaneously on 12 bifidobacterial strains. For quantitative consumption, deuterated and reduced human milk oligosaccharide (HMO) standards were used. A custom software suite developed in house called Glycolyzer was used to process the large amounts of oligosaccharide mass spectra automatically with ¹³C corrections based on de‐isotoping protocols. High growth on HMOs was characteristic of Bifidobacterium longum biovar infantis strains, which consumed nearly all available substrates, while other bifidobacterial strains tested, B. longum bv. longum, B. adolescentis, B. breve and B. bifidum, showed low or only moderate growth ability. Total oligosaccharide consumption ranged from a high of 87% for B. infantis JCM 7009 to only 12% for B. adolescentis ATCC 15703. A detailed analysis of consumption glycoprofiles indicated strain‐specific capabilities towards differential metabolism of milk oligosaccharides. This method overcomes previous limitations in the quantitative, multi‐strain analysis of bacterial metabolism of HMOs and represents a novel approach towards understanding bacterial consumption of complex prebiotic oligosaccharides.