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PubMed2004

Increased poliovirus-specific intestinal antibody response coincides with promotion of Bifidobacterium longum-infantis and Bifidobacterium breve in infants: a randomized, double-blind, placebo-controlled trial.

To determine whether the size of the intestinal bifidobacterial population can influence the immune response to poliovirus vaccination in infants, we set up a randomized, placebo-controlled trial. From birth to 4 mo, infants were given a fermented infant formula (FIF) or a standard formula (placebo). Bifidobacteria were quantified monthly in infant stools. Antipoliovirus IgA response to Pentacoq was assessed before and 1 mo after the second vaccine injection. Thirty infants were randomized, and 20 completed the study (nine in the placebo group and 11 in the FIF group). Fecal bifidobacterial level was significantly higher with the FIF group at 4 mo of age (p=0.0498). Furthermore, B. longum/B. infantis carriage was higher at 4 mo in the FIF group (p=0.0399). Antipoliovirus IgA titers increased after Pentacoq challenge (p <0.001), and the rise was significantly higher in the FIF group (p <0.02). Antibody titers correlated with bifidobacteria, especially with B. longum/B. infantis and B. breve levels (p <0.002). Infants who harbored B. longum/B. infantis also exhibited higher levels of antipoliovirus IgAs (p <0.002). In conclusion, the present results indicate that antipoliovirus response can be triggered with a fermented formula that is able to favor intestinal bifidobacteria. Whether this effect on the immune system is achieved through the bifidogenic effect of the formula (mainly through B. longum/B. infantis and B. breve stimulation) or directly linked to compounds (i.e. peptides) produced by milk fermentation remains to be investigated.

Bifidobacterium↗
PubMed2001

Molecular microbial analysis of Bifidobacterium isolates from different environments by the species-specific amplified ribosomal DNA restriction analysis (ARDRA).

One hundred and six isolates of the genus Bifidobacterium, isolated from different environments (mainly gastrointestinal), were identified and classified taxonomically to species level by amplified ribosomal DNA restriction analysis. Two restriction endonucleases (Sau3AI and BamHI) were chosen for aligning the 16S rRNA sequences of 16 bifidobacterial species retrieved from various databases, to obtain species-specific restriction patterns. A rapid and accurate identification scheme was obtained by comparing the resulting 16S rDNA digestion profiles of 16 Bifidobacterium type-strains and 90 strains of various origins. All of the investigated strains were previously confirmed at the species level as belonging to the genus Bifidobacterium by fluorescence in-situ hybridisation and by polymerase chain reaction amplification with genus- and species-specific primers. The present work demonstrates that species-specific detection of Bifidobacterium adolescentis, Bifidobacterium animalis, Bifidobacterium bifidum, Bifidobacterium breve, Bifidobacterium catenulatum, Bifidobacterium coryneforme, Bifidobacterium cuniculi, Bifidobacterium dentium, Bifidobacterium infantis, Bifidobacterium lactis, Bifidobacterium longum, Bifidobacterium suis, Bifidobacterium magnum, Bifidobacterium pseudolongum, Bifidobacterium pseudocatenulatum and Bifidobacterium pullorum present in different micro-ecological environments (e.g. gastrointestinal tract) can be accomplished in a reliable, rapid and accurate manner, circumventing the recognised deficiencies of traditional identification techniques.

Journal Article↗
PubMed2005

Quantitative real-time PCR assays to identify and quantify fecal Bifidobacterium species in infants receiving a prebiotic infant formula.

A healthy intestinal microbiota is considered to be important for priming of the infants' mucosal and systemic immunity. Breast-fed infants typically have an intestinal microbiota dominated by different Bifidobacterium species. It has been described that allergic infants have different levels of specific Bifidobacterium species than healthy infants. For the accurate quantification of Bifidobacterium adolescentis, Bifidobacterium angulatum, Bifidobacterium bifidum, Bifidobacterium breve, Bifidobacterium catenulatum, Bifidobacterium dentium, Bifidobacterium infantis, and Bifidobacterium longum in fecal samples, duplex 5' nuclease assays were developed. The assays, targeting rRNA gene intergenic spacer regions, were validated and compared with conventional PCR and fluorescent in situ hybridization methods. The 5' nuclease assays were subsequently used to determine the relative amounts of different Bifidobacterium species in fecal samples from infants receiving a standard formula or a standard formula supplemented with galacto- and fructo-oligosaccharides (OSF). A breast-fed group was studied in parallel as a reference. The results showed a significant increase in the total amount of fecal bifidobacteria (54.8% +/- 9.8% to 73.4% +/- 4.0%) in infants receiving the prebiotic formula (OSF), with a diversity of Bifidobacterium species similar to breast-fed infants. The intestinal microbiota of infants who received a standard formula seems to resemble a more adult-like distribution of bifidobacteria and contains relatively more B. catenulatum and B. adolescentis (2.71% +/- 1.92% and 8.11% +/- 4.12%, respectively, versus 0.15% +/- 0.11% and 1.38% +/- 0.98% for the OSF group). In conclusion, the specific prebiotic infant formula used induces a fecal microbiota that closely resembles the microbiota of breast-fed infants also at the level of the different Bifidobacterium species.

Bifidobacterium↗
PubMed2001

New approach to phylogenetic analysis of the genus Bifidobacterium based on partial HSP60 gene sequences.

The partial 60 kDa heat-shock protein (HSP60) genes of 36 Bifidobacterium strains representing 30 different Bifidobacterium species and subspecies and of the type strain of Gardnerella vaginalis were cloned and sequenced using a pair of universal degenerate HSP60 PCR primers. The HSP60 DNA sequence similarities were determined for the taxa at various ranks as follows: 99.4-100% within the same species, 96% at the subspecies level, and 73-96% (mean 85%) at the interspecies level (and 98% in the case of two groups of closely related species, Bifidobacterium animalis and Bifidobacterium lactis, Bifidobacterium infantis, Bifidobacterium longum and Bifidobacterium suis, whose 165 rRNA sequence similarities are all above 99%). The HSP60 DNA sequence similarities between different Bifidobacterium species and G. vaginalis, a closely related bacterium according to 16S rRNA analysis, ranged from 71 to 79% (mean 75%). Although the topology of the phylogenetic tree constructed using the HSP60 sequences determined was basically similar to that for 16S rRNA, it seemed to be more clear-cut for species delineation, and the clustering was better correlated with the DNA base composition (mol% G+C) than that of the 16S rRNA tree. In the HSP60 phylogenetic tree, all of the high-G+C (55-67 mol%) bifidobacteria were grouped into one cluster, whereas the low-G+C species Bifidobacterium inopinatum (45 mol %) formed a separate cluster with G. vaginalis (42 mol%) and Bifidobacterium denticolens (55 mol%); a Bifidobacterium species of intermediate G+C content formed another cluster between the two. This study demonstrates that the highly conserved and ubiquitous HSP60 gene is an accurate and convenient tool for phylogenetic analysis of the genus Bifidobacterium.

Animals↗
PubMed2004

Evaluation of three different forward primers by terminal restriction fragment length polymorphism analysis for determination of fecal bifidobacterium spp. in healthy subjects.

The 27F forward primer is frequently used in 16S rRNA gene libraries and T-RFLP analysis. However, Bifidobacterium spp. were barely detected with this primer in human fecal samples. In this study, fecal microbiota were analyzed using the T-RFLP method with three different forward primers (27F, 35F, and 529F) in conjunction with one reverse primer (1492R). T-RFLP analysis of fecal microbiota using 35F and 529F detected higher proportions of the terminal restriction fragment (T-RF) corresponding to Bifidobacterium spp. than that using 27F. 27F is in imperfect agreement while 35F and 529F are in good concordance with the 16S rRNA gene sequences of Bifidobacterium spp., and the latter primers allowed for the detection of T-RFs of Bifidobacterium spp. in fecal samples from five healthy subjects. The T-RFs presumed to be Bifidobacterium spp. were cloned and sequenced, and found to match the 16S rRNA gene sequences of Bifidobacterium spp. Among the five fecal samples, two samples with low frequencies of T-RFs of Bifidobacterium spp. were detected using these forward primers. This probably reflects a low prevalence of Bifidobacterium spp. in these two samples. Our study emphasizes the importance of selecting a suitable forward primer for detection of Bifidobacterium spp.

Adult↗
PubMed2004

Modulation of the effect of dextran sulfate sodium-induced acute colitis by the administration of different probiotic strains of Lactobacillus and Bifidobacterium.

The pathogenic mechanism of inflammatory bowel diseases is not fully understood but colonic microflora including Lactobacillus and Bifidobacterium species may affect the induction of colonic inflammation. In this study the relative efficacy of different probiotic organisms in the prevention of colitis was compared in an induced rat colitis model. Three Lactobacillus strains and two Bifidobacterium strains were fed to Sprague-Dawley rats for 7 days prior to offering the rats 5% dextran sulfate sodium (DSS) in their drinking water to induce colitis and the administration of the probiotics continued for 7 days with the DSS. Colitis severity was assessed daily using a disease activity index (DAI). Samples were collected 7 days after colitis induction for intestinal bacterial flora and bacterial translocation. The DAI decreased significantly on days 4, 5, 6, and 7 in the Lactobacillus plantarum DSM 9843, Bifidobacterium sp. 3B1, and Bifidobacterium infantis DSM 15158 groups compared to the colitis control. It decreased significantly on days 5, 6, and 7 in the Bifidobacterium infantis DSM 15158 group compared to the Lactobacillus paracaesi DSM 13434 and Lactobacillus gasseri 5B3 groups. It also decreased significantly on day 7 in the L. plantarum DSM 9843 group compared to the L. gasseri 5B3 group. Bacterial translocation to the mesenteric lymph nodes decreased significantly in all treatment groups compared to the colitis control. Enterobacteriaceae bacterial translocation to the liver decreased in all treatment groups compared to the colitis control. Administration of certain strains of Lactobacillus and Bifidobacterium significantly improves the DAI and reduces bacterial translocation, and L. plantarum DSM 9843, Bifidobacterium sp. 3B1, and Bifidobacterium infantis DSM 15158 seem to have the best effect.

Acute Disease↗
PubMed2005

Antitumor effect of cytosine deaminase/5-fluorocytosine suicide gene therapy system mediated by Bifidobacterium infantis on melanoma.

AIM: To construct a Bifidobacterium infantis/CD targeting gene therapy system and observe the antitumor effect of cytosine deaminase/5-fluorocytosine (CD/5-FC) suicide gene therapy system mediated by Bifidobacterium infantis on melanoma in vitro and in vivo. METHODS: A recombinant CD/pGEX-1LamdaT plasmid was transfected into Bifidobacterium infantis by electroporation. Bifidobacterium infantis transfected by recombinant CD/pGEX-1LamdaT plasmid was incubated with 5-FC anaerobically. Then the supernatant fluid was collected and added to melanoma B16-F10 cells to observe the killing effect for B16-F10 cells. Mice were inoculated with melanoma B16-F10 cells to establish animal models. The mice were then injected with 5-FC and Bifidobacterium infantis transfected by recombinant CD/pGEX-1LamdaT plasmid. RESULTS: Two segments of approximate 4.9 kb and 1.3 kb were extracted from the 6.2 kb recombinant plasmid, which were equal to the size of the pGEX-1LamdaT plasmid and CD gene, respectively. Sequencing results showed that the full length and sequence of nucleotide acid of the inserted gene in extracted recombinant plasmid was completely identical to the CD gene. In vitro, B16-F10 cells treated by supernatant fluid were remarkably damaged morphologically, and the cell growth was significantly inhibited. Experiments on the mice melanoma model showed that after treatment with a combination of transfected Bifidobacterium infantis and 5-FC, the tumor volume was significantly inhibited compared with controls. CONCLUSION: The foreign gene, CD gene, was correctly inserted into pGEX-1LambdaT plasmid and transferred into Bifidobacterium infantis. CD/5-FC suicide gene therapy system mediated by Bifidobacterium infantis demonstrated a good antitumor effect on melanoma in vitro and in vivo.

Animals↗
PubMed2006

Characterization of the genus Bifidobacterium by automated ribotyping and 16S rRNA gene sequences.

In order to characterize the genus Bifidobacterium, ribopatterns and approximately 500 bp (Escherichia coli positions 27 to 520) of 16S rRNA gene sequences of 28 type strains and 64 reference strains of the genus Bifidobacterium were determined. Ribopatterns obtained from Bifidobacterium strains were divided into nine clusters (clusters I-IX) with a similarity of 60%. Cluster V, containing 17 species, was further subdivided into 22 subclusters with a similarity of 90%. In the genus Bifidobacterium, four groups were shown according to Miyake et al.: (i) the Bifidobacterium longum infantis-longum-suis type group, (ii) the B. catenulatum-pseudocatenulatum group, (iii) the B. gallinarum-saeculare-pullorum group, and (iv) the B. coryneforme-indicum group, which showed higher than 97% similarity of the 16S rRNA gene sequences in each group. Using ribotyping analysis, unique ribopatterns were obtained from these species, and they could be separated by cluster analysis. Ribopatterns of six B. adolescentis strains were separated into different clusters, and also showed diversity in 16S rRNA gene sequences. B. adolescentis consisted of heterogeneous strains. The nine strains of B. pseudolongum subsp. pseudolongum were divided into five subclusters. Each type strain of B. pseudolongum subsp. pseudolongum and B. pseudolongum subsp. globosum and two intermediate groups, which were suggested by Yaeshima et al., consisted of individual clusters. B. animalis subsp. animalis and B. animalis subsp. lactis could not be separated by ribotyping using Eco RI. We conclude that ribotyping is able to provide another characteristic of Bifidobacterium strains in addition to 16S rRNA gene sequence phylogenetic analysis, and this information suggests that ribotyping analysis is a useful tool for the characterization of Bifidobacterium species in combination with other techniques for taxonomic characterization.

Bifidobacterium↗
PubMed2006

Population dynamics of Bifidobacterium species in human feces during raffinose administration monitored by fluorescence in situ hybridization-flow cytometry.

The population dynamics of bifidobacteria in human feces during raffinose administration were investigated at the species level by using fluorescence in situ hybridization (FISH) coupled with flow cytometry (FCM) analysis. Although double-staining FISH-FCM using both fluorescein isothiocyanate (FITC) and indodicarbocyanine (Cy5) as labeling dyes for fecal samples has been reported, the analysis was interfered with by strong autofluorescence at the FITC fluorescence region because of the presence of autofluorescence particles/debris in the fecal samples. We circumvented this problem by using only Cy5 fluorescent dye in the FISH-FCM analysis. Thirteen subjects received 2 g of raffinose twice a day for 4 weeks. Fecal samples were collected, and the bifidobacterial populations were monitored using the established FISH-FCM method. The results showed an increase in bifidobacteria from about 12.5% of total bacteria in the prefeeding period to about 28.7 and 37.2% after the 2-week and 4-week feeding periods, respectively. Bifidobacterium adolescentis, the Bifidobacterium catenulatum group, and Bifidobacterium longum were the major species, in that order, at the prefeeding period, and these bacteria were found to increase nearly in parallel during the raffinose administration. During the feeding periods, indigenous bifidobacterial populations became more diverse, such that minor species in human adults, such as Bifidobacterium breve, Bifidobacterium bifidum, Bifidobacterium dentium, and Bifidobacterium angulatum, proliferated. Four weeks after raffinose administration was stopped, the proportion of each major bifidobacterial species, as well as that of total bifidobacteria, returned to approximately the original values for the prefeeding period, whereas that of each minor species appeared to differ considerably from its original value. To the best of our knowledge, these results provide the first clear demonstration of the population dynamics of indigenous bifidobacteria at the species level in response to raffinose administration.

Adult↗
PubMed1992

Relationship between oxygen sensitivity and oxygen metabolism of Bifidobacterium species.

Bifidobacteria, which are obligate anaerobes, were studied to determine the relationship between their sensitivity to oxygen and oxygen metabolism. Among the four species tested, Bifidobacterium infantis, Bifidobacterium breve, and Bifidobacterium longum differed from Bifidobacterium adolescentis in sensitivity to oxygen. The former three species showed marked growth under conditions of partial aeration, whereas the growth of B. adolescentis was suppressed by low concentrations of oxygen. Bifidobacteria express reduced NAD-oxidase and -peroxidase activities, which function in a pathway for two-electron reduction of molecular oxygen, producing hydrogen peroxide and, subsequently, water. Activities of reduced NAD-oxidase and -peroxidase were inversely correlated with their sensitivities to oxygen. Bifidobacterium adolescentis exhibited lowered activities of these two enzymes; the activities were 10 to 20% of those observed with B. infantis, B. breve, and B. longum. These observations are compatible with the hypothesis that reduced NAD-oxidase and reduced NAD-peroxidase in Bifidobacterium species play a role in prevention of oxygen toxicity. Superoxide dismutase activity was also detected in Bifidobacterium species. Superoxide dismutase is probably not involved in detoxification of oxygen, because the activity of this enzyme was extremely low, and the sensitivity to oxygen varied independently of superoxide dismutase activity.

Bifidobacterium↗
PubMed2005

Production of bacteriocin-like inhibitory compounds by human fecal Bifidobacterium strains.

Acid- and bile-resistant Bifidobacterium strains were isolated from human feces and identified by genus-specific PCR and randomly amplified polymorphic DNA PCR. Twenty-four different strains were screened for possible production of proteinaceous antimicrobial compounds by assaying the inhibitory effects of their neutralized culture supernatants. Six Bifidobacterium strains (BIR-0304, BIR-0307, BIR-0312, BIR-0324, BIR-0326, and BIR-0349) were selected on the basis of their broad inhibitory spectra. These strains were active against gram-positive and gram-negative bacteria and yeasts relevant to food safety and human health. The antagonistic effects of the six selected Bifidobacterium strains were related to bacteriocin-like compounds, which were active at pH values between 3 and 10, stable at 100 degrees C for 10 min, resistant to alpha-amylase and lipase A, but sensitive to proteinases (trypsin, proteinase K, protease A, pepsin, and cathepsin B). The molecular masses of the antimicrobial compounds produced by Bifidobacterium BIR-0312 and BIR-0324 were in the range of 10 to 30 kDa, and those of the compounds produced by Bifidobacterium BIR-0304, BIR-0307, BIR-0326, and BIR-0349 were less than 10 kDa. All Bifidobacterium strains produced maximum antimicrobial activities in the late logarithmic phase of growth and in the presence of Tween 80. These results confirm that the synthesis of bacteriocin-like inhibitory compounds is a key factor in the in vitro inhibition of pathogen and spoilage bacteria by Bifidobacterium strains.

Antibiosis↗
PubMed2006

Bifidobacterium species isolated from animal feces and from beef and pork meat.

Bifidobacteria were isolated from 122 of 145 samples of animal feces (from cattle, swine, sheep, goats, horses, rabbits, chickens, geese, and pigeons) from farms in France and Austria and from 92 of 955 production and processing chain samples of beef and pork (obtained at slaughter, cutting, and retail). Bacterial strains were identified to species by phenotypic numerical classification based on API 50CH and ID 32A tests and DNA-DNA hybridization. Bifidobacterium pseudolongum was present in 81% (99 of 122 samples) of all Bifidobacterium-positive fecal samples and predominated in samples from all animal species except those from swine from Austria. In these Austrian swine samples, the majority of strains were identified as Bifidobacterium thermophilum (78%), followed by B. pseudolongum (48%). The distribution of B. thermophilum and B. pseudolongum differed significantly between Austrian swine and cattle samples such as those collected along beef and pork production and processing chains. Bifidobacterium animalis was isolated from swine feces, and Bifidobacterium ruminantium was isolated from cow dung. Six fecal isolates (from cattle, swine, rabbits, goats, and horses) were identified as belonging to Bifidobacterium species of predominantly human origin: B. adolescentis, B. bifidum, and B. catenulatum. Only one other species, Bifidobacterium choerinum, was detected with low frequency in a pork processing chain. B. pseudolongum subsp. pseudolongum was predominant in pig feces, whereas B. pseudolongum subsp. globosum was predominant in feces from other animal species. Four strains closely related to both subspecies (58 to 61% DNA reassociation) formed a distinct genomic group. PCR techniques, which are more rapid and sensitive than culture-based methods, could be used to detect directly B. pseudolongum and B. thermophilum as indicators of fecal contamination along the meat processing chain.

Animals↗
PubMed2006

Occurrence of Bifidobacterium in the intestine of newborns by fluorescence in situ hybridization.

Colonization by Bifidobacterium occurs generally within 4 days of life. The new method FISH has been applied for molecular detection of Bifidobacteria. The study was carried out on 26 healthy newborns delivered by vaginal delivery. Breast-fed infants harbor a gastrointestinal flora characterized by an increased concentration of Bifidobacterium cells (by a factor of 1.75). In artificial alimentation, some infants either did not harbor any Bifidobacterium or showed lower numbers of Bifidobacterium. Moreover, male newborns show higher numbers of Bifidobacterium, but in both sexes the predominance of Bifidobacterium is evident after maternal alimentation.

Adult↗
PubMed1996

16S rRNA and 16S to 23S internal transcribed spacer sequence analyses reveal inter- and intraspecific Bifidobacterium phylogeny.

In the last few years many attempts have been made to differentiate more than 20 Bifidobacterium species. It has been recognized that identification of bifidobacterial species is problematic because of phenetic and genetic heterogeneities. In order to contribute to our understanding of Bifidobacterium taxonomy, we studied Bifidobacterium phylogeny by performing both 16S rRNA and 16S to 23S (16S-23S) internally transcribed spacer (ITS) sequence analyses. In this study, we determined 16S rRNA sequences of five Bifidobacterium strains representing four species, and compared them with the sequences available in the GenBank database, and used them to construct a distance tree and for a bootstrap analysis. Moreover, we determined the ITS sequences of 29 bifidobacterial strains representing 18 species and compared these sequences with each other. We constructed a phylogenetic tree based on these sequence data and compared this tree with the tree based on 16S rRNA sequence data. We found that the two trees were similar topologically, suggesting that the two types of molecules provided the same kind of phylogenetic information. However, while 16S rRNA sequences are a good tool to infer interspecific links, the 16S-23S rDNA spacer data allowed us to determine intraspecific relationships. Each of the strains was characterized by its own ITS sequence; hence, 16S-23S rRNA sequences are a good tool for strain identification. Moreover, a comparison of the ITS sequences allowed us to estimate that the maximum level of ITS divergence between strains belonging to the same species was 13%. Our data allowed us to confirm the validity of most of the Bifidobacterium species which we studied and to identify some classification errors. Finally, our results showed that Bifidobacterium strains have no tRNA genes in the 16S-23S spacer region.

Base Sequence↗
PubMed1990

Growth promotion of Bifidobacterium species by whey and casein fractions from human and bovine milk.

An in vitro assay was used to study the growth-promotional activity of human milk (HM), cow's milk (CM), and whey and casein fractions of HM and CM for five strains of Bifidobacterium species isolated originally from stools of human infants. Whey- and casein-predominant CM-based infant formulas were studied as well. When compared on an equivalent protein basis, the growth promotion activity of HM was greater than that of CM for Bifidobacterium bifidum serovar pennsylvanicus and Bifidobacterium longum but comparable for B. bifidum, Bifidobacterium infantis, and Bifidobacterium breve. Pasteurization of HM and CM resulted in an increase of growth promotion activity for B. bifidum serovar pennsylvanicus and B. bifidum, a decrease for B. infantis, and no change for B. longum and B. breve. The growth promotion activity of HM whey was slightly higher than that of HM casein for four strains of bifidobacteria. When CM casein was a substrate, virtually no growth occurred for B. bifidum serovar pennsylvanicus, B. bifidum, B. infantis, and B. longum. The growth promotion activity of CM whey, however, was similar to that of HM whey. A similar trend was observed for CM-based infant formula. Whey-dominant formulas promoted better growth of B. bifidum serovar pennsylvanicus, B. bifidum, and B. infantis than casein-dominant formulas. The data suggest a direct relationship between amount of whey-specific factors and the ability to promote growth of clinically relevant strains of Bifidobacterium species by HM, CM, and CM-based infant formulas.

Animals↗