Enumeration of Bifidobacteria in Gastrointestinal Samples from Piglets

The population of Bifidobacterium spp. in fecal samples from suckling piglets was investigated, and Beerens, raffinose-bifidobacterium (RB), and modified Wilkins-Chalgren (MW) agar media were evaluated with regard to the enumeration of bifidobacteria in porcine intestinal samples. The results demonstrated that the population of bifidobacteria in the feces of suckling piglets is numerically low, and a phylogenetic analysis of the 16S rRNA gene from bifidobacterial isolates suggested that a possibly new Bifidobacterium species was isolated. Beerens, RB, and MW agar media were not selective for bifidobacteria in the fecal samples. The highest recovery and diversity of bifidobacteria were obtained for MW agar. Nonbifidobacterial isolates from the three agar media were identified and may contribute to the future formulation of improved selective media for the enumeration of bifidobacteria.     

In Vitro Utilization of Amylopectin and High-Amylose Maize (Amylomaize) Starch Granules by Human Colonic Bacteria

It has been well established that a certain amount of ingested starch can escape digestion in the human small intestine and consequently enters the large intestine, where it may serve as a carbon source for bacterial fermentation. Thirty-eight types of human colonic bacteria were screened for their capacity to utilize soluble starch, gelatinized amylopectin maize starch, and high-amylose maize starch granules by measuring the clear zones on starch agar plates. The six cultures which produced clear zones on amylopectin maize starch- containing plates were selected for further studies for utilization of amylopectin maize starch and high-amylose maize starch granules A (amylose; Sigma) and B (Culture Pro 958N). Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was used to detect bacterial starch-degrading enzymes. It was demonstrated that Bifidobacterium spp., Bacteroides spp., Fusobacterium spp., and strains of Eubacterium, Clostridium, Streptococcus, and Propionibacterium could hydrolyze the gelatinized amylopectin maize starch, while only Bifidobacterium spp. and Clostridium butyricum could efficiently utilize high-amylose maize starch granules. In fact, C. butyricum and Bifidobacterium spp. had higher specific growth rates in the autoclaved medium containing high-amylose maize starch granules and hydrolyzed 80 and 40% of the amylose, respectively. Starch-degrading enzymes were cell bound on Bifidobacterium and Bacteroides cells and were extracellular for C. butyricum. Active staining for starch-degrading enzymes on SDS-PAGE gels showed that the Bifidobacterium cells produced several starch-degrading enzymes with high relative molecular (M_r) weights (>160,000), medium-sized relative molecular weights (>66,000), and low relative molecular weights (<66,000). It was concluded that Bifidobacterium spp. and C. butyricum degraded and utilized granules of amylomaize starch.     

Evaluation of the Diversity of Probiotic <Emphasis Type="Italic">Bacillus</Emphasis>, <Emphasis Type="Italic">Clostridium</Emphasis>, and <Emphasis Type="Italic">Bifidobacterium</Emphasis> Using the Illumina-Based Sequencing Method

Bacterial species of Bacillus, Lactobacillus, and Bifidobacterium in the intestinal tract have been used as probiotics. Selections for probiotic candidates by the culture-based approaches are time-consuming and labor-consuming. The aim of this study was to develop a new method based on sequencing strategies to select the probiotic Bacillus, Lactobacillus, and Bifidobacterium. The Illumina-based sequencing strategies with different specific primers for Bacillus, Clostridium, and Bifidobacterium were applied to analyze diversity of the genera in goat feces. The average number of different Bacillus, Clostridium, and Bifidobacterium OTUs (operational taxonomic units) at the 97% similarity level ranged from 1922 to 63172. The coverage index values of Bacillus, Clostridium, and Bifidobacterium calculated from the bacterial OTUs were 0.89, 0.99, and 1.00, respectively. The most genera of Bacillus (37.9%), Clostridium (53%), and Bifidobacterium (99%) were detected in goat feces by the Illumina-based sequencing with the specific primers of the genera, respectively. Higher phylogenetic resolutions of the genera in goat feces were successfully established. The results suggest that the selection for probiotic Bacillus, Clostridium, and Bifidobacterium based on the Illumina sequencing with their specific primers is reliable and feasible, and the core Bacillus, Clostridium, and Bifidobacterium species of healthy goats possess the potentials as probiotic microbial consortia.     

Use of Bifidobacterium dentium as an Indicator of the Origin of Fecal Water Pollution

A new, simple, and specific protocol to discriminate between human and animal fecal pollution is described. The procedure is based on the detection of certain Bifidobacterium species in the samples. Two 16S rRNA gene-targeted probes are described. One of these probes (BDE) has as its target a region of the 16S rRNA gene of Bifidobacterium dentium, a Bifidobacterium species of exclusively human origin. The other probe (BAN) is based on the sequence of a region of 16S rRNA gene for several Bifidobacterium species related with animal origins. The specificity of both probes was evaluated by using 24 Bifidobacterium species, and their threshold detection limit was established by DNA-DNA hybridization. DNA-DNA hybridization with the BDE probe showed it to be specific for B. dentium, whereas that with the BAN probe showed it to be specific for B. animalis, B. asteroides, B. coryneforme, B. cuniculi, B. globosum, B. magnum, B. minimum, and B. subtile. A simple and specific protocol was also developed for the detection of their target species in environmental samples (sewage and feces). DNA-DNA hybridization with the BAN probe was only positive for samples from cattle and goats. Thus, this probe is not suitable for the identification of any animal fecal pollution. Whereas all samples with human fecal pollution showed a positive DNA-DNA hybridization result with the BDE probe, none of those with animal fecal pollution did. Therefore, this finding supports the potential use of this probe in detecting fecal pollution of human origin.     

Relationship between the resistance to bile salts and low pH with exopolysaccharide (EPS) production of Bifidobacterium spp. isolated from infants feces and breast milk

The purpose of this study was to investigate a possible relation between resistance to bile salts and low pH with exopolysaccharide (EPS) producing of Bifidobacterium spp. In this study, a total of 31 Bifidobacterium spp. were isolated from breast fed infants feces and breast milk samples. As a result of the identification tests, isolates were identified as Bifidobacterium breve (15 strains), B. bifidum (11 strains), B. pseudocatenulatum (3 strains) and B. longum (2 strains). Bifidobacterium spp. were determined exopolysaccharide (EPS) production. EPS productions observed at chance rations (38.00–97.64 mg/l) among of Bifidobacterium spp. Furthermore, Bifidobacterium spp. were determined resistance to bile salts and low pH. Positive correlations between production of exopolysaccharide and resistance to bile salts (p < 0.01) or low pH (p < 0.01) were found Bifidobacterium spp. This investigation showed that high EPS production of Bifidobacteria may be important in the selection of probiotic strains for resistance to bile salts and low pH.     

New method for selection of hydrogen peroxide adapted bifidobacteria cells using continuous culture and immobilized cell technology

Background  

Oxidative stress can severely compromise viability of bifidobacteria. Exposure of Bifidobacterium cells to oxygen causes accumulation of reactive oxygen species, mainly hydrogen peroxide, leading to cell death. In this study, we tested the suitability of continuous culture under increasing selective pressure combined with immobilized cell technology for the selection of hydrogen peroxide adapted Bifidobacterium cells. Cells of B. longum NCC2705 were immobilized in gellan-xanthan gum gel beads and used to continuously ferment MRS medium containing increasing concentration of H₂O₂ from 0 to 130 ppm.     

Investigation of the Evolutionary Development of the Genus Bifidobacterium by Comparative Genomics

The Bifidobacterium genus currently encompasses 48 recognized taxa, which have been isolated from different ecosystems. However, the current phylogeny of bifidobacteria is hampered by the relative paucity of genotypic data. Here, we reassessed the taxonomy of this bacterial genus using genome-based approaches, which demonstrated that the previous taxonomic view of bifidobacteria contained several inconsistencies. In particular, high levels of genetic relatedness were shown to exist between particular Bifidobacterium taxa which would not justify their status as separate species. The results presented are here based on average nucleotide identity analysis involving the genome sequences for each type strain of the 48 bifidobacterial taxa, as well as phylogenetic comparative analysis of the predicted core genome of the Bifidobacterium genus. The results of this study demonstrate that the availability of complete genome sequences allows the reconstruction of a more robust bifidobacterial phylogeny than that obtained from a single gene-based sequence comparison, thus discouraging the assignment of a new or separate bifidobacterial taxon without such a genome-based validation.     

Development of an Improved Selective Agar Medium for Isolation of Yersinia pestis

Existing media designed for selective isolation of clinically important members of the genus Yersinia were found to be unsatisfactory for the growth and isolation of Yersinia pestis. We report the development of a new selective agar medium (termed BIN) that supports the growth of Y. pestis. The development of the formulation of this medium was based on a fluorescence screening system designed for monitoring bacterial growth on semisolid media, using a green fluorescent protein-expressing strain. High-throughput combinatorial experiments can be conducted for the quantitative evaluation of the effect of different medium components on growth. Generation of fluorescence plots in this system, using microplates, allowed the quantitative evaluation of the growth rate of Y. pestis EV76 cultures in different agar compositions. The final BIN formulation is based on brain heart infusion agar, to which the selective agents irgasan, cholate salts, crystal violet, and nystatin were introduced. It was found that BIN agar is more efficient in supporting colony formation and recovery of Y. pestis than are the conventional semisolid media MacConkey agar and Yersinia-selective agar (cefsulodin-irgasan-novobiocin agar). The advantage of BIN over other media has been also demonstrated in recovering virulent Y. pestis from the mixed bacterial populations found in decaying carcasses of infected mice. The BIN medium is suggested as a selective medium for isolation and recovery of Y. pestis from various backgrounds.     

Quantitative PCR with 16S rRNA-Gene-Targeted Species-Specific Primers for Analysis of Human Intestinal Bifidobacteria

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 10⁶ 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 >10⁶ 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.     

Comparative Genomic Analysis of 45 Type Strains of the Genus Bifidobacterium: A Snapshot of Its Genetic Diversity and Evolution

Bifidobacteria are well known for their human health-promoting effects and are therefore widely applied in the food industry. Members of the Bifidobacterium genus were first identified from the human gastrointestinal tract and were then found to be widely distributed across various ecological niches. Although the genetic diversity of Bifidobacterium has been determined based on several marker genes or a few genomes, the global diversity and evolution scenario for the entire genus remain unresolved. The present study comparatively analyzed the genomes of 45 type strains. We built a robust genealogy for Bifidobacterium based on 402 core genes and defined its root according to the phylogeny of the tree of bacteria. Our results support that all human isolates are of younger lineages, and although species isolated from bees dominate the more ancient lineages, the bee was not necessarily the original host for bifidobacteria. Moreover, the species isolated from different hosts are enriched with specific gene sets, suggesting host-specific adaptation. Notably, bee-specific genes are strongly associated with respiratory metabolism and are potential in helping those bacteria adapt to the oxygen-rich gut environment in bees. This study provides a snapshot of the genetic diversity and evolution of Bifidobacterium, paving the way for future studies on the taxonomy and functional genomics of the genus.     

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.     

Selective Plating Underestimates Abundance and Shows Differential Recovery of Bifidobacterial Species from Human Feces

The aim of the present work was to compare the efficacies and levels of selectivity of different culture-dependent and -independent methods for analyzing bifidobacteria in human stool samples. The three different culture media used here significantly differed from each other, particularly with regard to the recovery of Bifidobacterium adolescentis. Bifidobacterium medium failed to recover B. adolescentis; Beerens medium recovered some B. adolescentis organisms (17% of total bifidobacteria), whereas tomato-Eugon medium recovered mainly B. adolescentis organisms (58% of total bifidobacteria). A culture-independent method that combines GC fractionation of bacterial community DNA and 16S rRNA sequencing indicated that B. adolescentis organisms accounted for 85% of all bifidobacteria. Methodological biases, such as those described in this paper, should be taken into account in interpreting earlier studies and designing future experiments.     

Bifidobacterium asteroides PRL2011 Genome Analysis Reveals Clues for Colonization of the Insect Gut

Bifidobacteria are known as anaerobic/microaerophilic and fermentative microorganisms, which commonly inhabit the gastrointestinal tract of various animals and insects. Analysis of the 2,167,301 bp genome of Bifidobacterium asteroides PRL2011, a strain isolated from the hindgut of Apis mellifera var. ligustica, commonly known as the honey bee, revealed its predicted capability for respiratory metabolism. Conservation of the latter gene clusters in various B. asteroides strains enforces the notion that respiration is a common metabolic feature of this ancient bifidobacterial species, which has been lost in currently known mammal-derived Bifidobacterium species. In fact, phylogenomic based analyses suggested an ancient origin of B. asteroides and indicates it as an ancestor of the genus Bifidobacterium. Furthermore, the B. asteroides PRL2011 genome encodes various enzymes for coping with toxic products that arise as a result of oxygen-mediated respiration.     

Enrichment Medium for Selection of Salmonella from Fish Homogenate

A new liquid medium, called “dulcitol selenite enrichment,” has been developed for the detection and enumeration of Salmonella in foods. The medium is not only highly selective in enriching Salmonella and inhibiting completely or appreciably other extraneous organisms commonly found in seafoods, but is also highly sensitive in recovering as low as 2 to 7 cells of Salmonella, even in the presence of large numbers (10⁴ to 10⁶ cells) of mixed flora common to these foods. The addition of seafood material does not seem to interfere with the sensitivity, selectivity, or productivity of the medium. Even physiologically debilitated cells of Salmonella were enriched well enough in this medium to be detected easily.     

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.     

Rationale for Using of Bifidobacterium Probiotic Strains-Fermented Milk Against Colitis Based on Animal Experiments and Clinical Trials

Probiotic foods such as probiotic strain-fermented milk or supplements proposing various health claims are now available. The beneficial effects of these probiotic foods on the digestive system are expected for not only healthy persons but also patients with diseases of the alimentary tract. This review focused on the rationale of using our Bifidobacterium strains-fermented milk as an adjunct for the prevention of recurrence or exacerbation of colitis. Animal experiments using gnotobiotic colitis or spontaneously colitis models and also human clinical trials of ulcerative colitis patients showed the potential of Bifidobacterium strains-fermented milk as a beneficial anti-colitis adjunct.     

Response of the Microaerophilic Bifidobacterium Species, B. boum and B. thermophilum, to Oxygen

We investigated the effects of O₂ on Bifidobacterium species using liquid shaking cultures under various O₂ concentrations. Although most of the Bifidobacterium species we selected showed O₂ sensitivity, two species, B. boum and B. thermophilum, demonstrated microaerophilic profiles. The growth of B. bifidum and B. longum was inhibited under high-O₂ conditions accompanied by the accumulation of H₂O₂ in the medium, and growth was restored by adding catalase to the medium. B. boum and B. thermophilum grew well even under 20% O₂ conditions without H₂O₂ accumulation, and growth was stimulated compared to anoxic growth. H₂O-forming NADH oxidase activities were detected dominantly in cell extracts of B. boum and B. thermophilum under acidic reaction conditions (pH 5.0 to 6.0).     

In Silico Assigned Resistance Genes Confer Bifidobacterium with Partial Resistance to Aminoglycosides but Not to Β-Lactams

Bifidobacteria have received significant attention due to their contribution to human gut health and the use of specific strains as probiotics. It is thus not surprising that there has also been significant interest with respect to their antibiotic resistance profile. Numerous culture-based studies have demonstrated that bifidobacteria are resistant to the majority of aminoglycosides, but are sensitive to β-lactams. However, limited research exists with respect to the genetic basis for the resistance of bifidobacteria to aminoglycosides. Here we performed an in-depth in silico analysis of putative Bifidobacterium-encoded aminoglycoside resistance proteins and β-lactamases and assess the contribution of these proteins to antibiotic resistance. The in silico-based screen detected putative aminoglycoside and β-lactam resistance proteins across the Bifidobacterium genus. Laboratory-based investigations of a number of representative bifidobacteria strains confirmed that despite containing putative β-lactamases, these strains were sensitive to β-lactams. In contrast, all strains were resistant to the aminoglycosides tested. To assess the contribution of genes encoding putative aminoglycoside resistance proteins in Bifidobacterium sp. two genes, namely Bbr_0651 and Bbr_1586, were targeted for insertional inactivation in B. breve UCC2003. As compared to the wild-type, the UCC2003 insertion mutant strains exhibited decreased resistance to gentamycin, kanamycin and streptomycin. This study highlights the associated risks of relying on the in silico assignment of gene function. Although several putative β-lactam resistance proteins are located in bifidobacteria, their presence does not coincide with resistance to these antibiotics. In contrast however, this approach has resulted in the identification of two loci that contribute to the aminoglycoside resistance of B. breve UCC2003 and, potentially, many other bifidobacteria.     

Amoxicillin treatment modifies the composition of Bifidobacterium species in infant intestinal microbiota

ObjectivesAmoxicillin is a beta-lactam antibiotic largely used in childhood. However only few studies described its impact on composition of children gut microbiota, in particular on Bifidobacterium populations considered as beneficial microorganisms. In this study, the impact on faecal Bifidobacterium species of a seven-day amoxicillin treatment was quantitatively and qualitatively assessed in infants during an episode of acute respiratory infection.MethodsFaecal samples from 31 infants were obtained on day 0 (just before amoxicillin therapy) and on day 7 (the end of therapy). Total DNA was extracted and bifidobacteria were quantified using real-time PCR. Predominant Bifidobacterium species were then identified using specific PCR-TTGE.ResultsBifidobacteria concentrations were not significantly altered by amoxicillin compared to the healthy group. However, amoxicillin treatment induced a complete disappearance of Bifidobacterium adolescentis species (occurrence rate of 0% versus 36.4% in healthy group, P < 0.001), a significant decrease in the occurrence rate of Bifidobacterium bifidum (23% versus 54.5% in healthy group, P < 0.05), but did not affect Bifidobacterium longum (93.5% versus 100% in healthy group) and Bifidobacterium pseudocatenulatum/B. catenulatum (about 55% in both groups). The number of Bifidobacterium species per microbiota significantly decreased from 2.5 ± 1 for healthy group to 1.8 ± 0.9 for treated infants (P < 0.05).ConclusionsThis study showed that a 7 day amoxicillin treatment did not alter the counts of Bifidobacterium. However amoxicillin can have an impact by changing the microbiota at the species level and decreased the diversity of this population.