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 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.     

双歧杆菌脂磷壁酸抗体的制备及其在双歧制品中的检测应用

目的制备双歧杆菌脂磷壁酸抗体并用以检测双歧制品中脂磷壁酸和双歧杆菌活菌的含量。方法提取两歧双歧杆菌脂磷壁酸,加甲基化牛血清白蛋白与佐剂免疫预先已用卡介苗进行多克隆激活的BALB／C小鼠,间接ELISA法检测抗体效价与特异性,用免疫血清经双抗体夹心ELISA法和免疫结合微量培养的方法分别检测酸奶中脂磷壁酸和双歧杆菌活菌量。结果免疫血清最高效价可达1：1280,与所测其他人体双歧杆菌种属存在较强交叉反应,与非双歧杆菌种属无交叉反应。对于脂磷壁酸和双歧杆菌活菌的含量检测取得良好结果,检测线可达10^5 CFU／ml。结论以双歧杆菌脂磷壁酸制备免疫血清,效价高,属特异性好,可用于双歧食品中脂磷壁酸和双歧杆菌活菌的含量检测。     

Multiplex PCR using 16S rRNA gene-targeted primers for the identification of bifidobacteria from human origin

Three multiplex polymerase chain reactions (PCRs) targeted on Bifidobacterium and related species were designed to identify human species. The selected primers yielded amplified products of various sizes, each specific for a species. Three to four pairs were gathered in one PCR reaction and their specificity under multiplex conditions was confirmed using DNA from 26 reference strains. Using this technique on unidentified faecal strains, B. bifidum, B. longum and B. breve species were commonly recovered in infants while B. adolescentis, B. catenulatum/B. pseudocatenulatum continuum and B. longum species were predominant in adults. Thus, a single PCR can provide the assignment of a strain to one these species, reducing the number of PCR reactions and hands-on time for the identification of human isolates of bifidobacteria. Moreover, this technique is also applicable for the in situ detection of bifidobacteria in DNA extracts from human stools.     

Genus- and species-specific PCR primers for the detection and identification of bifidobacteria

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.     

Physiology of consumption of human milk oligosaccharides by infant gut-associated bifidobacteria

The bifidogenic effect of human milk oligosaccharides (HMOs) has long been known, yet the precise mechanism underlying it remains unresolved. Recent studies show that some species/subspecies of Bifidobacterium are equipped with genetic and enzymatic sets dedicated to the utilization of HMOs, and consequently they can grow on HMOs; however, the ability to metabolize HMOs has not been directly linked to the actual metabolic behavior of the bacteria. In this report, we clarify the fate of each HMO during cultivation of infant gut-associated bifidobacteria. Bifidobacterium bifidum JCM1254, Bifidobacterium longum subsp. infantis JCM1222, Bifidobacterium longum subsp. longum JCM1217, and Bifidobacterium breve JCM1192 were selected for this purpose and were grown on HMO media containing a main neutral oligosaccharide fraction. The mono- and oligosaccharides in the spent media were labeled with 2-anthranilic acid, and their concentrations were determined at various incubation times using normal phase high performance liquid chromatography. The results reflect the metabolic abilities of the respective bifidobacteria. B. bifidum used secretory glycosidases to degrade HMOs, whereas B. longum subsp. infantis assimilated all HMOs by incorporating them in their intact forms. B. longum subsp. longum and B. breve consumed lacto-N-tetraose only. Interestingly, B. bifidum left degraded HMO metabolites outside of the cell even when the cells initiate vegetative growth, which indicates that the different species/subspecies can share the produced sugars. The predominance of type 1 chains in HMOs and the preferential use of type 1 HMO by infant gut-associated bifidobacteria suggest the coevolution of the bacteria with humans.     

Examination of bovine lactoferrin binding to bifidobacteria

In the present study, lactoferrin binding to bifidobacteria and detection of lactoferrin-binding protein in membrane fractions of several bifidobacteria have been demonstrated. This is the first report showing the binding of bovine lactoferrin to four Bifidobacterium spp. (B. infantis, B. breve, B. bifidum, B. longum) incubated with biotinylated lactoferrin and fluorescein conjugated-avidin and observed under an inverted confocal laser scanning microscope. Fluorescence staining showed lactoferrin binding at the pole of the bacterial cells. A lactoferrin-binding protein with a molecular weight of approximately 67 kDa was also detected in the membrane fraction of Bifidobacterium spp. by far western blotting technique using biotinylated lactoferrin and horseradish peroxidase-conjugated streptavidin. Based on the results of this and previously reported studies, we suggest that binding of lactoferrin to Bifidobacterium longum is strain-dependent.     

Effect of heat-shock and bile salts on protein synthesis of Bifidobacterium longum revealed by [35S]methionine labelling and two-dimensional gel electrophoresis

Experimental conditions for efficient protein radiolabelling and two-dimensional gel electrophoresis were developed for Bifidobacterium longum. Using these tools, protein synthesis in cells before and after heat-shock and bile salts treatment was investigated. Following heat-stress, 13 proteins were upregulated, of which HtrA, DnaK and GroEL were also moderately induced by bile salts, indicating close relationship between the heat and bile salts responses in bifidobacteria. Our work indicated that, as a consequence of prolonged heat-stress, HtrA undergoes sequential modification and proteolysis, and that this mechanism could be employed by bifidobacteria to respond to heat-stress.     

Novel putative galactose operon involving lacto-N-biose phosphorylase in Bifidobacterium longum

A lacto-N-biose phosphorylase (LNBP) was purified from the cell extract of Bifidobacterium bifidum. Its N-terminal and internal amino acid sequences were homologous with those of the hypothetical protein of Bifidobacterium longum NCC2705 encoded by the BL1641 gene. The homologous gene of the type strain B. longum JCM1217, lnpA, was expressed in Escherichia coli to confirm that it encoded LNBP. No significant identity was found with any proteins with known function, indicating that LNBP should be classified in a new family. The lnpA gene is located in a novel putative operon for galactose metabolism that does not contain a galactokinase gene. The operon seems to be involved in intestinal colonization by bifidobacteria mediated by metabolism of mucin sugars. In addition, it may also resolve the question of the nature of the bifidus factor in human milk as the lacto-N-biose structure found in milk oligosaccharides.     

Characteristics of the soluble antigens of bifidobacteria

The immunological study of aqueous buffer extracts obtained from 45 strains of bifidobacteria belonging to the species B. bifidum, B. longum, B. adducens, B. breve, B. infantis and B. parvulorum was made. This study revealed 3 levels of the immunological specificity of soluble bifidobacterial proteins: common to the genus Bifidobacterium, common to a limited number of strains belonging to one or several species of bifidobacteria and strain-specific.     

Growth-promoting effects of lactoferrin on L. acidophilus and Bifidobacterium spp.

We investigated the effects of lactoferrin on the growth of L. acidophilus CH-2, Bifidobacterium breve ATCC 15700, B. longum ATCC 15707, B. infantis ATCC 15697, and B. bifidum ATCC 15696. The growth of L. acidophilus was stimulated by bovine holo-lactoferrin but not by apo-lactoferrin. With bifidobacteria, bovine lactoferrin stimulated growth of three strains: B. breve, B. infantis and B. bifidum under certain conditions. Both apoprotein and holoprotein had similar effects. However, B. longum growth was not affected by lactoferrin. Thus, the mechanism of stimulating growth of bifidobacteria may be different from that of L. acidophilus. By far-western blotting using biotinylated lactoferrin and horseradish peroxidase-conjugated streptavidin, lactoferrin-binding proteins were detected in the membrane protein fraction of L. acidophilus, B. bifidum, B. infantis and B. breve. The molecular weights of lactoferrin-binding proteins of L. acidophilus were estimated from SDS-polyacrylamide gel electrophoresis to be 27, 41 and 67 kDa, and those of the three bifidobacterial strains were estimated to be 67-69 kDa. However, no such lactoferrin-binding components were detected in the membrane fraction of B. longum. It is interesting that the appearance of lactoferrin-binding proteins in the membrane fraction of these species corresponds to their growth stimulation by lactoferrin.     

Study of adhesion and survival of lactobacilli and bifidobacteria on table olives with the aim of formulating a new probiotic food

With the aim of developing new functional foods, a traditional product, the table olive, was used as a vehicle for incorporating probiotic bacterial species. Survival on table olives of Lactobacillus rhamnosus (three strains), Lactobacillus paracasei (two strains), Bifidobacterium bifidum (one strain), and Bifidobacterium longum (one strain) at room temperature was investigated. The results obtained using a selected olive sample demonstrated that bifidobacteria and one strain of L. rhamnosus (Lactobacillus GG) showed a good survival rate, with a recovery of about 10(6) CFU g(-1) after 30 days. The Lactobacillus GG population remained unvaried until the end of the experiment, while a slight decline (to about 10(5) CFU g(-1)) was observed for bifidobacteria. High viability, with more than 10(7) CFU g(-1), was observed throughout the 3-month experiment for L. paracasei IMPC2.1. This strain, selected for its potential probiotic characteristics and for its lengthy survival on olives, was used to validate table olives as a carrier for transporting bacterial cells into the human gastrointestinal tract. L. paracasei IMPC2.1 was recovered from fecal samples in four out of five volunteers fed 10 to 15 olives per day carrying about 10(9) to 10(10) viable cells for 10 days.     

Quantitative detection of probiotic Bifidobacterium strains in bacterial mixtures by using real-time PCR

Strain-specific rRNA-targeted primers were designed for the quantitative detection of Bifidobacterium infantis Y1, B. breve Y8 and B. longum Y10 used in a pharmaceutical probiotic product (VSL-3). PCR and real-time PCR techniques with the selected primers were employed for the direct enumeration of the bifidobacteria in the probiotic preparation and for studying their kinetic characteristics in batch cultures. These analysis revealed that B. infantis Y1 was the predominant strain in the probiotic product and that its growth rate was the highest. Since B. infantis Y1, B. breve Y8 and B. longum Y10 are co-cultured during the industrial production of VSL-3, the kinetic characteristics of these strains can explain their different concentrations in the probiotic preparation. A validation of the PCR quantification method was performed by identifying a representative number of isolates from the bacterial mixtures with automated ribotyping. The methodology described represents a useful tool for the specific quantitative detection of bacterial strains and species in complex mixtures such as pharmaceutical preparations, dairy starter cultures, faecal samples and biopsies.     

Identification, detection, and enumeration of human bifidobacterium species by PCR targeting the transaldolase gene

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.     

Endo-β-N-acetylglucosaminidases from Infant Gut-associated Bifidobacteria Release Complex N-glycans from Human Milk Glycoproteins

Breastfeeding is one of the main factors guiding the composition of the infant gut microbiota in the first months of life. This process is shaped in part by the high amounts of human milk oligosaccharides that serve as a carbon source for saccharolytic bacteria such as Bifidobacterium species. Infant-borne bifidobacteria have developed various molecular strategies for utilizing these oligosaccharides as a carbon source. We hypothesized that these species also interact with N-glycans found in host glycoproteins that are structurally similar to free oligosaccharides in human milk. Endo-β-N-acetylglucosaminidases were identified in certain isolates of Bifidobacterium longum subsp. longum, B. longum subsp. infantis, and Bifidobacterium breve, and their presence correlated with the ability of these strains to deglycosylate glycoproteins. An endoglycosidase from B. infantis ATCC 15697, EndoBI-1, was active toward all major types of N-linked glycans found in glycosylated proteins. Its activity was not affected by core fucosylation or extensive fucosylation, antenna number, or sialylation, releasing several N-glycans from human lactoferrin and immunoglobulins A and G. Extensive N-deglycosylation of whole breast milk was also observed after coincubation with this enzyme. Mutation of the active site of EndoBI-1 did not abolish binding to N-glycosylated proteins, and this mutant specifically recognized Man(3)GlcNAc(2)(α1-6Fuc), the core structure of human N-glycans. EndoBI-1 is constitutively expressed in B. infantis, and incubation of the bacterium with human or bovine lactoferrin led to the induction of genes associated to import and consumption of human milk oligosaccharides, suggesting linked regulatory mechanisms among these glycans. This work reveals an unprecedented interaction of bifidobacteria with host N-glycans and describes a novel endoglycosidase with broad specificity on diverse N-glycan types, potentially a useful tool for glycoproteomics studies.     

Quantitative determination of the spatial distribution of pure- and mixed-strain immobilized cells in gel beads by immunofluorescence

A new method was developed to detect and quantify two strains, Lactococcus lactis subsp. lactis biovar. diacetylactis MD and Bifidobacterium longum ATCC 15707, immobilized separately and co-immobilized in gel beads, using specific polyclonal antibodies and confocal laser-scanning microscopy. The establishment of biomass concentration profiles for each strain was measured during colonization of beads using successive pH-controlled batch fermentations. Growth occurred preferentially in 200- and 300-microm peripheral layers of the beads for L. diacetylactis and B. longum, respectively. Repeated-batch cultures with immobilized cells permitted the production of a mixed culture containing a non-competitive strain of bifidobacteria, as a result of immobilized-cell growth and high cell-release activity from the beads. During co-immobilized fermentations, there were no apparent interactions between the strains.     

Genetic heterogeneity and functional properties of intestinal bifidobacteria

AIMS: The aim of the present study was to compare several molecular methods for the identification and genotyping of bifidobacteria, and further to investigate genetic heterogeneity and functional properties of bifidobacterial isolates from intestinal samples of Finnish adult subjects. METHODS AND RESULTS: A total of 153 intestinal bifidobacterial isolates were included in initial screening and 34 isolates were further characterized. Identification results obtained with PCR-ELISA and ribotyping were well in accordance with each other, while randomly amplified polymorphic DNA (RAPD) gave tentative identification only to Bifidobacterium bifidum and to 65% of the B. longum isolates. The most commonly detected species were B. longum biotype longum followed by B. adolescentis and B. bifidum. In addition, B. animalis (lactis), B. angulatum and B. pseudocatenulatum were found. Ribotyping and pulsed-field gel electrophoresis (PFGE) proved to be discriminatory methods for bifidobacteria, but also RAPD revealed intraspecies heterogeneity. Besides two B. animalis (lactis) isolates with very close similarity to a commercially available probiotic strain, none of the intestinal isolates showed optimal survival in all tolerance (acid, bile and oxygen) or growth performance tests. CONCLUSIONS: Several species/strains of bifidobacteria simultaneously colonize the gastrointestinal tract of healthy Finnish adults and intestinal Bifidobacterium isolates were genetically heterogeneous. Functional properties of bifidobacteria were strain-dependent. SIGNIFICANCE AND IMPACT OF THE STUDY: Applicability of ribotyping with the automated RiboPrinter System for identification and genotyping of bifidobacteria was shown in the present study.     

Characterization of human intestinal bifidobacteria using competitive PCR and PCR-TTGE

In this study, a competitive PCR was developed to estimate the quantity of bifidobacteria in human faecal samples using two 16S rRNA gene Bifidobacterium genus-specific primers, Bif164f and Bif662r. A PCR-temporal temperature gradient gel electrophoresis (TTGE) with the same primers also allowed us to describe the Bifidobacterium species present in these faecal samples. The PCR product obtained from the competitor had 467 bp, and was 47 bp shorter than the PCR products obtained from Bifidobacterium strains. The number of bifidobacterial cells was linear from 10 to 10(8) cells per PCR assay. Taking into account the dilutions of the extracted DNA, the linear range was over 8 x 10(5) bifidobacteria g(-1) of faeces. Reproducibility was assessed from 10 independent DNA extractions from the same stool and the coefficient of variation was 0.5%. When the competitive PCR was compared with the culture method, a similar count of seven out of nine Bifidobacterium pure cultures were obtained, or had a difference inferior or equal to 1 log(10). In faecal samples, the enumeration of Bifidobacterium genus in most cases gave higher results with competitive PCR than with culture on selective Columbia-Beerens agar pH 5 (P < 0.05). In conclusion, this competitive PCR allows a rapid, highly specific and reproducible quantification of Bifidobacterium genus in faecal samples. TTGE fragments co-migrating with B. longum CIP64.63 fragment were found in 10 out of 11 faecal samples. Bifidobacterium adolescentis and B. bifidum were detected in five out of 11 subjects. Thus, cPCR and PCR-TTGE can be associated in order to characterize human faecal bifidobacteria.     

Quantitative fluorometric assay for rapid enzymatic characterization of Bifidobacterium longum and related bifidobacteria.

The quantitative, semi-automated assay described here is an alternative characterization method allowing for highly sensitive and specific detection of bifidobacterial enzymes. Twenty strains of Bifidobacterium longum, including the type strain ATCC 15707, and type strains of 15 other Bifidobacterium species were enzymatically characterized using 20 4-methylumbelliferyl conjugated substrates. Enzyme activities were determined by directly measuring the intensity of fluorescence derived from 4-methylumbelliferone, a fluorescent metabolic by-product. For this method, a Titertek Fluoroskan II fluorometer was used. Enzymes included glycosidases, an esterase, phosphatase, sulphatase, and neuraminidase. B. longum showed strong activity (greater than 1,000 absolute fluorescence units, afu) for alpha-L-Arabinopyranosidase and alpha-L-Arabinofuranosidase, beta-D-Fucosidase, alpha- and beta-D-Galactosidase, alpha-D-Glucosidase, and alpha-D-Mannosidase. No activity (less than or equal to 50 afu) was observed for beta-D-Cellobiosidase, alpha- and beta-L-Fucosidase, beta-D-Glucuronidase, beta-D-Mannosidase, Neuraminidase and Sulphatase. Enzymatic activity profiles in other bifidobacteria were different according to the species. This assay is simple and rapid (6 hr). Special cultural requirements are unnecessary. Results are objective and quantitative. This assay may be a useful tool for bifidobacterial taxonomy.     

Purification and N-terminal amino acid sequence of fructose-6-phosphate phosphoketolase from Bifidobacterium longum BB536

AIMS: The key enzyme in the fructose-6-phosphate shunt in bifidobacteria, Fructose-6-phosphate phosphoketolase (F6PPK; E.C. 4.1.2.22.), was purified to electrophoretic homogeneity for the first time from Bifidobacterium longum (BB536). METHODS AND RESULTS: A three-step procedure comprising acetone fractionation followed by fast protein liquid chromatography (FPLC) resulted in a 30-fold purification. The purified enzyme had a molecular mass of 300 +/- 5 kDa as determined by gel filtration. It is probably a tetramer containing two different subunits with molecular masses of 93 +/- 1 kDa and 59 +/- 0.5 kDa, as determined by SDS-PAGE. CONCLUSION: The deduced N-terminal amino acid sequences of the two subunits revealed no significant similarity between them and other proteins when compared to the data bases of EMBL and SWISS-PROT, indicating that this could be the first report on N-terminal amino acid sequence of F6PPK. SIGNIFICANCE AND IMPACT OF THE STUDY: The data from this study will be used to design oligonucleotide probe specific for bifidobacteria and to study the gene encoded F6PPK.     

两歧双歧杆菌中serpin基因的克隆、表达与功能分析

从Bifidobacterium bifidum WBBI02基因组中克隆了serpin基因片段,构建了重组Serpin蛋白的原核表达体系,实现了Serpin的表达与纯化。纯化的Serpin蛋白进行了抑制肠道蛋白酶活性检测,以及对双歧杆菌粘附作用影响的显微观察研究。结果表明:WBBI02中长度为768 bp的serpin基因序列,与GENEBANK中Bifidobacterium longum NCC2705 serpin序列同源性为99. 9 %。原核表达载体pBX₂-WBBI02表达的Serpin能有效地抑制糜蛋白酶和胰弹性蛋白酶的活性,最高抑制率分别为90%和97%,显微观察结果证实Serpin能促进双歧杆菌对HT-29细胞的粘附。