Study of the physiological and biochemical characteristics of bifidobacteria at the late stages of population development

An investigation of the physiological and biochemical characteristics of the Bifidobacterium bifidum no. 1, B. adolescentis MC-42, and B. adolescentis 94-BIM strains showed that bifidobacteria with a higher growth rate produced greater amounts of the end fermentation products, acetate and lactate. The growth of the strains in batch cultures was found to be inhibited by acidic fermentation products. The growth of B. bifidum no. 1 in a batch mode lasted 100 h at a population density of 10(6) CFU/ml and the growth of B. adolescentis MC-42 and 94-BIM lasted 96-120 h at population densities from 10(4) to 10(7) CFU/ml. Analysis of the bifidobacterial populations by light and electron microscopy showed that they represent conglomerates of cells with lysed cytoplasm in the cell center and intact cytoplasm in the apical parts of the cells. The maximum production of extra-cellular and cell-bound proteinases was observed in the logarithmic growth phase. By the 120th h of cultivation, the metabolic activity of cells, the production of proteinases, and the protein content of bifidobacterial cultures considerably decreased. In the first, second, and third subcultures of 96-h-old bifidobacterial cells on fresh nutrient media, the population density of bifidobacteria and their normal physiological and biochemical characteristics were restored after 48 to 72 h of cultivation.     

Patterns of growth and β-galactosidase production by Bifidobacteria

We investigated the patterns of growth and β-galactosidase production in the strains Bifidobacterium adolescentis GO-13, MS-42, 91-BIM, and 94-BIM and b. bifidum No.1, LVA-3, 791 on media with various carbon sources. The synthesis of β-galactosidase was shown to be associated with exponential growth of the cultures involved. The maximum specific rate of β-galactosidase synthesis of 0.20 U mg^?1 h^?1 was observed in B. bifidum LVA-3 after 3–6 h of cultivation. This value for B. adolescentis 91-BIM and 94-BIM was lower and amounted to 0.03–0.08 U mg^?1h^?1. On the medium with lactose, the highest specific growth rates for B. bifidum LVA-3 and B. bifidum No.1 were 0.38 and 0.60 h^?1, respectively, after 3–6 h of cultivation. For B. adolescentis 91-BIM and 94-BIM, this parameter peaked at 12–15 h of cultivation at 0.13 and 0.22 h^?1, respectively. The hydrolytic activity of β-galactosidase in the growth medium decreased during the stationary growth phase of the tested cultures.     

Production of Cell-Wall-Bound Proteinases in Bifidobacterium adolescentis 94-BIM

Bifidobacterium adolescentis 94-BIM was found to produce cell-wall-bound proteolytic enzymes active at acidic, neutral, and alkaline pH values. The solubilization of proteinases with 0.5% Triton X-100 substantially improved the yield of the enzymes. The most active accumulation of cell-bound proteinases was observed in the third hour of cultivation at rates of 156.7, 179.5, and 111.1 U/(mg h), measured at pH 2.5, 7.0, and 9.0, respectively. It is suggested that the cell-wall-bound proteinases of B. adolescentis 94-BIM are the precursors of the enzymes secreted into the medium.     

Production of hydrolases by lactic acid bacteria and bifidobacteria and their antibiotic resistance

It was demonstrated that bifidobacteria and lactic acid bacteria B. adolescentis and Lactobacillus sp. synthesized extracellular enzymes cleaving glycoside bonds in the molecules of dextran, pectic acid, and soluble starch. The maximal production of extracellular β-galactosidase by B. adolescentis 91-BIM and 94-BIM at a rate of 0.08 and 0.03 U/mg per h was observed during the exponential growth phase at 5 and 12 h of cultivation, respectively. The cultures of bifidobacteria retained 60–70% of β-galactosidase and α-amylase activities after six months of storage. The bifidobacterium strains studied were resistant to amphotericin and aminoglycosides (gentamicin, kanamycin, and netromycin). The lactam antibiotics (ampicillin, benzylpenicillin, bicillin 3, bicillin 5, and carbenicillin), the preparations inhibiting protein synthesis at the level of ribosomes (lincomycin), RNA polymerase inhibitors (rifampin), cephalosporin, and Maxipime inhibited the growth of bifidobacteria. Rifampin, erythromycin, amphotericin, Maxipime, Fortum, doxycycline, levomycetin, streptomycin, and the aminoglycosides netromycin, gentamicin, and kanamycin did not have an effect on the growth of Lactobacillus sp., whereas semisynthetic derivatives of penicillin, carbenicillin and ampicillin, inhibited its growth as well as Oxamp and lincomycin. The lactam antibiotics benzylpenicillin, bicillin 3, and bicillin 5 inhibited the growth of lactic acid bacilli by 30–90%.     

Isolation of Bifidobacteria from Breast Milk and Assessment of the Bifidobacterial Population by PCR-Denaturing Gradient Gel Electrophoresis and Quantitative Real-Time PCR

The objective of this work was to elucidate if breast milk contains bifidobacteria and whether they can be transmitted to the infant gut through breastfeeding. Twenty-three women and their respective infants provided samples of breast milk and feces, respectively, at days 4 to 7 after birth. Gram-positive and catalase-negative isolates from specific media with typical bifidobacterial shapes were identified to the genus level by F6PPK (fructose-6-phosphate phosphoketolase) assays and to the species level by 16S rRNA gene sequencing. Bifidobacterial communities in breast milk were assessed by PCR-denaturing gradient gel electrophoresis (PCR-DGGE), and their levels were estimated by quantitative real-time PCR (qRTi-PCR). Bifidobacteria were present in 8 milk samples and 21 fecal samples. Bifidobacterium breve, B. adolescentis, and B. bifidum were isolated from milk samples, while infant feces also contained B. longum and B. pseudocatenulatum. PCR-DGGE revealed the presence of one to four dominant bifidobacterial bands in 22 milk samples. Sequences with similarities above 98% were identified as Bifidobacterium breve, B. adolescentis, B. longum, B. bifidum, and B. dentium. Bifidobacterial DNA was detected by qRTi-PCR in the same 22 milk samples at a range between 40 and 10,000 16S rRNA gene copies per ml. In conclusion, human milk seems to be a source of living bifidobacteria for the infant gut.     

Inter-species differences in the growth of bifidobacteria cultured on human milk oligosaccharides

Human milk (HM) contains as the third most abundant component around 200 different structures of human milk oligosaccharides (HMOs). HMOs are the first and irreplaceable prebiotics for infants, supporting bifidobacteria as the most important bacterial group in an infant intestine. The aim of our study was to test the growth of bifidobacteria in HM and on HMOs. Bifidobacteria were isolated from two groups of infants. The first one (eight strains) were isolated from infants who had bifidobacteria in their feces but, after a short period of time (4 to 24 days), bifidobacteria were no longer detected in their feces (disappeared bifidobacteria [DB]). The second group of bifidobacteria (eight strains) originated from infants with continual presence of bifidobacteria in their feces (persistent bifidobacteria [PB]). There were significant differences (p?Bifidobacterium bifidum and B. breve species were able to utilize HMOs, while B. adolescentis and B. longum subsp. longum species did not. The ability to grow in HM and to utilize HMOs seem to be important properties of bifidobacteria which are able to colonize infant intestinal tract.     

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.     

Bifidobacterial Diversity in Human Feces Detected by Genus-Specific PCR and Denaturing Gradient Gel Electrophoresis

We describe the development and validation of a method for the qualitative analysis of complex bifidobacterial communities based on PCR and denaturing gradient gel electrophoresis (DGGE). Bifidobacterium genus-specific primers were used to amplify an approximately 520-bp fragment from the 16S ribosomal DNA (rDNA), and the fragments were separated in a sequence-specific manner in DGGE. PCR products of the same length from different bifidobacterial species showed good separation upon DGGE. DGGE of fecal 16S rDNA amplicons from five adult individuals showed host-specific populations of bifidobacteria that were stable over a period of 4 weeks. Sequencing of fecal amplicons resulted in Bifidobacterium-like sequences, confirming that the profiles indeed represent the bifidobacterial population of feces. Bifidobacterium adolescentis was found to be the most common species in feces of the human adult subjects in this study. The methodological approach revealed intragenomic 16S rDNA heterogeneity in the type strain of B. adolescentis, E-981074. The strain was found to harbor five copies of 16S rDNA, two of which were sequenced. The two 16S rDNA sequences of B. adolescentis E-981074^T exhibited microheterogeneity differing in eight positions over almost the total length of the gene.     

Susceptibility of bifidobacteria to lysozyme as a possible selection criterion for probiotic bifidobacterial strains

Resistance or susceptibility of bifidobacteria to lysozyme and growth of bifidobacteria in human milk were tested. Susceptible bifidobacterial strains stopped their growth almost immediately after the addition of lysozyme (400 μg/ml), moderately susceptible strains exhibited reduced growth rate, and growth curves of resistant strains were not affected. Strains of human origin were more resistant to lysozyme than animal strains. While strains of B. bifidum grew well in human milk samples, the growth B. animalis strains was inhibited after inoculation to human milk. The resistance to lysozyme seems to be a promising criterion for the selection of new probiotic bifidobacterial strains.     

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.     

Cholesterol-lowering probiotics: in vitro selection and in vivo testing of bifidobacteria

Thirty-four strains of bifidobacteria belonging to Bifidobacterium adolescentis, Bifidobacterium animalis, Bifidobacterium bifidum, Bifidobacterium breve, Bifidobacterium longum, and Bifidobacterium pseu-docatenulatum were assayed in vitro for the ability to assimilate cholesterol and for bile salt hydrolase (BSH) against glycocholic and taurodeoxycholic acids (GCA and TDCA). Cholesterol assimilation was peculiar characteristic of two strains belonging to the species B. bifidum (B. bifidum MB 107 and B. bifidum MB 109), which removed 81 and 50 mg of cholesterol per gram of biomass, being the median of specific cholesterol absorption by bifidobacteria 19 mg/g. Significant differences in BSH activities were not established among bifidobacterial species. However, the screening resulted in the selection of promising strains able to efficiently deconjugate GCA and TDCA. No relationship was recognized between BSH phenotype and the extent of cholesterol assimilation. On the basis of cholesterol assimilation or BSH_GCA and BSH_TDCA activities, B. bifidum MB 109 (DSMZ 23731), B. breve MB 113 (DSMZ 23732), and B. animalis subsp. lactis MB 2409 (DSMZ 23733) were combined in a probiotic mixture to be fed to hypercholesterolemic rats. The administration of this probiotic formulation resulted in a significant reduction of total cholesterol and low-density cholesterol (LDL-C), whereas it did not affect high-density cholesterol (HDL-C) and HDL-C/LDL-C ratio.     

Microelement Preparations Obtained During the Processing of Natural Flint Affect the Physiology and Biochemistry of Bifidobacteria

Microelement preparations obtained in the course of processing flint powder stimulate the biological activity of Bifidobacterium adolescentis94-BIM grown on complex and synthetic nutritive media. The composition of the microelement preparations differed in the content of cations and anions. Introduction of the preparations into cultures of physiologically active or anabiotic forms of bifidobacteria changed the parameters of exponential growth: compared to the controls, the cultures were characterized by an increased specific growth rate and a decreased generation time. In the presence of microelements, the development of populations of bifidobacteria was associated with a more pronounced accumulation of metabolic products (acetate, lactate, and ethanol). Introduction of microelement preparations increased the rate of synthesis of the extracellular proteinase (the maximum content of the enzyme was observed after 3 h, whereas control cultures attained this level only after 6 h).     

Influence of cell surface properties on adhesion ability of bifidobacteria

Adhesion ability of bifidobacteria to the intestinal mucosa is considered to be the prerequisite for colonization of bifidobacteria and can protect against gastrointestinal pathogens infection. The aim of this study was to investigate bifidobacterial surface traits related to adhesion ability in vitro and characterize the cell surface substances that may be involved in the adhesion process of bifidobacteria. Twelve strains of Bifidobacterium spp. were studied for the correlation among their adhesion ability, autoaggregation ability and surface hydrophobicity. The strain that exhibited good adhesion ability also showed high degree of hydrophobicity and strong autoaggregation ability. Pepsin treatment had negative effect on the surface traits and adhesion ability of B. bifidum KLDS2.0603 (P < 0.01), it revealed that hydrophobicity, autoaggregation and adhesion process maybe mediated by proteinaceous components on the surface of cell. Moreover, the adhesion and autoaggregation ability decreased after extraction of B. bifidum KLDS2.0603 with 5 mol l⁻¹ LiCl, and an unreported 50-kDa surface protein which can bind to Caco-2 cell was observed by western blotting. Our results indicated that surface hydrophobicity and autoaggregation ability can be used together for preliminary screening the strains with high adhesion ability, and the present of the surface proteinaceous components would contribute to understand the interactions between bifidobacteria and human intestinal mucosa.     

Identification and characterization of the serine/threonine protein kinases in Bifidobacterium

Six genes encoding the bifidobacterial Hanks-type (eukaryote-like) serine/threonine protein kinases (STPK) were identified and classified. The genome of each bifidobacterial strain contains four conserved genes and one species-specific gene. Bifidobacterium longum and Bifidobacterium bifidum possess the unique gene found only in these species. The STPK genes of Russian industrial probiotic strain B. longum B379M were cloned and sequenced. The expression of these genes in Escherichia coli and bifidobacteria was observed. Autophosphorylation of the conserved STPK Pkb5 and species-specific STPK Pkb2 was demonstrated. This is the first report on Hanks-type STPK in bifidobacteria.     

Bifidobacterial Succession and Correlation Networks in a Large Unselected Cohort of Mothers and Their Children

Bifidobacteria are a major microbial component of infant gut microbiota, which is believed to promote health benefits for the host and stimulate maturation of the immune system. Despite their perceived importance, very little is known about the natural development of and possible correlations between bifidobacteria in human populations. To address this knowledge gap, we analyzed stool samples from a randomly selected healthy cohort of 87 infants and their mothers with >90% of vaginal delivery and nearly 100% breast-feeding at 4 months. Fecal material was sampled during pregnancy, at 3 and 10 days, at 4 months, and at 1 and 2 years after birth. Stool samples were predicted to be rich in the species Bifidobacterium adolescentis, B. bifidum, B. dentium, B. breve, and B. longum. Due to high variation, we did not identify a clear age-related structure at the individual level. Within the population as a whole, however, there were clear age-related successions. Negative correlations between the B. longum group and B. adolescentis were detected in adults and in 1- and 2-year-old children, whereas negative correlations between B. longum and B. breve were characteristic for newborns and 4-month-old infants. The highly structured age-related development of and correlation networks between bifidobacterial species during the first 2 years of life mirrors their different or competing nutritional requirements, which in turn may be associated with specific biological functions in the development of healthy gut.     

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.     

Optimization of culture conditions of probiotic bifidobacteria for maximal adhesion to hexadecane

Culture growth conditions were optimized for adhesion to hexadecane of the probiotic Bifidobacterium bifidum HI 39 and HI 48. Among three growth media used, MILS lactose broth was the best medium to obtain maximum cell adhesion, followed by MRS and TPY lactose broth for B. bifidum HI 39 and HI 48. Increasing the incubation time from 6 to 18 h resulted in a gradual increase in percentage adhesion at 37 °C of both organisms in MILS, MRS and TPY media. Thereafter, incubation up to 48 h showed a marked reduction in adhesion of B. bifidum HI 39 and B. bifidum HI 48. When the test cultures were grown at pH values from 5.0 to 8.0 in MILS lactose broth at 37 °C for 18 h, there was a gradual enhancement in cell adhesion up to pH 7.0; but higher pH values retarded the bacterial adhesion. The study showed that the optimum conditions for adhesion to hexadecane of the selected bifidobacterial strains were pH 7.0 and incubation at 37 °C for 18 h in MILS broth.     

Bifidobacteria strains isolated from stools of iron deficient infants can efficiently sequester iron

Background

Bifidobacteria is one of the major gut commensal groups found in infants. Their colonization is commonly associated with beneficial effects to the host through mechanisms like niche occupation and nutrient competition against pathogenic bacteria. Iron is an essential element necessary for most microorganisms, including bifidobacteria and efficient competition for this micronutrient is linked to proliferation and persistence. For this research we hypothesized that bifidobacteria in the gut of iron deficient infants can efficiently sequester iron. The aim of the present study was to isolate bifidobacteria in fecal samples of iron deficient Kenyan infants and to characterize siderophore production and iron internalization capacity.

Results

Fifty-six bifidobacterial strains were isolated by streaking twenty-eight stool samples from Kenyan infants, in enrichment media. To target strains with high iron sequestration mechanisms, a strong iron chelator 2,2-dipyridyl was supplemented to the agar media. Bifidobacterial isolates were first identified to species level by 16S rRNA sequencing, yielding B. bifidum (19 isolates), B. longum (15), B. breve (11), B. kashiwanohense (7), B. pseudolongum (3) and B. pseudocatenulatum (1). While most isolated bifidobacterial species are commonly encountered in the infantile gut, B. kashiwanohense was not frequently reported in infant feces. Thirty strains from culture collections and 56 isolates were characterized for their siderophore production, tested by the CAS assay. Siderophore activity ranged from 3 to 89% siderophore units, with 35 strains (41%) exhibiting high siderophore activity, and 31 (36%) and 20 (23%) showing intermediate or low activity. The amount of internalized iron of 60 bifidobacteria strains selected for their siderophore activity, was in a broad range from 8 to118 μM Fe. Four strains, B. pseudolongum PV8-2, B. kashiwanohense PV20-2, B. bifidum PV28-2a and B. longum PV5-1 isolated from infant stool samples were selected for both high siderophore activity and iron internalization.

Conclusions

A broad diversity of bifidobacteria were isolated in infant stools using iron limited conditions, with some strains exhibiting high iron sequestration properties. The ability of bifidobacteria to efficiently utilize iron sequestration mechanism such as siderophore production and iron internalization may confer an ecological advantage and be the basis for enhanced competition against enteropathogens.

Electronic supplementary material

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