The range of antigenic specificity of Bifidobacterium peptidoglycan]

The antigenic relationships of Bifidobacterium bifidum 1 peptidoglycans with different strains of this species (LVA-3, 791, GO-4), bifidobacteria of other species (B. adolescentis GO-13, B. breve 79-38, B. lactentis 79-41, B. longum GO-3) and bacteria of remote taxonomic groups (Streptococcus faecalis 6-3. Staphylococcus aureus COM 885, S. epidermidis COM 2124. Lactobacillus plantarum 1, Escherichia coli M-17) were studied on the basis of a highly sensitive test system permitting the registration of normal human antibodies to peptidoglycans. The level of cross reactions with staphylococci and streptococci correspond to intraspecific and antigenic affinity to L. plantarum and E. coli was considerably less pronounced. Copying a number of epitopes of bifidobacteria, S. aureus peptidoglycan seems to possess additional antigenic determinants which participate in the formation of immunological responsiveness in man.     

Effect of Exposure to Air on 84 strains of Bifidobacteria

Among 84 strains of bifidobacteria tested, 62%, belonging to animal species, decreased in count by less than one log 10 unit after 4 days of contact with air at a temperature of 20°C. When 10 strains each of B. pseudolongum subsp. pseudolongum and B. thermophilum were tested at 4°C under the same conditions, eight of the former and five of the latter survived without any decline during 4 days.     

Search of promising strains of bifidobacteria and lactobacillus for the development of new biopreparations

For the first time the species composition of bifidobacteria and lactobacilli in clinically healthy young children has been studied. As revealed in this study, the dominating species of bifidobacteria are B. longum, B. adolescentis and B. infantis, while the dominating species of lactobacilli are Lactobacillus acidophilus and L. rhamnosus. In 83 isolated cultures of bifidobacteria and 34 isolated species of lactobacilli the activity of acid formation and the antagonistic activity with respect to Escherichia coli, Klebsiella pneumoniae, Staphylococcus aureus, Enterococcus faecalis, Clostridium perfringens have been tested. B. longum strain 58B, B. infantis strain 37B, L. rhamnosus strain 12L and L. acidophilus strain 27L, typical for children of this age group, having good antagonistic activity and pronounced acid-forming properties, have been selected. These strains hold good promise to be used as the basis for the development of a complex probiotic preparation for correcting intestinal microflora in young children.     

The biological properties of Bifidobacterium]

The possibility of the formation of exoenzymes, such as DNAase, RNAase and hemolysin, by bifidobacteria was studied in comparison with their acid-forming and adhesive activity. Bifidobacterium reference strains, originally isolated from healthy adults and children, were studied. The study involved altogether 73 strains of bifidobacteria, including 24 B. bifidum strains, 13 B. adolescentis strains, 7 B. infantis strains, 10 B. breve strains and 19 B. longum strains. The bifidobacteria under study were shown to differ not only in the presence and activity of properties useful for macroorganisms, but also in the presence of enzymes having depolymerizing activity (DNAase, hemolysin). Thus, out of 73 strains under study 9 proved to be DNAase-positive and 6, hemolysin positive. At the same time a specific feature of bifidobacteria was their high acid-forming activity with the complete absence of RNAase activity and insignificant DNAase- and hemolysin-forming activity.     

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

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

Phenotypic differentiation of bifidobacteria of human and animal origins.

The phenotypes of 153 strains belonging or related to the genus Bifidobacterium were studied. These organisms included 38 collection strains and 115 wild strains (41 strains of human origin, 56 strains of animal origin, and 18 strains obtained from rivers or sewage). Our phenotypic analysis revealed seven main groups that were subdivided into 20 subgroups. Seven subgroups contained no type or collection strain. Among the human strains, the type strains of Bifidobacterium pseudocatenulatum and B. catenulatum fell into group I, which contained the type strains of B. adolescentis (subgroup Ib), B. dentium (subgroup Ic), and B. angulatum (ungrouped). The type strain of B. breve belonged to subgroup IIIa1, and the type strains of B. infantis and B. longum fell into subgroup IIIb1. Group VII comprised only wild strains that were isolated from human infant feces. Among the animal strains, group II consisted mainly of bifidobacteria that were isolated from pig feces and contained the type strains of B. suis (subgroup IIb), B. thermophilum (subgroup IIf), B. choerinum, and B. boum (ungrouped). Wild strains belonging to group V were isolated from pig, calf, cow, and chicken feces; this included the type strains of B. animalis (subgroup Va), B. magnum (subgroup Vb), B. pseudolongum, and B. globosum (subgroup Vc). The strains of human origin (groups I, III, and VII) were well separated from the animal strains (groups II, IV, and V). It was not surprising that the wild strains isolated from surface water or sewage were distributed in the animal groups as well as the human groups. Thus, bifidobacteria can be considered to be successful indicators of human or animal fecal pollution when they are correctly classified. The acidification patterns were not adequate to differentiate Bifidobacterium species, as determined previously by Mitsuoka (Bifidobacteria Microflora 3:11-28, 1984) and Scardovi (p. 1418-1434, in P. H. A. Sneath, N. S. Mair, M. E. Sharpe, and J. G. Holt, ed., Bergey's Manual of Systematic Bacteriology, vol. 2, 1986). However, enzymatic tests furnished new taxonomic criteria for the genus.     

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.     

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.     

The vaginal Bifidobacterium flora in women of reproductive age

The composition of vaginal bifidoflora in 56 clinically healthy women of reproductive age was studied. The study revealed that four species of bifidobacteria, viz. Bifidobacterium bifidum, B. breve, B. adolescentis 2 and B. longum, dominated in the composition of this bifidobacterial population. Nine out of 11 isolated strains were found to be capable of inhibiting indicator microorganisms Staphylococcus aureus and Enterococcus faecalis when tested in vitro; in addition, strains B. adolescentis 2 F1, B. bifidum G1, B. breve P2 and B. longum Z4 inhibited Klebsiella ozaenae, Pseudomonas aeruginosa, Escherichia coli and were also active acid producers. Three of these 4 bifidobacterial strains were capable of adhesion to vaginal epitheliocytes, while B. bifidum G1 was practically incapable of adherence to these cells, similarly to B. bifidum strain 791 of intestinal origin. In addition, the spectra of antibiotic susceptibility varied from strain to strain, but all bifidobacterial strains were susceptible to benzylpenicillin and resistant to lomefloxacin, most of them being also resistant to cyprofloxacin and gentamicin. Thus the data presented in this work are indicative of the possibility and advantages of using bifidobacterial strains belonging to this ecological niche as probiotics for the correction of the microflora of the urogenital tract in females.     

Longitudinal analysis and genotyping of infant dominant bifidobacterial populations

Bifidobacterial population dynamics were investigated by the longitudinal analysis of the dominant population isolated from the feces of young infants. After molecular identification and fingerprinting comparison, clone identity of the consecutive strains belonging to the same species for one individual was performed by pulsed-field gel electrophoresis. The results, obtained from 15 individuals sampled four times over a five-week period suggested a turnover of the dominant bifidobacteria in the population not only at the species but also at its species representative levels. This study provides new insights of the in vivo dynamics of commensal bifidobacteria. It highlights the need to take into consideration the fluctuation of bifidobacterial populations that may occur in one individual in order to investigate reliably the impact of dietary components, such as probiotics or prebiotics, on the intestinal ecosystem.     

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.     

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.     

Qualitative and quantitative composition of acids formed by bifidobacteria

The dynamics of acid production in 18 strains of bifidobacteria, belonging to 5 different species, has been studied Bifidobacteria have been found to produce 3 acids, lactic, acetic and formic, in the process of their metabolism. The lactic acid/acetic acid quantitative ratio varies, depending on the culture medium on the average, 1:2 in Blaurock medium, 1:5 in milk hydrolysate medium. Various strains have also been found to differ in the dynamics of acid production with respect to the amounts of lactic and acetic acids. The study has shown that, despite the active production of acids for a period of up to 72 hours, a decrease in the pH of the medium is observed for not more than 48 hours. The existence of a specific mechanism permitting bifidobacteria to regulate the acidity of their environment is supposed.     

The Ability of Bifidobacteria To Degrade Arabinoxylan Oligosaccharide Constituents and Derived Oligosaccharides Is Strain Dependent

Arabinoxylan oligosaccharides (AXOS) are prebiotic carbohydrates with promising health-promoting properties that stimulate the activity of specific colon bacteria, in particular bifidobacteria. However, the mechanisms by which bifidobacterial strains break down these compounds in the colon is still unknown. This study investigates AXOS consumption of a large number of bifidobacterial strains (36), belonging to 11 different species, systematically. To determine their degradation mechanisms, all strains were grown on a mixture of arabinose and xylose, xylo-oligosaccharides, and complex AXOS molecules as the sole added energy sources. Based on principal component and cluster analyses of their different arabinose substituent and/or xylose backbone consumption patterns, five clusters that were species independent could be distinguished among the bifidobacterial strains tested. In parallel, the strains were screened for the presence of genes encoding several putative AXOS-degrading enzymes, but no clear-cut correlation could be made with the different degradation mechanisms. The intra- and interspecies differences in the consumption patterns of AXOS indicate that bifidobacterial strains could avoid competition among each other or even could cooperate jointly to degrade these complex prebiotics. The knowledge gained on the AXOS degradation mechanisms in bifidobacteria can be of importance in the rational design of prebiotics with tailor-made composition and thus increased specificity in the colon.     

Isolation, growth on prebiotics and probiotic potential of novel bifidobacteria from pigs

Bifidobacteria were isolated from the faeces of pigs of various ages and examined for their potential use as probiotics in combination with di- and oligosaccharides. Ninty-six per cent of the isolates were found to have characteristics in common with Bifidobacterium boum, B. thermophilum and B. choerinum. B. thermophilum was most commonly isolated from sows, whereas most of the other strains were isolated from piglets. A few strains of each species were able to grow in the presence of air. A microplate assay was modified to allow comparison of growth on different substrates. Di- and oligosaccharides considered to promote bifidobacteria were screened for their ability to support growth of selected isolates in vitro. Growth on these substrates varied within and between species. Of the fructose oligosaccharides tested, Actilight P supported the best growth of the widest range of strains. The strains which grew best on the disaccharide lactulose were related to B. choerinum and some of these strains grew on xylo-oligosaccharides. It seems that prebiotic di- and oligosaccharides may have both a species and intra-species/strain selective effect. B. choerinum appeared to be well adapted to the gut of pre-weaned piglets.     

Identification of bifidobacteria isolated from Asian elephant (Elephas maximus)

Bifidobacteria are considered as one of the key genera in intestinal tracts of animals, and their species composition vary depending on the host. The aim of this study was to identify faecal bifidobacteria from Asian elephants (Elephas maximus), housed in Zoological gardens (Ostrava, Czech Republic). Using culturing, bifidobacteria were found in counts 7.60?±?0.56 log CFU/g. Twenty-six pure strains were isolated from faeces of Asian elephant. The isolates were clustered into two groups according to fingerprinting profiles and fermentation characteristic. Bacteria were identified by a combination of MALDI-TOF MS, PCR methods and sequencing as B. boum (12 isolates) and B. adolescentis (14 isolates). Elephant strains showed different fingerprinting profiles than type and collection strains. Since these two species are frequently isolated from gastrointestinal tract of herbivores, they seem to be typical of animals fed plant diets.     

Specific detection of bifidobacterium strains in a pharmaceutical probiotic product and in human feces by polymerase chain reaction

For PCR specific detection of the strains Bifidobacterium longum Y 10, B. infantis Y 1 and B. breve Y 8 used in a new probiotic product (VSL-3), strains-specific rDNA primers have been developed. Spacer regions between the 16S and 23S rRNA genes (ITS) of the three strains were amplified by PCR with conserved primers and the nucleotide sequence of these ITSs were determined. On the basis of their comparison with the rDNA sequences retrieved from GenBank, we designed new primers which specifically recognize the species B. breve and the two strains B. infantis Y 1 and B. breve Y 8. Specificity of these primers was confirmed through the analysis of 60 bifidobacteria strains belonging to the more representative human species. The feasibility of this PCR method was investigated in commercial VSL-3 product and fecal samples collected from 4 patients affected by inflammatory bowel deseases and two healthy subjects before and after the VSL-3 administration. By PCR analysis of different VSL-3 commercial batches we were successful in differentiating and quantifying the strains B. longum Y 10, B. infantis Y 1 and B. breve Y 8. B. infantis Y 1 and B. breve Y 8 could be detected at high concentration in fecal specimens of both patients and subjects treated with the probiotic preparation, showing a different colonization behaviour. Seven days after the VSL-3 treatment suspension, no patients and subjects harbored B. infantis Y 1 and B. breve Y 8, indicating a transient presence of these exogenous strains.     

Sensitivity of bifidobacteria to antibacterial drugs

Twenty one strains of bifidobacteria belonging to various species were studied with respect to their sensitivity to antibacterial drugs most widely used in medical practice. The strains were isolated from practically healthy persons and from persons in contact with antibiotics in their production. It was found that sensitivity of the strains was the highest with respect to penicillins and tetracyclines. With respect to kanamycin, monomycin and levomycetin the strains were resistant. Strain differences in resistance to separate antibacterial drugs were observed. Increased streptomycin and tetracycline resistance of the strains isolated from the persons being for a prolonged period in contact with these antibiotics in their production, was stated.     

Toll-like receptor-2-activating bifidobacteria strains differentially regulate inflammatory cytokines in the porcine intestinal epithelial cell culture system: finding new anti-inflammatory immunobiotics

A total of 23 strains of bifidobacteria taxonomically belonging to five species were tested for their potent immunomodulatory effect using a combination of two methods: the NF-κB-reporter assay using a toll-like receptor 2-expressing transfectant (HEK(pTLR2) system) and the mitogenic assay using porcine Peyer's patches immunocompetent cells. Among the four preselected strains from different immunomodulatory groups, Bifidobacterium breve MCC-117 was able to efficiently modulate the inflammatory response triggered by enterotoxigenic Escherichia coli (ETEC) in a porcine intestinal epithelial (PIE) cell line. Moreover, using PIE cells and swine Peyer's patches immunocompetent cell co-culture system, we demonstrated that the immunoregulatory effect of B. breve MCC-117 was related to the capacity of the strain to influence PIE and immune cell interactions, leading to the stimulation of regulatory T cells. The results suggested that bifidobacteria that express high activity in both the HEK(pTLR2) and the mitogenic assays may behave like potential anti-inflammatory strains. The combination of the HEK(pTLR2) system, the evaluation of mitogenic activity and PIE cells will be of value for the development of new immunologically functional foods and feeds that could prevent inflammatory intestinal disorders. Although our findings should be proven in appropriate experiments in vivo, the results of the present work provide a scientific rationale for the use of B. breve MCC-117 to prevent ETEC-induced intestinal inflammation.