Comparison of mucosal adhesion and species identification of bifidobacteria isolated from healthy and allergic infants

Fifty bifidobacteria strains were isolated from fecal samples of allergic and age matched healthy infants. Allergic infants were found to have an adult type Bifidobacterium flora with high levels of Bifidobacterium adolescentis. Healthy infants had a typical infant Bifidobacterium flora with high levels of Bifidobacterium bifidum. These isolates were tested for their adhesive properties to human intestinal mucus. The adhesion of the fecal bifidobacteria from healthy infants was significantly higher (P<0.0001) than for allergic infants. This suggests a correlation between allergic disease and the composition of the intestinal bifidobacteria flora which has reduced adhesive abilities to the intestinal mucus. Therefore, dietary supplementation of bifidobacteria typical for healthy infants, may be beneficial in the treatment of allergic disorders.     

Differences in Composition and Mucosal Adhesion of Bifidobacteria Isolated from Healthy Adults and Healthy Seniors

Fifty-one Bifidobacterium strains were isolated from the feces of healthy adults (30–40 years old) and seniors (older than 70 years of age). B. adolescentis, B. breve, B. infantis, and B. longum were isolated from the healthy adults and B. adolescentis and B. longum from elderly subjects. The tested bacteria bound, in vitro, to intestinal mucus in a strain dependent manner. The strains isolated from healthy adults, and especially B. adolescentis, bound better to intestinal mucus than those isolated from seniors. These results indicate that the mucosal adhesive properties of the human Bifidobacterium flora were reduced with the aging of the host. This shift to a Bifidobacterium flora with reduced adhesive abilities may explain the decrease in bifidobacteria levels in the intestinal microflora of aging people. Received: 7 February 2001 / Accepted: 3 April 2001     

Adhesion of human bifidobacterial strains to cultured human intestinal epithelial cells and inhibition of enteropathogen-cell interactions.

Thirteen human bifidobacterial strains were tested for their abilities to adhere to human enterocyte-like Caco-2 cells in culture. The adhering strains were also tested for binding to the mucus produced by the human mucus-secreting HT29-MTX cell line in culture. A high level of calcium-independent adherence was observed for Bifidobacterium breve 4, for Bifidobacterium infantis 1, and for three fresh human isolates from adults. As observed by scanning electron microscopy, adhesion occurs to the apical brush border of the enterocytic Caco-2 cells and to the mucus secreted by the HT29-MTX mucus-secreting cells. The bacteria interacted with the well-defined apical microvilli of Caco-2 cells without cell damage. The adhesion to Caco-2 cells of bifidobacteria did not require calcium and was mediated by a proteinaceous adhesion-promoting factor which was present both in the bacterial whole cells and in the spent supernatant of bifidobacterium culture. This adhesion-promoting factor appeared species specific, as are the adhesion-promoting factors of lactobacilli. We investigated the inhibitory effect of adhering human bifidobacterial strains against intestinal cell monolayer colonization by a variety of diarrheagenic bacteria. B. breve 4, B. infantis 1, and fresh human isolates were shown to inhibit cell association of enterotoxigenic, enteropathogenic, diffusely adhering Escherichia coli and Salmonella typhimurium strains to enterocytic Caco-2 cells in a concentration-dependent manner. Moreover, B. breve 4 and B. infantis 1 strains inhibited, dose dependently, Caco-2 cell invasion by enteropathogenic E. coli, Yersinia pseudotuberculosis, and S. typhimurium strains.     

Bifidobacteria in Feces and Environmental Waters

Bifidobacteria have been recommended as potential indicators of human fecal pollution in surface waters even though very little is known about their presence in nonhuman fecal sources. The objective of this research was to shed light on the occurrence and molecular diversity of this fecal indicator group in different animals and environmental waters. Genus- and species-specific 16S rRNA gene PCR assays were used to study the presence of bifidobacteria among 269 fecal DNA extracts from 32 different animals. Twelve samples from three wastewater treatment plants and 34 water samples from two fecally impacted watersheds were also tested. The species-specific assays showed that Bifidobacterium adolescentis, B. bifidum, B. dentium, and B. catenulatum had the broadest host distribution (11.9 to 17.4%), whereas B. breve, B. infantis, and B. longum were detected in fewer than 3% of all fecal samples. Phylogenetic analysis of 356 bifidobacterial clones obtained from different animal feces showed that ca. 67% of all of the sequences clustered with cultured bifidobacteria, while the rest formed a supercluster with low sequence identity (i.e., <94%) to previously described Bifidobacterium spp. The B. pseudolongum subcluster (>97% similarity) contained 53 fecal sequences from seven different animal hosts, suggesting the cosmopolitan distribution of members of this clade. In contrast, two clades containing B. thermophilum and B. boum clustered exclusively with 37 and 18 pig fecal clones, respectively, suggesting host specificity. Using species-specific assays, bifidobacteria were detected in only two of the surface water DNA extracts, although other fecal anaerobic bacteria were detected in these waters. Overall, the results suggest that the use of bifidobacterial species as potential markers to monitor human fecal pollution in natural waters may be questionable.     

Growth promotion and cell binding ability of bovine lactoferrin to Bifidobacterium longum

Lactoferrin, a major whey protein of human milk, is considered as growth promoter for bifidobacteria, the predominant microorganisms of human intestine. In the present study, in vitro growth promotion and cell binding ability of bovine lactoferrin to several strains of Bifidobacterium longum have been demonstrated. A dose-dependent as well as strain-dependent growth promotion effect by lactoferrin was observed. Cell binding ability of lactoferrin was inspected under an inverted confocal laser scanning microscope by incubation bacterial cells with biotinylated bovine lactoferrin and FITC-conjugated avidin. Fluorescence staining showed bovine lactoferrin binding to all tested strains. A lactoferrin-binding protein with a molecular weight of approximately 67 kDa was also detected in the extracted membrane and cytosolic fraction of each B. longum strain by far-Western blot technique using biotinylated lactoferrin and horseradish peroxidase-conjugated streptavidin. Based on these results, we suggest that existence of lactoferrin-binding protein could be a common characteristic in bifidobacteria. It can also be hypothesized that lactoferrin-binding protein in bifidobacteria is not only involved in growth stimulation mechanism but also could play different roles.     

Good adhesion properties of probiotics: a potential risk for bacteremia?

The ability to adhere to human intestinal mucus was tested for lactic acid bacteria of clinical blood culture, human fecal and dairy origin. The blood culture isolates were found to adhere better than the dairy strains. Of the Lactobacillus rhamnosus strains (nine clinical, 10 fecal and three dairy), blood culture isolates adhered better than the fecal strains. Although these results indicate a trend for blood culture isolates to bind to intestinal mucus in higher numbers than strains of dairy and human fecal origin, other factors are also likely to be involved in the etiology of lactobacillemia since some of the clinical Lactobacillus isolates exhibited a relatively low level of adhesion.     

Microbiota Composition of the Intestinal Mucosa: Association with Fecal Microbiota?

The fecal and mucosal microbiota of infants with rectal bleeding and the fecal microbiota of healthy age-matched controls were investigated by fluorescent in situ hybridization. Bifidobacteria were the main genus in both the feces and mucosa. The other genera tested, Bacteroides, Clostridium, Escherichia coli and lactobacilli/enterococci, represented only minor constituents. No differences in fecal microbiota were observed between patients and controls. In the patients, however, four times greater numbers of bifidobacteria were observed in the feces when compared to the mucosa. Notwithstanding this difference, a strong positive correlation prevailed for bifidobacteria in feces and mucosal samples. The genera assessed accounted for 16% of total bacterial counts on mucosal samples and for 47% of total bacterial counts in feces. This indicates that the unidentified part of the microbiota, especially on the mucosa, deserves more attention.     

Characterization of the properties of human- and dairy-derived probiotics for prevention of infectious diseases in fish

The present study aimed to investigate the potential probiotic properties of six lactic acid bacteria (LAB) intended for human use, Lactobacillus rhamnosus ATCC 53103, Lactobacillus casei Shirota, Lactobacillus bulgaricus, L. rhamnosus LC 705, Bifidobacterium lactis Bb12, and Lactobacillus johnsonii La1, and one for animal use, Enterococcus faecium Tehobak, for use as a fish probiotic. The strains for human use were specifically chosen since they are known to be safe for human use, which is of major importance because the fish are meant for human consumption. The selection was carried out by five different methods: mucosal adhesion, mucosal penetration, inhibition of pathogen growth and adhesion, and resistance to fish bile. The adhesion abilities of the seven LAB and three fish pathogens, Vibrio anguillarum, Aeromonas salmonicida, and Flavobacterium psychrophilum, were determined to mucus from five different sites on the surface or in the gut of rainbow trout. Five of the tested LAB strains showed considerable adhesion to different fish mucus types (14 to 26% of the added bacteria). Despite their adhesive character, the LAB strains were not able to inhibit the mucus binding of A. salmonicida. Coculture experiments showed significant inhibition of growth of A. salmonicida, which was mediated by competition for nutrients rather than secretion of inhibitory substances by the probiotic bacteria as measured in spent culture liquid. All LAB except L. casei Shirota showed tolerance against fish bile. L. rhamnosus ATCC 53103 and L. bulgaricus were found to penetrate fish mucus better than other probiotic bacteria. Based on bile resistance, mucus adhesion, mucus penetration, and suppression of fish pathogen growth, L. rhamnosus ATCC 53103 and L. bulgaricus can be considered for future in vivo challenge studies in fish as a novel and safe treatment in aquaculture.     

Influence of carp intestinal mucus molecular size and glycosylation on bacterial adhesion

The first step of the pathogenesis of many infectious diseases is the colonisation of the mucosal surface by the pathogen. Bacterial colonisation of the mucosal surface is promoted by adherence to high molecular weight mucus glycoproteins. We examined the effect of carp intestinal mucus glycoproteins on the adhesion of different bacteria. The bacteria used were 3 strains of Aeromonas hydrophila, and A. salmonicida, Edwardsiella tarda and Yersinia ruckeri. All bacteria adhered to mucus, but at varying intensities. All tested bacteria adhered best to molecules of 670 to 2000 kDa in size, less to molecules larger than 2000 kDa and weakest to molecules of 30 to 670 kDa. In general, bacteria that showed a stronger adhesion to intestinal mucus were cytotoxic to cells in vitro, and bacteria that showed a weaker adhesion to intestinal mucus did not lead to alterations of monolayers of EPC-cells. Furthermore, the involvement of glycan side chains of the glycoproteins for bacterial adhesion was analysed for one A. hydrophila strain. After cleavage of terminal sugar residues by treatment of mucus glycoproteins with different glycosidases, binding of bacteria was modulated. When mannose was cleaved off, adhesion significantly increased. Blocking of glycan receptors by incubation of bacteria with different oligosaccharides had no clear effect on bacterial binding to mucus glycoproteins. Our results suggest that bacteria interact with carbohydrate side chains of mucus glycoproteins, and that the carbohydrates of the core region are involved in bacterial binding.     

Growth of infant fecal bacteria on commercial prebiotics

Fecal bacteria from 33 infants (aged 1 to 6 months) were tested for growth on commercial prebiotics. The children were born vaginally (20) or by caesarean section (13). Bifidobacteria, lactobacilli, gram-negative bacteria, Escherichia coli, and total anaerobes in fecal samples were enumerated by selective agars and fluorescence in situ hybridization. The total fecal bacteria were inoculated into cultivation media containing 2 % Vivinal® (galactooligosaccharides—GOS) or Raftilose® P95 (fructooligosaccharides—FOS) as a single carbon source and bacteria were enumerated again after 24 h of anaerobic cultivation. Bifidobacteria dominated, reaching counts of 9–10 log colony-forming units (CFU)/g in 17 children born vaginally and in seven children delivered by caesarean section. In these infants, lactobacilli were more frequently detected and a lower number of E. coli and gram-negative bacteria were determined compared to bifidobacteria-negative infants. Clostridia dominated in children without bifidobacteria, reaching counts from 7 to 9 log CFU/g. Both prebiotics supported all groups of bacteria tested. In children with naturally high counts of bifidobacteria, bifidobacteria dominated also after cultivation on prebiotics, reaching counts from 8.23 to 8.77 log CFU/mL. In bifidobacteria-negative samples, clostridia were supported by prebiotics, reaching counts from 7.17 to 7.69 log CFU/mL. There were no significant differences between bacterial growth on Vivinal® and Raftilose® P95 and counts determined by cultivation and FISH. Prebiotics should selectively stimulate the growth of desirable bacteria such as bifidobacteria and lactobacilli. However, our results showed that commercially available FOS and GOS may stimulate also other fecal bacteria.     

Adhesion of four Bifidobacterium strains to human intestinal mucus from subjects in different age groups

The number of bifidobacteria in faeces and intestinal contents has been shown to be reduced with increasing age of the subject. The adhesion of four Bifidobacterium strains was tested to mucus isolated from subjects of different age. All strains bound significantly less to mucus isolated from elderly subjects, compared to mucus from the other age groups. Two of the tested strains also showed decreased adhesion to mucus isolated from 6-month-old and adult subjects compared to the adhesion to mucus from 2-month-old subjects. The results suggest that reduced adhesion may be a factor involved in the decreasing colonisation of elderly subjects by bifidobacteria.     

The mucus binding of Bifidobacterium lactis Bb12 is enhanced in the presence of Lactobacillus GG and Lact. delbrueckii subsp. bulgaricus

The ability to adhere to mucosal surfaces is related to many probiotic health effects. In the presence of Lactobacillus GG or Lact. bulgaricus, the adhesion of Bifidobacterium lactis Bb12 to a mucus model was more than doubled. Other tested lactobacilli did not affect the adhesion, nor was the adhesion of the lactobacilli influenced by the bifidobacteria. Co-aggregation between Bif. lactis Bb12 and the tested lactobacilli was insignificant and does not explain the observed effect. The results suggest that combinations of probiotics strains may have synergistic adhesion effects. Such specific strain combinations should also be assessed in clinical studies.     

Enumeration,isolation, and identification of bifidobacteria from infant feces

Thirty-three fully breast-fed infants aged between 1 and 12 weeks were screened for bifidobacteria in feces. Bifidobacteria counts in most fecal samples determined both by TPY agar and FISH procedure ranged from 10(8) to 10(11) CFU/g. Three infants did not contain any bifidobacteria in their fecal samples. One child was delivered by caesarean section and the other two by normal vaginal delivery. All bifidobacteria-free infants possessed Gram-positive regular rods as a major group of their fecal flora. These bacteria were identified as clostridia using genus-specific FISH probe. In bifidobacteria-positive samples, B. longum (57.9% of the samples) was the most frequently found species, followed by B. adolescentis (31.6%), B. bifidum (21.0%), B. breve (10.5%), B. pseudocatenulatum (5.3%), and B. dentium (5.3%).     

Auto-aggregation and Co-aggregation ability in bifidobacteria and clostridia

A total of 142 human and 88 calf bifidobacteria were isolated and identified; approximately 12 % of all isolated strains exhibited auto-aggregation (Agg) phenotype (Agg+). Properties considered to be predicting for their adhesion to intestine, i.e. auto-aggregation, and hydrophobicity were determined by xylene extraction in 18 human and 8 calf origin bifidobacteria. Co-aggregation of 8 human bifidobacteria with 8 clostridia was also evaluated. Agg varied between 16.3 and 96.4 %, hydrophobicity values ranged from 0 to 82.8 %. The strongest Agg and hydrophobicity were observed in B. bifidum and B. merycicum isolates. However, there were no statistically significant correlations between these two properties. Variability in the percentage of Agg and hydrophobicity was observed after cultivation of bifidobacteria on different carbon sources. All bifidobacteria showed co-aggregation ability with clostridia tested but there were remarkable differences depending on specific combinations of strains. The bifidobacterial strains with the highest ability to co-aggregate with clostridia were B. bifidum I4 and B. longum I10 isolated from infants; these strains gave also high values of Agg. Agg properties together with co-aggregation ability with potential pathogen can be used for preliminary selection of probiotic bacteria.     

Stimulation of the secretion of pro-inflammatory cytokines by Bifidobacterium strains

To characterize the ability of bifidobacteria to affect the production of macrophage-derived cytokines, a murine macrophage-like cell line, J774.1, was cultured in the presence of 27 strains of heat-inactivated bifidobacteria. Bifidobacterium adolescentis and B. longum, known as adult-type bifidobacteria, induced significantly more pro-inflammatory cytokine secretion, IL-12 and TNF-alpha, by J774.1 cells, than did the infant-type bifidobacteria, B. bifidum, B. breve, and B. infantis (P<0.01). In contrast, B. adolescentis did not stimulate the production of anti-inflammatory IL-10 from J774.1 cells as the other tested bacteria did. The results suggest that the adult-type bifidobacteria, especially B. adolescentis, may be more potent to amplify but less able to down-regulate the inflammatory response.     

Analysis of fecal populations of bifidobacteria and lactobacilli and investigation of the immunological responses of their human hosts to the predominant strains.

The bifidobacterial and lactobacillus populations of fecal samples collected from 10 human subjects were studied. The numbers of bifidobacteria were similar in the fecal samples of all of the subjects, but lactobacillus numbers varied, even between samples collected from the same individual. Analysis of the composition of the bacterial populations by ribotyping and pulsed-field gel electrophoresis to differentiate between strains showed that, at least for the numerically predominant strains, each subject harbored a unique collection of bifidobacteria and lactobacilli. Predominant bifidobacterial and lactobacillus strains detected in the feces of each subject were used in immunological assays (lymphocyte transformation, serum antibody titers) to determine the influence of the bacteria on the immune system of their host. Immunoglobulin G antibodies reactive with lactobacilli were detected at high concentrations; antibodies reactive with bifidobacteria were present at lower concentrations. The antibodies appeared to be genus specific rather than strain specific. The results of the study emphasized the complexity of the relationship that exists between the intestinal microflora and the human host.     

Decreased colonic mucus in rats with loperamide-induced constipation

Constipation is a risk factor of colorectal cancer. Mucin is a major component of lumenal mucus, which protects the colorectal mucosa against mechanical and chemical damage. The aim of this study was to evaluate mucus production and to quantitate lumen mucus in a rat model of spastic constipation. We induced constipation with loperamide (1.5 mg/kg), and histochemically evaluated mucus production and the thickness of the mucus layer at the fecal surface. We quantitated the mucus attached to the mucosal surface using colonic perfusion with N-acetylcysteine. While more feces remained in the colon, there was less fecal excretion and lower fecal water content in loperamide-administered rats than in control rats. Crypt epithelial cells contained less mucus in constipated rats than in control rats. The mucus layer at the fecal surface was thinner and less mucus was recovered from the mucosal surface in constipated rats than in control rats. Mucus production of crypt epithelial cells and mucus at the fecal and mucosal surface were reduced by loperamide-induced constipation.     

Evaluation of using a short region of the recA gene for rapid and sensitive speciation of dominant bifidobacteria in the human large intestine

The feasibility of intragenerically characterizing bifidobacteria by a comparison of a short region within the recA gene was tested. An ∼300 bp fragment of the recA gene was PCR-amplified from six species from the genus Bifidobacterium using primers directed to two universally conserved regions of the recA gene. A phylogenetic analysis of the sequenced recA products compared favorably to classification based on the 16S rRNA sequences of the species tested. To apply this rapid methodology to unknown human intestinal bifidobacteria, 46 isolates were randomly chosen from the feces of four subjects and initially characterized by RFLP analysis of a PCR-amplified region of their 16S RNA genes. From a representative of the dominant RFLP family in each of the subjects, the recA segment was PCR-amplified, sequenced and phylogenetically analyzed. All four isolates were found to be related to one another and to B. longum and B. infantis. These results illustrate that the recA gene may be useful for intrageneric phylogenetic analysis as well as for the identification of unknown fecal bifidobacteria.     

Lactoferrin and bifidobacteria

We herein summarized the effects of lactoferrin (LF) on bifidobacteria. Many in vitro studies previously reported the growth-promoting (bifidogenic) effects of LF on bifidobacteria. The involvement of bound iron, sugar chains, and LF peptides has been proposed in this bifidogenic mechanism. Peptides in the LF pepsin hydrolysate (LFH) showed stronger bifidogenic activity than natural LF; therefore, we speculated that peptides may be the bifidogenic active principle of LF. LF or its peptides may be recognized by LF-binding proteins on the surface of bifidobacterial cells, and the cationic nature or disulfide bonds of LF or its peptides may play a crucial role in its recognition by these proteins. Of the bifidobacterial species so far identified, human LF and peptides in human LFH were more likely to show bifidogenic activity especially to Bifidobacterium bifidum, and bovine LF (bLF) and peptides in bovine LFH (bLFH) to B. breve and B. infantis. In animal studies, the administration of LF to mice or piglets increased bifidobacteria levels in the intestine. In human trials, the administration of LF-containing formula to infants increased bifidobacteria levels in the feces; however, human milk achieved better results than LF-containing formula. In the case of breast-fed infants, LF may show bifidogenic activity synergistically with other milk components such as human milk oligosaccharides. As bLFH showed stronger bifidogenic activity than natural bLF, especially to B. breve and B. infantis in vitro, and these species are known to be infant-specific species, bLFH may be a beneficial ingredient in formula.     

Integrated analysis of established and novel microbial and chemical methods for microbial source tracking

Several microbes and chemicals have been considered as potential tracers to identify fecal sources in the environment. However, to date, no one approach has been shown to accurately identify the origins of fecal pollution in aquatic environments. In this multilaboratory study, different microbial and chemical indicators were analyzed in order to distinguish human fecal sources from nonhuman fecal sources using wastewaters and slurries from diverse geographical areas within Europe. Twenty-six parameters, which were later combined to form derived variables for statistical analyses, were obtained by performing methods that were achievable in all the participant laboratories: enumeration of fecal coliform bacteria, enterococci, clostridia, somatic coliphages, F-specific RNA phages, bacteriophages infecting Bacteroides fragilis RYC2056 and Bacteroides thetaiotaomicron GA17, and total and sorbitol-fermenting bifidobacteria; genotyping of F-specific RNA phages; biochemical phenotyping of fecal coliform bacteria and enterococci using miniaturized tests; specific detection of Bifidobacterium adolescentis and Bifidobacterium dentium; and measurement of four fecal sterols. A number of potentially useful source indicators were detected (bacteriophages infecting B. thetaiotaomicron, certain genotypes of F-specific bacteriophages, sorbitol-fermenting bifidobacteria, 24-ethylcoprostanol, and epycoprostanol), although no one source identifier alone provided 100% correct classification of the fecal source. Subsequently, 38 variables (both single and derived) were defined from the measured microbial and chemical parameters in order to find the best subset of variables to develop predictive models using the lowest possible number of measured parameters. To this end, several statistical or machine learning methods were evaluated and provided two successful predictive models based on just two variables, giving 100% correct classification: the ratio of the densities of somatic coliphages and phages infecting Bacteroides thetaiotaomicron to the density of somatic coliphages and the ratio of the densities of fecal coliform bacteria and phages infecting Bacteroides thetaiotaomicron to the density of fecal coliform bacteria. Other models with high rates of correct classification were developed, but in these cases, higher numbers of variables were required.