Application of a single‐colony coculture technique to the isolation of hitherto unculturable gut bacteria

Molecular studies have led to postulation of a relationship between gut microbiota and certain diseases. However, because studies of hitherto uncultured species in vivo are essential for characterizing the biology and pathogenic properties of gut bacteria, techniques for culturing and isolating such bacteria must be developed. Here, a technique is described that partially overcomes the obstacles that prevent detection of interbacterial communication in vitro and are thus responsible for the failure to culture certain bacterial species. For this purpose, a ring with a membrane filter at the bottom was designed and a relatively simple nutrient medium was used instead of conventional media. Gut bacteria were cocultivated in soft agar separated by the membrane filter to simulate interbacterial communication in vitro. Use of this soft agar coculture technique led to the successful isolation of hitherto uncultured bacteria and the demonstration of multistage interbacterial communication among gut bacteria in vitro. Cultivation and isolation of single colonies of bacteria that require other bacteria for growth will enhance efforts to better understand the physiological and pathogenic roles of gut microbiota.     

Comparative genomics of Bifidobacterium species isolated from marmosets and humans

The genus Bifidobacterium is purported to have beneficial consequences for human health and is a major component of many gastrointestinal probiotics. Although species of Bifidobacterium are generally at low relative frequency in the adult human gastrointestinal tract, they can constitute high proportions of the gastrointestinal communities of adult marmosets. To identify genes that might be important for the maintenance of Bifidobacterium in adult marmosets, ten strains of Bifidobacterium were isolated from the feces of seven adult marmosets, and their genomes were sequenced. There were six B. reuteri strains, two B. callitrichos strains, one B. myosotis sp. nov. and one B. tissieri sp. nov. among our isolates. Phylogenetic analysis showed that three of the four species we isolated were most closely related to B. bifidum, B. breve and B. longum, which are species found in high abundance in human infants. There were 1357 genes that were shared by at least one strain of B. reuteri, B. callitrichos, B. breve, and B. longum, and 987 genes that were found in all strains of the four species. There were 106 genes found in B. reuteri and B. callitrichos but not in human bifidobacteria, and several of these genes were involved in nutrient uptake. These pathways for nutrient uptake appeared to be specific to Bifidobacterium from New World monkeys. Additionally, the distribution of Bifidobacterium in fecal samples from captive adult marmosets constituted as much as 80% of the gut microbiome, although this was variable between individuals and colonies. We suggest that nutrient transporters may be important for the maintenance of Bifidobacterium during adulthood in marmosets.     

Comparative Analysis of the Gut Microbiota in People with Different Levels of Ginsenoside Rb1 Degradation to Compound K

Panax ginseng (family Araliaceae) which contains ginsenoside Rb1 as a main constituent is traditionally used as a remedy for cancer, inflammation, stress, and ageing. The ginsenoside Rb1 in orally administered ginseng is metabolized to bioactive compounds by gut microbiota before their absorptions to the blood. However, its metabolizing activities in individuals are significantly different as we previously demonstrated. Here, we selected 5 samples with fecal activity potently metabolizing ginsenoside Rb1 to compound K (FPG; metabolic activity, 0.058±0.029 pmol/min/mg) and 5 samples with fecal activity non-metabolizing ginsenoside Rb1 to compound K (FNG) from a pool of 100 subjects investigated in a previous study and analyzed fecal microbiota by 16S rRNA gene pyrosequencing. Taxonomy-based analysis showed that the population levels of Firmicutes and Proteobacteria in FPG were lower than in FNG, but those of Bacteroidetes and Tenericutes in FPG were higher than in FNG. At the genus level, the population levels of Clostridiales_uc_g, Oscillibacter, Ruminococcus, Holdemania, and Sutterella in FPG were significantly higher than in FNG, but that of Leuconostoc in FPG was lower than in FNG. The population levels of Bacteroides and Bifidobacterium, which potently metabolizes ginsenoside Rb1 to compound K were dramatically increased in FPG. The gut microbiota compositions of FPG and FNG were segregated on PCO2 by Principal Coordinate Analysis. Intestinal bacterial metabolism of ginseng, particularly ginsenoside Rb1, may be dependent on the composition of gut microbiota, such as Ruminococcus spp., Bacteroides spp. and Bifidobacterium spp.     

Recent insight into oligosaccharide uptake and metabolism in probiotic bacteria

Abstract

In recent years, a plethora of studies have demonstrated the paramount physiological importance of the gut microbiota on various aspects of human health and development. Particular focus has been set on probiotic members of this community, the best studied of which are assigned into the Lactobacillus and Bifidobacterium genera. Effects such as pathogen exclusion, alleviation of inflammation and allergies, colon cancer, and other bowel disorders are attributed to the activity of probiotic bacteria, which selectively ferment prebiotics comprising mainly non-digestible oligosaccharides. Thus, glycan metabolism is an important attribute of probiotic action and a factor influencing the composition of the gut microbiota.

In the quest to understand the molecular mechanism of this selectivity for certain glycans, we have explored the routes of uptake and utilization of a variety of oligosaccharides differing in size, composition, and glycosidic linkages. A combination of “omics” technologies bioinformatics, enzymology and protein characterization proved fruitful in elucidating the protein transport and catabolic machinery conferring the utilization of glucosides, galactosides, and xylosides in the two clinically validated probiotic strains Lactobacillus acidophilus NCFM and Bifidobacterium animalis subsp. lactis Bl-04. Importantly, we have been able to identify and in some cases validate the specificity of several transport systems, which are otherwise poorly annotated. Further, we have demonstrated for the first time that non-naturally occurring tri- and tetra-saccharides are internalized and efficiently utilized by probiotic bacteria in some cases better than well-established natural prebiotics.

Selected highlights of these data are presented, emphasising the importance and the diversity of oligosaccharide transport in probiotic bacteria.     

Fecal Bacterial Composition of Sichuan Snub-Nosed Monkeys (<Emphasis Type="Italic">Rhinopithecus roxellana</Emphasis>)

The intestinal microbiota plays an important role in maintaining the health of its host, including human and nonhuman primates. Little is known about the intestinal bacterial composition of the Sichuan snub-nosed monkey (Rhinopithecus roxellana), which has been classified as Endangered on the International Union for Conservation of Nature Red List since 2003. We evaluated the fecal bacterial compositions of 11 Sichuan snub-nosed monkeys, including six young captive individuals (one sample from each), three adult captive individuals (four samples each), and two adult provisioned free-ranging individuals (four samples each). We also quantified fecal Bacteroides vulgatus, Bifidobacterium spp., and Lactobacillus spp., which are defined as probiotics in humans, using real-time polymerase chain reaction. We identified five major phyla in the collected samples, including Firmicutes (32.4 %), Bacteroidetes (14.7 %), Verrucomicrobia (8.8 %), Actinobacteria (4.4 %), and unclassified microbacteria (39.7 %). Fecal bacteria composition varied with age and different seasons. The fecal bacterial composition of the captive monkeys was less variable than that of provisioned free-ranging monkeys. B. vulgatus amounts were almost 100 times higher in the provisioned free-ranging monkeys (10¹²) than in the captive monkeys (10¹⁰). Our results provide an initial catalogue of gut microbiota in the Sichuan snub-nosed monkey, which helps to enrich our knowledge of gut microbiota in nonhuman primates.     

Impact of a synbiotic food on the gut microbial ecology and metabolic profiles

Background  

The human gut harbors a diverse community of microorganisms which serve numerous important functions for the host wellbeing. Functional foods are commonly used to modulate the composition of the gut microbiota contributing to the maintenance of the host health or prevention of disease. In the present study, we characterized the impact of one month intake of a synbiotic food, containing fructooligosaccharides and the probiotic strains Lactobacillus helveticus Bar13 and Bifidobacterium longum Bar33, on the gut microbiota composition and metabolic profiles of 20 healthy subjects.     

Diversity of Intestinal Bifidobacteria in Patients with Japanese Cedar Pollinosis and Possible Influence of Probiotic Intervention

This study was conducted to evaluate the potential association between intestinal bifidobacteria and Japanese cedar pollinosis (JCPsis) and possible influences of probiotic intervention. In this study, fecal samples were the collected from 29 JCPsis patients. The qualitative and quantitative analyses of fecal bifidobacteria were conducted by quantitative real-time PCR with 16S rRNA-gene-targeted species-specific primers before cedar pollen spread and after a 10-week intervention with fermented milk prepared with Lactobacillus GG and L. gasseri TMC0356 during pollen spread. Each JCPsis patient had a unique diversity of bifidobacteria, which varied qualitatively and quantitatively in an individual-dependent manner during pollen spread. The serum IgE concentration of JCPsis patients with more than 3 detectable Bifidobacterium species was significantly lower than that of patients with less than 2 detected species. The prevalence of B. adolescentis, B. longum, and B. catenulatum increased after probiotic intervention, although the changes were not statistically significant. These results suggest that lower diversity of intestinal Bifidobacterium species might be a pathological aspect of JCPsis. The diversity of intestinal bifidobacteria could be a prospective target for using probiotics in the management of IgE-mediated allergic disorders including JCPsis.     

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.     

Antibiotic Susceptibility of Lactobacillus and Bifidobacterium Species from the Human Gastrointestinal Tract

One hundred and twenty-two strains of Bifidobacterium and Lactobacillus species have been tested against 12 antibiotics and two antibiotic mixtures by a commercial system (Sensititre Anaero3; Treck Diagnostic Systems). The upper limits of some minimum inhibitory concentrations (MICs) were completed on MRS agar plates by the NCCLS procedure. All strains were sensitive to chloramphenicol and imipenem and most of the strains were resistant to metronidazole. Bifidobacteria isolates were susceptible to cefoxitin, whereas about half of the lactobacilli were resistant. Approximately 30% of the Bifidobacterium isolates were resistant to tetracycline, as well as five Lactobacillus strains belonging to four different species. None of the tested Bifidobacterium isolates was resistant to vancomycin, whereas a species-dependent resistance was found among the lactobacilli. Single strains of Bifidobacterium longum, Bifidobacterium pseudocatenulatum, Lactobacillus acidophilus, Lactobacillus rhamnosus, and Lactobacillus brevis were resistant to erythromycin and/or clindamycin. Most of the observed resistances seemed to be intrinsic, but some others could be compatible with transmissible determinants.     

Live and heat-inactivated lactobacilli from feces inhibit <Emphasis Type="Italic">Salmonella typhi</Emphasis> and <Emphasis Type="Italic">Escherichia coli</Emphasis> adherence to caco-2 cells

A quantitative approach has been proposed to evaluate the competitive inhibition of Escherichia coli and Salmonella typhi by live and heat-inactivated laboratory isolated Lactobacillus sp. on adhesion to monolayer of Caco-2 cells. Three species of Lactobacillus (L. casei, L. acidophilus, L. agilis) isolated from human neonate feces and two commercial probiotic strains (L. casei, L. acidophilus) have been compared for probiotic activity. All lactobacilli were able to attach to the Caco-2 cells, however, the degree of adhesion was bacterial strain-dependent. The adhesion indices of the two commercial probiotic strains were not significantly different from the values obtained for the other two similar fecal strains (p > 0.01). The inhibition of attachment of the pathogenic bacteria by inactivated cells of fecal L. acidophilus was examined and compared to the results of live bacteria. The inhibition pattern was similar for live and heat-inactivated L. acidophilus (p > 0.01). The number of attached pathogenic bacteria to the Caco-2 cells decreased when the number of L. acidophilus increased from 10⁶ to 10⁹ CFU/mL. The heat-inactivated L. acidophilus displayed similar probiotic activity compared to the live bacteria.     

Bifidobacterium longum supplementation improves age-related delays in fracture repair

Age-related delays in bone repair remains an important clinical issue that can prolong pain and suffering. It is now well established that inflammation increases with aging and that this exacerbated inflammatory response can influence skeletal regeneration. Recently, simple dietary supplementation with beneficial probiotic bacteria has been shown to influence fracture repair in young mice. However, the contribution of the gut microbiota to age-related impairments in fracture healing remains unknown. Here, we sought to determine whether supplementation with a single beneficial probiotic species, Bifidobacterium longum (B. longum), would promote fracture repair in aged (18-month-old) female mice. We found that B. longum supplementation accelerated bony callus formation which improved mechanical properties of the fractured limb. We attribute these pro-regenerative effects of B. longum to preservation of intestinal barrier, dampened systemic inflammation, and maintenance of the microbiota community structure. Moreover, B. longum attenuated many of the fracture-induced systemic pathologies. Our study provides evidence that targeting the gut microbiota using simple dietary approaches can improve fracture healing outcomes and minimize systemic pathologies in the context of aging.     

Quantitative Real-Time PCR Assays To Identify and Quantify Fecal Bifidobacterium Species in Infants Receiving a Prebiotic Infant Formula

A healthy intestinal microbiota is considered to be important for priming of the infants' mucosal and systemic immunity. Breast-fed infants typically have an intestinal microbiota dominated by different Bifidobacterium species. It has been described that allergic infants have different levels of specific Bifidobacterium species than healthy infants. For the accurate quantification of Bifidobacterium adolescentis, Bifidobacterium angulatum, Bifidobacterium bifidum, Bifidobacterium breve, Bifidobacterium catenulatum, Bifidobacterium dentium, Bifidobacterium infantis, and Bifidobacterium longum in fecal samples, duplex 5′ nuclease assays were developed. The assays, targeting rRNA gene intergenic spacer regions, were validated and compared with conventional PCR and fluorescent in situ hybridization methods. The 5′ nuclease assays were subsequently used to determine the relative amounts of different Bifidobacterium species in fecal samples from infants receiving a standard formula or a standard formula supplemented with galacto- and fructo-oligosaccharides (OSF). A breast-fed group was studied in parallel as a reference. The results showed a significant increase in the total amount of fecal bifidobacteria (54.8% ± 9.8% to 73.4% ± 4.0%) in infants receiving the prebiotic formula (OSF), with a diversity of Bifidobacterium species similar to breast-fed infants. The intestinal microbiota of infants who received a standard formula seems to resemble a more adult-like distribution of bifidobacteria and contains relatively more B. catenulatum and B. adolescentis (2.71% ± 1.92% and 8.11% ± 4.12%, respectively, versus 0.15% ± 0.11% and 1.38% ± 0.98% for the OSF group). In conclusion, the specific prebiotic infant formula used induces a fecal microbiota that closely resembles the microbiota of breast-fed infants also at the level of the different Bifidobacterium species.     

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

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

Kinetics of microbial hydrogenation of free linoleic acid to conjugated linoleic acids

Aims: To investigate the ability of selected probiotic bacterial strains to produce conjugated linoleic acid (CLA) and also to estimate the biohydrogenation kinetics of Lactobacillus acidophilus on the production of CLA from free linoleic acid (LA). Methods and Results: Six probiotic bacteria, Lact. paracasei, Lact. rhamnosus GG, Lact. acidophilus ADH, and Bifidobacterium longum B6, Lact. brevis, and Lact. casei, were used to examine their ability to convert LA to CLA. LA tolerance was evaluated by addition of different LA concentrations in MRS broth. Lact. acidophilus showed the major tolerant to LA and the greatest CLA‐producing ability (36–48 μg ml^?1 of CLA). The rate‐controlling steps were k₂ and k₁ for the addition of 1 and 3 mg ml^?1 of LA, respectively. The percentage of CLA conversion was higher in MRS broth supplemented with 1 mg ml^?1 (65%) than 3 mg ml^?1 (26%). Conclusion: The results provide useful information and new approach for understanding the biohydrogenation mechanisms of CLA production. Significance and Impact of the Study: This study would help elucidate the pathway from LA to stearic acid (SA), known as biohydrogenation. In addition, the use of selected probiotic bacteria might lead to a significant improvement in food safety.     

Microbiota profile in feces of breast- and formula-fed newborns by using fluorescence in situ hybridization (FISH)

The development of the gut is controlled and modulated by different interacting mechanisms such as, genetic endowment, intrinsic biological regulatory functions, environment influences and last but no least, the diet influence. Considered together with other endogenous and exogenous factors the type of feeding may interfere greatly in the regulation of the intestinal microbiota. During the last years molecular methods offer a complementarity to the classic culture-based knowledge. FISH has been applied for molecular evaluation of the microbiota in newborns delivered by vaginal delivery. Eleven probes/probe combinations for specific groups of faecal bacteria were used to determine the bacterial composition in faecal samples of newborns infants under different types of feeding. Breast-fed infants harbor a fecal microbiota by more than two times increased in numbers of Bifidobacterium cells when compared to formula-fed infants. After formula-feeding, Atopobium was found in significant counts and the numbers of Bifidobacterium dropped followed by increasing numbers in Bacteroides population. Moreover, under formula feeding the infants microbiota was more diverse.     

Robustness of Gut Microbiota of Healthy Adults in Response to Probiotic Intervention Revealed by High-Throughput Pyrosequencing

Probiotics are live microorganisms that potentially confer beneficial outcomes to host by modulating gut microbiota in the intestine. The aim of this study was to comprehensively investigate effects of probiotics on human intestinal microbiota using 454 pyrosequencing of bacterial 16S ribosomal RNA genes with an improved quantitative accuracy for evaluation of the bacterial composition. We obtained 158 faecal samples from 18 healthy adult Japanese who were subjected to intervention with 6 commercially available probiotics containing either Bifidobacterium or Lactobacillus strains. We then analysed and compared bacterial composition of the faecal samples collected before, during, and after probiotic intervention by Operational taxonomic units (OTUs) and UniFrac distances. The results showed no significant changes in the overall structure of gut microbiota in the samples with and without probiotic administration regardless of groups and types of the probiotics used. We noticed that 32 OTUs (2.7% of all analysed OTUs) assigned to the indigenous species showed a significant increase or decrease of ≥10-fold or a quantity difference in >150 reads on probiotic administration. Such OTUs were found to be individual specific and tend to be unevenly distributed in the subjects. These data, thus, suggest robustness of the gut microbiota composition in healthy adults on probiotic administration.     

Supportive Role of Probiotic Strains in Protecting Rats from Ovariectomy-Induced Cortical Bone Loss

Osteoporosis is a major health problem that occurs as a result of an imbalance between bone formation and bone resorption. Different approaches have been established for treating osteoporosis. Recently, because of their health benefits and also low adverse reaction, probiotics have been receiving considerable attention. In this study, we compared the effectiveness of five probiotic strains, Lactobacillus acidophilus, Lactobacillus reuteri, Lactobacillus casei, Bifidobacterium longum, and Bacillus coagulans, in protecting rats from ovariectomized (OVX)-induced bone loss. Forty-nine adult female Sprague-Dawley rats were allocated into seven groups as follows: group 1, control; group 2, OVX; group 3, OVX + Lactobacillus acidophilus; group 4, OVX + Lactobacillus casei; group 5, OVX + Bacillus coagulans; group 6, OVX + Bifidobacterium longum; and group 7, OVX + Lactobacillus reuteri. Probiotics were fed to OVX groups at the concentration of (1 × 10⁹ CFU/ml/day) for 4 weeks. Then, biochemical parameters, including vitamin D, calcium (Ca), phosphorus (P), and alkaline phosphatase (ALP), were assessed. Dual-energy X-ray absorptiometry (DEXA) scans were used that assess bone mineral density (BMD), bone marrow concentration (BMC), and area of global, femur, spine, and tibia. Lactobacillus acidophilus and Lactobacillus casei significantly increased Ca and ALP and decreased P in treated groups. Lactobacillus casei, Lactobacillus reuteri, and Bifidobacterium longum increased vitamin D significantly. Lactobacillus acidophilus and Lactobacillus casei indicated the most effects on BMD. In terms of BMC, and bone area, Lactobacillus acidophilus, Lactobacillus reuteri, and Lactobacillus casei demonstrated the significant enhancement in OVX groups treated with. Among the probiotics used in this study, Lactobacillus acidophilus and Lactobacillus casei showed the most effects in terms of BMD, BMC, bone area, and biochemical parameters. It seems that probiotics effects on bone health are strain dependent, but further studies should be done to prove these findings.

     

In vitro Effects of Prebiotics and Synbiotics on Apis cerana Gut Microbiota

This study aimed to investigate in vitro effects of the selected prebiotics alone, and in combination with two potential probiotic Lactobacillus strains on the microbial composition of Apis cerana gut microbiota and acid production. Four prebiotics, inulin, fructo-oligosaccharides, xylo-oligosaccharides, and isomalto-oligosaccharides were chosen, and glucose served as the carbon source. Supplementation of this four prebiotics increased numbers of Bifidobacterium and lactic acid bacteria while decreasing the pH value of in vitro fermentation broth inoculated with A. cerana gut microbiota compared to glucose. Then, two potential probiotics derived from A. cerana gut at different dosages, Lactobacillus helveticus KM7 and Limosilactobacillus reuteri LP4 were added with isomalto-oligosaccharides in fermentation broth inoculated with A. cerana gut microbiota, respectively. The most pronounced impact was observed with isomalto-oligosaccharides. Compared to isomalto-oligosaccharides alone, the combination of isomalto-oligosaccharides with both lactobacilli strains induced the growth of Bifidobacterium, LAB, and total bacteria and reduced the proliferation of Enterococcus and fungi. Consistent with these results, the altered metabolic activity was observed as lowered pH in in vitro culture of gut microbiota supplemented with isomalto-oligosaccharides and lactobacilli strains. The symbiotic impact varied with the types and concentration of Lactobacillus strains and fermentation time. The more effective ability was observed with IMO combined with L. helveticus KM7. These results suggested that isomalto-oligosaccharides could be a potential prebiotic and symbiotic with certain lactobacilli strains on A. cerana gut microbiota.     

Immunology and probiotic impact of the newborn and young children intestinal microflora

Human body has developed a holistic defence system, which mission is either to recognize and destroy the aggressive invaders or to evolve mechanisms permitting to minimize or restore the consequences of harmful actions. The host immune system keeps the capital role to preserve the microbial intestinal balance via the barrier effect. Specifically, pathogenic invaders such as, bacteria, parasites, viruses and other xenobiotic invaders are rejected out of the body via barriers formed by the skin, mucosa and intestinal flora. In case physical barriers are breached, the immune system with its many components comes into action in order to fence infection. The intestine itself is considered as an “active organ” due to its abundant bacterial flora and to its large metabolic activity. The variation among different species or even among different strains within a species reflects the complexity of the genetic polymorphism which regulates the immune system functions. Additionally factors such as, gender, particular habits, smoking, alcohol consumption, diet, religion, age, gender, precedent infections and vaccinations must be involved. Hormonal profile and stress seems to be associated to the integrity microbiota and inducing immune system alterations. Which bacterial species are needed for inducing a proper barrier effect is not known, but it is generally accepted that this barrier function can be strongly supported by providing benefic alimentary supplements called functional foods. In this vein it is stressed the fact that early intestinal colonization with organisms such as Lactobacilli and Bifidobacteria and possibly subsequent protection from many different types of diseases. Moreover, this benefic microflora dominated but Bifidobacteria and Lactobacilli support the concept of their ability to modify the gut microbiota by reducing the risk of cancer following their capacity to decrease β-glucoronidase and carcinogen levels. Because of their beneficial roles in the human gastrointestinal tract, LAB are referred to as “probiotics”, and efforts are underway to employ them in modern nutrition habits with so-called functional foods. Members of Lactobacillus and Bifidobacterium genera are normal residents of the microbiota in the human gastrointestinal tract, in which they developed soon after birth. But, whether such probiotic strains derived from the human gut should be commercially employed in the so-called functional foods is a matter of debate between scientists and the industrial world. Within a few hours from birth the newborn develops its normal bacterial flora. Indeed human milk frequently contains low amounts of non-pathogenic bacteria like Streptococcus, Micrococcus, Lactobacillus, Staphylococcus, Corynebacterium and Bifidobacterium. In general, bacteria start to appear in feces within a few hours after birth. Colonization by Bifidobacterium occurs generally within 4 days of life. Claims have been made for positive effects of Bifidobacterium on infant growth and health. The effect of certain bacteria having a benefic action on the intestinal ecosystem is largely discussed during the last years by many authors. Bifidobacterium is reported to be a probiotic bacterium, exercising a beneficial effect on the intestinal flora. An antagonism has been reported between B. bifidum and C. perfringens in the intestine of newborns delivered by cesarian section. The aim of the probiotic approach is to repair the deficiencies in the gut flora and restore the protective effect. However, the possible ways in which the gut microbiota is being influenced by probiotics is yet unknown.     

Prevention of <Emphasis Type="Italic">Escherichia coli</Emphasis> O157:H7 infection in gnotobiotic mice associated with <Emphasis Type="Italic">Bifidobacterium</Emphasis> strains

Previous reports have shown that Escherichia coli O157:H7 infection is strongly modified by intestinal microbes. In this paper, we examined whether bifidobacteria protect against E. coli O157:H7 infections using gnotobiotic mice di-associated with Bifidobacterium strains (6 species, 9 strains) and E. coli O157:H7. Seven days after oral administration of each Bifidobacterium strain, the mice were orally infected with E. coli O157:H7 and their mortality was examined. Bifidobacterium longum subsp. infantis 157F-4-1 (B. infantis 157F) and B. longum subsp. longum NCC2705 (B. longum NS) protected against the lethal infection, while mice associated with all other Bifidobacterium strains, including type strains of B. longum subsp. infantis and B. longum subsp. longum, died. There were no significant differences in the numbers of E. coli O157:H7 in the faeces among the Bifidobacterium-associated mouse groups. However, the Shiga toxin concentrations in the cecal contents and sera of the GB mice associated with B. infantis 157F and B. longum NS were significantly lower than those of the other groups. However, there were no significant differences in the volatile fatty acid concentrations and histopathological lesions between these two groups. These data suggest that some strains of B. longum subsp. longum/infantis can protect against the lethal infections of E. coli O157:H7 by preventing Shiga toxin production in the cecum and/or Shiga toxin transfer from the intestinal lumen to the bloodstream.