Cross-feeding between Bifidobacterium longum BB536 and acetate-converting, butyrate-producing colon bacteria during growth on oligofructose

In vitro coculture fermentations of Bifidobacterium longum BB536 and two acetate-converting, butyrate-producing colon bacteria, Anaerostipes caccae DSM 14662 and Roseburia intestinalis DSM 14610, with oligofructose as the sole energy source, were performed to study interspecies interactions. Two clearly distinct types of cross-feeding were identified. A. caccae DSM 14662 was not able to degrade oligofructose but could grow on the fructose released by B. longum BB536 during oligofructose breakdown. R. intestinalis DSM 14610 could degrade oligofructose, but only after acetate was added to the medium. Detailed kinetic analyses of oligofructose breakdown by the last strain revealed simultaneous degradation of the different chain length fractions, in contrast with the preferential degradation of shorter fractions by B. longum BB536. In a coculture of both strains, initial oligofructose degradation and acetate production by B. longum BB536 took place, which in turn also allowed oligofructose breakdown by R. intestinalis DSM 14610. These and similar cross-feeding mechanisms could play a role in the colon ecosystem and contribute to the combined bifidogenic/butyrogenic effect observed after addition of inulin-type fructans to the diet.     

In Vitro Kinetic Analysis of Oligofructose Consumption by Bacteroides and Bifidobacterium spp. Indicates Different Degradation Mechanisms

The growth of pure cultures of Bacteroides thetaiotaomicron LMG 11262 and Bacteroides fragilis LMG 10263 on fructose and oligofructose was examined and compared to that of Bifidobacterium longum BB536 through in vitro laboratory fermentations. Gas chromatography (GC) analysis was used to determine the different fractions of oligofructose and their degradation during the fermentation process. Both B. thetaiotaomicron LMG 11262 and B. fragilis LMG 10263 were able to grow on oligofructose as fast as on fructose, succinic acid being the major metabolite produced by both strains. B. longum BB536 grew slower on oligofructose than on fructose. Acetic acid and lactic acid were the main metabolites produced when fructose was used as the sole energy source. Increased amounts of formic acid and ethanol were produced when oligofructose was used as an energy source at the cost of lactic acid. Detailed kinetic analysis revealed a preferential metabolism of the short oligofructose fractions (e.g., F₂ and F₃) for B. longum BB536. After depletion of the short fractions, the larger oligofructose fractions (e.g., F₄, GF₄, F₅, GF₅, and F₆) were metabolized, too. Both Bacteroides strains did not display such a preferential metabolism and degraded all oligofructose fractions simultaneously, transiently increasing the fructose concentration in the medium. This suggests a different mechanism for oligofructose breakdown between the strain of Bifidobacterium and both strains of Bacteroides, which helps to explain the bifidogenic nature of inulin-type fructans.     

Two Routes of Metabolic Cross-Feeding between Bifidobacterium adolescentis and Butyrate-Producing Anaerobes from the Human Gut

Dietary carbohydrates have the potential to influence diverse functional groups of bacteria within the human large intestine. Of 12 Bifidobacterium strains of human gut origin from seven species tested, four grew in pure culture on starch and nine on fructo-oligosaccharides. The potential for metabolic cross-feeding between Bifidobacterium adolescentis and lactate-utilizing, butyrate-producing Firmicute bacteria related to Eubacterium hallii and Anaerostipes caccae was investigated in vitro. E. hallii L2-7 and A. caccae L1-92 failed to grow on starch in pure culture, but in coculture with B. adolescentis L2-32 butyrate was formed, indicating cross-feeding of metabolites to the lactate utilizers. Studies with [¹³C]lactate confirmed carbon flow from lactate, via acetyl coenzyme A, to butyrate both in pure cultures of E. hallii and in cocultures with B. adolescentis. Similar results were obtained in cocultures involving B. adolescentis DSM 20083 with fructo-oligosaccharides as the substrate. Butyrate formation was also stimulated, however, in cocultures of B. adolescentis L2-32 grown on starch or fructo-oligosaccharides with Roseburia sp. strain A2-183, which produces butyrate but does not utilize lactate. This is probably a consequence of the release by B. adolescentis of oligosaccharides that are available to Roseburia sp. strain A2-183. We conclude that two distinct mechanisms of metabolic cross-feeding between B. adolescentis and butyrate-forming bacteria may operate in gut ecosystems, one due to consumption of fermentation end products (lactate and acetate) and the other due to cross-feeding of partial breakdown products from complex substrates.     

In vitro kinetic analysis of oligofructose consumption by Bacteroides and Bifidobacterium spp. indicates different degradation mechanisms

The growth of pure cultures of Bacteroides thetaiotaomicron LMG 11262 and Bacteroides fragilis LMG 10263 on fructose and oligofructose was examined and compared to that of Bifidobacterium longum BB536 through in vitro laboratory fermentations. Gas chromatography (GC) analysis was used to determine the different fractions of oligofructose and their degradation during the fermentation process. Both B. thetaiotaomicron LMG 11262 and B. fragilis LMG 10263 were able to grow on oligofructose as fast as on fructose, succinic acid being the major metabolite produced by both strains. B. longum BB536 grew slower on oligofructose than on fructose. Acetic acid and lactic acid were the main metabolites produced when fructose was used as the sole energy source. Increased amounts of formic acid and ethanol were produced when oligofructose was used as an energy source at the cost of lactic acid. Detailed kinetic analysis revealed a preferential metabolism of the short oligofructose fractions (e.g., F2 and F3) for B. longum BB536. After depletion of the short fractions, the larger oligofructose fractions (e.g., F4, GF4, F5, GF5, and F6) were metabolized, too. Both Bacteroides strains did not display such a preferential metabolism and degraded all oligofructose fractions simultaneously, transiently increasing the fructose concentration in the medium. This suggests a different mechanism for oligofructose breakdown between the strain of Bifidobacterium and both strains of Bacteroides, which helps to explain the bifidogenic nature of inulin-type fructans.     

In Vitro Kinetics of Prebiotic Inulin-Type Fructan Fermentation by Butyrate-Producing Colon Bacteria: Implementation of Online Gas Chromatography for Quantitative Analysis of Carbon Dioxide and Hydrogen Gas Production

Kinetic analyses of bacterial growth, carbohydrate consumption, and metabolite production of five butyrate-producing clostridial cluster XIVa colon bacteria grown on acetate plus fructose, oligofructose, inulin, or lactate were performed. A gas chromatography method was set up to assess H₂ and CO₂ production online and to ensure complete coverage of all metabolites produced. Method accuracy was confirmed through the calculation of electron and carbon recoveries. Fermentations with Anaerostipes caccae DSM 14662^T, Roseburia faecis DSM 16840^T, Roseburia hominis DSM 16839^T, and Roseburia intestinalis DSM 14610^T revealed similar patterns of metabolite production with butyrate, CO₂, and H₂ as the main metabolites. R. faecis DSM 16840^T and R. intestinalis DSM 14610^T were able to degrade oligofructose, displaying a nonpreferential breakdown mechanism. Lactate consumption was only observed with A. caccae DSM 14662^T. Roseburia inulinivorans DSM 16841^T was the only strain included in the present study that was able to grow on fructose, oligofructose, and inulin. The metabolites produced were lactate, butyrate, and CO₂, without H₂ production, indicating an energy metabolism distinct from that of other Roseburia species. Oligofructose degradation was nonpreferential. In a coculture of R. inulinivorans DSM 16841^T with the highly competitive strain Bifidobacterium longum subsp. longum LMG 11047 on inulin, hardly any production of butyrate and CO₂ was detected, indicating a lack of competitiveness of the butyrate producer. Complete recovery of metabolites during fermentations of clostridial cluster XIVa butyrate-producing colon bacteria allowed stoichiometric balancing of the metabolic pathway for butyrate production, including H₂ formation.The implementation of 16S rRNA gene-based analytical techniques in the ongoing exploration of the microbial diversity of the human colon ecosystem has both broadened and sharpened the prevailing image of its population (17, 24, 32). While a rather conservative perception of the composition of the colon microbiota has dominated gut research for several decades (36), recent studies have revealed the importance of previously largely neglected bacterial groups and have reduced historically numerically overestimated subpopulations to their actual (marginal) size (8, 22, 52). The human colon has been shown to be a remarkably selective environment, which is reflected by a rather shallow microbial diversity (32). Species belonging to the bacterial divisions Firmicutes, Bacteroidetes, Proteobacteria, and Actinobacteria make up more than 98% of the bacterial population of the human colon (2, 17, 24). However, this superficial uniformity only covers an overwhelming diversity at the lower taxonomic levels; the human colon has been estimated to harbor between 500 and 1,000 species, representing over 7,000 strains, with up to 80% of them considered uncultivable using presently available methodologies (14, 28, 53).Assessing identity and abundance of the major microbial groups composing the colon microbiota is a first and indispensable step toward a better understanding of the ecosystem of the large intestine (48). However, defining a complex ecosystem such as the human colon requires more than the construction of a catalog of its members (32). A major challenge of gastrointestinal microbiology lies in linking phylogenetic subgroups with particular ecological habitats and niches (7, 8, 23). The latter requires further development of highly discriminating 16S rRNA gene-targeted probes to monitor spatial bacterial distribution, combined with renewed efforts toward species isolation through the application of innovative cultivation methods and media, and extensive metabolic characterization of representative strains (19, 35, 48).Recently, a global ecological approach, combining efforts in probe development (1, 27), species isolation (3), and metabolic characterization (4, 11, 15, 20), has led to the identification of a functional group of microorganisms, composed of species belonging to the clostridial clusters IV and XIVa, that are responsible for colon butyrate production. As butyrate is regarded as a key metabolite for the maintenance of colon health, this functional subunit of the colon microbiota could have a major influence on human well-being and might be considered as a target for prebiotic dietary interventions (25, 35, 45). Some recently described lactate- and/or acetate-converting colon butyrate producers have been reported to be able to degrade prebiotic inulin-type fructans, although the kinetics of their respective breakdown mechanisms have hardly been investigated (10, 20). The enhancement of colon butyrate production observed after consumption of oligofructose or inulin (6, 31, 40)—the so-called butyrogenic effect—as well as the limited stimulatory effect of these prebiotics on the clostridial cluster IV and XIVa colon populations (16, 30) have been attributed to cross-feeding with bifidobacteria, which are still considered the primary fructan degraders (5, 38). Anaerostipes caccae as well as Roseburia spp. have been shown to be able to (co)metabolize end products of bifidobacterial fructan fermentation (lactate and/or acetate) or to grow on short oligosaccharides and monosaccharides released by Bifidobacterium spp. during fructan degradation (4, 20).Recently, many clostridial cluster IV and XIVa butyrate producers characterized in detail have been shown to produce gases, mainly CO₂ and H₂ (12, 15, 20, 46). Consequently, they might be responsible for an enhancement of gas production as a result of fructan fermentation, through either cross-feeding or direct degradation of inulin-type fructans (15, 16). Indeed, inulin-type fructan consumption has been reported to cause some gastrointestinal discomfort related to gas production—essentially, flatulence and bloating (43)—while bifidobacteria, the main beneficiaries of dietary fructan intake, do not produce gases (19, 49). Although CO₂ and H₂ production by colon butyrate producers could have implications for human intestinal well-being, (in vitro) production has not been satisfactorily monitored up to now, probably due to limited availability of a performant apparatus for (online) gas analysis (15, 20). Moreover, the currently proposed pathway for colon butyrate production does not provide a conclusive quantitative link between bacterial (co)substrate metabolism and H₂ formation (11).This study investigated the kinetics of inulin-type fructan degradation by representatives of the genera Anaerostipes and Roseburia. A method based on online gas chromatography (GC) was developed to assess gas production qualitatively and quantitatively in a continuously sparged fermentation vessel for complete coverage of metabolite production. The competitiveness of inulin-degrading butyrate producers was investigated through coculture fermentations with Bifidobacterium longum subsp. longum LMG 11047, a strain representing a highly competitive cluster of bifidobacteria that share both high fructose consumption and oligofructose degradation rates and are able to perform partial breakdown of inulin (18, 20). A stoichiometrically balanced pathway for butyrate production, including H₂ production, is proposed.     

In Vitro Kinetic Analysis of Fermentation of Prebiotic Inulin-Type Fructans by Bifidobacterium Species Reveals Four Different Phenotypes

Kinetic analyses of bacterial growth, carbohydrate consumption, and metabolite production of 18 Bifidobacterium strains grown on fructose, oligofructose, or inulin were performed. A principal component analysis of the data sets, expanded with the results of a genetic screen concerning the presence of a β-fructofuranosidase gene previously encountered in Bifidobacterium animalis subsp. lactis DSM 10140^T, revealed the existence of four clusters among the bifidobacteria tested. Strains belonging to a first cluster could not degrade oligofructose or inulin. Strains in a second cluster could degrade oligofructose, displaying a preferential breakdown mechanism, but did not grow on inulin. Fructose consumption was faster than oligofructose degradation. A third cluster was composed of strains that degraded all oligofructose fractions simultaneously and could partially break down inulin. Oligofructose degradation was substantially faster than fructose consumption. A fourth, smaller cluster consisted of strains that shared high fructose consumption and oligofructose degradation rates and were able to perform partial breakdown of inulin. For all strains, a metabolic shift toward more acetate, formate, and ethanol production, at the expense of lactate production, was observed during growth on less readily fermentable energy sources. No correlation between breakdown patterns and the presence of the β-fructofuranosidase gene could be detected. These variations indicate niche-specific adaptation of bifidobacteria and could have in vivo implications on the strain specificity of the stimulatory effect of inulin-type fructans on bifidobacteria.     

Mutual Cross-Feeding Interactions between Bifidobacterium longum subsp. longum NCC2705 and Eubacterium rectale ATCC 33656 Explain the Bifidogenic and Butyrogenic Effects of Arabinoxylan Oligosaccharides

Arabinoxylan oligosaccharides (AXOS) are a promising class of prebiotics that have the potential to stimulate the growth of bifidobacteria and the production of butyrate in the human colon, known as the bifidogenic and butyrogenic effects, respectively. Although these dual effects of AXOS are considered beneficial for human health, their underlying mechanisms are still far from being understood. Therefore, this study investigated the metabolic interactions between Bifidobacterium longum subsp. longum NCC2705 (B. longum NCC2705), an acetate producer and arabinose substituent degrader of AXOS, and Eubacterium rectale ATCC 33656, an acetate-converting butyrate producer. Both strains belong to prevalent species of the human colon microbiota. The strains were grown on AXOS during mono- and coculture fermentations, and their growth, AXOS consumption, metabolite production, and expression of key genes were monitored. The results showed that the growth of both strains and gene expression in both strains were affected by cocultivation and that these effects could be linked to changes in carbohydrate consumption and concomitant metabolite production. The consumption of the arabinose substituents of AXOS by B. longum NCC2705 with the concomitant production of acetate allowed E. rectale ATCC 33656 to produce butyrate (by means of a butyryl coenzyme A [CoA]:acetate CoA-transferase), explaining the butyrogenic effect of AXOS. Eubacterium rectale ATCC 33656 released xylose from the AXOS substrate, which favored the B. longum NCC2705 production of acetate, explaining the bifidogenic effect of AXOS. Hence, those interactions represent mutual cross-feeding mechanisms that favor the coexistence of bifidobacterial strains and butyrate producers in the same ecological niche. In conclusion, this study provides new insights into the bifidogenic and butyrogenic effects of AXOS.     

Safety evaluation of probiotic bifidobacteria by analysis of mucin degradation activity and translocation ability

Although probiotic-containing nutrient formulas for infants and toddlers have become very popular, some adverse effects related to translocation of probiotic strains have been reported. We assessed the safety of probiotic bifidobacteria that have been used in clinical investigations and proven to have beneficial effects, by analyzing mucin degradation activity and translocation ability. Mucin degradation activities of three probiotic bifidobacteria strains; Bifidobacterium longum BB536, Bifidobacterium breve M-16V and Bifidobacterium infantis M-63, were evaluated by three in vitro tests comprising growth in liquid medium, SDS-PAGE analysis of degraded mucin residues, and degradation assay in Petri dish. All test strains and control type strains failed to grow in the liquid medium containing mucin as the only carbon source, although good growth was obtained from fecal sample. In the SDS-PAGE analyses of mucin residues and observation of mucinolytic zone in agar plate, the three test strains also showed no mucin degradation activity as the type strains, although fecal sample yielded positive results. In another study, a high dose of B. longum BB536 was administered orally to conventional mice to examine the translocation ability. No translocation into blood, liver, spleen, kidney and mesenteric lymph nodes was observed and no disturbance of epithelial cells and mucosal layer in the ileum, cecum and colon was detected, indicating that the test strain had no translocation ability and induced no damage to intestinal surface. These results resolve the concern about bacterial translocation when using bifidobacteria strains as probiotics, which have been tested in various clinical trials, supporting the continuous use of these probiotic strains without anxiety.     

Summer Meeting 2013: growth and physiology of bifidobacteria

Bifidobacteria are a minor fraction of the human colon microbiota with interesting properties for carbohydrate degradation. Monosaccharides such as glucose and fructose are degraded through the bifid shunt, a dedicated pathway involving phosphoketolase activity. Its stoechiometry learns that three moles of acetate and two moles of lactate are produced per two moles of glucose or fructose that are degraded. However, deviations from this 3 : 2 ratio occur, depending on the rate of substrate consumption. Slower growth rates favour the production of acetate and pyruvate catabolites (such as formate) at the cost of lactate. Interestingly, bifidobacteria are capable to degrade inulin‐type fructans (ITF) (oligofructose and inulin) and arabinoxylan‐oligosaccharides (AXOS). Beta‐fructofuranosidase activity enables bifidobacteria to degrade ITF. However, this property is strain‐dependent. Some strains consume both fructose and oligofructose, with different preferences and degradation rates. Small oligosaccharides (degree of polymerization or DP of 2–7) are taken up, in a sequential order, indicating intracellular degradation and as such giving these bacteria a competitive advantage towards other inulin‐type fructan degraders such as lactobacilli, bacteroides and roseburias. Other strains consume long fractions of oligofructose and inulin. Exceptionally, oligosaccharides with a DP of up to 20 (long‐chain inulin) are consumed by specific strains. Also, the degradation of AXOS by α‐arabinofuranosidase and β‐xylosidase is strain‐dependent. Particular strains consume the arabinose substituents, whether or not together with a consumption of the xylose backbones of AXOS, either up to xylotetraose or higher and either extra‐ or intracellularly. The production of high amounts of acetate that accompanies inulin‐type fructan degradation by bifidobacteria cross‐feeds other colon bacteria involved in the production of butyrate. However, bifidobacterial strain‐dependent differences in prebiotic degradation indicate the existence of niche‐specific adaptations and hence mechanisms to avoid competition among each other and to favour coexistence with other colon bacteria.     

Resistance to Simulated Gastrointestinal Conditions and Adhesion to Mucus as Probiotic Criteria for Bifidobacterium longum Strains

Eight Bifidobacterium longum strains, including reported probiotic strains (commercial and noncommercial), collection strains, and laboratory isolates, were investigated for their ability to adhere to mucin as well as their ability to tolerate acid and bile. Strains could be discriminated based on their sensitivity at pH values of 2.0 to 2.5 and bile concentrations of 0.5% to 2.0%. B. longum NCC 2705, a strain known for its probiotic properties, showed the highest resistance to gastrointestinal conditions, whereas the commercial probiotic strains B. longum BB 536 and SP 07/3 were the least resistant. In parallel, the human isolate B. longum BIF 53 showed the highest adhesion to mucin, whereas the commercial probiotic strains B. longum W 11, BB 536, and SP 07/3 were the least adhesive. The bacterial adhesion to mucin of strains B. longum NCC 2705 and BIF 53 could be reduced by lysozyme, indicating that cell-wall components are involved in the adhesion process. These results showed that there is no obvious link between adhesion and resistance to gastrointestinal conditions and the probiotic status of the studied strains. This calls for a definition of conditions for in vitro tests that better predict the in vivo functionality of probiotic strains.     

Cloning and sequence analysis of bile salt hydrolase (bsh) gene from Bifidobacterium longum

A pair of PCR primers for the rapid detection of bile salt hydrolase (bsh) gene from Bifidobacterium longum BB536 has been synthesised and have revealed the bsh gene of approx 970 bp in Bifidobacterium longum BB 536 but not in other species of bacteria tested. The bsh gene was cloned and sequenced showing a high similarity to bsh gene previously published. The resulting nucleotide sequence encodes a predicted protein of 317 amino acids, Mw = 35 kDa.     

Lactobacillus paracasei subsp. paracasei 8700:2 Degrades Inulin-Type Fructans Exhibiting Different Degrees of Polymerization

Ten strains of lactobacilli were assessed for their capacity to degrade inulin-type fructans, which are well-known prebiotics. Both oligofructose and inulin were tested. The dairy isolate Lactobacillus acidophilus IBB 801 degraded only oligofructose. The human isolate Lactobacillus paracasei subsp. paracasei 8700:2 degraded oligofructose and long-chain inulin and grew rapidly on both energy sources. In both cases, fractions of different degrees of polymerization were fermented. Moreover, large and short fractions of oligofructose were degraded simultaneously. When L. paracasei subsp. paracasei 8700:2 grew on oligofructose-enriched inulin, oligofructose was preferentially metabolized. In all cases, lactic acid was the main metabolic end product. Significant amounts of acetic acid, formic acid, and ethanol were produced when long-chain inulin or oligofructose-enriched inulin was used as the sole energy source.     

Two routes of metabolic cross-feeding between Bifidobacterium adolescentis and butyrate-producing anaerobes from the human gut

Dietary carbohydrates have the potential to influence diverse functional groups of bacteria within the human large intestine. Of 12 Bifidobacterium strains of human gut origin from seven species tested, four grew in pure culture on starch and nine on fructo-oligosaccharides. The potential for metabolic cross-feeding between Bifidobacterium adolescentis and lactate-utilizing, butyrate-producing Firmicute bacteria related to Eubacterium hallii and Anaerostipes caccae was investigated in vitro. E. hallii L2-7 and A. caccae L1-92 failed to grow on starch in pure culture, but in coculture with B. adolescentis L2-32 butyrate was formed, indicating cross-feeding of metabolites to the lactate utilizers. Studies with [(13)C]lactate confirmed carbon flow from lactate, via acetyl coenzyme A, to butyrate both in pure cultures of E. hallii and in cocultures with B. adolescentis. Similar results were obtained in cocultures involving B. adolescentis DSM 20083 with fructo-oligosaccharides as the substrate. Butyrate formation was also stimulated, however, in cocultures of B. adolescentis L2-32 grown on starch or fructo-oligosaccharides with Roseburia sp. strain A2-183, which produces butyrate but does not utilize lactate. This is probably a consequence of the release by B. adolescentis of oligosaccharides that are available to Roseburia sp. strain A2-183. We conclude that two distinct mechanisms of metabolic cross-feeding between B. adolescentis and butyrate-forming bacteria may operate in gut ecosystems, one due to consumption of fermentation end products (lactate and acetate) and the other due to cross-feeding of partial breakdown products from complex substrates.     

Oral administration of Bifidobacterium longum prevents gut-derived Pseudomonas aeruginosa sepsis in mice

Aims: The aim of the study was to evaluate the efficacy of probiotics on gut‐derived sepsis caused by Pseudomonas aeruginosa in immunocompromised mice. Methods and Results: After oral inoculation of P. aeruginosa, mice were treated with cyclophosphamide to induce leucopenia and translocation of the intestinal P. aeruginosa into blood, thereby producing gut‐derived sepsis. In this model, administration of 1 × 10⁹ CFU of Bifidobacterium longum strain BB536 for 10 days significantly (P < 0·01) increased the survival rate compared with groups of mice administered either with Bifidobacterium breve strain ATCC 15700 or excipients contained in the probiotic bacterial powder. Administration of B. longum significantly decreased viable counts of P. aeruginosa in the liver and blood compared with other groups. Culture of intestinal contents revealed a significantly lower viable count of P. aeruginosa in the jejunum of B. longum‐treated mice compared with other groups of mice. Furthermore, in vitro data demonstrated that B. longum possessed apparently higher adherent activity to Caco‐2 cell monolayers and significantly suppressed the adherence of P. aeruginosa to the monolayers of cells compared with other groups. Conclusion: Oral administration of B. longum protects mice against gut‐derived sepsis caused by P. aeruginosa, and the effect may be due to interference of P. aeruginosa adherence to intestinal epithelial cells. Significance and Impact of this Study: This study demonstrated that oral administration of B. longum BB536 is effective to protect against opportunistic infection with drug‐resistant bacteria such as P. aeruginosa. The results suggest that probiotics may play an important role even in the immunocompromised patients.     

Coculture Fermentations of Bifidobacterium Species and Bacteroides thetaiotaomicron Reveal a Mechanistic Insight into the Prebiotic Effect of Inulin-Type Fructans

Four bifidobacteria, each representing a cluster of strains with specific inulin-type-fructan degradation capacities, were grown in coculture fermentations with Bacteroides thetaiotaomicron LMG 11262, a strain able to metabolize both oligofructose and inulin. In a medium for colon bacteria with inulin as the sole added energy source, the ability of the bifidobacteria to compete for this substrate reflected phenotypical variation. Bifidobacterium breve Yakult, a strain that was not able to degrade oligofructose or inulin, was outcompeted by B. thetaiotaomicron LMG 11262. Bifidobacterium adolescentis LMG 10734, a strain that could degrade oligofructose (displaying a preferential breakdown mechanism) but that did not grow on inulin, managed to become competitive when oligofructose and short fractions of inulin started to accumulate in the fermentation medium. Bifidobacterium angulatum LMG 11039^T, a strain that was previously shown to degrade all oligofructose fractions simultaneously and to be able to partially break down inulin, was competitive from the beginning of the fermentation, consuming short fractions of inulin from the moment they appeared. Bifidobacterium longum LMG 11047, representing a cluster of bifidobacteria that shared both high fructose consumption and oligofructose degradation rates and were able to perform partial breakdown of inulin, was the dominating strain in a coculture with B. thetaiotaomicron LMG 11262. These observations indicate that distinct subgroups within the large-intestinal Bifidobacterium population will be stimulated by different groups of prebiotic inulin-type fructans, a variation that could be reflected in differences concerning their health-promoting effects.The vast complexity of the human colon microbiota, the key element of the large-intestinal ecosystem, has inspired researchers to describe it as a postnatally acquired microbial organ located inside a host organ (1, 46). The microbial colon community is estimated to be composed of up to 100 trillion microorganisms, a number exceeding 10 times the total number of somatic and germ cells of a human adult (18, 38). The human microbiome is thought to contain more than 100 times the total number of human genes (1, 18). It not only broadens the digestive abilities of the host (18, 22, 40) but also influences body processes far beyond digestion (7, 33). In spite of its fundamental impact on human health and disease, the human gastrointestinal ecosystem remains largely unexplored (7, 8).Despite the fact that the present knowledge of the composition of the human large-intestinal microbiota is partial, fragmented, and undetailed, the consistency of some observations allows them to be generalized as facts (8, 28, 47). Notwithstanding the huge diversity at the strain level, up to 87% of the human colon inhabitants belong to only two bacterial phyla, the Bacteroidetes and the Firmicutes (1, 8, 14). Within the group of large-intestinal Bacteroidetes, large variations between individuals have been reported (8). However, Bacteroides spp. generally seem to account for up to 20% of the human colon microbiota (26, 32). Moreover, the presence of Bacteroides thetaiotomicron appears to be universal (8, 21). This species, which has been isolated only from human and rodent intestines or feces up to now, has gained importance as a perfect example of a flexible, niche-adapted, human symbiont with a wide carbohydrate consumption range (3, 4, 40).Although B. thetaiotaomicron is considered a human symbiont contributing to the stability of the colon ecosystem, the Bacteroides genus also harbors some notorious pathogens that are linked with severe extraintestinal infections and that have been mentioned as causal agents of acute diarrhea (30, 35). Moreover, besides their enormous saccharolytic potential, Bacteroides spp. are also capable of proteolytic fermentation (22). These considerations make them unsuited as target organisms for stimulation by prebiotics such as inulin-type fructans (23, 31).Most in vivo studies regarding the effect of the addition of inulin or oligofructose to the diet on the composition of the human colon microbiota reveal that Bacteroides spp. are neither stimulated nor repressed through administration of these prebiotics (34). However, at least some Bacteroides spp. are able to degrade inulin-type fructans, including B. thetaiotaomicron (13, 44). Since this species accounts for up to 6% of the colon microbiota (8), it is at least surprising that its numbers are hardly influenced by an increased availability of these prebiotics as substrates for large-intestinal fermentation. A possible explanation for these contradicting observations is to be found in the mechanism of inulin degradation, which in the case of Bacteroides is presumed to be periplasmic or even extracellular (37, 44). Leakage of free fructose toward the extracellular environment appears to be inherent in such breakdown mechanisms (10, 25, 44). Hence, extracellular fructan degraders inevitably provide opportunistic competitors, which are not able to degrade inulin-type fructans themselves, with a valuable source of energy (2, 10, 19). In contrast, a cell-associated or intracellular degradation mechanism is thought to be widespread among Bifidobacterium spp., which are still considered the main target organisms for prebiotic stimulation by inulin-type fructans (15, 16, 39, 44). This mechanism is often reflected in a clearly preferential breakdown of different-chain-length fractions of oligofructose, which approaches degradation of the long fractions only when short ones are depleted (10, 42, 44). The main disadvantage of such a cell-associated or intracellular degradation strategy seems to be the bifidobacterial incapacity to grow on long-chain-length fractions of inulin (36). Reports of the latter are indeed scarce: kinetic pure culture studies report an upper chain length limit for inulin degradation by Bifidobacterium spp., a disadvantage that will presumably not affect extracellular fructan degraders, such as Bacteroides spp. (9). Although the prebiotic effect of inulin-type fructans on the colon Bifidobacterium population is well documented, in vivo stimulation studies usually tend to consider the bifidobacterial community as a whole, ignoring interspecies differences (23). However, since the early days of in vitro prebiotic studies, a large variation in fructan degradation capacities of different Bifidobacterium strains has been reported (17, 36). It is likely that this variety is translated to the in vivo environment, implying that not all bifidobacteria are equally subject to prebiotic stimulation (5, 45). In a recent study, the kinetics of growth, carbohydrate consumption, and metabolite production of 18 Bifidobacterium spp., 17 of which were human intestinal isolates, have been statistically analyzed (9). The existence of four phenotypically distinct clusters among the tested strains, probably reflecting niche-specific adaptation, has been revealed. This rather limited variation was hypothesized to influence the susceptibilities of various bifidobacteria toward prebiotic stimulation by inulin-type fructans and their fitness to compete for these substrates in a complex environment, such as the colon ecosystem (44).The present study aimed at mapping the fructan degradation capacity of B. thetaiotaomicron LMG 11262 growing on oligofructose or inulin. In vitro competitiveness trials with bifidobacterial strains belonging to the different phenotypical clusters mentioned above were designed to investigate the abilities of these strains to compete for inulin in a coculture with an inulin-degrading B. thetaiotaomicron strain.     

Cross-Feeding of Lactate Between Streptococcus lactis and Bacteroides sp. Isolated from Termite Hindguts

Streptococcus lactis and Bacteroides sp., isolated from hindguts of Reticulitermes flavipes termites, were grown anaerobically in monoculture and coculture. When grown in a glucose medium, S. lactis monoculture produced lactate as the major fermentation product, with small amounts of formate, acetate, ethanol, and CO₂. In coculture, glucose was completely consumed during growth of S. lactis. Lactate, produced by S. lactis, then supported much of the growth of Bacteroides and was fermented to propionate, acetate, and CO₂. Small amounts of succinate were formed during growth of Bacteroides in the coculture, but little change in the formate or ethanol concentration was observed. Monoculture growth of Bacteroides in a tryptone-yeast extract medium revealed that incorporation of 20 to 40 mM lactate increased cell yields and production of organic acids. However, initial lactate concentrations greater than 40 mM suppressed not only growth of Bacteroides but also acidic product formation. Results suggest that cross-feeding of lactate between streptococci and bacteroides constitutes one aspect of the overall hindgut fermentation in termites.     

Effects of Xylo-Oligosaccharides on Broiler Chicken Performance and Microbiota

In broiler chickens, feed additives, including prebiotics, are widely used to improve gut health and to stimulate performance. Xylo-oligosaccharides (XOS) are hydrolytic degradation products of arabinoxylans that can be fermented by the gut microbiota. In the current study, we aimed to analyze the prebiotic properties of XOS when added to the broiler diet. Administration of XOS to chickens, in addition to a wheat-rye-based diet, significantly improved the feed conversion ratio. XOS significantly increased villus length in the ileum. It also significantly increased numbers of lactobacilli in the colon and Clostridium cluster XIVa in the ceca. Moreover, the number of gene copies encoding the key bacterial enzyme for butyrate production, butyryl-coenzyme A (butyryl-CoA):acetate CoA transferase, was significantly increased in the ceca of chickens administered XOS. In this group of chickens, at the species level, Lactobacillus crispatus and Anaerostipes butyraticus were significantly increased in abundance in the colon and cecum, respectively. In vitro fermentation of XOS revealed cross-feeding between L. crispatus and A. butyraticus. Lactate, produced by L. crispatus during XOS fermentation, was utilized by the butyrate-producing Anaerostipes species. These data show the beneficial effects of XOS on broiler performance when added to the feed, which potentially can be explained by stimulation of butyrate-producing bacteria through cross-feeding of lactate and subsequent effects of butyrate on gastrointestinal function.     

Immunoregulatory Effect of Bifidobacteria Strains in Porcine Intestinal Epithelial Cells through Modulation of Ubiquitin-Editing Enzyme A20 Expression

Background

We previously showed that evaluation of anti-inflammatory activities of lactic acid bacteria in porcine intestinal epithelial (PIE) cells is useful for selecting potentially immunobiotic strains.

Objective

The aims of the present study were: i) to select potentially immunomodulatory bifidobacteria that beneficially modulate the Toll-like receptor (TLR)-4-triggered inflammatory response in PIE cells and; ii) to gain insight into the molecular mechanisms involved in the anti-inflammatory effect of immunobiotics by evaluating the role of TLR2 and TLR negative regulators in the modulation of proinflammatory cytokine production and activation of mitogen-activated protein kinase (MAPK) and nuclear factor-κB (NF-κB) pathways in PIE cells.

Results

Bifidobacteria longum BB536 and B. breve M-16V strains significantly downregulated levels of interleukin (IL)-8, monocyte chemotactic protein (MCP)-1 and IL-6 in PIE cells challenged with heat-killed enterotoxigenic Escherichia coli. Moreover, BB536 and M-16V strains attenuated the proinflammatory response by modulating the NF-κB and MAPK pathways. In addition, our findings provide evidence for a key role for the ubiquitin-editing enzyme A20 in the anti-inflammatory effect of immunobiotic bifidobacteria in PIE cells.

Conclusions

We show new data regarding the mechanism involved in the anti-inflammatory effect of immunobiotics. Several strains with immunoregulatory capabilities used a common mechanism to induce tolerance in PIE cells. Immunoregulatory strains interacted with TLR2, upregulated the expression of A20 in PIE cells, and beneficially modulated the subsequent TLR4 activation by reducing the activation of MAPK and NF-κB pathways and the production of proinflammatory cytokines. We also show that the combination of TLR2 activation and A20 induction can be used as biomarkers to screen and select potential immunoregulatory bifidobacteria strains.     

Acetate Inhibition of Methanogenic, Syntrophic Benzoate Degradation

Acetate inhibited benzoate degradation by a syntrophic coculture of an anaerobic benzoate degrader (strain BZ-2) and Methanospirillum strain PM-1; the apparent K_i for acetate was approximately 40 mM. The addition of acetate resulted in a decrease in the hydrogen concentration in the coculture, indicating that phenomena related to interspecies hydrogen transfer affected this value and that the effect of acetate on the benzoate-degrading partner was probably greater than the apparent K_i for the coculture suggests.     

Interactions between Bifidobacterium and Bacteroides Species in Cofermentations Are Affected by Carbon Sources,Including Exopolysaccharides Produced by Bifidobacteria

Cocultures of strains from two Bifidobacterium and two Bacteroides species were performed with exopolysaccharides (EPS) previously purified from bifidobacteria, with inulin, or with glucose as the carbon source. Bifidobacterium longum NB667 and Bifidobacterium breve IPLA20004 grew in glucose but showed poor or no growth in complex carbohydrates (inulin, EPS E44, and EPS R1), whereas Bacteroides grew well in the four carbon sources tested. In the presence of glucose, the growth of Bacteroides thetaiotaomicron DSM-2079 was inhibited by B. breve, whereas it remained unaffected in the presence of B. longum. Ba. fragilis DSM-2151 contributed to a greater survival of B. longum, promoting changes in the synthesis of short-chain fatty acids (SCFA) and organic acids in coculture with respect to monocultures. In complex carbohydrates, cocultures of bifidobacterium strains with Ba. thetaiotaomicron did not modify the behavior of Bacteroides nor improve the poor growth of bifidobacteria. The metabolic activity of Ba. fragilis in coculture with bifidobacteria was not affected by EPS, but greater survival of bifidobacteria at late stages of incubation occurred in cocultures than in monocultures, leading to a higher production of acetic acid than in monocultures. Therefore, cocultures of Bifidobacterium and Bacteroides can behave differently against fermentable carbohydrates as a function of the specific characteristics of the strains from each species. These results stress the importance of considering specific species and strain interactions and not simply higher taxonomic divisions in the relationship among intestinal microbial populations and their different responses to probiotics and prebiotics.