Highly conserved bifidobacteria in the human gut: Bifidobacterium longum subsp. longum as a potential modulator of elderly innate immunity.

Aging is a physiological and immunological process involving the deterioration of human health, characterised by the progressive alteration of organs and their functions. The speed and extent of such decline are dependent on lifestyle, environment, and genetic factors. Moreover, with advancing age, humans become progressively more fragile and prone to acute and chronic diseases. Although the intestinal microbiota is predisposed to perturbations that accompany aging and frailty, it is generally accepted that the gut microbiota engages in multiple interactions that affect host health throughout the host life span. In the current study, an exhaustive in silico investigation of gut-associated bifidobacteria in healthy individuals from birth to old age revealed that Bifidobacterium longum subsp. longum is the most prevalent member, especially during infancy and in centenarians. Moreover, B. longum subsp. longum genome reconstruction and strain tracing among human gut microbiomes allowed the identification of prototypes of this taxon in the human gut microbiota of healthy elderly individuals. Such analyses guided culturomics attempts to isolate B. longum subsp. longum strains that matched the genomic content of B. longum subsp. longum prototypes from healthy elderly individuals. The molecular effects of selected B. longum subsp. longum strains on the human host were further investigated using in vitro microbe-host interactions, revealing differences in the host immune system transcriptome, with a reduction in gene expression of inflammation-related cytokines. These intriguing findings support the potential anti-aging effects of elderly associated prototypes of B. longum subsp. longum.

Beer spoilage and low pH tolerance is linked to manganese homeostasis in selected Lactobacillus brevis strains.

AIMS: Beer is a harsh medium for bacteria to survive, however, lactic acid bacteria including Lactobacillus brevis have evolved the ability to grow in beer. Here, the influence of environmental factors such as low pH, ethanol or hop content was assessed. METHODS AND RESULTS: A transcriptomic analysis of two Lact. brevis beer-spoiling strains was performed comparing growth in nutritive media with or without the imposition of a stressor related to the beer environment. This allowed the identification of a manganese transporter encoding gene that contributes to low pH tolerance. CONCLUSIONS: We report on the importance of a manganese transporter associated with pH tolerance and beer spoilage in Lact. brevis. The importance of manganese for Lact. brevis growth in a low pH environment was highlighted. SIGNIFICANCE AND IMPACT OF THE STUDY: Bacterial spoilage of beer may result in product withdrawal with concomitant economic losses for the brewing industry. A limited number of genes involved in beer spoilage have been identified but none of them are universal. It is clear that other molecular players are involved in beer spoilage. The study highlights the complexity of the genetic requirements to facilitate beer spoilage and the role of multiple key players in this process.

Exopolysaccharide from Bifidobacterium longum subsp. longum 35624™ modulates murine allergic airway responses.

Interactions between the host and the microbiota are thought to significantly influence immunological tolerance mechanisms at mucosal sites. We recently described that the loss of an exopolysaccharide (EPS) from Bifidobacterium longum 35624™ eliminated its protective effects in colitis and respiratory allergy murine models. Our goal was to investigate the immune response to purified EPS from B. longum 35624, determine if it has protective effects within the lung and identify the protective mechanisms. Isolated EPS from B. longum 35624 cultures was used for in vitro, ex vivo and in vivo studies. Human monocyte-derived dendritic cells (MDDCs) were used to investigate in vitro immunological responses to EPS. Cytokine secretion, expression of surface markers and signalling pathways were examined. The ovalbumin (OVA) respiratory allergy murine model was used to evaluate the in vivo immunomodulatory potential of EPS. In addition, interleukin (IL)-10 knockout (KO) mice and anti-Toll-like receptor (TLR)-2 blocking antibody were used to examine the underlying protective mechanisms of intranasal EPS administration. Stimulation of human MDDCs with EPS resulted in IL-10 secretion, but not proinflammatory cytokines. IL-10 secretion was TLR-2-dependent. Eosinophil recruitment to the lungs was significantly decreased by EPS intranasal exposure, which was associated with decreased expression of the Th2-associated markers C-C motif chemokine 11 (CCL11), C-C chemokine receptor type 3 (CCR3), IL-4 and IL-13. TLR-2-mediated IL-10 secretion was shown to be required for the reduction in eosinophils and Th2 cytokines. EPS-treatment reduced eosinophil recruitment within the lung in a respiratory inflammation mouse model, which is both TLR-2 and IL-10 mediated. EPS can be considered as a novel molecule potentially reducing the severity of chronic eosinophil-related airway disorders.

Extraction of the same novel homoglycan mixture from two different strains of Bifidobacterium animalis and three strains of Bifidobacterium breve.

Three strains of Bifidobacterium breve (JCM 7017, JCM 7019 and JCM 2258) and two strains of Bifidobacterium animalis subsp. lactis (AD011 and A1dOxR) were grown in broth cultures or on plates, and a standard exopolysaccharide extraction method was used in an attempt to recover exocellular polysaccharides. When the extracted materials were analysed by NMR it was clear that mixtures of polysaccharides were being isolated including exopolysaccharides (EPS) cell wall polysaccharides and intracellular polysaccharides. Treatment of the cell biomass from the B. breve strains, or the B. animalis subsp. lactis AD011 strain, with aqueous sodium hydroxide provided a very similar mixture of polysaccharides but without the EPS. The different polysaccharides were partially fractionated by selective precipitation from an aqueous solution upon the addition of increasing percentages of ethanol. The polysaccharides extracted from B. breve JCM 7017 grown in HBM media supplemented with glucose (or isotopically labelled D-glucose-1-13C) were characterised using 1D and 2D-NMR spectroscopy. Addition of one volume of ethanol generated a medium molecular weight glycogen (Mw=1×105 Da, yield 200 mg/l). The addition of two volumes of ethanol precipitated an intimate mixture of a low molecular weight ß-(1→6)-glucan and a low molecular weight ß-(1→6)-galactofuranan which could not be separated (combined yield 46 mg/l). When labelled D-glucose-1-13C was used as a carbon supplement, the label was incorporated into >95% of the anomeric carbons of each polysaccharide confirming they were being synthesised in situ. Similar 1H NMR profiles were obtained for polysaccharides recovered from the cells of B. animalis subsp. lactis AD011and A1dOxR (in combination with an EPS), B. breve JCM 7017, B. breve JCM 7019, B. breve JCM 2258 and from an EPS (-ve) mutant of B. breve 7017 (a non-EPS producer).

Bifidobacterium breve reduces apoptotic epithelial cell shedding in an exopolysaccharide and MyD88-dependent manner.

Certain members of the microbiota genus Bifidobacterium are known to positively influence host well-being. Importantly, reduced bifidobacterial levels are associated with inflammatory bowel disease (IBD) patients, who also have impaired epithelial barrier function, including elevated rates of apoptotic extrusion of small intestinal epithelial cells (IECs) from villi-a process termed 'cell shedding'. Using a mouse model of pathological cell shedding, we show that mice receiving Bifidobacterium breve UCC2003 exhibit significantly reduced rates of small IEC shedding. Bifidobacterial-induced protection appears to be mediated by a specific bifidobacterial surface exopolysaccharide and interactions with host MyD88 resulting in downregulation of intrinsic and extrinsic apoptotic responses to protect epithelial cells under highly inflammatory conditions. Our results reveal an important and previously undescribed role for B. breve, in positively modulating epithelial cell shedding outcomes via bacterial- and host-dependent factors, supporting the notion that manipulation of the microbiota affects intestinal disease outcomes.

The human gut microbiota and its interactive connections to diet.

The microbiota of the gastrointestinal tract plays an important role in human health. In addition to their metabolic interactions with dietary constituents, gut bacteria may also be involved in more complex host interactions, such as modulation of the immune system. Furthermore, the composition of the gut microbiota may be important in reducing the risk of contracting particular gut infections. Changes in the microbiota during an individual's lifespan are accompanied by modifications in multiple health parameters, and such observations have prompted intense scientific efforts aiming to understand the complex interactions between the microbiota and its human host, as well as how this may be influenced by diet.

Pangenome analysis of Bifidobacterium longum and site-directed mutagenesis through by-pass of restriction-modification systems.

BACKGROUND: Bifidobacterial genome analysis has provided insights as to how these gut commensals adapt to and persist in the human GIT, while also revealing genetic diversity among members of a given bifidobacterial (sub)species. Bifidobacteria are notoriously recalcitrant to genetic modification, which prevents exploration of their genomic functions, including those that convey (human) health benefits. METHODS: PacBio SMRT sequencing was used to determine the whole genome seqeunces of two B. longum subsp. longum strains. The B. longum pan-genome was computed using PGAP v1.2 and the core B. longum phylogenetic tree was constructed using a maximum-likelihood based approach in PhyML v3.0. M.blmNCII was cloned in E. coli and an internal fragment if arfBarfB was cloned into pORI19 for insertion mutagenesis. RESULTS: In this study we present the complete genome sequences of two Bifidobacterium longum subsp. longum strains. Comparative analysis with thirty one publicly available B. longum genomes allowed the definition of the B. longum core and dispensable genomes. This analysis also highlighted differences in particular metabolic abilities between members of the B. longum subspecies infantis, longum and suis. Furthermore, phylogenetic analysis of the B. longum core genome indicated the existence of a novel subspecies. Methylome data, coupled to the analysis of restriction-modification systems, allowed us to substantially increase the genetic accessibility of B. longum subsp. longum NCIMB 8809 to a level that was shown to permit site-directed mutagenesis. CONCLUSIONS: Comparative genomic analysis of thirty three B. longum representatives revealed a closed pan-genome for this bifidobacterial species. Phylogenetic analysis of the B. longum core genome also provides evidence for a novel fifth B. longum subspecies. Finally, we improved genetic accessibility for the strain B. longum subsp. longum NCIMB 8809, which allowed the generation of a mutant of this strain.

Structural investigation of cell wall polysaccharides of Lactobacillus delbrueckii subsp. bulgaricus 17.

Lactobacilli are valuable strains for commercial (functional) food fermentations. Their cell surface-associated polysaccharides (sPSs) possess important functional properties, such as acting as receptors for bacteriophages (bacterial viruses), influencing autolytic characteristics and providing protection against antimicrobial peptides. The current report provides an elaborate molecular description of several surface carbohydrates of Lactobacillus delbrueckii subsp. bulgaricus strain 17. The cell surface of this strain was shown to contain short chain poly(glycerophosphate) teichoic acids and at least two different sPSs, designated here as sPS1 and sPS2, whose chemical structures were examined by 2D nuclear magnetic resonance spectroscopy and methylation analysis. Neutral branched sPS1, extracted with n-butanol, was shown to be composed of hexasaccharide repeating units (-[α-d-Glcp-(1-3)-]-4-ß-l-Rhap2OAc-4-ß-d-Glcp-[α-d-Galp-(1-3)]-4-α-Rhap-3-α-d-Galp-), while the major component of the TCA-extracted sPS2 was demonstrated to be a linear d-galactan with the repeating unit structure being (-[Gro-3P-(1-6)-]-3-ß-Galf-3-α-Galp-2-ß-Galf-6-ß-Galf-3-ß-Galp-).

Selective carbohydrate utilization by lactobacilli and bifidobacteria.

AIM: To evaluate the ability of specific carbohydrates, including commercially available products, to support the growth of representatives of two well-known groups of gut commensals, namely lactobacilli and bifidobacteria. METHODS AND RESULTS: Sixty-eight bacterial strains, representing 29 human-derived lactobacilli and 39 bifidobacteria (both human- and animal-derived), were tested for their ability to metabolize 10 different carbohydrates. Analysis of growth and metabolic activity was performed using a combination of diagnostic parameters, such as final OD600 , final pH, fermentation end products and growth rate. CONCLUSIONS: The data assembled in this study provide significant complementary and comparative information on the growth-promoting properties of a range of carbohydrates, while also investigating interspecies differences between lactobacilli and/or bifidobacteria with regard to their carbohydrate utilization abilities. Galacto-oligosaccharides (GOS) and lactulose were shown to support the most favourable growth characteristics, whereas relatively poor growth of lactobacilli and bifidobacteria was observed on inulin, maltodextrin and polydextrose. GOS/inulin (9 : 1) and fructo-oligosaccharides (FOS)/inulin mixtures supported mostly similar growth abilities to those obtained for GOS and FOS, respectively. Microbial consumption of GOS, as determined by high-performance anion-exchange chromatography with pulsed amperometric detection, was evident for both lactobacilli and bifidobacteria. SIGNIFICANCE AND IMPACT OF THE STUDY: These results may allow for the rational prediction of lactobacilli and/or bifidobacteria to be used in conjunction with prebiotics, such as GOS, as synbiotics.

Comparative analysis of two antifungal Lactobacillus plantarum isolates and their application as bioprotectants in refrigerated foods.

AIMS: To compare the technological robustness of two antifungal Lactobacillus plantarum isolates and to assess their ability to inhibit growth of the spoilage yeast Rhodotorula mucilaginosa in two different refrigerated foods. METHODS AND RESULTS: The effects of freeze-drying, thermal treatments and varying salt concentrations on the viability of two antifungal lactic acid bacteria (LAB) were examined. Antifungal compound(s) contained in the supernatant of both isolates were compared to commercially available food preservatives. Both isolates were used as dairy starter adjuncts in yoghurt and inoculants in orange juice to determine the antiyeast activity towards R. mucilaginosa. Yeast growth was retarded by the tested isolates in both food settings with one of the isolates, Lact. plantarum 16, being the most potent inhibitor. CONCLUSIONS: Both lactobacilli exhibited considerable robustness to withstand processing treatments commonly encountered in a food industrial setting. The isolates were shown to possess potent antifungal activity in both in vivo and in vitro food models. SIGNIFICANCE AND IMPACT OF THE STUDY: The studied antifungal lactobacilli may represent safer and consumer-friendly alternatives to the use of chemical preservatives. This is the first report of antifungal Lact. plantarum exerting protective potential in yoghurt and orange juice.

B-group vitamin production by lactic acid bacteria--current knowledge and potential applications.

Although most vitamins are present in a variety of foods, human vitamin deficiencies still occur in many countries, mainly because of malnutrition not only as a result of insufficient food intake but also because of unbalanced diets. Even though most lactic acid bacteria (LAB) are auxotrophic for several vitamins, it is now known that certain strains have the capability to synthesize water-soluble vitamins such as those included in the B-group (folates, riboflavin and vitamin B(12) amongst others). This review article will show the current knowledge of vitamin biosynthesis by LAB and show how the proper selection of starter cultures and probiotic strains could be useful in preventing clinical and subclinical vitamin deficiencies. Here, several examples will be presented where vitamin-producing LAB led to the elaboration of novel fermented foods with increased and bioavailable vitamins. In addition, the use of genetic engineering strategies to increase vitamin production or to create novel vitamin-producing strains will also be discussed. This review will show that the use of vitamin-producing LAB could be a cost-effective alternative to current vitamin fortification programmes and be useful in the elaboration of novel vitamin-enriched products.

Genome analysis of food grade lactic Acid-producing bacteria: from basics to applications.

Whole-genome sequencing has revolutionized and accelerated scientific research that aims to study the genetics, biochemistry and molecular biology of bacteria. Lactic acid-producing bacteria, which include lactic acid bacteria (LAB) and bifidobacteria, are typically Gram-positive, catalase-negative organisms, which occupy a wide range of natural plant- and animal-associated environments. LAB species are frequently involved in the transformation of perishable raw materials into more stable, pleasant, palatable and safe fermented food products. LAB and bifidobacteria are also found among the resident microbiota of the gastrointestinal and/or genitourinary tracts of vertebrates, where they are believed to exert health-promoting effects. At present, the genomes of more than 20 LAB and bifidobacterial species have been completely sequenced. Their genome content reflects its specific metabolism, physiology, biosynthetic capabilities, and adaptability to varying conditions and environments. The typical LAB/bifidobacterial genome is relatively small (from 1.7 to 3.3 Mb) and thus harbors a limited assortment of genes (from around 1,600 to over 3,000). These small genomes code for a broad array of transporters for efficient carbon and nitrogen assimilation from the nutritionally-rich niches they usually inhabit, and specify a rather limited range of biosynthetic and degrading capabilities. The variation in the number of genes suggests that the genome evolution of each of these bacterial groups involved the processes of extensive gene loss from their particular ancestor, diversification of certain common biological activities through gene duplication, and acquisition of key functions via horizontal gene transfer. The availability of genome sequences is expected to revolutionize the exploitation of the metabolic potential of LAB and bifidobacteria, improving their use in bioprocessing and their utilization in biotechnological and health-related applications.

Lactic acid bacteria with potential to eliminate fungal spoilage in foods.

AIMS: To investigate antifungal activity produced by lactic acid bacteria (LAB) isolated from malted cereals and to determine if such LAB have the capacity to prevent fungal growth in a particular food model system. METHODS AND RESULTS: The effect of pH, temperature and carbon source on production of antifungal activity by four LAB was determined. Pediococcus pentosaceus was used to conduct a trial to determine if it is feasible to eliminate Penicillium expansum, the mould responsible for apple rot, using an apple model. Penicillium expansum was incapable of growth during the trial on apple-based agar plates inoculated with the antifungal-producing culture, whereas the mould did grow on apple plates inoculated with an LAB possessing no antifungal activity. CONCLUSION: Partial characterization of the antifungal compounds indicates that their activity is likely to be because of production of antifungal peptides. The trial conducted showed that the antifungal culture has the ability to prevent growth of the mould involved in apple spoilage, using apples as a model. SIGNIFICANCE AND IMPACT OF THE STUDY: The ability of an LAB to prevent growth of Pen. expansum using the apple model suggests that these antifungal LAB have potential applications in the food industry to prevent fungal spoilage of food.

Characterization of plasmid pASV479 from Bifidobacterium pseudolongum subsp. globosum and its use for expression vector construction.

Bifidobacterium pseudolongum subsp. globosum DPC479 is an intestinally-derived strain which contains a plasmid, pASV479, 4.8 kb in size. This plasmid has a G + C content of 59% and contains six open reading frames (ORFs), four of which are cryptic. The other two ORFs have 47% and 54% identity, respectively, to the replication and FtsK-like proteins found in a Bifidobacterium breve NCFB 2258 plasmid, indicating that these plasmids, though isolated from differing Bifidobacterium species, are related. Using this plasmid as a backbone, an expression vector, pBIFRIBO, was constructed which exploits a bifidobacteria rRNA promoter.

Ingestion of milk fermented by genetically modified Lactococcus lactis improves the riboflavin status of deficient rats.

Riboflavin deficiency is common in many parts of the world, particularly in developing countries. The use of riboflavin-producing strains in the production of dairy products such as fermented milks, yogurts, and cheeses is feasible and economically attractive because it would decrease the costs involved during conventional vitamin fortification and satisfy consumer demands for healthier foods. The present study was conducted to assess in a rat bioassay the response of administration of milk fermented by modified Lactococcus lactis on the riboflavin status of deficient rats. Rats were fed a riboflavin-deficient diet during 21 d after which this same diet was supplemented with milk fermented by Lactoccus lactis pNZGBAH, a strain that overproduces riboflavin during fermentation. The novel fermented product, with increased levels of riboflavin, was able to eliminate most physiological manifestations of ariboflavinosis, such as stunted growth, elevated erythrocyte glutathione reductase activation coefficient values and hepatomegaly, that were observed using a riboflavin depletion-repletion model, whereas a product fermented with a nonriboflavin-producing strain did not show similar results. A safety assessment of this modified strain was performed by feeding rodents with the modified strain daily for 4 wk. This strain caused no detectable secondary effects. These results pave the way for analyzing the effect of similar riboflavin-overproducing lactic acid bacteria in human trials. The regular consumption of products with increased levels of riboflavin could help prevent deficiencies of this essential vitamin.

In situ activity of a bacteriocin-producing Lactococcus lactis strain. Influence on the interactions between lactic acid bacteria during sourdough fermentation.

AIMS: To biochemically characterize the bacteriocin produced by Lactococcus lactis ssp. lactis M30 and demonstrate its effect on lactic acid bacteria (LAB) during sourdough propagation. METHODS AND RESULTS: A two-peptide bacteriocin produced by L. lactis ssp. lactis M30 was purified by ion exchange, hydrophobic interaction and reversed phase chromatography. Mass spectrometry of the two peptides and sequence analysis of the ltnA2 gene showed that the bacteriocin was almost identical to lacticin 3147. During a 20-day period of sourdough propagation the stability of L. lactis M30 was demonstrated, with concomitant inhibition of the indicator strain Lactobacillus plantarum 20, as well as the non-interference with the growth of the starter strain Lact. sanfranciscensis CB1. CONCLUSIONS: In situ active bacteriocins influence the microbial consortium of sourdough LAB and can "support" the dominance of insensitive strains during sourdough fermentation. SIGNIFICANCE AND IMPACT OF THE STUDY: The in situ bacteriocinogenic activity of selected lactococci enables the persistence of insensitive Lact. sanfranciscensis strains, useful to confer good characteristics to the dough, at a higher cell concentration with respect to other LAB of the same ecosystem.

In vitro inhibition of Eimeria tenella invasion by indigenous chicken Lactobacillus species.

The aim of this study was to determine the effects of indigenous chicken Lactobacillus species isolates from different parts of the gastrointestinal tract on Eimeria tenella invasion in vitro and to characterise the nature of inhibition, if any. The effects of competitive exclusion, steric interference and bacterial extracellular factors on E. tenella invasion were examined in an MDBK cell model. Several Lactobacillus species were initially isolated from chickens and identified by biochemical characteristics and 16S-rRNA. All Lactobacillus species isolates tested, significantly inhibited E. tenella invasion. Steric interference did not affect parasite invasion. Extracellular metabolic factors secreted by Lactobacillus species isolates into the surrounding media were shown to inhibit parasite invasion and these factors appeared to be heat stable. These results show that the natural microflora of poultry can provide a source of E. tenella-inhibiting Lactobacillus species in vitro, and thus may contribute to the control of Eimeria infection.

Characterization of bacteriocin-like inhibitory substances (BLIS) from sourdough lactic acid bacteria and evaluation of their in vitro and in situ activity.

AIMS: To identify and characterize bacteriocion-producing lactic acid bacteria (LAB) in sourdoughs and to compare in vitro and in situ bacteriocin activity of sourdough- and nonsourdough LAB. METHODS AND RESULTS: Production of antimicrobial compounds by 437 Lactobacillus strains isolated from 70 sourdoughs was investigated. Five strains (Lactobacillus pentosus 2MF8 and 8CF, Lb. plantarum 4DE and 3DM and Lactobacillus spp. CS1) were found to produce distinct bacteriocin-like inhibitory substances (BLIS). BLIS-producing Lactococcus lactis isolated from raw barley showed a wider inhibitory spectrum than sourdough LAB, but they did not inhibit all strains of the key sourdough bacterium Lb. sanfranciscensis. Antimicrobial production by Lb. pentosus 2MF8 and Lc. lactis M30 was also demonstrated in situ. CONCLUSIONS: BLIS production by sourdough LAB appears to occur at a low frequency, showing limited inhibitory spectrum when compared with BLIS-producing Lc. lactis. Nevertheless, they are active BLIS producers under sourdough and bread-making conditions. SIGNIFICANCE AND IMPACT OF THE STUDY: The activity of BLIS has been demonstrated in situ. It may influence the complex sourdough microflora and support the implantation and stability of selected insensitive bacteria, such as Lb. sanfranciscensis, useful to confer good characteristics to the dough.

Purification and characterisation of a lactococcal aminoacylase.

The amd1-encoded aminoacylase from Lactococcus lactis MG1363 was cloned and overexpressed in Escherichia coli and purified. The assumed dimeric enzyme has a subunit molecular mass of about 42 kDa and contains 2.0+/-0.1 g-atoms of zinc and cobalt, in equimolar amounts, per subunit of Amd1. The enzyme was characterised with respect to substrate specificity, pH, temperature and metal dependence. Amd1 exhibited a broad activity range towards N-acetylated- l-amino acids with a strong preference towards those containing neutral aliphatic and aromatic side chains. It hydrolysed N-acetyl- l-alanine most efficiently, and exhibited temperature and pH optima of 30 degrees C and 7.0, respectively. The activity of Amd1 towards N-acetyl- l-alanine was enhanced by the divalent cation Co(2+), while Cd(2+ )inhibited activity. Interestingly, Amd1 was shown to catalyse the hydrolysis of several dipeptides at pH 7.0, although with reduced V(max) values as compared to hydrolysis of N-acetylated- l-amino acids. This characteristic has also biological significance since Amd1 was able to complement a growth deficiency in a L. lactis triple peptidase mutant.