Short chain fatty acids in human gut and metabolic health.

Evidence is accumulating that short chain fatty acids (SCFA) play an important role in the maintenance of gut and metabolic health. The SCFA acetate, propionate and butyrate are produced from the microbial fermentation of indigestible carbohydrates and appear to be key mediators of the beneficial effects elicited by the gut microbiome. Microbial SCFA production is essential for gut integrity by regulating the luminal pH, mucus production, providing fuel for epithelial cells and effects on mucosal immune function. SCFA also directly modulate host metabolic health through a range of tissue-specific mechanisms related to appetite regulation, energy expenditure, glucose homeostasis and immunomodulation. Therefore, an increased microbial SCFA production can be considered as a health benefit, but data are mainly based on animal studies, whereas well-controlled human studies are limited. In this review an expert group by ILSI Europe's Prebiotics Task Force discussed the current scientific knowledge on SCFA to consider the relationship between SCFA and gut and metabolic health with a particular focus on human evidence. Overall, the available mechanistic data and limited human data on the metabolic consequences of elevated gut-derived SCFA production strongly suggest that increasing SCFA production could be a valuable strategy in the preventing gastro-intestinal dysfunction, obesity and type 2 diabetes mellitus. Nevertheless, there is an urgent need for well controlled longer term human SCFA intervention studies, including measurement of SCFA fluxes and kinetics, the heterogeneity in response based on metabolic phenotype, the type of dietary fibre and fermentation site in fibre intervention studies and the control for factors that could shape the microbiome like diet, physical activity and use of medication.

Effect of fructans, prebiotics and fibres on the human gut microbiome assessed by 16S rRNA-based approaches: a review.

The inherent and diverse capacity of dietary fibres, nondigestible oligosaccharides (NDOs) and prebiotics to modify the gut microbiota and markedly influence health status of the host has attracted rising interest. Research and collective initiatives to determine the composition and diversity of the human gut microbiota have increased over the past decade due to great advances in high-throughput technologies, particularly the 16S ribosomal RNA (rRNA) sequencing. Here we reviewed the application of 16S rRNA-based molecular technologies, both community wide (sequencing and phylogenetic microarrays) and targeted methodologies (quantitative PCR, fluorescent in situ hybridisation) to study the effect of chicory inulin-type fructans, NDOs and specific added fibres, such as resistant starches, on the human intestinal microbiota. Overall, such technologies facilitated the monitoring of microbiota shifts due to prebiotic/fibre consumption, though there are limited community-wide sequencing studies so far. Molecular studies confirmed the selective bifidogenic effect of fructans and galactooligosaccharides (GOS) in human intervention studies. Fructans only occasionally decreased relative abundance of Bacteroidetes or stimulated other groups. The sequencing studies for various resistant starches, polydextrose and beta-glucan showed broader effects with more and different types of gut microbial species being enhanced, often including phylotypes of Ruminococcaceae. There was substantial variation in terms of magnitude of response and in individual responses to a specific fibre or NDO which may be due to numerous factors, such as initial presence and relative abundance of a microbial type, diet, genetics of the host, and intervention parameters, such as intervention duration and fibre dose. The field will clearly benefit from a more systematic approach that will support defining the impact of prebiotics and fibres on the gut microbiome, identify biomarkers that link gut microbes to health, and address the personalised response of an individual's microbiota to prebiotics and dietary fibres.

Gut microbial metabolism of polyphenols from black tea and red wine/grape juice is source-specific and colon-region dependent.

The colonic microbial degradation of a polyphenol-rich black tea extract (BTE) and red wine/grape juice extract (RWGE) was compared in a five-stage in vitro gastrointestinal model (TWINSHIME). Microbial metabolism of BTE and RWGE polyphenols in the TWINSHIME was studied subsequently in single- and continuous-dose experiments. A combination of liquid or gas chromatography with mass spectrometry (LC-MS or GC-MS) and NMR-based metabolic profiling was used to measure selected parent polyphenols, their microbial degradation into phenolic acids, and the production of short-chain fatty acids (SCFAs) in different colon compartments. Acetate production was increased by continuous feeding of BTE but not RWGE. During RWGE feeding, gallic acid and 4-hydroxyphenylpropionic acid remained elevated throughout the colon, while during BTE feeding, they were consumed in the distal colon, while 3-phenylpropionic acid was strongly produced. Gut microbial production of phenolics and SCFAs is dependent on colon location and polyphenol source, which may influence potential health benefits.

Pretreatment of endothelial progenitor cells with osteopontin enhances cell therapy for peripheral vascular disease.

Tissue necrosis resulting from critical limb ischemia (CLI) leads to amputation in a significant number of patients. Autologous cell therapy using angiogenic cells such as endothelial progenitor cells (EPCs) holds promise as a treatment for CLI but a limitation of this treatment is that the underlying disease etiology that resulted in CLI may also contribute to dysfunction of the therapeutic EPCs. This study aimed to elucidate the mechanism of EPC dysfunction using diabetes mellitus as a model and to determine whether correction of this defect in dysfunctional EPCs ex vivo would improve the outcome after cell transplantation in the murine hind limb ischemia model. EPC dysfunction was confirmed in a homogenous population of patients with type 1 diabetes mellitus and a microarray study was preformed to identify dysregulated genes. Notably, the secreted proangiogenic protein osteopontin (OPN) was significantly downregulated in diabetic EPCs. Furthermore, OPN-deficient mice showed impaired recovery following hind limb ischemia, suggesting a critical role for OPN in postnatal neovascularization. EPCs isolated from OPN KO mice showed decreased ability to adhere to endothelial cells as well as impaired angiogenic potential. However, this dysfunction was reversed upon exposure to recombinant OPN, suggesting that OPN may act in an autocrine manner on EPCs. Indeed, exposure of OPN knockout (KO) EPCs to OPN was sufficient to induce the secretion of angiogenic proteins (IL-6, TGF-α, and FGF-α). We also demonstrated that vascular regeneration following hind limb ischemia in OPN KO mice was significantly improved upon injection of EPCs preexposed to OPN. We concluded that OPN acts in an autocrine manner on EPCs to induce the secretion of angiogenic proteins, thereby playing a critical role in EPC-mediated neovascularization. Modification of cells by exposure to OPN may improve the efficacy of autologous EPC transplantation via the enhanced secretion of angiogenic proteins.

Transcription factor plasmid binding modulates microtubule interactions and intracellular trafficking during gene transfer.

For non-viral gene delivery to be successful, plasmids must move through the cytoplasm to the nucleus in order to be transcribed. While the cytoskeletal meshwork acts as a barrier to plasmid DNA movement in the cytoplasm, the microtubule network is required for directed plasmid trafficking to the nucleus. We have shown previously that plasmid-microtubule interactions require cytoplasmic adapter proteins such as molecular motors, transcription factors (TFs) and importins. However, not all plasmid sequences support these interactions to allow movement to the nucleus. We now demonstrate that microtubule-DNA interactions can show sequence specificity with promoters containing binding sites for cyclic AMP response-element binding protein (CREB), including the cytomegalovirus immediate early promoter (CMV(iep)). Plasmids containing CREB-binding sites showed stringent interactions in an in vitro microtubule-binding assay. Using microinjection and real-time particle tracking, we show that the inclusion of TF binding sites within plasmids permits cytoplasmic trafficking of plasmids during gene transfer. We found that CREB-binding sites are bound by CREB in the cytoplasm during transfection, and allow for enhanced rates of movement and subsequent nuclear accumulation. Moreover, small interfering RNA knockdown of CREB prevented this enhanced trafficking. Therefore, TF binding sites within plasmids are necessary for interactions with microtubules and enhance movement to the nucleus.

Novel approaches for analysing gut microbes and dietary polyphenols: challenges and opportunities.

Polyphenols, ubiquitously present in the food we consume, may modify the gut microbial composition and/or activity, and moreover, may be converted by the colonic microbiota to bioactive compounds that influence host health. The polyphenol content of fruit and vegetables and derived products is implicated in some of the health benefits bestowed on eating fruit and vegetables. Elucidating the mechanisms behind polyphenol metabolism is an important step in understanding their health effects. Yet, this is no trivial assignment due to the diversity encountered in both polyphenols and the gut microbial composition, which is further confounded by the interactions with the host. Only a limited number of studies have investigated the impact of dietary polyphenols on the complex human gut microbiota and these were mainly focused on single polyphenol molecules and selected bacterial populations. Our knowledge of gut microbial genes and pathways for polyphenol bioconversion and interactions is poor. Application of specific in vitro or in vivo models mimicking the human gut environment is required to analyse these diverse interactions. A particular benefit can now be gained from next-generation analytical tools such as metagenomics and metatranscriptomics allowing a wider, more holistic approach to the analysis of polyphenol metabolism. Understanding the polyphenol-gut microbiota interactions and gut microbial bioconversion capacity will facilitate studies on bioavailability of polyphenols in the host, provide more insight into the health effects of polyphenols and potentially open avenues for modulation of polyphenol bioactivity for host health.

Potential probiotic Lactobacillus strains from fermented sausages: Further investigations on their probiotic properties.

A rational selection of probiotic microorganisms is an important challenge and requires the definition of fundamental information about the physiology and genetics of candidate strains. In this study, selected Lactobacillus (Lact.) strains already characterized in a previous study for their capability to resist low pH and to grow in conditions simulating the intestinal environment, were further investigated to explore their probiotic properties, such as the adhesion capability to intestinal human Caco-2 cell lines and their growth behaviour in the presence of various prebiotic carbohydrates. At first 25 Lactobacillus strains were characterized by pulsed field gel electrophoresis using the endonuclease NotI. Among them, 13 strains belonging to the Lact. plantarum-group were identified at species level by a multiplex PCR assay. Subsequently 11 Lactobacillus strains showing different PFGE restriction pattern and the best acid- and bile-resistances, were chosen to investigate their in vitro adhesion capability to human intestinal epithelial cells and their fermentation properties of five prebiotic substances (FOS, Inulin, IMO, GOS and lactulose) at a concentration of 2%. The 11 strains analysed in this study possessed good adhesion capability to Caco-2 cell layers and, in particular, the eight strains belonging to the Lact. plantarum-group showed the higher final number of viable adhering cells. Moreover a species-related fermentative behaviour was pointed out and the strain Lact. paracasei EL7 was the only one able to grow in the presence of all prebiotics tested. In conclusion the strains of Lactobacillus studied in this research could be further investigated to assess possible in vivo human health benefits.

Double blind, placebo controlled trial of two probiotic strains in interleukin 10 knockout mice and mechanistic link with cytokine balance.

BACKGROUND: Prophylactic efficacy against colitis following lactobacillus consumption in interleukin 10 (IL-10) knockout (KO) mice has been reported. Whether this applies equally to other probiotic strains is unknown, and the mechanism is unclear. AIMS: (1) To compare the effect of feeding Lactobacillus salivarius subspecies salivarius 433118 and Bifidobacterium infantis 35624 against placebo on enterocolitis, the intestinal microflora, and (2) to compare the systemic immunological response to in vitro microbial challenge in probiotic fed and control IL-10 KO mice. METHODS: Three groups of 10 IL-10 KO mice were fed fermented milk products containing Lb salivarius 433118 at 10(9) CFU/ml, B infantis 35624 at 10(8) CFU/ml, and unmodified milk, respectively, for 19 weeks. Faecal samples were taken at regular intervals to confirm gut transit, recovery of fed probiotics, and to assess the impact on the microflora. At sacrifice, the bowels were histologically scored. Cytokine production from Peyers' patches and splenocytes was measured in vitro by ELISA. RESULTS: Faecal recovery of probiotics was confirmed in all probiotic fed mice but not in controls. Colonic and caecal inflammatory scores were significantly decreased in both groups of probiotic fed mice (p<0.05). Proinflammatory cytokine production by Peyers' patches and splenocytes was significantly reduced in probiotic fed animals whereas transforming growth factor beta (TGF-beta) levels were maintained. CONCLUSION: Both Lactobacillus salivarius 433118 and Bifidobacterium infantis 35624 significantly attenuate colitis in this murine model. Attenuation of colitis is associated with a reduced ability to produce Th1-type cytokines systemically and mucosally, while levels of TGF-beta are maintained.

Polymerase chain reaction and denaturing gradient gel electrophoresis monitoring of fecal bifidobacterium populations in a prebiotic and probiotic feeding trial.

A culture-independent approach based on genus-specific PCR and denaturing gradient gel electrophoresis (DGGE) was used to monitor qualitative changes in fecal bifidobacterial communities in a human feeding trial. DNA was extracted directly from feces and bifidobacterial 16S rDNA sequences were amplified using genus-specific PCR. The PCR fragments were subsequently separated in a sequence-specific manner by DGGE in order to obtain a profile of bifidobacterial fragments. The DGGE profiles revealed that in general, administration for two weeks of galactooligosaccharide and/or Bifidobacterium lactis Bb-12 (8 g and 3 x 10(10) cfu per day, respectively) did not affect the qualitative composition of the indigenous Bifidobacterium population, while B. lactis Bb-12 transiently colonised the gut.

Bifidobacterial diversity in human feces detected by genus-specific PCR and denaturing gradient gel electrophoresis.

We describe the development and validation of a method for the qualitative analysis of complex bifidobacterial communities based on PCR and denaturing gradient gel electrophoresis (DGGE). Bifidobacterium genus-specific primers were used to amplify an approximately 520-bp fragment from the 16S ribosomal DNA (rDNA), and the fragments were separated in a sequence-specific manner in DGGE. PCR products of the same length from different bifidobacterial species showed good separation upon DGGE. DGGE of fecal 16S rDNA amplicons from five adult individuals showed host-specific populations of bifidobacteria that were stable over a period of 4 weeks. Sequencing of fecal amplicons resulted in Bifidobacterium-like sequences, confirming that the profiles indeed represent the bifidobacterial population of feces. Bifidobacterium adolescentis was found to be the most common species in feces of the human adult subjects in this study. The methodological approach revealed intragenomic 16S rDNA heterogeneity in the type strain of B. adolescentis, E-981074. The strain was found to harbor five copies of 16S rDNA, two of which were sequenced. The two 16S rDNA sequences of B. adolescentis E-981074(T) exhibited microheterogeneity differing in eight positions over almost the total length of the gene.

Activation of silent gal genes in the lac-gal regulon of Streptococcus thermophilus.

Streptococcus thermophilus strain CNRZ 302 is unable to ferment galactose, neither that generated intracellularly by lactose hydrolysis nor the free sugar. Nevertheless, sequence analysis and complementation studies with Escherichia coli demonstrated that strain CNRZ 302 contained structurally intact genes for the Leloir pathway enzymes. These were organized into an operon in the order galKTE, which was preceded by a divergently transcribed regulator gene, galR, and followed by a galM gene and the lactose operon lacSZ. Results of Northern blot analysis showed that the structural gal genes were transcribed weakly, and only in medium containing lactose, by strain CNRZ 302. However, in a spontaneous galactose-fermenting mutant, designated NZ302G, the galKTE genes were well expressed in cells grown on lactose or galactose. In both CNRZ 302 and the Gal(+) mutant NZ302G, the transcription of the galR gene was induced by growth on lactose. Disruption of galR indicated that it functioned as a transcriptional activator of both the gal and lac operons while negatively regulating its own expression. Sequence analysis of the gal promoter regions of NZ302G and nine other independently isolated Gal(+) mutants of CNRZ 302 revealed mutations at three positions in the galK promoter region, which included substitutions at positions -9 and -15 as well as a single-base-pair insertion at position -37 with respect to the main transcription initiation point. Galactokinase activity measurements and analysis of gusA reporter gene fusions in strains containing the mutated promoters suggested that they were gal promoter-up mutations. We propose that poor expression of the gal genes in the galactose-negative S. thermophilus CNRZ 302 is caused by naturally occurring mutations in the galK promoter.

A molecular view of the intestinal ecosystem.

This review describes the state of the art as well as the initial results of molecular methodologies used to study the ecology of the complex microflora of the human intestinal tract. The detection and identification of many of these organisms has largely been hampered by the incomplete knowledge of their culture conditions. Many of the molecular methodologies are rooted in the use of ribosomal RNA (rRNA) and its encoding genes to describe the relationship between the bacteria in such communities and their individual identity. This approach permits the elucidation both qualitatively as well as quantitatively of the abundance of bacterial species and how their presence interacts with diet and health. Emphasis is given to the analysis of complex communities rather than detection of individual groups of bacteria. The potential of novel advances in molecular technologies such as DNA arrays for analysis of the intestinal ecosystem are also discussed.

Functionality of probiotics and intestinal lactobacilli: light in the intestinal tract tunnel.

The commercial interest in functional foods that contain live microorganisms, also termed probiotics, is paralleled by increasing scientific attention to their functionality in the digestive tract. Most studies are focused on intestinal Lactobacillus species, which are part of the natural gastro-intestinal microbiota, and include analysis of colonisation factors and other interactions with the host, the design of novel or improved strains with specific health benefits, and the application of sophisticated molecular tools to determine their fate and activity in situ.

Probiotics in the new millennium.

Beneficial effects on human health by specific probiotic microorganisms such as prevention of gastrointestinal tract infections immune stimulation, and balancing of the intestinal microflora have been established in numerous clinical trials. The successful probiotic strains which are mainly members of Lactobacillus and more recently Bifidobacterium naturally found in the human intestinal tract, have been traditionally incorporated into fermented milk products but have excellent potential for further inclusion in functional foods and health-related products. While the health claims are generally accepted by both scientists and consumers, often the molecular mechanisms underlying the probiotic properties remains controversial. Further progress concerning the molecular basis of probiotic traits will give vital reinforcement to the probiotic concept and is a prerequisite for rational development into further applications. In this review, current research on the genetics of properties of the intestinal lactobacilli that may contribute to the activity and effectiveness of probiotics is described. The potential of molecular biology for future probiotic applications is addressed and a probiotic strains developed by modern biotechnology with advantages for specific consumers is presented.

Transcriptional regulation and evolution of lactose genes in the galactose-lactose operon of Lactococcus lactis NCDO2054.

The genetics of lactose utilization within the slow-lactose-fermenting Lactococcus lactis strain NCDO2054 was studied with respect to the organization, expression, and evolution of the lac genes. Initially the beta-galactosidase gene (lacZ) was cloned by complementation of an Escherichia coli mutant on a 7-kb HpaI fragment. Nucleotide sequence analysis of the complete fragment revealed part of a gal-lac operon, and the genes were characterized by inactivation and complementation analyses and in vitro enzyme activity measurements. The gene order is galK-galT-lacA-lacZ-galE; the gal genes encode enzymes of the Leloir pathway for galactose metabolism, and lacA encodes a galactoside acetyltransferase. The galT and galE genes of L. lactis LM0230 (a lactose plasmid-cured derivative of the fast-lactose-fermenting L. lactis C2) were highly similar at the nucleotide sequence level to their counterparts in strain NCDO2054 and, furthermore, had the same gene order except for the presence of the intervening lacA-lacZ strain NCDO2054. Analysis of mRNA for the gal and lac genes revealed an unusual transcriptional organization for the operon, with a surprisingly large number of transcriptional units. The regulation of the lac genes was further investigated by using fusions consisting of putative promoter fragments and the promoterless beta-glucuronidase gene (gusA) from E. coli, which identified three lactose-inducible intergenic promoters in the gal-lac operon. The greater similarity of the lacA and lacZ genes to homologs in gram-negative organisms than to those of gram-positive bacteria, in contrast to the homologies of the gal genes, suggests that the genes within the gal operon of L. lactis NCDO2054 have been recently acquired. Thus, the lacA-lacZ genes appear to have engaged the promoters of the gal operon in order to direct and control their expression.

Controlled gene expression systems for lactic acid bacteria: transferable nisin-inducible expression cassettes for Lactococcus, Leuconostoc, and Lactobacillus spp.

A transferable dual-plasmid inducible gene expression system for use in lactic acid bacteria that is based on the autoregulatory properties of the antimicrobial peptide nisin produced by Lactococcus lactis was developed. Introduction of the two plasmids allowed nisin-inducible gene expression in Lactococcus lactis MG1363, Leuconostoc lactis NZ6091, and Lactobacillus helveticus CNRZ32. Typically, the beta-glucuronidase activity (used as a reporter in this study) remained below the detection limits under noninducing conditions and could be raised to high levels, by addition of subinhibitory amounts of nisin to the growth medium, while exhibiting a linear dose-response relationship. These results demonstrate that the nisin-inducible system can be functionally implemented in lactic acid bacteria other than Lactococcus lactis.

The lactose transporter in Leuconostoc lactis is a new member of the LacS subfamily of galactoside-pentose-hexuronide translocators.

The gene encoding the lactose transport protein (lacS) of Leuconostoc lactis NZ6009 has been cloned from its native lactose plasmid, pNZ63, by functional complementation of lactose permease-deficient Escherichia coli mutants. Nucleotide sequence analysis revealed an open reading frame with the capacity to encode a protein of 639 amino acids which had limited but significant identity to the lactose transport carriers (LacS) of Streptococcus thermophilus (34.5%) and Lactobacillus bulgaricus (35.6%). This similarity was present both in the amino-terminal hydrophobic carrier domain, which is homologous to the E. coli melibiose transporter, and in the carboxy-terminal enzyme IIA-like regulatory domain. The flanking regions of DNA surrounding lacS were also sequenced. Preceding the lacS gene was a small open reading frame in the same orientation encoding a deduced 95-amino-acid protein with a sequence similar to the amino-terminal portion of beta-galactosidase I from Bacillus stearothermophilus. The lacS gene was separated from the downstream beta-galactosidase genes (lacLM) by 2 kb of DNA containing an IS3-like insertion sequence, which is a novel arrangement for lac genes in comparison with that in other lactic acid bacteria. The lacS gene was cloned in an E. coli-Streptococcus shuttle vector and was expressed both in a lacS deletion derivative of S. thermophilus and in a pNZ63-cured strain, L. lactis NZ6091. The role of the LacS protein was confirmed by uptake assays in which substantial uptake of radiolabeled lactose or galactose was observed with L. lactis or S. thermophilus plasmids harboring an intact lacS gene. Furthermore, galactose uptake was observed in NZ6091, suggesting the presence of at least one more transport system for galactose in L. lactis.

Characterization of plasmid-encoded citrate permease (citP) genes from Leuconostoc species reveals high sequence conservation with the Lactococcus lactis citP gene.

The citrate permease determinant (citP) in several Leuconostoc strains was demonstrated to be plasmid encoded by curing experiments and hybridization studies with a DNA fragment containing the citP gene from Lactococcus lactis subsp. lactis biovar diacetylactis NCDO176. Cloning and nucleotide sequence analysis of Leuconostoc lactis NZ6070 citP revealed almost complete identity to lactococcal citP.

Identification and characterization of the insertion element IS1070 from Leuconostoc lactis NZ6009.

A novel insertion sequence, designated IS1070, was identified on the lactose plasmid of Leuconostoc lactis NZ6009 by nucleotide sequence analysis. The 1027-bp sequence contains partially matched (24 of 28 bp) inverted repeats and has one long open reading frame. The deduced 305-amino-acid sequence demonstrated homology to transposases of IS30 from Escherichia coli, IS4351 from Bacteroides fragilis, IS1086 from Alcaligenes eutrophus, IS1161 from Streptococcus salivarius, ISAS2 from Aeromonas salmonicida and a putative protein encoded by ORF3 of virus SpV1-R8A2 B from Spiroplasma citri. At least fifteen IS1070-like sequences were detected in the genome of the parent Lc. lactis strain and five of these were situated on plasmids. Analysis of the direct repeats of two of these copies with that of IS1070 revealed differences in the target duplication lengths.

Genetics of lactose utilization in lactic acid bacteria.

Lactose utilization is the primary function of lactic acid bacteria used in industrial dairy fermentations. The mechanism by which lactose is transported determines largely the pathway for the hydrolysis of the internalized disaccharide and the fate of the glucose and galactose moieties. Biochemical and genetic studies have indicated that lactose can be transported via phosphotransferase systems, transport systems dependent on ATP binding cassette proteins, or secondary transport systems including proton symport and lactose-galactose antiport systems. The genetic determinants for the group translocation and secondary transport systems have been identified in lactic acid bacteria and are reviewed here. In many cases the lactose genes are organized into operons or operon-like structures with a modular organization, in which the genes encoding lactose transport are tightly linked to those for lactose hydrolysis. In addition, in some cases the genes involved in the galactose metabolism are linked to or co-transcribed with the lactose genes, suggesting a common evolutionary pathway. The lactose genes show characteristic configurations and very high sequence identity in some phylogenetically distant lactic acid bacteria such as Leuconostoc and Lactobacillus or Lactococcus and Lactobacillus. The significance of these results for the adaptation of lactic acid bacteria to the industrial milk environment in which lactose is the sole energy source is discussed.