The aging phenotype in humans has been thoroughly studied but a detailed metabolic profiling capable of shading light on the underpinning biological processes of longevity is still missing. Here using a combined metabonomics approach compromising holistic 1H-NMR profiling and targeted MS approaches, we report for the first time the metabolic phenotype of longevity in a well characterized human aging cohort compromising mostly female centenarians, elderly, and young individuals. With increasing age, targeted MS profiling of blood serum displayed a marked decrease in tryptophan concentration, while an unique alteration of specific glycerophospholipids and sphingolipids are seen in the longevity phenotype. We hypothesized that the overall lipidome changes specific to longevity putatively reflect centenarians' unique capacity to adapt/respond to the accumulating oxidative and chronic inflammatory conditions characteristic of their extreme aging phenotype. Our data in centenarians support promotion of cellular detoxification mechanisms through specific modulation of the arachidonic acid metabolic cascade as we underpinned increased concentration of 8,9-EpETrE, suggesting enhanced cytochrome P450 (CYP) enzyme activity. Such effective mechanism might result in the activation of an anti-oxidative response, as displayed by decreased circulating levels of 9-HODE and 9-oxoODE, markers of lipid peroxidation and oxidative products of linoleic acid. Lastly, we also revealed that the longevity process deeply affects the structure and composition of the human gut microbiota as shown by the increased extrection of phenylacetylglutamine (PAG) and p-cresol sulfate (PCS) in urine of centenarians. Together, our novel approach in this representative Italian longevity cohort support the hypothesis that a complex remodeling of lipid, amino acid metabolism, and of gut microbiota functionality are key regulatory processes marking exceptional longevity in humans.

Over the past years, we synthesized a series of new molecules that are hybrids of spirocyclic ketones as complexity-bearing cores with bi- and ter-phenyls as privileged fragments. Some of these newly-shaped small molecules showed antiproliferative, pro-apoptotic and differentiating activity in leukemia cell lines. In the present study, to investigate more in depth the mechanisms of action of these molecules, the protein expression profiles of K562 cells treated with or without the compounds IND_S1, MEL_T1, IND_S7 and MEL_S3 were analyzed using two-dimensional gel electrophoresis coupled with mass spectrometry. Proteome comparisons revealed several differentially expressed proteins, mainly related to cellular metabolism, chaperone activity, cytoskeletal organization and RNA biogenesis. The major results were validated by Western blot and qPCR. To attempt integrating findings into a cellular signaling context, proteomic data were explored using MetaCore. Network analysis highlighted relevant relationships between the identified proteins and additional potential effectors. Notably, qPCR validation of central hubs showed that the compound MEL_S3 induced high mRNA levels of the transcriptional factors EGR1 and HNF4-alpha; the latter to our knowledge is reported here for the first time to be present in K562 cells. Consistently with the known EGR1 involvement in the regulation of differentiation along megakaryocyte lineage, MEL_S3-treated leukemia cells showed a marked expression of glycoprotein IIb/IIIa (CD41) and glycoprotein Ib (CD42), two important cell markers in megakaryocytic differentiation, together with morphological aspects of megakaryoblasts and megakaryocytes.

Bacterial vaginosis (BV) is a common vaginal disorder characterized by an alteration of the vaginal bacterial morphotypes, associated with sexually transmitted infections and adverse pregnancy outcomes. The purpose of the present study was to evaluate the impact of different doses of rifaximin vaginal tablets (100 mg/day for 5 days, 25 mg/day for 5 days, and 100 mg/day for 2 days) on the vaginal microbiota of 102 European patients with BV enrolled in a multicenter, double-blind, randomized, placebo-controlled study. An integrated molecular approach based on quantitative PCR (qPCR) and PCR-denaturing gradient gel electrophoresis (PCR-DGGE) was used to investigate the effects of vaginal tablets containing the antibiotic. An increase in members of the genus Lactobacillus and a decrease in the BV-related bacterial groups after the antibiotic treatment were demonstrated by qPCR. PCR-DGGE profiles confirmed the capability of rifaximin to modulate the composition of the vaginal microbial communities and to reduce their complexity. This molecular analysis supported the clinical observation that rifaximin at 25 mg/day for 5 days represents an effective treatment to be used in future pivotal studies for the treatment of BV.

Background

The vaginal microbiota of healthy women consists of a wide variety of anaerobic and aerobic bacterial genera and species dominated by the genus Lactobacillus. The activity of lactobacilli helps to maintain the natural healthy balance of the vaginal microbiota. This role is particularly important during pregnancy because vaginal dismicrobism is one of the most important mechanisms for preterm birth and perinatal complications. In the present study, we characterized the impact of a dietary supplementation with the probiotic VSL#3, a mixture of Lactobacillus, Bifidobacterium and Streptococcus strains, on the vaginal microbiota and immunological profiles of healthy women during late pregnancy.

Results

An association between the oral intake of the probiotic VSL#3 and changes in the composition of the vaginal microbiota of pregnant women was revealed by PCR-DGGE population profiling. Despite no significant changes were found in the amounts of the principal vaginal bacterial populations in women administered with VSL#3, qPCR results suggested a potential role of the probiotic product in counteracting the decrease of Bifidobacterium and the increase of Atopobium, that occurred in control women during late pregnancy. The modulation of the vaginal microbiota was associated with significant changes in some vaginal cytokines. In particular, the decrease of the anti-inflammatory cytokines IL-4 and IL-10 was observed only in control women but not in women supplemented with VSL#3. In addition, the probiotic consumption induced the decrease of the pro-inflammatory chemokine Eotaxin, suggesting a potential anti-inflammatory effect on the vaginal immunity.

Conclusion

Dietary supplementation with the probiotic VSL#3 during the last trimester of pregnancy was associated to a modulation of the vaginal microbiota and cytokine secretion, with potential implications in preventing preterm birth.

Trial registration

ClinicalTrials.gov NCT01367470

The capacity to intervene with the host plasminogen system has recently been considered an important component in the interaction process between Bifidobacterium animalis subsp. lactis and the human host. However, its significance in the bifidobacterial microecology within the human gastrointestinal tract is still an open question. Here we demonstrate that human plasminogen favors the B. animalis subsp. lactis BI07 adhesion to HT29 cells. Prompting the HT29 cell capacity to activate plasminogen, tumor necrosis factor alpha (TNF-α) modulated the plasminogen-mediated bacterium-enterocyte interaction, reducing the bacterial adhesion to the enterocytes and enhancing migration to the luminal compartment.

Considering the increase in the consumption of yeasts as human probiotics, the aim of this study was to broadly investigate the beneficial properties of the lactic yeast Kluyveromyces marxianus (formerly Kluyveromyces fragilis) B0399. Several potential probiotic traits of K. marxianus B0399 were investigated by using in vitro assays, including adhesion and immune modulation, and the effect of the administration of 107 CFU/day of K. marxianus B0399 on the composition and metabolic activity of the human intestinal microbiota was investigated in a 3-stage continuous-culture system simulating the human colon. We demonstrated that this strain was highly adhesive to human enterocyte-like Caco-2 cells and modulated the immune response, inducing proinflammatory cytokines in peripheral blood mononuclear cells (PBMCs). In the presence of inflammatory stimulation with lipopolysaccharide (LPS), K. marxianus B0399 provoked decreases in the levels of production of proinflammatory cytokines in PBMCs and Caco-2 cells, thus ameliorating the inflammatory response. Furthermore, K. marxianus B0399 impacted the colonic microbiota, increasing the bifidobacterial concentration in the stages of the colonic model system simulating the proximal and transverse colon. The amounts of the short-chain fatty acids acetate and propionate also increased following yeast supplementation. Finally, K. marxianus B0399 was found to induce a decrease of the cytotoxic potential of the culture supernatant from the first stage of the colonic model system. The effects of K. marxianus B0399 on adhesion, immune function, and colonic microbiota demonstrate that this strain possesses a number of beneficial and strain-specific properties desirable for a microorganism considered for application as a probiotic.

Background

Playing a strategic role in the host immune function, the intestinal microbiota has been recently hypothesized to be involved in the etiology of atopy. In order to investigate the gastrointestinal microbial ecology of atopic disease, here we performed a pilot comparative molecular analysis of the faecal microbiota in atopic children and healthy controls.

Results

Nineteen atopic children and 12 healthy controls aged 4–14 years were enrolled. Stools were collected and the faecal microbiota was characterized by means of the already developed phylogenetic microarray platform, HTF-Microbi.Array, and quantitative PCR. The intestinal microbiota of atopic children showed a significant depletion in members of the Clostridium cluster IV, Faecalibacterium prausnitzii, Akkermansia muciniphila and a corresponding increase of the relative abundance of Enterobacteriaceae.

Conclusion

Depleted in key immunomodulatory symbionts, the atopy-associated microbiota can represent an inflammogenic microbial consortium which can contribute to the severity of the disease. Our data open the way to the therapeutic manipulation of the intestinal microbiota in the treatment of atopy by means of pharmaceutical probiotics.

Human plasmin(ogen) is regarded as a component of the molecular cross talk between the probiotic species Bifidobacterium animalis subsp. lactis and the human host. However, up to now, only in vitro studies have been reported. Here, we demonstrate that the probiotic strain B. animalis subsp. lactis BI07 is capable of recruiting plasmin(ogen) present at physiological concentrations in crude extracts from human feces. Our results provide evidence that supports the significance of the B. lactis-plasmin(ogen) interaction in the human gastrointestinal tract.

Human beings have been recently reviewed as ‘metaorganisms’ as a result of a close symbiotic relationship with the intestinal microbiota. This assumption imposes a more holistic view of the ageing process where dynamics of the interaction between environment, intestinal microbiota and host must be taken into consideration. Age-related physiological changes in the gastrointestinal tract, as well as modification in lifestyle, nutritional behaviour, and functionality of the host immune system, inevitably affect the gut microbial ecosystem. Here we review the current knowledge of the changes occurring in the gut microbiota of old people, especially in the light of the most recent applications of the modern molecular characterisation techniques. The hypothetical involvement of the age-related gut microbiota unbalances in the inflamm-aging, and immunosenescence processes will also be discussed. Increasing evidence of the importance of the gut microbiota homeostasis for the host health has led to the consideration of medical/nutritional applications of this knowledge through the development of probiotic and prebiotic preparations specific for the aged population. The results of the few intervention trials reporting the use of pro/prebiotics in clinical conditions typical of the elderly will be critically reviewed.

Oxalic acid occurs extensively in nature and plays diverse roles, especially in pathological processes. Due to its highly oxidizing effects, hyperabsorption or abnormal synthesis of oxalate can cause serious acute disorders in mammals and can be lethal in extreme cases. Intestinal oxalate-degrading bacteria could therefore be pivotal in maintaining oxalate homeostasis and reducing the risk of kidney stone development. In this study, the oxalate-degrading activities of 14 bifidobacterial strains were measured by a capillary electrophoresis technique. The oxc gene, encoding oxalyl-coenzyme A (CoA) decarboxylase, a key enzyme in oxalate catabolism, was isolated by probing a genomic library of Bifidobacterium animalis subsp. lactis BI07, which was one of the most active strains in the preliminary screening. The genetic and transcriptional organization of oxc flanking regions was determined, unraveling the presence of two other independently transcribed open reading frames, potentially responsible for the ability of B. animalis subsp. lactis to degrade oxalate. pH-controlled batch fermentations revealed that acidic conditions were a prerequisite for a significant oxalate degradation rate, which dramatically increased in cells first adapted to subinhibitory concentrations of oxalate and then exposed to pH 4.5. Oxalate-preadapted cells also showed a strong induction of the genes potentially involved in oxalate catabolism, as demonstrated by a transcriptional analysis using quantitative real-time reverse transcription-PCR. These findings provide new insights into the characterization of oxalate-degrading probiotic bacteria and may support the use of B. animalis subsp. lactis as a promising adjunct for the prophylaxis and management of oxalate-related kidney disease.

Background

Age-related physiological changes in the gastrointestinal tract, as well as modifications in lifestyle, nutritional behaviour, and functionality of the host immune system, inevitably affect the gut microbiota, resulting in a greater susceptibility to infections.

Methodology/Principal Findings

By using the Human Intestinal Tract Chip (HITChip) and quantitative PCR of 16S rRNA genes of Bacteria and Archaea, we explored the age-related differences in the gut microbiota composition among young adults, elderly, and centenarians, i.e subjects who reached the extreme limits of the human lifespan, living for over 100 years. We observed that the microbial composition and diversity of the gut ecosystem of young adults and seventy-years old people is highly similar but differs significantly from that of the centenarians. After 100 years of symbiotic association with the human host, the microbiota is characterized by a rearrangement in the Firmicutes population and an enrichment in facultative anaerobes, notably pathobionts. The presence of such a compromised microbiota in the centenarians is associated with an increased inflammatory status, also known as inflammageing, as determined by a range of peripheral blood inflammatory markers. This may be explained by a remodelling of the centenarians' microbiota, with a marked decrease in Faecalibacterium prauznitzii and relatives, symbiotic species with reported anti-inflammatory properties. As signature bacteria of the long life we identified specifically Eubacterium limosum and relatives that were more than ten-fold increased in the centenarians.

Conclusions/Significance

We provide evidence for the fact that the ageing process deeply affects the structure of the human gut microbiota, as well as its homeostasis with the host's immune system. Because of its crucial role in the host physiology and health status, age-related differences in the gut microbiota composition may be related to the progression of diseases and frailty in the elderly population.

Background

Affecting the core functional microbiome, peculiar high level taxonomic unbalances of the human intestinal microbiota have been recently associated with specific diseases, such as obesity, inflammatory bowel diseases, and intestinal inflammation.

Results

In order to specifically monitor microbiota unbalances that impact human physiology, here we develop and validate an original DNA-microarray (HTF-Microbi.Array) for the high taxonomic level fingerprint of the human intestinal microbiota. Based on the Ligase Detection Reaction-Universal Array (LDR-UA) approach, the HTF-Microbi.Array enables specific detection and approximate relative quantification of 16S rRNAs from 30 phylogenetically related groups of the human intestinal microbiota. The HTF-Microbi.Array was used in a pilot study of the faecal microbiota of eight young adults. Cluster analysis revealed the good reproducibility of the high level taxonomic microbiota fingerprint obtained for each of the subject.

Conclusion

The HTF-Microbi.Array is a fast and sensitive tool for the high taxonomic level fingerprint of the human intestinal microbiota in terms of presence/absence of the principal groups. Moreover, analysis of the relative fluorescence intensity for each probe pair of our LDR-UA platform can provide estimation of the relative abundance of the microbial target groups within each samples. Focusing the phylogenetic resolution at division, order and cluster levels, the HTF-Microbi.Array is blind with respect to the inter-individual variability at the species level.

AIM: To profile protein expression in mucosal biopsies from patients with chronic refractory pouchitis following antibiotic or probiotic treatment, using a comparative proteomic approach.

METHODS: Two-dimensional polyacrylamide gel electrophoresis and matrix-assisted laser desorption/ionization-time of flight mass spectrometry were used to characterize the changes related to antibiotic therapy in the protein expression profiles of biopsy samples from patients with chronic refractory pouchitis. The same proteomic approach was applied to identify differentially expressed proteins in the non-inflamed pouch before and after probiotic administration.

RESULTS: In the first set of 2D gels, 26 different proteins with at least 2-fold changes in their expression levels between the pouchitis condition and antibiotic-induced remission were identified. In the second set of analysis, the comparison between mucosal biopsy proteomes in the normal and probiotic-treated pouch resulted in 17 significantly differently expressed proteins. Of these, 8 exhibited the same pattern of deregulation as in the pouchitis/pouch remission group.

CONCLUSION: For the first time, 2D protein maps of mucosal biopsies from patients with ileal pouch-anal anastomosis were provided, and differentially expressed proteins following antibiotic/probiotic treatment were identified.

Background

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

Results

The synbiotic food did not modify the overall structure of the gut microbiome, as indicated by Polymerase Chain Reaction-Denaturing Gradient Gel Electrophoresis (PCR-DGGE). The ability of the probiotic L. helveticus and B. longum strains to pass through the gastrointestinal tract was hypothesized on the basis of real-time PCR data. In spite of a stable microbiota, the intake of the synbiotic food resulted in a shift of the fecal metabolic profiles, highlighted by the Gas Chromatography Mass Spectrometry Solid Phase Micro-Extraction (GC-MS/SPME) analysis. The extent of short chain fatty acids (SCFA), ketones, carbon disulfide and methyl acetate was significantly affected by the synbiotic food consumption. Furthermore, the Canonical discriminant Analysis of Principal coordinates (CAP) of GC-MS/SPME profiles allowed a separation of the stool samples recovered before and after the consumption of the functional food.

Conclusion

In this study we investigated the global impact of a dietary intervention on the gut ecology and metabolism in healthy humans. We demonstrated that the intake of a synbiotic food leads to a modulation of the gut metabolic activities with a maintenance of the gut biostructure. In particular, the significant increase of SCFA, ketones, carbon disulfide and methyl acetate following the feeding period suggests potential health promoting effects of the synbiotic food.

The microbial flora of the vagina plays a major role in preventing genital infections, including bacterial vaginosis (BV) and candidiasis (CA). An integrated approach based on PCR-denaturing gradient gel electrophoresis (PCR-DGGE) and real-time PCR was used to study the structure and dynamics of bacterial communities in vaginal fluids of healthy women and patients developing BV and CA. Universal eubacterial primers and Lactobacillus genus-specific primers, both targeted at 16S rRNA genes, were used in DGGE and real-time PCR analysis, respectively. The DGGE profiles revealed that the vaginal flora was dominated by Lactobacillus species under healthy conditions, whereas several potentially pathogenic bacteria were present in the flora of women with BV. Lactobacilli were the predominant bacterial population in the vagina for patients affected by CA, but changes in the composition of Lactobacillus species were observed. Real-time PCR analysis allowed the quantitative estimation of variations in lactobacilli associated with BV and CA diseases. A statistically significant decrease in the relative abundance of lactobacilli was found in vaginal fluids of patients with BV compared to the relative abundance of lactobacilli in the vaginal fluids of healthy women and patients with CA.

Bifidobacteria constitute up to 3% of the total microbiota and represent one of the most important health-promoting bacterial groups of the human intestinal microflora. The presence of Bifidobacterium in the human gastrointestinal tract has been directly related to several health-promoting activities; however, to date, no information about the specific mechanisms of interaction with the host is available. In order to provide some insight into the molecular mechanisms involved in the interaction with the host, we investigated whether Bifidobacterium was able to capture human plasminogen on the cell surface. By using flow cytometry, we demonstrated a dose-dependent human plasminogen-binding activity for four strains belonging to three bifidobacterial species: Bifidobacterium lactis, B. bifidum, and B. longum. The binding of human plasminogen to Bifidobacterium was dependent on lysine residues of surface protein receptors. By using a proteomic approach, we identified five putative plasminogen-binding proteins in the cell wall fraction of the model strain B. lactis BI07. The data suggest that plasminogen binding to B. lactis is due to the concerted action of a number of proteins located on the bacterial cell surface, some of which are highly conserved cytoplasmic proteins which have other essential cellular functions. Our findings represent a step forward in understanding the mechanisms involved in the Bifidobacterium-host interaction.

Oxalyl coenzyme A (CoA) decarboxylase (Oxc) is a key enzyme in the catabolism of the highly toxic compound oxalate, catalyzing the decarboxylation of oxalyl-CoA to formyl-CoA. The gene encoding a novel oxalyl-CoA decarboxylase from Bifidobacterium lactis DSM 10140 (oxc) was identified and characterized. This strain, isolated from yogurt, showed the highest oxalate-degrading activity in a preliminary screening with 12 strains belonging to Bifidobacterium, an anaerobic intestinal bacterial group largely used in probiotic products. The oxc gene was isolated by probing a B. lactis genomic library with a probe obtained by amplification of the oxalyl-CoA decarboxylase gene from Oxalobacter formigenes, an anaerobic bacterium of the human intestinal microflora. The oxc DNA sequence analysis revealed an open reading frame of 1,773 bp encoding a deduced 590-amino-acid protein with a molecular mass of about 63 kDa. Analysis of amino acid sequence showed a significant homology (47%) with oxalyl-CoA decarboxylase of O. formigenes and a typical thiamine pyrophosphate-binding site that has been reported for several decarboxylase enzymes. Primer extension experiments with oxc performed by using RNA isolated from B. lactis identified the transcriptional start site 28 bp upstream of the ATG start codon, immediately adjacent to a presumed promoter region. The protein overexpressed in Escherichia coli cross-reacted with an anti-O. formigenes oxalyl-CoA decarboxylase antibody. Enzymatic activity, when evaluated by capillary electrophoresis analysis, demonstrated that the consumption substrate oxalyl-CoA was regulated by a product inhibition of the enzyme. These findings suggest a potential role for Bifidobacterium in the intestinal degradation of oxalate.