Gut barrier-microbiota imbalances in early life lead to higher sensitivity to inflammation in a murine model of C-section delivery.

BACKGROUND: Most interactions between the host and its microbiota occur at the gut barrier, and primary colonizers are essential in the gut barrier maturation in the early life. The mother-offspring transmission of microorganisms is the most important factor influencing microbial colonization in mammals, and C-section delivery (CSD) is an important disruptive factor of this transfer. Recently, the deregulation of symbiotic host-microbe interactions in early life has been shown to alter the maturation of the immune system, predisposing the host to gut barrier dysfunction and inflammation. The main goal of this study is to decipher the role of the early-life gut microbiota-barrier alterations and its links with later-life risks of intestinal inflammation in a murine model of CSD. RESULTS: The higher sensitivity to chemically induced inflammation in CSD mice is related to excessive exposure to a too diverse microbiota too early in life. This early microbial stimulus has short-term consequences on the host homeostasis. It switches the pup's immune response to an inflammatory context and alters the epithelium structure and the mucus-producing cells, disrupting gut homeostasis. This presence of a too diverse microbiota in the very early life involves a disproportionate short-chain fatty acids ratio and an excessive antigen exposure across the vulnerable gut barrier in the first days of life, before the gut closure. Besides, as shown by microbiota transfer experiments, the microbiota is causal in the high sensitivity of CSD mice to chemical-induced colitis and in most of the phenotypical parameters found altered in early life. Finally, supplementation with lactobacilli, the main bacterial group impacted by CSD in mice, reverts the higher sensitivity to inflammation in ex-germ-free mice colonized by CSD pups' microbiota. CONCLUSIONS: Early-life gut microbiota-host crosstalk alterations related to CSD could be the linchpin behind the phenotypic effects that lead to increased susceptibility to an induced inflammation later in life in mice. Video Abstract.

Influence of body lesion severity on oxidative status and gut microbiota of weaned pigs.

Body lesions in pigs are a common welfare concern, particularly during the weaning period. These lesions can lead to pain, infection, and impaired mobility, resulting in reduced growth performance and increased mortality. Moreover, weaning stress can affect gut microbiota, immune response and increase the oxidative stress of piglets during this transition period. It has been hypothesised that social stress and body lesions could contribute to affect the gut microbiota, physiological and immune response of piglets. The study aims to evaluate the impact of the body lesions due to social stress on microbial profile, immune response, and oxidative status of weaned piglets. Lesion score (LS) on skin, tail, ear, neck, middle trunk, and hind quarters was measured 1 week (28 days of age, T1) and 7 weeks postweaning (T2) on 45 tail-docked pigs according to the method suggested from the Walfer Quality® (2009) on a scale from 0 to 2. Based on the LS, at T1, piglets were classified as High LS (n = 16), when LS was >1 in at least two of the areas considered, or Low LS (n = 29). At T2, based on the same scoring system and to the LS observed at T1, piglets were divided into four groups: High to Low LS (H-L, n = 11), High to High LS (H-H, n = 5), Low to Low LS (L-L, n = 21) and Low to High LS (L-H, n = 8). Blood and faecal samples were collected at T1 and T2. At T1, pigs with a high LS had a lower biological antioxidant potential compared with the L group (P < 0.02). At T2, the L-H group had a lower Reactive Oxygen Metabolites concentration compared with the H-H group (P = 0.03) while the L-L group had a lower concentration of Immunoglobulin A compared with H-H and L-H groups (P = 0.02 and P = 0.04, respectively). At T1, piglets with high LS had a different microbiota compared to piglets with low LS (R2 = 0.04, P < 0.01). Low LS pigs were characterised by a higher abundance of Firmicutes, Blautia, Eubacterium coprostanoligenes, Faecalibacterium, Megasphaera, Subdoligranulum (P.adj < 0.05), while pigs with high LS were characterised by higher abundance of Bacteroidota, Rikenellaceae RC9, Prevotellaceae UCG-003, uncultured-Lachnospiraceae and uncultured-Oscillospiraceae (P.adj < 0.05). At T2, the H-H group were characterised by Oscillospirales-UCG-010, H-L by Agatobachter and L-L by Alloprevotella (P.adj < 0.05). Overall, this study provides valuable insights into the relationship between body lesions, oxidative stress, and gut microbiota in weaned pigs.

Early colonisation and temporal dynamics of the gut microbial ecosystem in Standardbred foals.

BACKGROUND: Even if horses strictly depend on the gut microbiota for energy homeostasis, only a few molecular studies have focused on its characterisation and none on the perinatal gut microbial colonisation process. OBJECTIVES: To explore the perinatal colonisation process of the foal gut microbial ecosystem and the temporal dynamics of the ecosystem assembly during the first days of life. STUDY DESIGN: Longitudinal study. METHODS: Thirteen Standardbred mare-foal pairs were included in the study. For each pair, at delivery we collected the mare amniotic fluid, faeces and colostrum, and the foal meconium. Milk samples and faeces of both mare and foal were also taken longitudinally, until day 10 post-partum. Samples were analysed by means of next-generation sequencing of the 16S rRNA gene on Illumina MiSeq. RESULTS: Our findings suggest that microbial components derived from the mare symbiont communities establishes in the foal gut since fetal life. After birth, an external transmission route of mare microorganisms takes place. This involves a rapid and dynamic process of assembling the mature foal gut microbiome, in which the founder microbial species are derived from both the milk and the gut microbial ecosystems of the mare. MAIN LIMITATIONS: The inability to discriminate between live and dead cells, the possible presence of contaminating bacteria in low biomass samples (e.g. meconium and amniotic fluid), the limits of the phylogenetic assignment down to species level, and the presence of unassigned operational taxonomic units. CONCLUSIONS: Our data highlight the importance of mare microbiomes as a key factor for the establishment of the gut microbial ecosystem of the foal.

Gut microbiota and hematopoietic stem cell transplantation: where do we stand?

Advances in biological techniques have potentiated great progresses in understanding the interaction between human beings and the ∼10 to 100 trillion microbes living in their gastrointestinal tract: gut microbiota (GM). In this review, we describe recent emerging data on the role of GM in hematopoietic stem cell transplantation, with a focus on immunomodulatory properties in the immune system recovery and the impact in the development of the main complications, as GvHD and infections.

Impact of diet and nutraceutical supplementation on inflammation in elderly people. Results from the RISTOMED study, an open-label randomized control trial.

BACKGROUND & AIMS: Eating habits may influence the life span and the quality of ageing process by modulating inflammation. The RISTOMED project was developed to provide a personalized and balanced diet, enriched with or without nutraceutical compounds, to decrease and prevent inflammageing, oxidative stress and gut microbiota alteration in healthy elderly people. This paper focused on the effect on inflammation and metabolism markers after 56 days of RISTOMED diet alone or supplementation with three nutraceutical compounds. METHODS: A cohort of 125 healthy elderly subjects was recruited and randomized into 4 arms (Arm A, RISTOMED diet; Arm B, RISTOMED diet plus VSL#3 probiotic blend; Arm C, RISTOMED diet plus AISA d-Limonene; Arm D, RISTOMED diet plus Argan oil). Inflammatory and metabolism parameters as well as the ratio between Clostridium cluster IV and Bifidobacteria (CL/B) were collected before and after 56 days of dietary intervention, and their evolution compared among the arms. Moreover, participants were subdivided according to their baseline inflammatory parameters (erythrocytes sedimentation rate (ESR), C-Reactive Protein, fibrinogen, Tumor Necrosis Factor-alfa (TNF-α), and Interleukin 6) in two clusters with low or medium-high level of inflammation. The evolution of the measured parameters was then examined separately in each cluster. RESULTS: Overall, RISTOMED diet alone or with each nutraceutical supplementation significantly decreased ESR. RISTOMED diet supplemented with d-Limonene resulted in a decrease in fibrinogen, glucose, insulin levels and HOMA-IR. The most beneficial effects were observed in subjects with a medium-high inflammatory status who received RISTOMED diet with AISA d-Limonene supplementation. Moreover, RISTOMED diet associated with VSL#3 probiotic blend induced a decrease in the CL/B ratio. CONCLUSIONS: Overall, this study emphasizes the beneficial anti-inflammageing effect of RISTOMED diet supplemented with nutraceuticals to control the inflammatory status of elderly individuals.

Gut microbiota trajectory in pediatric patients undergoing hematopoietic SCT.

Acute GvHD (aGvHD) is the main complication of hematopoietic SCT (HSCT) during the treatment of hematological disorders. We carried out the first longitudinal study to follow the gut microbiota trajectory, from both the phylogenetic and functional points of view, in pediatric patients undergoing HSCT. Gut microbiota trajectories and short-chain fatty acid production profiles were followed starting from before HSCT and through the 3-4 months after transplant in children developing and not developing aGvHD. According to our findings, HSCT procedures temporarily cause a structural and functional disruption of the gut microbial ecosystem, describing a trajectory of recovery during the following 100 days. The onset of aGvHD is associated with specific gut microbiota signatures both along the course of gut microbiota reconstruction immediately after transplant and, most interestingly, prior to HSCT. Indeed, in pre-HSCT samples, non-aGvHD patients showed higher abundances of propionate-producing Bacteroidetes, highly adaptable microbiome mutualists that showed to persist during the HSCT-induced ecosystem disruption. Our data indicate that structure and temporal dynamics of the gut microbial ecosystem can be a relevant factor for the success of HSCT and opens the perspective to the manipulation of the pre-HSCT gut microbiota configuration to favor mutualistic persisters with immunomodulatory properties in the gut.

Effect of a new synbiotic supplement on symptoms, stool consistency, intestinal transit time and gut microbiota in patients with severe functional constipation: a pilot randomized double-blind, controlled trial.

BACKGROUND: Data on the benefits of synbiotics in functional constipation are conflicting. The aim of this study was to assess whether the administration of the synbiotic supplement Psyllogel Megafermenti(®) normalized stool consistency and decreased intestinal transit time (ITT) in patients with severe functional constipation, based on its ability to impact on the gut microbiota. METHODS: We conducted a pilot randomized, double-blind, controlled trial. After a 2-week run-in period, patients from a tertiary care setting with severe functional constipation fulfilling the Rome III Diagnostic Criteria in the past year were randomly assigned to receive by mouth 2 bags/day of Psyllogel Megafermenti(®) (Group A) or 2.8 g of maltodextrin twice daily (Group B) for 8 weeks. Primary endpoints were increase of bowel evacuations with normal stool consistency and volume, and ITT reduction. Secondary endpoints included symptom improvement according to the Rome III Diagnostic Criteria, reduction of the Agachan-Wexner score and changes in gut microbiota composition. RESULTS: Twenty-nine patients completed the study: 17 were allocated to Group A and 12 to Group B. A statistically significant increase in stools with normal consistency was observed only in Group A (p = 0.001), even when considering patients with normal stools ≤50 % of time at baseline. In Group A, a significant reduction in ITT was also found (p = 0.022). According to polymerase chain reaction-denaturing gradient gel electrophoresis profiling of stool samples, 50 % of the patients treated with synbiotics harbored all the probiotic species of the study product. CONCLUSIONS: An 8-week treatment with Psyllogel Megafermenti(®) improved the main clinical parameters of functional constipation in patients extremely homogeneous for disorder severity and underlying pathophysiology ( Eudract.ema.europa.eu , No. 2008-000913-30).

A probiotics-containing biscuit modulates the intestinal microbiota in the elderly.

OBJECTIVES: Evaluation of the impact of a biscuit containing the probiotics Bifidobacterium longum Bar33 and Lactobacillus helveticus Bar13 on the intestinal microbiota in the elderly. DESIGN: Randomized double-blind placebo-controlled trial. PARTICIPANTS: Thirty-two elderly volunteers living in Italy. The group was composed of 19 women and 13 men aged between 71 and 88 years (mean 76). INTERVENTION: Subjects were randomized in two groups consuming one dose of the probiotics-containing biscuit or placebo once a day for 30 days. MEASUREMENTS: For each subject the intestinal microbiota was characterized using the phylogenetic microarray platform HTF-Microbi.Array before and after intervention. RESULTS: Our data demonstrated that one-month consumption of a probiotics-containing biscuit was effective in redressing some of the age-related dysbioses of the intestinal microbiota. In particular, the probiotic treatment reverted the age-related increase of the opportunistic pathogens Clostridium cluster XI, Clostridium difficile, Clostridium perfringens, Enterococcus faecium and the enteropathogenic genus Campylobacter. CONCLUSION: The present study opens the way to the development of elderly-tailored probiotic-based functional foods to counteract the age-related dysbioses of the intestinal microbiota.

Development of a high-performance affinity chromatography-based method to study the biological interaction between whole micro-organisms and target proteins.

AIMS: The bacteria-host molecular cross-talk is the matter of primary importance both in pathogenesis and in commensalism. Principally based on immunological methods, the methodologies commonly utilized for these studies are laborious and require specific antibodies. Here, we developed a new high-performance affinity chromatography (HPAC)-based approach that allows a direct measure of the interaction between whole bacterial cells and host molecules. METHODS AND RESULTS: Bifidobacterium lactis BI07 cells immobilized on amino-derivatized silica beads were utilized as stationary phase in a high-performance affinity chromatography approach. The analytes plasminogen, collagen I and collagen IV were injected, and interactions were evaluated by the insertion in an HPLC system with UV detection. According to our data, Bif. lactis BI07 is capable of interacting with plasminogen, while it does not exhibit any binding activity to collagen I and IV. CONCLUSIONS: In this study, we implemented a high-performance affinity chromatography-based method to characterize the biological interaction between whole micro-organisms and target proteins. SIGNIFICANCE AND IMPACT OF THE STUDY: With respect to the approaches commonly utilized to study the interaction between bacteria and host proteins, this HPAC-based approach is fast and cheaper than other methods and allows a direct measure of the interaction between bacterial cells and target molecules.

Quantitative variations in the vaginal bacterial population associated with asymptomatic infections: a real-time polymerase chain reaction study.

The real-time polymerase chain reaction (PCR) quantification of several vaginal bacterial groups in healthy women and patients developing asymptomatic bacterial vaginosis (BV) and candidiasis (CA) was performed. Statistical analysis revealed that the BV condition is characterised by a great variability among subjects and that it is associated with a significant increase of Prevotella, Atopobium, Veillonella and Gardnerella vaginalis, and a drop in Lactobacillus. On the contrary, the vaginal microflora of healthy women and patients developing CA was found to be homogeneous and stable over time.

Interaction of probiotic Lactobacillus and Bifidobacterium strains with human intestinal epithelial cells: adhesion properties, competition against enteropathogens and modulation of IL-8 production.

The human intestinal microbiota plays a pivotal role in human nutrition and health by promoting the supply of nutrients, preventing pathogen colonization and shaping and maintaining normal mucosal immunity. The depletion of the individual microbiota can result in a higher susceptibility to enteropathogenic bacteria infection. In order to reduce this risk, the use of food supplements containing probiotic bacteria has been recently addressed. In this paper, we investigate the protective role toward enteropathogen infection of probiotic strains belonging to Lactobacillus and Bifidobacterium. According to our experimental data, Lactobacillus acidophilus Bar13, L. plantarum Bar10, Bifidobacterium longum Bar33 and B. lactis Bar30 were effective in displacing the enteropathogens Salmonella typhimurium and Escherichia coli H10407 from a Caco-2 cell layer. Moreover, L. acidophilus Bar13 and B. longum Bar33 have been assessed for their immunomodulatory activity on IL-8 production by HT29 cells. Both strains showed the potential to protect enterocytes from an acute inflammatory response. These probiotic strains are potential candidates for the development of new functional foods helpful in counteracting enteropathogen infections.

Oxalate consumption by lactobacilli: evaluation of oxalyl-CoA decarboxylase and formyl-CoA transferase activity in Lactobacillus acidophilus.

AIMS: This study was undertaken to evaluate the oxalate-degrading activity in several Lactobacillus species widely used in probiotic dairy and pharmaceutical preparations. Functional characterization of oxalyl-CoA decarboxylase and formyl-CoA transferase in Lactobacillus acidophilus was performed in order to assess the possible contribution of Lactobacillus in regulating the intestinal oxalate homeostasis. METHODS AND RESULTS: In order to determine the oxalate-degrading ability in 60 Lactobacillus strains belonging to 12 species, a screening was carried out by using an enzymatic assay. A high variability in the oxalate-degrading capacity was found in the different species. Strains of Lact. acidophilus and Lactobacillus gasseri showed the highest oxalate-degrading activity. Oxalyl-CoA decarboxylase and formyl-CoA transferase genes from Lact. acidophilus LA14 were cloned and sequenced. The activity of the recombinant enzymes was assessed by capillary electrophoresis. CONCLUSIONS: Strains of Lactobacillus with a high oxalate-degrading activity were identified. The function and significance of Lact. acidophilus LA14 oxalyl-CoA decarboxylase and formyl-CoA transferase in oxalate catabolism were demonstrated. These results suggest the potential use of Lactobacillus strains for the degradation of oxalate in the human gut. SIGNIFICANCE AND IMPACT OF THE STUDY: Identification of probiotic strains with oxalate-degrading activity can offer the opportunity to provide this capacity to individuals suffering from an increased body burden of oxalate and oxalate-associated disorders.

Modulation of human dendritic cell phenotype and function by probiotic bacteria.

BACKGROUND: "Probiotic" bacteria are effective in treating some inflammatory bowel diseases. However which bacteria confer benefit and mechanisms of action remain poorly defined. Dendritic cells, which are pivotal in early bacterial recognition, tolerance induction, and shaping of T cell responses, may be central in mediating the effects of these bacteria. AIMS: To assess effects of different probiotic bacteria on dendritic cell function. METHODS: Human intestinal lamina propria mononuclear cells, whole blood, or an enriched blood dendritic cell population were cultured with cell wall components of the eight bacterial strains in the probiotic preparation VSL#3 (four lactobacilli, three bifidobacteria, and one streptococcal strains). Dendritic cells were identified and changes in dendritic cell maturation/costimulatory markers and cytokine production in response to probiotic bacteria were analysed by multicolour flow cytometry, in addition to subsequent effects on T cell polarisation. RESULTS: VSL#3 was a potent inducer of IL-10 by dendritic cells from blood and intestinal tissue, and inhibited generation of Th1 cells. Individual strains within VSL#3 displayed distinct immunomodulatory effects on dendritic cells; the most marked anti-inflammatory effects were produced by bifidobacteria strains which upregulated IL-10 production by dendritic cells, decreased expression of the costimulatory molecule CD80, and decreased interferon-gamma production by T cells. VSL#3 diminished proinflammatory effects of LPS by decreasing LPS induced production of IL-12 while maintaining IL-10 production. CONCLUSIONS: Probiotic bacteria differ in their immunomodulatory activity and influence polarisation of immune responses at the earliest stage of antigen presentation by dendritic cells.

Identification method based on PCR combined with automated ribotyping for tracking probiotic Lactobacillus strains colonizing the human gut and vagina.

AIMS: A molecular methodology based on PCR-associated automated ribotyping was developed to specifically detect the Lactobacillus strains of two probiotic products (an orally administered lyophilized preparation and vaginal tablets) in human faeces and vaginal swabs. METHODS AND RESULTS: The 16S-23S rDNA sequences and the ribotype profiles of the probiotic lactobacilli were characterized and new species-specific primer sets were designed. The identification of faecal and vaginal lactobacilli isolated from subjects administered with the probiotic products was performed by using PCR with species-specific primers followed by strain-specific automated ribotyping. CONCLUSIONS: The PCR-ribotyping identification allowed to study the colonization patterns of the probiotic lactobacilli in the human gut and vagina evidencing the strains with the best survival capability. SIGNIFICANCE AND IMPACT OF THE STUDY: The proposed molecular method represents a powerful tool of strain-specific identification, useful for differentiating exogenous from indigenous strains in any microbial ecosystem and for rationally choosing probiotic bacteria with the best chance of survival in the host.

Evaluation of the rrn operon copy number in Bifidobacterium using real-time PCR.

AIMS: A real-time PCR-based method was developed to evaluate the Bifidobacterium rRNA operon copy number. As a result of their repetitive nature, rRNA operons are very suitable targets for chromosomal integration of heterologous genes. METHODS AND RESULTS: The rrn operon multiplicity per chromosome was determined by real-time PCR quantification of the 16S rRNA amplicons obtained from genomic DNA. The values obtained in several bifidobacterial strains of human origin ranged from 1 to 5. The reliability of the method developed was confirmed by Southern hybridization technique. CONCLUSIONS: In the Bifidobacterium genus the rrn operon copies showed variability at species and strain level. The identification of Bifidobacterium strains with high rRNA multiplicity allowed the selection of potential hosts for chromosomal integration. SIGNIFICANCE AND IMPACT OF THE STUDY: The methodology here proposed represents a rapid, reliable and sensitive new tool for the quantification of rrn operon copy number in bacteria.

Characterization and selection of vaginal Lactobacillus strains for the preparation of vaginal tablets.

AIMS: To characterize and select Lactobacillus strains for properties that would make them a good alternative to the use of antibiotics to treat human vaginal infections. METHODS AND RESULTS: Ten Lactobacillus strains belonging to four different Lactobacillus species were analysed for properties relating to mucosal colonization or microbial antagonism (adhesion to human epithelial cells, hydrogen peroxide production, antimicrobial activity towards Gardnerella vaginalis and Candida albicans and coaggregation with pathogens). The involvement of electrostatic interactions and the influence of bacterial metabolic state in the binding of lactobacilli to the cell surface were also studied. Adherence to epithelial cells varied greatly among the Lactobacillus species and among different strains belonging to the same Lactobacillus species. The reduction in surface negative electric charge promoted the binding of several Lactobacillus strains to the cell membrane whereas lyophilization reduced the adhesion capacity of many isolates. The antimicrobial activity of lactobacilli culture supernatant fluids was not directly related to the production of H2O2. CONCLUSIONS: Three strains (Lactobacillus brevis CD2, Lact. salivarius FV2 and Lact. gasseri MB335) showed optimal properties and were, therefore, selected for the preparation of vaginal tablets. The selected strains adhered to epithelial cells displacing vaginal pathogens; they produced high levels of H2O2, coaggregated with pathogens and inhibited the growth of G. vaginalis. SIGNIFICANCE AND IMPACT OF THE STUDY: The dosage formulation developed in this study appears to be a good candidate for the probiotic prophylaxis and treatment of human vaginal infections.

Effects of rifaximin administration on the intestinal microbiota in patients with ulcerative colitis.

The effect of rifaximin on the intestinal bacterial population was studied in a clinical trial. Twelve patients with ulcerative colitis were administered rifaximin 1800 mg/day in 3 treatment periods of 10 days, each followed by 25 days of wash-out. Fecal samples were collected at the beginning and at the end of each treatment period to perform microbiological examinations. Titer variations of enterococci, coliforms, lactobacilli, bifidobacteria, Bacteroides spp., and Clostridium perfringens as well as their susceptibility to rifaximin during the different phases of the study were evaluated. The presence of Candida spp. was also monitored. After each wash-out period, concentrations of the intestinal microbial groups tested returned to initial values, showing that the administration of high doses of rifaximin does not significantly modify the colonic microbiota. Rifaximin-resistant isolates were also found, particularly in bacteria belonging to Bifidobacterium genus, included as probiotics in several fermented foods and in pharmaceutical preparations.

Effect of probiotic strains on interleukin 8 production by HT29/19A cells.

OBJECTIVES: Promising results from clinical studies on the effect of probiotics as maintenance therapy in inflammatory bowel disease and in the prevention of onset of pouchitis ask for studies to unravel the still poorly understood mechanism of action of probiotics. METHODS: To evaluate whether the probiotic bacteria that were used in the clinical studies (VSL#3, Escherichia coli Nissle 1917, and Lactobacillus GG) are able to induce chemokine production in epithelial cells, HT29/19A monolayers were incubated with cell debris and cell extract fractions of single strains of the probiotic bacteria in doses ranging from 10(3) to 10(9) colony-forming units/ml for 32 h. Supernatants were measured for interleukin 8 by ELISA. RESULTS: Lactobacilli and bifidobacteria strains from VSL#3 and Lactobacillus GG did not induce interleukin 8, whereas both cell debris and cell extracts from E. coli Nissle 1917 induced interleukin 8 production in a dose-dependent way. Cell extracts from streptococcal strains induced interleukin 8 when applied at high concentrations. CONCLUSIONS: Probiotic Gram-positive bacteria did not induce interleukin 8, whereas the nonpathogenic, Gram-negative E. coli Nissle 1917 strain induced interleukin 8 in a dose-dependent way in this culture model. These results suggest that probiotic Gram-positive bacteria and E. coli Nissle 1917 may exert their beneficial effects on the host by a different mechanism of action.

Effects of probiotic administration upon the composition and enzymatic activity of human fecal microbiota in patients with irritable bowel syndrome or functional diarrhea.

In a clinical trial, 10 patients suffering from irritable bowel syndrome or functional diarrhea were administered the probiotic preparation VSL-3. Preliminary results indicated that administration of VSL-3 improved the clinical picture and changed the composition and biochemistry of fecal microbiota. Titer variations of intestinal bacterial groups were evaluated by culture and PCR techniques. A significant increase in lactobacilli, bifidobacteria and Streptococcus thermophilus was observed as a consequence of probiotic treatment, while enterococci, coliforms, Bacteroides and Clostridium perfringens did not change significantly. The strains Bifidobacterium infantis Y1 and Bifidobacterium breve Y8, included in VSL-3, were specifically detected in feces of patients treated with the probiotic by using strain-specific PCR primers. In addition, fecal beta-galactosidase increased and urease activities decreased as a result of changes in the intestinal microbiota induced by VSL-3 administration.