Teemu Rinttilä

Development of an extensive set of 16S rDNA‐targeted primers for quantification of pathogenic and indigenous bacteria in faecal samples by real‐time PCR

Aims:  The microbiota of the human intestinal tract constitutes a complex ecosystem. We report the design and optimization of an extensive set of 16S rDNA‐targeted species‐ and group‐specific primers for more accurate quantification of bacteria from faecal samples with real‐time PCR.

Analysis of the Fecal Microbiota of Irritable Bowel Syndrome Patients and Healthy Controls with Real-Time PCR

OBJECTIVE:The gut microbiota may contribute to the onset and maintenance of irritable bowel syndrome (IBS). In this study, the microbiotas of patients suffering from IBS were compared with a control group devoid of gastrointestinal (GI) symptoms.METHODS:Fecal microbiota of patients (n = 27) fulfilling the Rome II criteria for IBS was compared with age- and gender-matched control subjects (n = 22). Fecal samples were obtained at 3 months intervals. Total bacterial DNA was analyzed by 20 quantitative real-time PCR assays covering approximately 300 bacterial species.RESULTS:Extensive individual variation was observed in the GI microbiota among both the IBS- and control groups. Sorting of the IBS patients according to the symptom subtypes (diarrhea, constipation, and alternating predominant type) revealed that lower amounts of Lactobacillus spp. were present in the samples of diarrhea predominant IBS patients wheras constipation predominant IBS patients carried increased amounts of Veillonella spp. Average results from three fecal samples suggested differences in the Clostridium coccoides subgroup and Bifidobacterium catenulatum group between IBS patients (n = 21) and controls (n = 15). Of the intestinal pathogens earlier associated with IBS, no indications of Helicobacter spp. or Clostridium difficile were found whereas one case of Campylobacter jejuni was identified by sequencing.CONCLUSIONS:With these real-time PCR assays, quantitative alterations in the GI microbiota of IBS patients were found. Increasing microbial DNA sequence information will further allow designing of new real-time PCR assays for a more extensive analysis of intestinal microbes in IBS.

Effects of multispecies probiotic supplementation on intestinal microbiota in irritable bowel syndrome

Background  A multispecies probiotic has shown beneficial effects in irritable bowel syndrome. In addition, certain other probiotics have demonstrated advantageous effects, but the mechanisms behind this are poorly understood.

Real-time PCR analysis of enteric pathogens from fecal samples of irritable bowel syndrome subjects

BackgroundGrowing amount of scientific evidence suggests that microbes are involved in the pathophysiology of irritable bowel syndrome (IBS). The predominant fecal microbiota composition of IBS subjects has been widely studied with DNA-based techniques but less research has been focused on the intestinal pathogens in this disorder. Here, we optimized a highly sensitive panel of 12 quantitative real-time PCR (qPCR) assays to shed light on the putative presence of intestinal pathogens in IBS sufferers. The panel was used to screen fecal samples from 96 IBS subjects and 23 healthy controls.ResultsFifteen IBS samples (17%) tested positive for Staphylococcus aureus with a thermonuclease (nuc) gene-targeting qPCR assay, whereas none of the healthy controls were positive for S. aureus (p < 0.05). The S. aureus -positive IBS samples were confirmed by sequencing of the PCR amplicons. Clostridium perfringens was detected from IBS and control groups with a similar frequency (13% and 17%, respectively) with α-toxin (plc) gene -targeting qPCR assay while none of the samples tested positive for the Cl. perfringens enterotoxin-encoding gene (cpe).ConclusionsThe qPCR panel consisting of 12 assays for an extensive set of pathogenic microorganisms provides an efficient alternative to the conventional detection of gastrointestinal pathogens and could accelerate the initiation of targeted antibiotic therapy reducing the risk of post-infectious IBS (PI-IBS). S. aureus has not been previously reported to be associated with the onset of IBS. Although we discovered significant differences in the prevalence of S. aureus between the study groups, its importance in giving rise to IBS symptoms requires further studies.

Effect of high contents of dietary animal-derived protein or carbohydrates on canine faecal microbiota

BackgroundConsiderable evidence suggests that food impacts both the gastro-intestinal (GI) function and the microbial ecology of the canine GI tract. The aim of this study was to evaluate the influence of high-carbohydrate (HC), high-protein (HP) and dry commercial (DC) diets on the canine colonic microbiota in Beagle dogs. Diets were allocated according to the Graeco-Latin square design. For this purpose, microbial DNA was isolated from faecal samples and separated by density gradient centrifugation, resulting in specific profiling based on the guanine-cytosine content (%G + C). In addition, 16 S rRNA gene amplicons were obtained from the most abundant %G + C peaks and analysed by sequence analysis, producing a total of 720 non-redundant sequences (240 sequences per diet).ResultsThe DC diet sample showed high abundance of representatives of the orders Clostridiales, Lactobacillales, Coriobacteriales and Bacteroidales. Sequence diversity was highest for DC diet samples and included representatives of the orders Lactobacillales and Bacteroidales, which were not detected in samples from the HP and HC diets. These latter two diets also had reduced levels of representatives of the family Lachnospiraceae, specifically Clostridial cluster XIVa. The HC diet favoured representatives of the order Erysipelotrichales, more specifically the Clostridial cluster XVIII, while the HP diet favoured representatives of the order Fusobacteriales.ConclusionsThis study detected Coriobacteriales in dog faeces, possibly due to the non-selective nature of the %G + C profiling method used in combination with sequencing. Moreover, our work demonstrates that the effect of diet on faecal microbiota can be explained based on the metabolic properties of the detected microbial taxa.

Effect of Lactobacillus brevis ATCC 8287 as a feeding supplement on the performance and immune function of piglets.

Lactobacillus brevis ATCC 8287, a surface (S-layer) strain, possesses a variety of functional properties that make it both a potential probiotic and a good vaccine vector candidate. With this in mind, our aim was to study the survival of L. brevis in the porcine gut and investigate the effect of this strain on the growth and immune function of recently weaned piglets during a feeding trial. For this, 20 piglets were divided evenly into a treatment and a control group. Piglets in the treatment group were fed L. brevis cells (1×10(10)) daily for three weeks, whereas those in the control group were provided an equivalent amount of probiotic-free placebo. For assessing the impact of L. brevis supplementation during the feeding trial, health status and weight gain of the piglets were monitored, pre- and post-trial samples of serum and feces were obtained, and specimens of the small and large intestinal mucosa and digesta were collected at slaughter. The results we obtained indicated that L. brevis-supplemented feeding induced a non-significant increase in piglet body weight and caused no change in the morphology of the intestinal mucosa. L. brevis cells were found to localize mainly in the large intestine, but they could not be isolated from feces. To a lesser extent, L. brevis was detected in the small intestine, although there was no specific attachment to the Peyers patches. Changes in total serum IgG and IgA concentrations were not caused by supplemented L. brevis and no measurable rise in L. brevis-specific IgG was observed. However, analysis of cytokine gene expression in intestinal mucosa revealed downregulation of TGF-β1 in the ileum and upregulation of IL-6 in the cecum in the L. brevis-supplemented group. Based on the results from this study, we conclude that whereas L. brevis appears to have some intestinal immunomodulatory effects, the ability of this strain to survive and colonize within the porcine gut appears to be limited.