Kajsa Kajander

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.

Global and Deep Molecular Analysis of Microbiota Signatures in Fecal Samples From Patients With Irritable Bowel Syndrome

BACKGROUND & AIMS Irritable bowel syndrome (IBS) has been associated with disruptions to the intestinal microbiota, but studies have had limited power, coverage, and depth of analysis. We aimed to define microbial populations that can be used discriminate the fecal microbiota of patients with IBS from that of healthy subjects and correlate these with IBS intestinal symptom scores. METHODS The microbiota composition was assessed by global and deep molecular analysis of fecal samples from 62 patients with IBS patients and 46 healthy individuals (controls). We used a comprehensive and highly reproducible phylogenetic microarray in combination with quantitative polymerase chain reaction. RESULTS The intestinal microbiota of IBS patients differed significantly (P = .0005) from that of controls. The microbiota of patients, compared with controls, had a 2-fold increased ratio of the Firmicutes to Bacteroidetes (P = .0002). This resulted from an approximately 1.5-fold increase in numbers of Dorea, Ruminococcus, and Clostridium spp (P < .005); a 2-fold decrease in the number of Bacteroidetes (P < .0001); a 1.5-fold decrease in numbers of Bifidobacterium and Faecalibacterium spp (P < .05); and, when present, a 4-fold lower average number of methanogens (3.50 × 10(7) vs 8.74 × 10(6) cells/g feces; P = .003). Correlation analysis of the microbial groups and IBS symptom scores indicated the involvement of several groups of Firmicutes and Proteobacteria in the pathogenesis of IBS. CONCLUSIONS Global and deep molecular analysis of fecal samples indicates that patients with IBS have a different composition of microbiota. This information might be used to develop better diagnostics and ultimately treatments for IBS.

Development and application of the human intestinal tract chip, a phylogenetic microarray: analysis of universally conserved phylotypes in the abundant microbiota of young and elderly adults

In this paper we present the in silico assessment of the diversity of variable regions of the small subunit ribosomal RNA (SSU rRNA) gene based on an ecosystem-specific curated database, describe a probe design procedure based on two hypervariable regions with minimal redundancy and test the potential of such probe design strategy for the design of a flexible microarray platform. This resulted in the development and application of a phylogenetic microarray for studying the human gastrointestinal microbiota – referred as the human intestinal tract chip (HITChip). Over 4800 dedicated tiling oligonucleotide probes were designed based on two hypervariable regions of the SSU rRNA gene of 1140 unique microbial phylotypes (< 98% identity) following analysis of over 16 000 human intestinal SSU rRNA sequences. These HITChip probes were hybridized to a diverse set of human intestinal samples and SSU rRNA clones to validate its fingerprinting and quantification potential. Excellent reproducibility (median Pearsons correlation of 0.99) was obtained following hybridization with T7 polymerase transcripts generated in vitro from SSU rRNA gene amplicons. A linear dose–response was observed with artificial mixtures of 40 different representative amplicons with relative abundances as low as 0.1% of total microbiota. Analysis of three consecutively collected faecal samples from ten individuals (five young and five elderly adults) revealed temporal dynamics and confirmed that the adult intestinal microbiota is an individual-specific and relatively stable ecosystem. Further analysis of the stable part allowed for the identification of a universal microbiota core at the approximate genus level (90% sequence similarity). This core consists of members of Actinobacteria, Bacteroidetes and Firmicutes. Used as a phylogenetic fingerprinting tool with the possibility for relative quantification, the HITChip has the potential to bridge the gaps in our knowledge in the quantitative and qualitative description of the human gastrointestinal microbiota composition.

A probiotic mixture alleviates symptoms in irritable bowel syndrome patients: a controlled 6-month intervention

Background : Irritable bowel syndrome is a gastrointestinal disorder of unknown aetiology. The effect of probiotics in this syndrome remains unclear.

Clinical trial: multispecies probiotic supplementation alleviates the symptoms of irritable bowel syndrome and stabilizes intestinal microbiota

Background  Irritable bowel syndrome is the most common diagnosis in gastroenterology. Trials suggest certain probiotics to be beneficial.

Microbial community analysis reveals high level phylogenetic alterations in the overall gastrointestinal microbiota of diarrhoea-predominant irritable bowel syndrome sufferers

BackgroundA growing amount of scientific evidence suggests that microbes are involved in the aetiology of irritable bowel syndrome (IBS), and the gastrointestinal (GI) microbiota of individuals suffering from diarrhoea-predominant IBS (IBS-D) is distinguishable from other IBS-subtypes. In our study, the GI microbiota of IBS-D patients was evaluated and compared with healthy controls (HC) by using a high-resolution sequencing method. The method allowed microbial community analysis on all levels of microbial genomic guanine plus cytosine (G+C) content, including high G+C bacteria.MethodsThe collective faecal microbiota composition of ten IBS-D patients was analysed by examining sequences obtained using percent G+C (%G+C) -based profiling and fractioning combined with 16S rRNA gene clone library sequencing of 3267 clones. The IBS-D library was compared with an analogous healthy-control library of 23 subjects. Real-time PCR analysis was used to identify phylotypes belonging to the class Gammaproteobacteria and the order Coriobacteriales.ResultsSignificant differences were found between clone libraries of IBS-D patients and controls. The microbial communities of IBS-D patients were enriched in Proteobacteria and Firmicutes, but reduced in the number of Actinobacteria and Bacteroidetes compared to control. In particular, 16S rDNA sequences belonging to the family Lachnospiraceae within the phylum Firmicutes were in greater abundance in the IBS-D clone library.ConclusionsIn the microbiota of IBS-D sufferers, notable differences were detected among the prominent bacterial phyla (Firmicutes, Actinobacteria, Bacteroidetes, and Proteobacteria) localized within the GI tract.

The Causal Element for the Lactase Persistence/ non-persistence Polymorphism is Located in a 1 Mb Region of Linkage Disequilibrium in Europeans

Expression of lactase in the intestine persists into adult life in some people and not others, and this is due to a cis‐acting regulatory polymorphism. Previous data indicated that a mutation leading to lactase persistence had occurred on the background of a 60 kb 11‐site LCT haplotype known as A ( Hollox et al. 2001 ). Recent studies reported a 100% correlation of lactase persistence with the presence of the T allele at a CT SNP at −14 kb from LCT, in individuals of Finnish origin, suggesting that this SNP may be causal of the lactase persistence polymorphism, and also reported a very tight association with a second SNP (GA –22 kb) ( Enattah et al. 2002 ). Here we report the existence of a one megabase stretch of linkage disequilibrium in the region of LCT and show that the –14 kb T allele and the –22 kb A allele both occur on the background of a very extended A haplotype. In a series of Finnish individuals we found a strong correlation (40/41 people) with lactose digestion and the presence of the T allele. The T allele was present in all 36 lactase persistent individuals from the UK (phenotyped by enzyme assay) studied, 31/36 of whom were of Northern European ancestry, but not in 11 non‐persistent individuals who were mainly of non‐UK ancestry. However, the CT heterozygotes did not show intermediate lactase enzyme activity, unlike those previously phenotyped by determining allelic transcript expression. Furthermore the one lactase persistent homozygote identified by having equally high expression of A and B haplotype transcripts, was heterozygous for CT at the −14 kb site. SNP analysis across the 1 megabase region in this person showed no evidence of recombination on either chromosome between the –14 kb SNP and LCT. The combined data shows that although the –14 kb CT SNP is an excellent candidate for the cause of the lactase persistence polymorphism, linkage disequilibrium extends far beyond the region searched so far. In addition, the CT SNP does not, on its own, explain all the variation in expression of LCT, suggesting the possibility of genetic heterogeneity.

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.

Effect of a multispecies probiotic supplement on quantity of irritable bowel syndrome-related intestinal microbial phylotypes.

BackgroundProbiotics can alleviate the symptoms of irritable bowel syndrome (IBS), possibly by stabilizing the intestinal microbiota. Our aim was to determine whether IBS-associated bacterial alterations were reduced during multispecies probiotic intervention consisting of Lactobacillus rhamnosus GG, L. rhamnosus Lc705, Propionibacterium freudenreichii ssp. shermanii JS and Bifidobacterium breve Bb99. The intervention has previously been shown to successfully alleviate gastrointestinal symptoms of IBS.MethodsThe faecal microbiotas of 42 IBS subjects participating in a placebo-controlled double-blind multispecies probiotic intervention were analysed using quantitative real-time polymerase chain reaction (qPCR). Eight bacterial targets within the gastrointestinal microbiota with a putative IBS association were measured.ResultsA phylotype with 94% similarity to Ruminococcus torques remained abundant in the placebo group, but was decreased in the probiotic group during the intervention (P = 0.02 at 6 months). In addition, the clostridial phylotype, Clostridium thermosuccinogenes 85%, was stably elevated during the intervention (P = 0.00 and P = 0.02 at 3 and 6 months, respectively). The bacterial alterations detected were in accordance with previously discovered alleviation of symptoms.ConclusionsThe probiotic supplement was thus shown to exert specific alterations in the IBS-associated microbiota towards the bacterial 16S rDNA phylotype quantities described previously for subjects free of IBS. These changes may have value as non-invasive biomarkers in probiotic intervention studies.

In School-Aged Children a Combination of Galacto-Oligosaccharides and Lactobacillus GG Increases Bifidobacteria More than Lactobacillus GG on Its Own

The aim of this study was to compare a combination of Lactobacillus GG (LGG) and galacto-oligosaccharides (GOS) with LGG on its own, and their effects on the intestinal microbiota in school-aged children. The randomized, double-blinded, crossover study comprised 30 healthy children. There were two 3-week study periods with a 4-week wash-out period in between. The children ingested daily 65 ml of milk-based fruit juice containing either LGG alone (6.5 × 109 CFU) or LGG plus 2 g of GOS. Symptom diaries were filled during the study periods. Fecal samples were collected at the beginning and end of both study periods. At the end of both study periods, the amount of bifidobacteria was significantly greater after the ingestion of LGG + GOS compared with LGG alone (geometric mean 9.33 × 109 vs. 4.28 × 109 CFU/g, p < 0.001). No significant differences were seen in the amount of lactobacilli or LGG, nor did gastrointestinal symptoms, defecation frequency, consistency of stools or ease of defecation differ between the two study periods. Ingestion of LGG combined with 2 g of GOS increased the bifidobacteria more than LGG on its own and thus GOS clearly has a prebiotic effect in children. The children tolerated well a daily intake of 2 g of GOS.