Faecal microbiota composition in adults is associated with the FUT2 gene determining the secretor status.

The human intestine is colonised with highly diverse and individually defined microbiota, which likely has an impact on the host well-being. Drivers of the individual variation in the microbiota compositions are multifactorial and include environmental, host and dietary factors. We studied the impact of the host secretor status, encoded by fucosyltransferase 2 (FUT2) -gene, on the intestinal microbiota composition. Secretor status determines the expression of the ABH and Lewis histo-blood group antigens in the intestinal mucosa. The study population was comprised of 14 non-secretor (FUT2 rs601338 genotype AA) and 57 secretor (genotypes GG and AG) adult individuals of western European descent. Intestinal microbiota was analyzed by PCR-DGGE and for a subset of 12 non-secretor subjects and 12 secretor subjects additionally by the 16S rRNA gene pyrosequencing and the HITChip phylogenetic microarray analysis. All three methods showed distinct clustering of the intestinal microbiota and significant differences in abundances of several taxa representing dominant microbiota between the non-secretors and the secretors as well as between the FUT2 genotypes. In addition, the non-secretors had lower species richness than the secretors. The soft clustering of microbiota into enterotypes (ET) 1 and 3 showed that the non-secretors had a higher probability of belonging to ET1 and the secretors to ET3. Our study shows that secretor status and FUT2 polymorphism are associated with the composition of human intestinal microbiota, and appears thus to be one of the key drivers affecting the individual variation of human intestinal microbiota.

Self-supervised MRI tissue segmentation by discriminative clustering.

The study of brain lesions can benefit from a clear identification of transitions between healthy and pathological tissues, through the analysis of brain imaging data. Current signal processing methods, able to address these issues, often rely on strong prior information. In this article, a new method for tissue segmentation is proposed. It is based on a discriminative strategy, in a self-supervised machine learning approach. This method avoids the use of prior information, which makes it very versatile, and able to cope with different tissue types. It also returns tissue probabilities for each voxel, crucial for a good characterization of the evolution of brain lesions. Simulated as well as real benchmark data were used to validate the accuracy of the method and compare it against other segmentation algorithms.

Transient proteolytic modification of mesenchymal stromal cells increases lung clearance rate and targeting to injured tissue.

Systemic infusion of therapeutic cells would be the most practical and least invasive method of administration in many cellular therapies. One of the main obstacles especially in intravenous delivery of cells is a massive cell retention in the lungs, which impairs homing to the target tissue and may decrease the therapeutic outcome. In this study we showed that an alternative cell detachment of mesenchymal stromal/stem cells (MSCs) with pronase instead of trypsin significantly accelerated the lung clearance of the cells and, importantly, increased their targeting to an area of injury. Cell detachment with pronase transiently altered the MSC surface protein profile without compromising cell viability, multipotent cell characteristics, or immunomodulative and angiogenic potential. The transient modification of the cell surface protein profile was sufficient to produce effective changes in cell rolling behavior in vitro and, importantly, in the in vivo biodistribution of the cells in mouse, rat, and porcine models. In conclusion, pronase detachment could be used as a method to improve the MSC lung clearance and targeting in vivo. This may have a major impact on the bioavailability of MSCs in future therapeutic regimes.

Microarray analysis reveals marked intestinal microbiota aberrancy in infants having eczema compared to healthy children in at-risk for atopic disease.

BACKGROUND: Deviations in composition and diversity of intestinal microbiota in infancy have been associated with both the development and recurrence of atopic eczema. Thus, we decided to use a deep and global microarray-based method to characterize the diversity and temporal changes of the intestinal microbiota in infancy and to define specific bacterial signatures associated with eczema. Faecal microbiota at 6 and 18 months of age were analysed from 34 infants (15 with eczema and 19 healthy controls) selected from a prospective follow-up study based on the availability of faecal samples. The infants were originally randomized to receive either Lactobacillus rhamnosus GG or placebo. RESULTS: Children with eczema harboured a more diverse total microbiota than control subjects as assessed by the Simpson's reciprocal diversity index of the microarray profiles. Composition of the microbiota did not differ between study groups at age of 6 months, but was significantly different at age of 18 months as assessed by MCPP (p=0.01). At this age healthy children harboured 3 -fold greater amount of members of the Bacteroidetes (p=0.01). Microbiota of children suffering from eczema had increased abundance of the Clostridium clusters IV and XIVa, which are typically abundant in adults. Probiotic Lactobacillus rhamnosus GG supplementation in early infancy was observed to have minor long-term effects on the microbiota composition. CONCLUSION: A diverse and adult-type microbiota in early childhood is associated with eczema and it may contribute to the perpetuation of eczema.

Aging bone marrow mesenchymal stromal cells have altered membrane glycerophospholipid composition and functionality.

Human mesenchymal stem/stromal cells (hMSC) are increasingly used in advanced cellular therapies. The clinical use of hMSCs demands sequential cell expansions. As it is well established that membrane glycerophospholipids (GPL) provide precursors for signaling lipids that modulate cellular functions, we studied the effect of the donor's age and cell doublings on the GPL profile of human bone marrow MSC (hBMSC). The hBMSCs, which were harvested from five young and five old adults, showed clear compositional changes during expansion seen at the level of lipid classes, lipid species, and acyl chains. The ratio of phosphatidylinositol to phosphatidylserine increased toward the late-passage samples. Furthermore, 20:4n-6-containing species of phosphatidylcholine and phosphatidylethanolamine accumulated while the species containing monounsaturated fatty acids (FA) decreased during passaging. Additionally, in the total FA pool of the cells, 20:4n-6 increased, which happened at the expense of n-3 polyunsaturated FAs, especially 22:6n-3. The GPL and FA correlated with the decreased immunosuppressive capacity of hBMSCs during expansion. Our observations were further supported by alterations in the gene expression levels of several enzymes involved in lipid metabolism and immunomodulation. The results show that extensive expansion of hBMSCs harmfully modulates membrane GPLs, affecting lipid signaling and eventually impairing functionality.

Cytokine response of human mononuclear cells induced by intestinal Clostridium species.

Altered composition of intestinal microbiota has been associated with various immunological disorders such as inflammatory bowel disease. Although Clostridium species are major inhabitants of the intestinal tract, their interaction with the host immunological system is yet poorly characterized. In this study, cytokine responses of human monocytic cell line THP-1 and peripheral blood mononuclear cells (PBMC) to six type strains representing common intestinal clostridial species were determined. The strains induced diverse cytokine responses in both THP-1 cells and PBMC. Clostridium perfringens was the most potent inducer of both tumour necrosis factor alpha (TNF-alpha) and interleukin-10 (IL-10), as compared to Clostridium histolyticum, Clostridium clostridioforme, Clostridium leptum, Clostridium sporosphaeroides and Blautia coccoides. Interleukin-8 (IL-8) production in PBMC was most efficiently stimulated by C. sporosphaeroides. The same PBMC preparations that responded strongly to Escherichia coli lipopolysaccharide (LPS) also responded strongly to bacterial stimulation. This indicates that the level of responsiveness is an individual feature of mononuclear cell preparations, and that the overall cytokine response is composed by a combination of host factors and microbial structures affecting them. This work supports the idea that the composition of the intestinal clostridial population influences immune responses and is likely to play an important role in intestinal homeostasis.

Association between the ABO blood group and the human intestinal microbiota composition.

BACKGROUND: The mucus layer covering the human intestinal epithelium forms a dynamic surface for host-microbial interactions. In addition to the environmental factors affecting the intestinal equilibrium, such as diet, it is well established that the microbiota composition is individually driven, but the host factors determining the composition have remained unresolved. RESULTS: In this study, we show that ABO blood group is involved in differences in relative proportion and overall profiles of intestinal microbiota. Specifically, the microbiota from the individuals harbouring the B antigen (secretor B and AB) differed from the non-B antigen groups and also showed higher diversity of the Eubacterium rectale-Clostridium coccoides (EREC) and Clostridium leptum (CLEPT) -groups in comparison with other blood groups. CONCLUSIONS: Our novel finding indicates that the ABO blood group is one of the genetically determined host factors modulating the composition of the human intestinal microbiota, thus enabling new applications in the field of personalized nutrition and medicine.

Comparative metaproteomics and diversity analysis of human intestinal microbiota testifies for its temporal stability and expression of core functions.

The human intestinal tract is colonized by microbial communities that show a subject-specific composition and a high-level temporal stability in healthy adults. To determine whether this is reflected at the functional level, we compared the faecal metaproteomes of healthy subjects over time using a novel high-throughput approach based on denaturing polyacrylamide gel electrophoresis and liquid chromatography-tandem mass spectrometry. The developed robust metaproteomics workflow and identification pipeline was used to study the composition and temporal stability of the intestinal metaproteome using faecal samples collected from 3 healthy subjects over a period of six to twelve months. The same samples were also subjected to DNA extraction and analysed for their microbial composition and diversity using the Human Intestinal Tract Chip, a validated phylogenetic microarray. Using metagenome and single genome sequence data out of the thousands of mass spectra generated per sample, approximately 1,000 peptides per sample were identified. Our results indicate that the faecal metaproteome is subject-specific and stable during a one-year period. A stable common core of approximately 1,000 proteins could be recognized in each of the subjects, indicating a common functional core that is mainly involved in carbohydrate transport and degradation. Additionally, a variety of surface proteins could be identified, including potential microbes-host interacting components such as flagellins and pili. Altogether, we observed a highly comparable subject-specific clustering of the metaproteomic and phylogenetic profiles, indicating that the distinct microbial activity is reflected by the individual composition.

Gut microbiota of healthy elderly NSAID users is selectively modified with the administration of Lactobacillus acidophilus NCFM and lactitol.

Ageing changes gut microbiota composition and alters immune system function. Probiotics, prebiotics and synbiotics may improve the health status of elderly individuals by modifying the intestinal environment and the microbiota composition, and by stimulating the immune system. In this work, we studied the effects of synbiotic supplementation on the gut microbiota of healthy elderly volunteers. Fifty-one elders were randomly assigned to consume either a synbiotic dietary supplement or a placebo in addition to their usual diet for a 2-week period. The synbiotic product consisted of the probiotic Lactobacillus acidophilus NCFM and the prebiotic lactitol and was ingested twice a day, with a total daily dose of 10 g lactitol and 2 × 10(10) cells of probiotic bacteria. Before, during and after the intervention period fecal quantities of six phylogenetic bacterial groups were determined using quantitative PCR, and relative changes in total microbiota composition were assessed by percent guanine-plus-cytosine profiling. The microbiota profiles showed certain relative changes within the microbial community, and indicated an increase of bifidobacteria levels during synbiotic supplementation. Quantification by PCR confirmed the in changes in the microbiota composition; for example increases in total levels of endogenous bifidobacteria and lactobacilli were recorded. Throughout the 6-week study period there was a decrease unrelated to intervention in the Blautia coccoides-Eubacterium rectale bacterial group levels and Clostridium cluster XIVab levels, but this decrease appeared to be halted during the synbiotic intervention. In conclusion, putatively beneficial changes in microbiota were observed in the elderly subjects supplemented with the synbiotic product.

Metabolic regulation in progression to autoimmune diabetes.

Recent evidence from serum metabolomics indicates that specific metabolic disturbances precede ß-cell autoimmunity in humans and can be used to identify those children who subsequently progress to type 1 diabetes. The mechanisms behind these disturbances are unknown. Here we show the specificity of the pre-autoimmune metabolic changes, as indicated by their conservation in a murine model of type 1 diabetes. We performed a study in non-obese prediabetic (NOD) mice which recapitulated the design of the human study and derived the metabolic states from longitudinal lipidomics data. We show that female NOD mice who later progress to autoimmune diabetes exhibit the same lipidomic pattern as prediabetic children. These metabolic changes are accompanied by enhanced glucose-stimulated insulin secretion, normoglycemia, upregulation of insulinotropic amino acids in islets, elevated plasma leptin and adiponectin, and diminished gut microbial diversity of the Clostridium leptum group. Together, the findings indicate that autoimmune diabetes is preceded by a state of increased metabolic demands on the islets resulting in elevated insulin secretion and suggest alternative metabolic related pathways as therapeutic targets to prevent diabetes.

Intestinal microbiota in healthy adults: temporal analysis reveals individual and common core and relation to intestinal symptoms.

BACKGROUND: While our knowledge of the intestinal microbiota during disease is accumulating, basic information of the microbiota in healthy subjects is still scarce. The aim of this study was to characterize the intestinal microbiota of healthy adults and specifically address its temporal stability, core microbiota and relation with intestinal symptoms. We carried out a longitudinal study by following a set of 15 healthy Finnish subjects for seven weeks and regularly assessed their intestinal bacteria and archaea with the Human Intestinal Tract (HIT) Chip, a phylogenetic microarray, in conjunction with qPCR analyses. The health perception and occurrence of intestinal symptoms was recorded by questionnaire at each sampling point. PRINCIPAL FINDINGS: A high overall temporal stability of the microbiota was observed. Five subjects showed transient microbiota destabilization, which correlated not only with the intake of antibiotics but also with overseas travelling and temporary illness, expanding the hitherto known factors affecting the intestinal microbiota. We identified significant correlations between the microbiota and common intestinal symptoms, including abdominal pain and bloating. The most striking finding was the inverse correlation between Bifidobacteria and abdominal pain: subjects who experienced pain had over five-fold less Bifidobacteria compared to those without pain. Finally, a novel computational approach was used to define the common core microbiota, highlighting the role of the analysis depth in finding the phylogenetic core and estimating its size. The in-depth analysis suggested that we share a substantial number of our intestinal phylotypes but as they represent highly variable proportions of the total community, many of them often remain undetected. CONCLUSIONS/SIGNIFICANCE: A global and high-resolution microbiota analysis was carried out to determine the temporal stability, the associations with intestinal symptoms, and the individual and common core microbiota in healthy adults. The findings provide new approaches to define intestinal health and to further characterize the microbial communities inhabiting the human gut.

Secretor genotype (FUT2 gene) is strongly associated with the composition of Bifidobacteria in the human intestine.

Intestinal microbiota plays an important role in human health, and its composition is determined by several factors, such as diet and host genotype. However, thus far it has remained unknown which host genes are determinants for the microbiota composition. We studied the diversity and abundance of dominant bacteria and bifidobacteria from the faecal samples of 71 healthy individuals. In this cohort, 14 were non-secretor individuals and the remainders were secretors. The secretor status is defined by the expression of the ABH and Lewis histo-blood group antigens in the intestinal mucus and other secretions. It is determined by fucosyltransferase 2 enzyme, encoded by the FUT2 gene. Non-functional enzyme resulting from a nonsense mutation in the FUT2 gene leads to the non-secretor phenotype. PCR-DGGE and qPCR methods were applied for the intestinal microbiota analysis. Principal component analysis of bifidobacterial DGGE profiles showed that the samples of non-secretor individuals formed a separate cluster within the secretor samples. Moreover, bifidobacterial diversity (p<0.0001), richness (p<0.0003), and abundance (p<0.05) were significantly reduced in the samples from the non-secretor individuals as compared with those from the secretor individuals. The non-secretor individuals lacked, or were rarely colonized by, several genotypes related to B. bifidum, B. adolescentis and B. catenulatum/pseudocatenulatum. In contrast to bifidobacteria, several bacterial genotypes were more common and the richness (p<0.04) of dominant bacteria as detected by PCR-DGGE was higher in the non-secretor individuals than in the secretor individuals. We showed that the diversity and composition of the human bifidobacterial population is strongly associated with the histo-blood group ABH secretor/non-secretor status, which consequently appears to be one of the host genetic determinants for the composition of the intestinal microbiota. This association can be explained by the difference between the secretor and non-secretor individuals in their expression of ABH and Lewis glycan epitopes in the mucosa.

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

BACKGROUND: Probiotics 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. METHODS: The 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. RESULTS: A 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. CONCLUSIONS: The 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.

Association of symptoms with gastrointestinal microbiota in irritable bowel syndrome.

AIM: To investigate the correlations between self-reported symptoms of irritable bowel syndrome (IBS) and the gastrointestinal (GI) microbiota composition. METHODS: Fecal samples were collected from a total of 44 subjects diagnosed with IBS. Their symptoms were monitored with a validated inflammatory bowel disease questionnaire adjusted for IBS patients. Thirteen quantitative real-time polymerase chain reaction assays were applied to evaluate the GI microbiota composition. Eubacteria and GI bacterial genera (Bifidobacterium, Lactobacillus and Veillonella), groups (Clostridium coccoides/Eubacterium rectale, Desulfovibrio desulfuricans) and distinct bacterial phylotypes [closest 16S rDNA sequence resemblance to species Bifidobacterium catenulatum, Clostridium cocleatum, Collinsella aerofaciens (C. aerofaciens), Coprococcus eutactus (C. eutactus), Ruminococcus torques and Streptococcus bovis] with a suspected association with IBS were quantified. Correlations between quantities or presence/absence data of selected bacterial groups or phylotypes and various IBS-related symptoms were investigated. RESULTS: Associations were observed between subjects' self-reported symptoms and the presence or quantities of certain GI bacteria. A Ruminococcus torques (R. torques)-like (94% similarity in 16S rRNA gene sequence) phylotype was associated with severity of bowel symptoms. Furthermore, among IBS subjects with R. torques 94% detected, the amounts of C. cocleatum 88%, C. aerofaciens-like and C. eutactus 97% phylotypes were significantly reduced. Interesting observations were also made concerning the effect of a subject's weight on GI microbiota with regard to C. aerofaciens-like phylotype, Bifidobacterium spp. and Lactobacillus spp. CONCLUSION: Bacteria seemingly affecting the symptom scores are unlikely to be the underlying cause or cure of IBS, but they may serve as biomarkers of the condition.

Advanced approaches to characterize the human intestinal microbiota by computational meta-analysis.

GOALS: We describe advanced approaches for the computational meta-analysis of a collection of independent studies, including over 1000 phylogenetic array datasets, as a means to characterize the variability of human intestinal microbiota. BACKGROUND: The human intestinal microbiota is a complex microbial community, consisting of several thousands of phylotypes, is specific for each individual, and impacts health and disease. We have developed a phylogenetic microarray, the Human Intestinal Tract Chip, to address the microbial diversity of the intestinal microbiota and used this tool to generate large datasets. It is of significant interest to use these datasets to be able to provide relations between microbial taxa, describe the extent and type of variability of the microbiota in the human gut, and establish relations between microbial taxa and their interaction with the host, intestinal location, or genotype. RESULTS: We present an advanced computational meta-analysis approach for studying human intestinal microbiota, outline the advantages and disadvantages of such a meta-analysis, and reflect it to analogous approaches in other fields. Finally, we illustrate the potential of this meta-analysis by identifying salient signatures of site-specific microbial communities, describe impact of genotype, and provide first examples of relevant relations between microbial taxa. DISCUSSION: We are in the process of designing and applying appropriate methods for carrying out a full meta-analysis of the present data. Beyond that, the next large challenges in future meta-analyses lie in the integration of data from several heterogeneous measurement methods such as next generation sequencing techniques, metaproteomics, or metabolomics. CONCLUSION: We have shown the feasibility of an advanced computational meta-analysis of the large datasets derived from the human intestinal microbiota.

Multivariate multi-way analysis of multi-source data.

MOTIVATION: Analysis of variance (ANOVA)-type methods are the default tool for the analysis of data with multiple covariates. These tools have been generalized to the multivariate analysis of high-throughput biological datasets, where the main challenge is the problem of small sample size and high dimensionality. However, the existing multi-way analysis methods are not designed for the currently increasingly important experiments where data is obtained from multiple sources. Common examples of such settings include integrated analysis of metabolic and gene expression profiles, or metabolic profiles from several tissues in our case, in a controlled multi-way experimental setup where disease status, medical treatment, gender and time-series are usual covariates. RESULTS: We extend the applicability area of multivariate, multi-way ANOVA-type methods to multi-source cases by introducing a novel Bayesian model. The method is capable of finding covariate-related dependencies between the sources. It assumes the measurements consist of groups of similarly behaving variables, and estimates the multivariate covariate effects and their interaction effects for the discovered groups of variables. In particular, the method partitions the effects to those shared between the sources and to source-specific ones. The method is specifically designed for datasets with small sample sizes and high dimensionality. We apply the method to a lipidomics dataset from a lung cancer study with two-way experimental setup, where measurements from several tissues with mostly distinct lipids have been taken. The method is also directly applicable to gene expression and proteomics. AVAILABILITY: An R-implementation is available at http://www.cis.hut.fi/projects/mi/software/multiWayCCA/.

Through ageing, and beyond: gut microbiota and inflammatory status in seniors and centenarians.

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.

Comparative analysis of fecal DNA extraction methods with phylogenetic microarray: effective recovery of bacterial and archaeal DNA using mechanical cell lysis.

Several different protocols are used for fecal DNA extraction, which is an integral step in all phylogenetic and metagenomic approaches to characterize the highly diverse intestinal ecosystem. We compared four widely used methods, and found their DNA yields to vary up to 35-fold. Bacterial, archaeal and human DNA was quantified by real-time PCR, and a compositional analysis of different extracts was carried out using the Human Intestinal Tract Chip, a 16S rRNA gene-based phylogenetic microarray. The overall microbiota composition was highly similar between the methods in contrast to the profound differences between the subjects (Pearson correlations >0.899 and 0.735, respectively). A detailed comparative analysis of mechanical and enzymatic methods showed that despite their overall similarity, the mechanical cell disruption by repeated bead beating showed the highest bacterial diversity and resulted in significantly improved DNA extraction efficiency of archaea and some bacteria, including Clostridium cluster IV. By applying the mechanical disruption method a high prevalence (67%) of methanogenic archaea was detected in healthy subjects (n=24), exceeding the typical values reported previously. The assessment of performance differences between different methodologies serves as a concrete step towards the comparison and reliable meta-analysis of the results obtained in different laboratories.

Diarrhoea-predominant irritable bowel syndrome distinguishable by 16S rRNA gene phylotype quantification.

AIM: To study whether selected bacterial 16S ribosomal RNA (rRNA) gene phylotypes are capable of distinguishing irritable bowel syndrome (IBS). METHODS: The faecal microbiota of twenty volunteers with IBS, subdivided into eight diarrhoea-predominant (IBS-D), eight constipation-predominant (IBS-C) and four mixed symptom-subtype (IBS-M) IBS patients, and fifteen control subjects, were analysed at three time-points with a set of fourteen quantitative real-time polymerase chain reaction assays. All assays targeted 16S rRNA gene phylotypes putatively associated with IBS, based on 16S rRNA gene library sequence analysis. The target phylotypes were affiliated with Actinobacteria, Bacteroidetes and Firmicutes. Eight of the target phylotypes had less than 95% similarity to cultured bacterial species according to their 16S rRNA gene sequence. The data analyses were made with repeated-measures ANCOVA-type modelling of the data and principle component analysis (PCA) with linear mixed-effects models applied to the principal component scores. RESULTS: Bacterial phylotypes Clostridium cocleatum 88%, Clostridium thermosuccinogenes 85%, Coprobacillus catenaformis 91%, Ruminococcus bromii-like, Ruminococcus torques 91%, and R. torques 93% were detected from all samples analysed. A multivariate analysis of the relative quantities of all 14 bacterial 16S rRNA gene phylotypes suggested that the intestinal microbiota of the IBS-D patients differed from other sample groups. The PCA on the first principal component (PC1), explaining 30.36% of the observed variation in the IBS-D patient group, was significantly altered from all other sample groups (IBS-D vs control, P = 0.01; IBS-D vs IBS-M, P = 0.00; IBS-D vs IBS-C, P = 0.05). Significant differences were also observed in the levels of distinct phylotypes using relative values in proportion to the total amount of bacteria. A phylotype with 85% similarity to C. thermosuccinogenes was quantified in significantly different quantities among the IBS-D and control subjects (-4.08 +/- 0.90 vs -3.33 +/- 1.16, P = 0.04) and IBS-D and IBS-M subjects (-4.08 +/- 0.90 vs -3.08 +/- 1.38, P = 0.05). Furthermore, a phylotype with 94% similarity to R. torques was more prevalent in IBS-D patients' intestinal microbiota than in that of control subjects (-2.43 +/- 1.49 vs -4.02 +/- 1.63, P = 0.01). A phylotype with 93% similarity to R. torques was associated with control samples when compared with IBS-M (-2.41 +/- 0.53 vs -2.92 +/- 0.56, P = 0.00). Additionally, a R. bromii-like phylotype was associated with IBS-C patients in comparison to control subjects (-1.61 +/- 1.83 vs -3.69 +/- 2.42, P = 0.01). All of the above mentioned phylotype specific alterations were independent of the effect of time. CONCLUSION: Significant phylotype level alterations in the intestinal microbiotas of IBS patients were observed, further emphasizing the possible contribution of the gastrointestinal microbiota in IBS.