Subjects fulfilling the Rome I criteria for IBS[15] were recruited from the district of Kuopio in Eastern Finland by the Kuopio University Hospital and Harjula Hospital during years 2004-2005. The participants (n = 44; 11 men, 33 women) were 20-72 years old and their general condition was confirmed as good by medical experts (see Table 1 for subject characteristics). Exclusion criteria for participation included presence of organic GI diseases, inadequately treated hypertension or pharmaceutically treated diabetes. Use of statins, pharmaceutically treated hypertension or coronary artery disease were not considered exclusion criteria if medication had been used for at least six months prior to the study with no changes in dosage.

Participants were subjected to a standard medical examination and laboratory tests, including blood cell counts (B-Leuk, B-Trom, B-Eryt), hemoglobin (B-Hb), hematocrit (B-Hcr), erythrocyte mean cell volume (E-MCV), mean cell hemoglobin (E-MCH) and mean corpuscular hemoglobin concentration (E-MCHC), erythrocyte sedimentation rate, glycosylated hemoglobin (B-GHb_Alc) and blood lipids (fS-Chol, fS-Chol-HDL, S-Trigly). Lactose absorption was ensured with a DNA test for a mutation in the lactase gene. The participants were also tested for the presence of IgA antibodies against gliadin, followed by a duodenal biopsy if celiac disease was suspected. Subjects over 45 years were examined for the presence of carcinoembryonic antigen (CEA). In addition, presence of occult blood in the feces was evaluated.

Participants filled in a questionnaire regarding their quality of life and symptoms. The survey was based on an internationally approved and validated questionnaire for inflammatory bowel disease questionnaire (IBDQ)[16]. The questions in the IBDQ were adapted to suit IBS patients. The query contained 30 questions clustered into four groups, comprising “bowel symptoms”, “systemic symptoms”, “social function” and “emotional function”. The seven alternative answers for each question were assigned numerical values from 1 to 7 (1 = no problem at all, 7 = very severe problem). Two questions regarding the form of feces and the frequency of defecation were treated as separate variables. The participants also answered two questions about prior antibiotic treatments and GI infections.

Each subject gave a fecal sample for bacteriological studies. The samples were stored at -80°C prior to analysis. Bacterial DNA was isolated from 1 g of fecal material by removing the undigested particles from the fecal material with three rounds of low-speed centrifugation, collection of the bacterial cells with high-speed centrifugation, enzymatic and mechanical cell lysis and DNA extraction and precipitation[17]. DNA concentrations were measured with the multilabel plate reader Victor3TM (PerkinElmer Life and Analytical Sciences, Boston, MA, USA). With the method, the average yield of DNA was 342 μg/g of fecal material (median: 290 μg/g, SD: 167 μg/g). Quantitative real-time polymerase chain reaction (qPCR) was used to assess the amounts of selected GI bacteria or bacterial phylotypes (Table 2) in the fecal samples.

In total, 13 qPCR assays were performed to analyze the GI microbiota in fecal samples (Table 2). The applied assays targeted quantitatively IBS-associated human GI bacteria (Lactobacillus spp., Clostridium coccoides/Eubacterium rectale-group, Veillonella spp. and Bifidobacterium spp.)[5] and bacterial phylotypes [Collinsella aerofaciens-like, Clostridium cocleatum 88%, Coprococcus eutactus 97%, Ruminococcus torques 91% and Ruminococcus torques (R. torques) 94%][4,18] or bacteria associated with IBS in semi-quantitative sequence data analyses (Bifidobacterium catenulatum/Pseudocatenulatum-like)[4] or with intestinal disturbances according to the literature (Desulfovibrio desulfuricans-group)[19]. The iCycler iQ Real-Time Detection System (Bio-Rad, Hercules, CA, USA) in conjunction with the iCycler Optical System Interface software (version 2.3; Bio-Rad) were used to analyze the samples as described previously[4,5]. Two technical replicates were used for the samples and standard reactions. Depending on the assay, 0.5 or 50 ng of fecal DNA was applied in the reactions.

Basic statistical analysis of the data was performed using the SPSS program, version 14.0 (SPSS Inc., Chicago, IL, USA). R program, version 2.8.0[20] and the package Rcmdr, version 1.4-10[21] were used to perform the principal component analysis (PCA), to describe the categorical data and to statistically test this data. Linear models were used to describe the relationship between variables and were applied to each bacterial genus and phylotype quantified.

The health-related quality of life questionnaire yielded ordinal data; thus, non-parametric statistical methods were used for analysis. However, PCA analysis was performed for the questionnaire data. Results are presented as medians and interquartile ranges. The χ² test was applied to compare nominal data, and the Mann-Whitney U test to compare continuous data when two patient groups were analyzed.

Logarithmic transformation was performed on the bacterial data prior to further analyses. Within the data, undetected abundances possibly caused by technical limitations instead of the absence of the target phylotype were imputed with the mean values obtained from the qPCR runs with the same primer applied to water. If these also were undetected for a certain assay, the minimum of all detected water runs was used. Bacterial qPCR data were also inspected to ascertain the presence or absence of certain phylotypes in the patient samples. Binary data were then used to evaluate whether the phylotypes had any relationship with various traits of the study subjects.

The study protocol was approved by the Kuopio University Hospital Ethical Committee. Participation in the study was voluntary, and patients were allowed to withdraw at any point without giving an explanation.

Characteristics of the IBS patients are listed in Table 1. In general, the clinical studies revealed no major issues regarding the health status of participants. However, of the 44 subjects studied, 7/11 men and 15/33 women had a body mass index (BMI) value above 25, which is considered borderline between normal weight and slightly overweight[22]. Presence of Helicobacter had been confirmed previously in 8 patients; interestingly, in some cases, treatment of the infection had remained incomplete (data not shown). Helicobacter infection or the way it had been treated was not, however, reflected in the symptoms (data not shown). Origin of IBS as a result of GI inflammation was not given support by this study, as only one subject recalled suffering from gastroenteritis but could not remember whether the onset of IBS occurred before or after the ailment.

The modified IBDQ symptom questionnaire consisted of 28 questions divided into the four categories of bowel symptoms, systemic symptoms, social function and emotional function (Table 3). High median values along with a narrow interquartile range can be considered characteristic of the questions central for ascertaining symptoms of IBS (Table 3). Correlations between the four categories were all significant (Table 4).

Abundance and prevalence of the 13 qPCR target bacteria or phylotypes in patient samples are shown in Tables 5 and 6, respectively. An association between R. torques 94% phylotype and symptom scores (emotional function, social function, systemic symptoms, bowel symptoms) was observed in a PCA visualization of the results as they correlated significantly with the same dimension, whereas a weaker negative association was observed for Coprococcus eutactus (C. eutactus) 97%, Bifidobacterium spp., Veillonella spp. and Desulfovibrio desulfuricans (D. desulfuricans)-group and the symptom scores (Figure 1A). When the bowel symptoms (bloating, passing gas, increased need to defecate or need to defecate when bowel is empty, abdominal cramps, abdominal pain, soiling) were analyzed in a PCA, a similar effect was observed: R. torques 94% and all bowel symptoms except soiling correlated with the same dimension, whereas Collinsella aerofaciens (C. aerofaciens)-like, C. eutactus 97%, Veillonella spp., Bifidobacterium spp., and Lactobacillus spp. were negatively associated (Figure 1B).

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Figure 1 Principal component analysis of bacterial data and patient symptom scores. A: Dimension 1 explains 20.4% of the observed variation among test subjects when bacterial data and symptom groups are studied. The four symptom groups have correlations with Dimension 1 of 0.77-0.63, all with P < 0.001, and Ruminococcus torques 94% has a correlation of 0.58, with P < 0.001. Coprococcus eutactus 97%, Bifidobacterium spp., Veillonella spp. and Desulfovibrio desulfuricans-group also present significant, albeit smaller, negative correlations with Dimension 1; B: When the bowel symptom sum is studied question-wise, individual symptoms (except for soiling) as well as Ruminococcus torques 94% show a significant correlation of 0.57-0.75, with P < 0.001, with Dimension 1. Negative associations are observed for Collinsella aerofaciens-like, Coprococcus eutactus 97%, Veillonella spp., Lactobacillus spp. and Bifidobacterium spp.

In linear modeling of continuous data, R. torques 94% was associated with an increase in self-reported bowel symptoms [analysis of variance (ANOVA), P < 0.05]. When the IBS subjects were grouped according to whether R. torques 94% was detected in their fecal samples (Table 7), self-reported bowel symptoms tended to be higher among subjects with R. torques 94% present (ANOVA, P = 0.056). Interestingly, presence of R. torques 94% had a negative effect on the abundance of C. eutactus 97% (P < 0.01), C. aerofaciens-like (P < 0.05) and C. cocleatum 88% (P < 0.05) phylotypes.

No other bacterial associations with symptom scores could be verified. Although in particular the C. aerofaciens-like phylotype had a negative association with R. torques 94%, its relationship with self-reported symptoms remained obscure. The phylotype was, however, associated with lower BMI values (Mann-Whitney test for present-absent data; P < 0.01) and lower blood pressure (Mann-Whitney test for present-absent data for systolic and diastolic blood pressure; P < 0.01 and 0.01, respectively), and the data also suggested a link to lower blood sugar levels (P = 0.06). The C. aerofacien-like phylotype was less frequently observed in subjects with BMI above 25 (Table 6) and none of the six subjects with BMI values over 30 had the C. aerofaciens-like phylotype in their feces. Similary to C. aerofaciens, C. eutactus 97% was also associated with lower blood pressure (P < 0.05 and 0.05 for diastolic and systolic blood pressure, respectively). A positive association was present between the presence of C. aerofaciens and amounts of C. eutactus 97% (P < 0.01), whereas a strong negative effect on R. torques 94% was observed (P < 0.001). In addition, when the IBS subjects were categorized according to their BMI, subjects with a BMI value over 25 had more bifidobacteria than normal-weight subjects, but less lactobacilli in an almost significant manner (Table 5).

Irritable bowel syndrome (IBS) is a common functional gastrointestinal (GI) disorder and results in a reduced quality of life. Alterations in the human GI microbiota have been detected among patients suffering from the syndrome. The abnormalities in the GI microbiota are suggested to contribute to IBS symptoms.

The role of GI microbiota in IBS has been under investigation and studies suggest that microbes associated with IBS possess potential as non-invasive biomarkers. Since the majority of the GI bacteria are uncultivable, molecular methods are crucial in this field and have enabled a deeper study of the disturbed microbiota. The authors examined whether the quantities, or presence or absence, of certain microbes previously linked to either IBS or healthy microbiota, correlated with the symptoms experienced by IBS patients.

Alterations in the overall microbiota and certain microbial phylotypes have been detected in IBS. The results of this study suggest that there is a connection between IBS-related microbiota and severity of self-reported symptoms.

The findings in this study indicate that certain bacterial phylotypes are associated with symptom severity in IBS. These bacteria may serve as biomarkers of the course of the condition.

Human intestinal microbiota is the ensemble of all microbes in the gastrointestinal tract. The term bacterial phylotype stands for an operative taxonomic unit determined by the 16S rRNA gene sequence similarity. Quantitative real-time polymerase chain reaction (qPCR) is a method that enables quantification of target DNA molecules in a sample. In this study 16S rDNA sequences of known bacterial genera and of bacterial phylotypes were quantified using qPCR.

This paper represents a large amount of work. Although the results are largely negative, they should be published, since IBS is such an important issue.