Original Article
Copyright ©2009 The WJG Press and Baishideng. All rights reserved.
World J Gastroenterol. Dec 21, 2009; 15(47): 5936-5945
Published online Dec 21, 2009. doi: 10.3748/wjg.15.5936
Diarrhoea-predominant irritable bowel syndrome distinguishable by 16S rRNA gene phylotype quantification
Anna Lyra, Teemu Rinttilä, Janne Nikkilä, Lotta Krogius-Kurikka, Kajsa Kajander, Erja Malinen, Jaana Mättö, Laura Mäkelä, Airi Palva
Anna Lyra, Teemu Rinttilä, Lotta Krogius-Kurikka, Erja Malinen, Janne Nikkilä, Laura Mäkelä, Airi Palva, Department of Basic Veterinary Sciences, Faculty of Veterinary Medicine, University of Helsinki, Helsinki 00014, Finland
Teemu Rinttilä, Alimetrics Ltd., Espoo, Espoo 02920, Finland
Kajsa Kajander, Valio Ltd., Research Centre, Helsinki 00370, Finland; Institute of Biomedicine, Faculty of Medicine, University of Helsinki, Helsinki 00014, Finland
Jaana Mättö, VTT Biotechnology, VTT, Espoo 02044, Finland; The Finnish Red Cross, Blood Service, Helsinki 00310, Finland
Laura Mäkelä, Institute of Clinical Medicine, University of Helsinki, Helsinki 00014, Finland
Author contributions: Lyra A, Rinttilä T, Krogius-Kurikka L, Kajander K, Malinen E, Mättö J and Palva A designed the research; Lyra A, Rinttilä T, Nikkilä J, Krogius-Kurikka L, Kajander K, Malinen E, Mättö J, Mäkelä L and Palva A took part in writing of the manuscript; Kajander K and Mättö J recruited the study subjects and planned and coordinated collection of samples; Lyra A, Rinttilä T and Mäkelä L designed the qPCR assays; Nikkilä J did the data analysis; Mäkelä L, Krogius-Kurikka L, Lyra A and Rinttilä T performed the experiments; Lyra A wrote the paper.
Supported by The Finnish Funding Agency for Technology and Innovation, Tekes, grants No. 945/401/00 and 40160/05, the Finnish Graduate School of Applied Biosciences, the Academy of Finland, Grant No. 214 157 and the Centre of Excellence on Microbial Food Safety Research, Academy of Finland
Correspondence to: Airi Palva, Professor, Department of Basic Veterinary Sciences, Faculty of Veterinary Medicine, University of Helsinki, PO Box 66, Helsinki 00014, Finland. airi.palva@helsinki.fi
Telephone: +358-9-19157058 Fax: +358-9-19157033
Received: August 13, 2009
Revised: September 29, 2009
Accepted: October 6, 2009
Published online: December 21, 2009

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.

Keywords: Irritable bowel syndrome, Diarrhoea-predominant irritable bowel syndrome, Intestinal microbiota, Quantitative real-time polymerase chain reaction, 16S ribosomal RNA