Ulcerative colitis and irritable bowel patients exhibit distinct abnormalities of the gut microbiota

Abstract

Background: Previous studies suggest a link between gut microbiota and the development of ulcerative colitis (UC) and irritable bowel syndrome (IBS). Our aim was to investigate any quantitative differences in faecal bacterial compositions in UC and IBS patients compared to healthy controls, and to identify individual bacterial species that contribute to these differences.

Methods: Faecal microbiota of 13 UC patients, 11 IBS patients and 22 healthy volunteers were analysed by PCR-Denaturing Gradient Gel Electrophoresis (DGGE) using universal and Bacteroides specific primers. The data obtained were normalized using in-house developed statistical method and interrogated by multivariate approaches. The differentiated bands were excised and identified by sequencing the V3 region of the 16S rRNA genes.

Results: Band profiles revealed that number of predominant faecal bacteria were significantly different between UC, IBS and control group (p < 10-4). By assessing the mean band numbers in UC (37 ± 5) and IBS (39 ± 6), compared to the controls (45 ± 3), a significant decrease in bacterial species is suggested (p = 0.01). There were no significant differences between IBS and UC. Biodiversity of the bacterial species was significantly lower in UC (μ = 2.94, σ = 0.29) and IBS patients (μ = 2.90, σ = 0.38) than controls (μ = 3.25, σ = 0.16; p = 0.01). Moreover, similarity indices revealed greater biological variability of predominant bacteria in UC and IBS compared to the controls (median Dice coefficients 76.1% (IQR 70.9 - 83.1), 73.8% (IQR 67.0 - 77.5) and 82.9% (IQR 79.1 - 86.7) respectively). DNA sequencing of discriminating bands suggest that the presence of Bacteroides vulgatus, B. ovatus, B. uniformis, and Parabacteroides sp. in healthy volunteers distinguishes them from IBS and UC patients. DGGE profiles of Bacteroides species revealed a decrease of Bacteroides community in UC relative to IBS and controls.

Conclusion: Molecular profiling of faecal bacteria revealed abnormalities of intestinal microbiota in UC and IBS patients, while different patterns of Bacteroides species loss in particular, were associated with UC and IBS.

Figure 1

DGGE profiles and band numbers. (a) DGGE profiles of faecal samples obtained from IBS, UC and control subjects showing the number of bands corresponding to amplicons of the V3 region of 16S rRNA of faecal bacteria. (b) Box plots presenting the median and range of band counts of all faecal samples taken from UC, IBS, and controls. (Boxes have lines at the lower quartile, median, and upper quartile values; whiskers show extent of the rest of the data. Notches display the variability of the median between samples; notches that do not overlap have different medians at the 5% significance level).

Figure 2

Synthetic Standards (SS) alignment. SS lanes from a single typical gel, (a) before alignment and (b) after alignment, showing relative stretching and shrinking of piecewise regions in the bespoke alignment routine. Images of all SS lanes from all gels: (c) before, and (d) after the complete alignment procedure.

Figure 3

Statistical analysis of DGGE profiles. (a) Classification success rate (cross-validated) versus number of PLS dimensions used. A local maximum of ~64% was obtained from a 4-factor model. Also shown are compact boxplots summarizing the results of PLS-DA permutation tests (using y-scrambling, 10,000 re-samples). (b) CVA scores plot derived from PLS scores (4 dimensions) of the DGGE data of faecal bacteria showing the relative positions of the UC, IBS, and control groups. (NB: fully-leave-sample-out cross-validated.)

Figure 4

Bands analysis of DGGE profiles. (a) Absolute values of first PLS loading, peak-picked to show bands that are most responsible for distinguishing between the groups. Greyed area indicates range of this loading across the training segments. (b) Greyscale image view of complete aligned dataset, on the same Rf axis.

Figure 5

Top-ranked discriminating bands. Boxplots summarizing the normalized intensities of the four top-ranked discriminating bands, measured separately for each of the three groups.

Figure 6

Bacteroides specific DGGE profiles. (a) Example of DGGE profiles obtained with Bacteroides specific primes in faecal samples obtained from IBS, UC and control subjects. (b) Boxplots showing band numbers in DGGE profiles of faecal samples taken from UC, IBS, and control.