Dominant fecal microbiota in newly diagnosed untreated inflammatory bowel disease patients

Abstract

Our knowledge about the microbiota associated with the onset of IBD is limited. The aim of our study was to investigate the correlation between IBD and the fecal microbiota for early diagnosed untreated patients. The fecal samples used were a part of the Inflammatory Bowel South-Eastern Norway II (IBSEN II) study and were collected from CD patients (n = 30), UC patients (n = 33), unclassified IBD (IBDU) patients (n = 3), and from a control group (n = 34). The bacteria associated with the fecal samples were analyzed using a direct 16S rRNA gene-sequencing approach combined with a multivariate curve resolution (MCR) analysis. In addition, a 16S rRNA gene clone library was prepared for the construction of bacteria-specific gene-targeted single nucleotide primer extension (SNuPE) probes. The MCR analysis resulted in the recovery of five pure components of the dominant bacteria present: Escherichia/Shigella, Faecalibacterium, Bacteroides, and two components of unclassified Clostridiales. Escherichia/Shigella was found to be significantly increased in CD patients compared to control subjects, and Faecalibacterium was found to be significantly reduced in CD patients compared to both UC patients and control subjects. Furthermore, a SNuPE probe specific for Escherichia/Shigella showed a significant overrepresentation of Escherichia/Shigella in CD patients compared to control subjects. In conclusion, samples from CD patients exhibited an increase in Escherichia/Shigella and a decrease in Faecalibacterium indicating that the onset of the disease is associated with an increase in proinflammatory and a decrease in anti-inflammatory bacteria.

Figure 1

Schematic outline of the methodology.

Figure 2

Spectra of the six components resolved by MCR analysis. Visual examination reveal components 2, 5, and 6 ((a)–(c)) to have well resolved spectra with low background sequences. Components 1 and 4 ((d) and (e)) also have well resolved spectra, although with somewhat higher background sequences than components 2, 5, and 6. Component 3 (f) has two high peaks (black arrows) and a poorly resolved spectrum.

Figure 3

Data matrix of the amount of components 1, 2, 4, 5, and 6 in the stool samples. The graded colors indicate the abundance of a particular component in a sample. Red color indicates a high amount of the component, and blue color indicates a low amount of the component. To the left of the matrix, sample numbers are shown together with the diagnosis UC (Ulcerative colitis), CD (Crohn's disease), and Con (control).

Figure 4

Comparison of the average amount of each component in the three disease states. The averages are calculated based on MCR analysis of stool samples from 33 subjects in the control group, 30 subjects diagnosed with CD (Crohn's Disease), and 33 subjects diagnosed with UC (Ulcerative Colitis). Standard error of arithmetic mean is shown. The significance of the differences between the averages of the three groups, control, CD, and UC, was tested using the t-test where the statistical significance was accepted at P < 0.05. Only the statistically significant P values are shown in the figure.

Figure 5

Comparison between the average amount of specific bacteria groups in the different disease states. The averages are calculated based on the height of the probe signals normalized by the signal height of the universal bacterial probe. Further, the averages are normalized to one because of Probe 18-Lactobacillus in particular, which gave much higher signals. There are 34 subjects in the control group, and the CD (Crohn's Disease) and UC (Ulcerative colitis) group have 30 and 33 subjects, respectively. Standard error of arithmetic mean is shown. The significance of the differences between the averages were tested using a t-test where the statistical significance was accepted at P < 0.05. Only the statistically significant P-value is shown in the figure.