Role of Intestinal Microbiota in Ulcerative Colitis
Role of Intestinal Microbiota in Ulcerative Colitis Role of Intestinal Microbiota in Ulcerative Colitis
Statistics PCA were generated by SAS JMP data analytical software (version 6.0.2; SAS Institute Inc., North Carolina, USA). Univariate ANOVA was used to analyze differences in ages and differences in DGGE bands of the three groups (version 5.03; GraphPad Software Inc., California, USA). Statistical analysis of the quantitative PCR data was performed with OriginPro software (version 8.1; OriginLab Corporation, Northampton, USA). Data was analyzed by univariate ANOVA. Where ANOVA indicated a significant difference multiple comparisons of means (Fisher’s least significant different test) were used to determine significant differences among disease groups, assuming equal variances. The mean of the triplicate feces sample for each subject was used for the statistical calculations. PCR measurements were log‐transformed before statistical analysis, if the data did not follow a normal distribution. Normal distribution was assessed using D'Agostino & Pearson omnibus normality test (GraphPad Prism version 5.03). Tests were considered statistically significant if P‐values lower than 0.05 were obtained. Results DGGE analysis with universal primers and sequencing of DGGE bands Microbial diversity of fecal samples was assessed by DGGE of 16S rRNA genes. The average number of bands (mean±SD) in the fecal profiles from the three groups (healthy 10.0±2.0, UC patients in remission 9.0±2.7 and UC patients in relapse 9.8±2.7) did not differ significantly (P > 0.10). Additionally, Dice cluster analysis revealed that the triplicates taken from each individual fecal sample were clustered together, demonstrating that the three extractions from each sample were similar (data not shown). Additionally, Dice cluster analysis was performed using one of the triplicate samples from each individual (See additional file 1). The dendrogram from this Dice cluster analysis showed two large clusters revealing that the DGGE profiles from the UC patients are more similar to each other than to the healthy controls. Cluster I contains ten samples all from UC patients, Cluster II contains five samples from healthy controls and two from UC patients, and a single (healthy) sample does not belong to either of the clusters. Part of this effect could be explained by the occurrence of three prominent bands present in either the profile of the healthy 8
controls or the UC patients. Band no. 1 was more prevalent in the profiles of the UC patients in relapse (5 out of 6 individuals) compared to the profiles of the healthy controls (2 out of 6 individuals) and the UC patients in remission (1 out of 6 individuals). Sequencing of the band revealed that it represented a species within the Gram‐negative genus Bacteroides (See additional file 2). Band no. 2 was more prevalent in the healthy control profiles (4 out of 6 individuals) compared to the other two UC profiles (remission; 2 out of 6 individuals and relapse; 2 out of 6 individuals). Sequencing of the band revealed that it represented a species belonging to the gram‐ negative genus Alistipes (See additional file 2). Band no. 3 was more prevalent in the UC profiles (remission; 4 out of 6 individuals and relapse; 4 out of 6 individuals) compared to the profiles of the healthy controls (0 out of 6 individuals). The band was found to represent members of the Gram‐negative genus Prevotella (See additional file 2). Principal component analysis of the relative quantities of the fecal microbiota QPCR was applied for measurement of 16S rRNA gene and 16S‐23S intergenic spacer region content of selected bacterial taxa. The data were analysed using PCA. As seen on Figure 1, the PCA plots of UC patients in remission indicated only low levels of systematic variation, with two patients showing similar profile as the healthy controls, and four patients showing similar profile as the UC patients in relapse. However, a separation between the healthy controls and the UC patients in relapse could be observed from PC1 and PC2 (29.37% and 21.82% of explained variance, respectively). This was attributed to Prevotella spp. in the first direction (PC1) in combination with Bacteroides spp., Bacteroides uniformis, Bacteroides fragilis and Bacteroides thetaiotaomicron versus Bacteroidetes, Bacteroides distasonis, Akk. muciniphila and Lactobacillus spp. in the second direction (PC2). PCA of the Gram‐negative bacteria and Gram‐positive bacteria showed a complete separation between the healthy control group and UC patients in relapse with respect to the composition of the Gram‐negative bacteria (Figure 2). The variation was mainly attributed in the second direction (PC2) with a higher positive score for Bacteroidetes, Akk. muciniphila, Desulfovibrio spp., and Bac. distasonis in the healthy controls, versus Bacteroides spp., Prevotella spp., Bac. thetaiotaomicron and B. uniformis in the UC patients in relapse. PCA of the UC patients in remission showed no clear grouping of the subjects. 9
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controls or the UC patients. Band no. 1 was more prevalent <strong>in</strong> the pr<strong>of</strong>iles <strong>of</strong> the UC patients <strong>in</strong><br />
relapse (5 out <strong>of</strong> 6 <strong>in</strong>dividuals) compared to the pr<strong>of</strong>iles <strong>of</strong> the healthy controls (2 out <strong>of</strong> 6<br />
<strong>in</strong>dividuals) and the UC patients <strong>in</strong> remission (1 out <strong>of</strong> 6 <strong>in</strong>dividuals). Sequenc<strong>in</strong>g <strong>of</strong> the band<br />
revealed that it represented a species with<strong>in</strong> the Gram‐negative genus Bacteroides (See additional<br />
file 2). Band no. 2 was more prevalent <strong>in</strong> the healthy control pr<strong>of</strong>iles (4 out <strong>of</strong> 6 <strong>in</strong>dividuals)<br />
compared to the other two UC pr<strong>of</strong>iles (remission; 2 out <strong>of</strong> 6 <strong>in</strong>dividuals and relapse; 2 out <strong>of</strong> 6<br />
<strong>in</strong>dividuals). Sequenc<strong>in</strong>g <strong>of</strong> the band revealed that it represented a species belong<strong>in</strong>g to the gram‐<br />
negative genus Alistipes (See additional file 2). Band no. 3 was more prevalent <strong>in</strong> the UC pr<strong>of</strong>iles<br />
(remission; 4 out <strong>of</strong> 6 <strong>in</strong>dividuals and relapse; 4 out <strong>of</strong> 6 <strong>in</strong>dividuals) compared to the pr<strong>of</strong>iles <strong>of</strong><br />
the healthy controls (0 out <strong>of</strong> 6 <strong>in</strong>dividuals). The band was found to represent members <strong>of</strong> the<br />
Gram‐negative genus Prevotella (See additional file 2).<br />
Pr<strong>in</strong>cipal component analysis <strong>of</strong> the relative quantities <strong>of</strong> the fecal microbiota<br />
QPCR was applied for measurement <strong>of</strong> 16S rRNA gene and 16S‐23S <strong>in</strong>tergenic spacer region<br />
content <strong>of</strong> selected bacterial taxa. The data were analysed us<strong>in</strong>g PCA. As seen on Figure 1, the PCA<br />
plots <strong>of</strong> UC patients <strong>in</strong> remission <strong>in</strong>dicated only low levels <strong>of</strong> systematic variation, with two<br />
patients show<strong>in</strong>g similar pr<strong>of</strong>ile as the healthy controls, and four patients show<strong>in</strong>g similar pr<strong>of</strong>ile<br />
as the UC patients <strong>in</strong> relapse. However, a separation between the healthy controls and the UC<br />
patients <strong>in</strong> relapse could be observed from PC1 and PC2 (29.37% and 21.82% <strong>of</strong> expla<strong>in</strong>ed<br />
variance, respectively). This was attributed to Prevotella spp. <strong>in</strong> the first direction (PC1) <strong>in</strong><br />
comb<strong>in</strong>ation with Bacteroides spp., Bacteroides uniformis, Bacteroides fragilis and Bacteroides<br />
thetaiotaomicron versus Bacteroidetes, Bacteroides distasonis, Akk. muc<strong>in</strong>iphila and Lactobacillus<br />
spp. <strong>in</strong> the second direction (PC2).<br />
PCA <strong>of</strong> the Gram‐negative bacteria and Gram‐positive bacteria showed a complete separation<br />
between the healthy control group and UC patients <strong>in</strong> relapse with respect to the composition <strong>of</strong><br />
the Gram‐negative bacteria (Figure 2). The variation was ma<strong>in</strong>ly attributed <strong>in</strong> the second direction<br />
(PC2) with a higher positive score for Bacteroidetes, Akk. muc<strong>in</strong>iphila, Desulfovibrio spp., and Bac.<br />
distasonis <strong>in</strong> the healthy controls, versus Bacteroides spp., Prevotella spp., Bac. thetaiotaomicron<br />
and B. uniformis <strong>in</strong> the UC patients <strong>in</strong> relapse. PCA <strong>of</strong> the UC patients <strong>in</strong> remission showed no clear<br />
group<strong>in</strong>g <strong>of</strong> the subjects.<br />
9
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