Increase of faecal tryptic activity relates to changes in the intestinal microbiome: analysis of Crohn's disease with a multidisciplinary platform

Abstract

Objective: To investigate-by molecular, classical and functional methods-the microbiota in biopsies and faeces from patients with active Crohn's disease (CD) and controls.

Design: The microbiota in biopsies was investigated utilizing a novel molecular method and classical cultivation technology. Faecal samples were investigated by classical technology and four functional methods, reflecting alterations in short chain fatty acids pattern, conversion of cholesterol and bilirubin and inactivation of trypsin.

Results: By molecular methods we found more than 92% similarity in the microbiota on the biopsies from the two groups. However, 4.6% of microbes found in controls were lacking in CD patients. Furthermore, NotI representation libraries demonstrate two different clusters representing CD patients and controls, respectively. Utilizing conventional technology, Bacteroides (alt. Parabacteroides) was less frequently detected in the biopsies from CD patients than from controls. A similar reduction in the number of Bacteroides was found in faecal samples. Bacteroides is the only group of bacteria known to be able to inactivate pancreatic trypsin. Faecal tryptic activity was high in CD patients, and inversely correlated to the levels of Bacteroides.

Conclusions: CD patients have compositional and functional alterations in their intestinal microbiota, in line with the global description hypothesis rather than the candidate microorganism theory. The most striking functional difference was high amount of faecal tryptic activity in CD patients, inversely correlated to the levels of Bacteroides in faeces.

Figure 1. Hybridization of control and CD patients “NotI representation” libraries to oligonucleotide-probes in biopsies.

Numbers reflect strength of signal after hybridization. Corresponding bacterial names are shown in the bottom row. Ruminococcus flavefaciens demonstrating the largest difference in the signal strength is shown in grey. The signals were clustered into a dendrogram using Euclidean distances depicted to the left. Crohn patients (C1–C4); controls (H1–H5).

Figure 2. Bacterial DNA unique to Crohn patients (C) or controls (H) as demonstrated by PCR-amplification using primers according to table 2.

PCR group 1 bacterial DNA was found in all four controls, while groups 3–5 and VPI were only detected in biopsy samples from the Crohn patients. By sequencing of the PCR-products (not shown) it could be shown that groups 1–5 represent bacteria not previously identified, while VPI represents Bacteroides thetaiotaomicron VPI-5482.

Figure 3. Cfu∶s of Bacteroides spp. and of E. coli per biopsy or per gram faeces.

CD patients versus controls. Horisontal line depicts mean number of log cfu∶s ( = geometric mean of cfu∶s) and the upper T depicts SEM. Note that three out of five biopsies from CD patients did not show any growth of Bacteroides, while all control biopsies did (p = 0.054, Mann-Whitney).

Figure 4. Levels of three MAC∶s in faecal samples from Crohn patients versus healthy controls.

Horizontal line depicts mean value.

Figure 5. Relation between log nr of Bacteroides in faeces and faecal tryptic activity (FTA).

Numbers denote FTA activity, p-value = 0.0011 (Person). It is to be noted that all control samples had high numbers of Bacteroides and low levels of FTA (Figures 3 and 4). *denotes a CD patient with ileo-caecal resection, why he was not included in other data in this manuscript. One CD patient was not included since his faecal sample was not subjected to cultivation for technical reasons.