Increase of Faecal Tryptic Activity Relates to Changes in the Intestinal Microbiome: Analysis of Crohn's Disease with a Multidisciplinary Platform

<div><p>Objective</p><p>To investigate—by molecular, classical and functional methods—the microbiota in biopsies and faeces from patients with active Crohn's disease (CD) and controls.</p><p>Design</p><p>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.</p><p>Results</p><p>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.</p><p>Conclusions</p><p>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 <i>Bacteroides</i> in faeces.</p></div

Microflora associated characteristics (MACs) of the CD patients (n = 4) and controls (n = 5). Concentrations of relevant short chain fatty acids (SCFAs) in faeces (mmol/kg) (mean and range).

<p>Microflora associated characteristics (MACs) of the CD patients (n = 4) and controls (n = 5). Concentrations of relevant short chain fatty acids (SCFAs) in faeces (mmol/kg) (mean and range).</p

Oligonucleotides used for preparation of probes.

<p>Oligonucleotides used for preparation of probes.</p

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

<p>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 (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0066074#pone-0066074-g003" target="_blank">Figures 3</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0066074#pone-0066074-g004" target="_blank">4</a>). *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.</p

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

<p>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 <i>Bacteroides thetaiotaomicron</i> VPI-5482.</p