Temperature gradient gel electrophoresis of fecal 16S rRNA reveals active Escherichia coli in the microbiota of patients with ulcerative colitis

Abstract

Previous studies of the endogenous microbiota in patients with ulcerative colitis (UC) have not taken bacterial activity into account, yet bacteria with high transcriptional activity might have a more important pathophysiological role than inactive bacteria. We therefore analyzed the biodiversity of active bacteria in the fecal microbiota of UC patients, in comparison with that of healthy subjects. Feces were collected from nine patients with active UC and from nine healthy controls. Total DNA and RNA were extracted, and 16S ribosomal DNA and RNA were amplified by PCR and reverse transcription-PCR, respectively. Amplification products were compared by means of temporal temperature gradient gel electrophoresis (TTGE). Bands of interest were excised, sequenced, and identified by comparison with the GenBank database (NCBI). The dominant-species diversity based on RNA-derived TTGE profiles was significantly lower for UC patients than for healthy controls (P = 0.01). The mean similarity index between the "present" and "active" microbiota was 74% +/- 18% for UC patients. Comparison of the individual "active" microbiota identified a band that was present for eight UC patients and only two controls (89% versus 22%; P = 0.008). The band was sequenced for 6 patients and always corresponded to Escherichia coli. The biodiversity of active bacteria in the dominant fecal microbiota of patients with UC is lower than that of healthy subjects. E. coli is more represented in the active microbiota of UC patients. The possible pathophysiological role of this difference remains to be determined.

FIG. 1.

Dendrogram representation of the TTGE profiles of 16S rRNa gene and rRNA amplicons (obtained using primers for the V6 to V8 regions) from fecal samples of nine UC patients and nine healthy controls. The dendrogram represents a statistically optimal representation of the similarities between TTGE profiles based on the matrix of Pearson correlation coefficients and by applying the unweighted pair group method using arithmetic averages. RNA- and DNA-derived TTGE profiles from a given patient did not cluster together, except for two patients with UC (UC1 and UC7). Samples tended to cluster on the basis of their clinical origin (UC versus control).

FIG. 2.

TTGE of 16S rRNA gene and rRNA amplicons (obtained using primers for the V6 to V8 regions) from fecal samples from three UC patients and one control. Right side: similarity indexes (%) of paired samples. Black arrow: band present in the DNA-derived but not the RNA-derived TTGE profile. White arrow: band present in the RNA-derived but not the DNA-derived TTGE profile.

FIG. 3.

TTGE of 16S rRNA amplicons (obtained using primers for the V6 to V8 regions) from fecal samples from nine UC patients and nine controls. One band was present for eight UC patients (framed) and only two controls.