An assessment of bacterial dysbiosis in pouchitis using terminal restriction fragment length polymorphisms of 16S ribosomal DNA from pouch effluent microbiota.
OBJECTIVE
Previous studies on dysbiosis and pouchitis using conventional culture techniques have been disappointing because of inherent limitations associated with the technique. This study was designed to use terminal restriction fragment length polymorphism to evaluate patients with and without pouchitis.


METHODS
Bacterial microbiota in 20 pouch patients (15 healthy and 5 with inflamed) were studied. DNA was extracted from feces, and polymerase chain reaction was performed using primers (V6-V8 region) that were modified at the 5' end with cyanine dyes. Amplicons were digested with merozoite surface protein-1 enzyme. The restricted fragments were analyzed by capillary electrophoresis, and the electrophenograms were studied. Electrophenograms provide information about operational taxonomic units, which correspond to specific organisms. Principal component analysis was performed to identify dominant and important operational taxonomic units in the 20 patients. Bacterial diversity and counts of these operational taxonomic units were compared in the two groups of patients.


RESULTS
Total bacterial diversity in patients with pouchitis was similar to that in patients with healthy pouches (16 (11-20) vs. 12 (9-13), P = 0.279). Using principal component analysis, 29 operational taxonomic units were found to be important. Bacterial counts of seven dominant organisms (operational taxonomic unit 79 (enterococci), 85 (Pantoea), 88 (Enterobacteriaceae), 90 (eubacteria), 91 (Pseudomonas), 146 (clostridia), and 148 (bacilli)) were similar in patients with pouchitis and those with a healthy pouch (P > 0.05). Seventeen (operational taxonomic unit 73 (Leptospira), 93 (Pseudoalteromonas), 96, 100 (Desulfosporosinus), 114, 121, 134, 137, 141 (Microcystis), 159, 174 (Methylobacter), 193 (uncultured proteobacteria), 232, 376, 381, 414, and 465) of the remaining 22 nondominant organisms were seen exclusively in patients with pouchitis. The majority of these organisms were novel.


CONCLUSIONS
Terminal restriction fragment length polymorphism can be used to identify candidate organisms that may be associated with pouchitis.
Lim
Michael
M
John Goligher Colorectal Unit, The General Infirmary at Leeds, Leeds, United Kingdom.
Adams
John D W
JD
Wilcox
Mark
M
Finan
Paul
P
Sagar
Peter
P
Burke
Dermot
D
D000328:Adult; D001419:Bacteria / Q000235:genetics* / Q000254:growth & development / Q000302:isolation & purification; D001424:Bacterial Infections / Q000382:microbiology*; D039021:Colonic Pouches / Q000009:adverse effects / Q000382:microbiology*; D015169:Colony Count, Microbial; D004275:DNA, Ribosomal / Q000032:analysis*; D005260:Female; D006801:Humans; D007413:Intestinal Mucosa / Q000382:microbiology; D008297:Male; D008875:Middle Aged; D016133:Polymerase Chain Reaction; D012150:Polymorphism, Restriction Fragment Length; D019449:Pouchitis / Q000382:microbiology*; D012329:RNA, Bacterial / Q000032:analysis*; D018570:Risk Assessment
D004275:DNA, Ribosomal; D012329:RNA, Bacterial