Identification of bacteria from blood in febrile patients with ulcerative colitis by terminal restriction fragment length polymorphism profile analysis of 16S rRNA gene

Abstract

Objective. Recently, highly sensitive molecular-biological approaches using the 16S rRNA gene sequence have been carried out for the detection of bacteria. The aim of this study was to detect bacteremia in febrile patients with ulcerative colitis (UC) using a new molecular approach. Material and methods. Fifteen febrile patients with relapsing UC were enrolled, and 15 healthy volunteers participated as normal controls. Blood samples were analyzed for bacteremia using nested polymerase chain reaction (PCR) with universal primers (27F, 529F, 1492R). We investigated the bacterial DNA by means of terminal restriction fragment length polymorphism (T-RFLP) with five restriction enzymes (Alu I, Hha I, Hae III, Msp I, and Rsa I). A terminal restriction fragment (TRF) profile database was created with the predicted profiles of 63 common bacteria isolated from blood cultures, using computer simulation based on sequence information. TRF lengths were analyzed using the TRF profile database and a T-RFLP profiler. Results. The bacterial gene was detected in 9 out of 15 UC patients (60%) and 8 of out 15 controls (53%). The numbers of Hae III- and Rsa I-digested T-RFs and the average number of five restriction enzyme-digested T-RFs were significantly higher in UC patients than in controls (p=0.0189, 0.0151, 0.0092, respectively). In UC patients, the most prevalent species included the 7 common species in controls and 10 other species. Conclusions. In febrile UC patients with relapse, bacteremia undetected by culture was found at high frequency by the PCR method. Therefore, antibiotic treatment for UC can be approved on the basis of the finding of bacteremia in this study.