Application of quantitative real-time PCR for rapid identification of Bacteroides fragilis group and related organisms in human wound samples

Abstract

Our goal was to establish a quantitative real-time PCR (QRT-PCR) method to detect Bacteroides fragilis group and related organisms from clinical specimens. Compared to conventional anaerobic culture, QRT-PCR can provide accurate and more rapid detection and identification of B. fragilis group and similar species. B. fragilis group and related organisms are the most frequently isolated anaerobic pathogens from clinical samples. However, culture and phenotypic identification is quite time-consuming. We designed specific primers and probes based on the 16S rRNA gene sequences of Bacteroides caccae, Bacteroides eggerthii, B. fragilis, Bacteroides ovatus, Bacteroides stercoris, Bacteroides thetaiotaomicron, Bacteroides uniformis, Bacteroides vulgatus, Odoribacter splanchnicus (Bacteroides splanchnicus), Parabacteroides distasonis (Bacteroides distasonis) and Parabacteroides merdae (Bacteroides merdae), and detected these species by means of QRT-PCR in 400 human surgical wound infection samples or closed abscesses. The target bacteria were detected from 31 samples (8%) by culture, but from 132 samples (33%) by QRT-PCR (p-value < 0.001). B. uniformis was the most common species (44 positive samples) according to QRT-PCR while culture showed it to be B. fragilis (16 positive samples). Additionally, for each species QRT-PCR detected higher counts than culture did; this may reflect detecting DNA of dead organisms by QRT-PCR. QRT-PCR is a rapid and sensitive method which has great potential for detection of B. fragilis group and related organisms in wound samples.