Fecal detection of enterotoxigenic Bacteroides fragilis

Abstract

Bacteroides fragilis is a common colonic symbiote of which one subtype, enterotoxigenic Bacteroides fragilis (ETBF), causes inflammatory diarrhea. However, asymptomatic ETBF colonization is common. Through its primary virulence factor, B. fragilis toxin (BFT), ETBF causes asymptomatic, chronic colitis in C57BL/6 mice and increased colon tumorigenesis in multiple intestinal neoplasia mice. Human studies suggest an association between ETBF infection, inflammatory bowel disease, and colon cancer. Additional studies on ETBF epidemiology are, therefore, crucial. The goal of this study is to develop a reliable fecal diagnostic for ETBF. To develop a sensitive assay for ETBF, we tested multiple protocols on mouse stools spiked with serially diluted ETBF. Each assay was based on either touchdown or quantitative polymerase chain reaction (qPCR) and used primers targeted to bft to detect ETBF. Using touchdown PCR or qPCR, the mean ETBF detection limit was 1.55 × 10(6) colony-forming units (CFU)/g stool and 1.33 × 10(4) CFU/g stool, respectively. Augmentation of Bacteroides spp. growth in fecal samples using PYGB (Peptone Yeast Glucose with Bile) broth enhanced ETBF detection to 2.93 × 10(2) CFU/g stool using the touchdown PCR method and 2.63 × 10(2) CFU/g stool using the qPCR method. Fecal testing using combined culture-based amplification and bft touchdown PCR is a sensitive assay for the detection of ETBF colonization and should be useful in studying the role of ETBF colonization in intestinal diseases, such as inflammatory bowel disease and colon cancer. We conclude that touchdown PCR with culture-based amplification may be the optimal ETBF detection strategy, as it performs as well as qPCR with culture-based amplification, but is a less expensive technique.

Fig. 1

bft-specific touchdown polymerase chain reaction (PCR) and qPCR assays. a–c Touchdown PCR. Representative images from one parallel experiment demonstrating touchdown PCR detection of bft in enterotoxigenic Bacteroides fragilis (ETBF) culture alone (a), ETBF culture spiked into glioblastoma multiforme (GBM) carrier DNA (b), and ETBF culture spiked into mouse stool (c). The numbered lanes refer to the log value in CFU/mL (a, b) or CFU/g of ETBF spiked into mouse stool (c). For example, the lane numbered “7” indicates that there were 10⁷ CFU/mL or 10⁷ CFU/g in that sample. “+” indicates the positive control lane, which contains pure ETBF DNA extract. The underlined numbers demonstrate the last sample where a band can be detected. d–e qPCR amplification of 10-fold dilutions of ETBF culture DNA extracts. d Standard curve generated from our bft-specific qPCR assay. e ΔRn demonstrating the magnitude of the signal generated by the qPCR conditions

Fig. 2

Touchdown PCR demonstrating increased ETBF detection when ETBF-spiked samples are pre-incubated in Peptone Yeast Glucose with Bile (PYGB) broth for 2 days prior to PCR analysis. a In this representative experiment, the detection limit without broth amplification is 3.55 × 10⁴ CFU/g stool. b The assay sensitivity is increased to 3.55 × 10² CFU/g after samples have been incubated for 2 days in PYGB broth, which promotes growth of Bacteroides spp. “+” indicates the positive control lane, which contains an ETBF culture spiked into mouse stool (10⁹ CFU/g). The spiked positive control is also pre-incubated for 2 days in PYGB broth. The numbered lanes refer to the log value in ETBF CFU/g spiked into mouse stool (see also the caption for Fig. 1). The underlined numbers demonstrate the last sample where a band can be detected

Fig. 3

bft detection using touchdown PCR on ETBF-spiked mouse stool stored for 6 months at −80 °C. “+” indicates the positive control lane, which contains ETBF DNA extract. The numbered lanes refer to the log value in CFU/g of ETBF spiked into mouse stool (see also the caption for Fig. 1). “−” is ETBF-negative stool and “ø” is no DNA added