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Fecal detection of enterotoxigenic Bacteroides fragilis

European Journal of Clinical Microbiology & Infectious Diseases volume 34, pages 1871–1877 (2015)Cite this article

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 × 106 colony-forming units (CFU)/g stool and 1.33 × 104 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 × 102 CFU/g stool using the touchdown PCR method and 2.63 × 102 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.

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References

  1. Arumugam M, Raes J, Pelletier E, Le Paslier D, Yamada T, Mende DR, Fernandes GR, Tap J, Bruls T, Batto JM, Bertalan M, Borruel N, Casellas F, Fernandez L, Gautier L, Hansen T, Hattori M, Hayashi T, Kleerebezem M, Kurokawa K, Leclerc M, Levenez F, Manichanh C, Nielsen HB, Nielsen T, Pons N, Poulain J, Qin J, Sicheritz-Ponten T, Tims S, Torrents D, Ugarte E, Zoetendal EG, Wang J, Guarner F, Pedersen O, de Vos WM, Brunak S, Doré J; MetaHIT Consortium, Antolín M, Artiguenave F, Blottiere HM, Almeida M, Brechot C, Cara C, Chervaux C, Cultrone A, Delorme C, Denariaz G, Dervyn R, Foerstner KU, Friss C, van de Guchte M, Guedon E, Haimet F, Huber W, van Hylckama-Vlieg J, Jamet A, Juste C, Kaci G, Knol J, Lakhdari O, Layec S, Le Roux K, Maguin E, Mérieux A, Melo Minardi R, M’rini C, Muller J, Oozeer R, Parkhill J, Renault P, Rescigno M, Sanchez N, Sunagawa S, Torrejon A, Turner K, Vandemeulebrouck G, Varela E, Winogradsky Y, Zeller G, Weissenbach J, Ehrlich SD, Bork P (2011) Enterotypes of the human gut microbiome. Nature 473:174–180

    PubMed Central  Article  CAS  PubMed  Google Scholar 

  2. Namavar F, Theunissen EBM, Verweij-Van Vught AMJJ, Peerbooms PGH, Bal M, Hoitsma HFW, Maclaren DM (1989) Epidemiology of the Bacteroides fragilis group in the colonic flora in 10 patients with colonic cancer. J Med Microbiol 29:171–176

    Article  CAS  PubMed  Google Scholar 

  3. Wexler HM (2007) Bacteroides: the good, the bad, and the nitty-gritty. Clin Microbiol Rev 20:593–621

    PubMed Central  Article  CAS  PubMed  Google Scholar 

  4. Sack RB, Myers LL, Almeido-Hill J, Shoop DS, Bradbury WC, Reid R, Santosham M (1992) Enterotoxigenic Bacteroides fragilis: epidemiologic studies of its role as a human diarrhoeal pathogen. J Diarrhoeal Dis Res 10:4–9

    CAS  PubMed  Google Scholar 

  5. Sears CL, Islam S, Saha A, Arjumand M, Alam NH, Faruque ASG, Salam MA, Shin J, Hecht D, Weintraub A, Sack RB, Qadri F (2008) Association of enterotoxigenic Bacteroides fragilis infection with inflammatory diarrhea. Clin Infect Dis 47:797–803

    PubMed Central  Article  CAS  PubMed  Google Scholar 

  6. Prindiville TP, Sheikh RA, Cohen SH, Tang YJ, Cantrell MC, Silva J Jr (2000) Bacteroides fragilis enterotoxin gene sequences in patients with inflammatory bowel disease. Emerg Infect Dis 6:171–174

    PubMed Central  Article  CAS  PubMed  Google Scholar 

  7. Basset C, Holton J, Bazeos A, Vaira D, Bloom S (2004) Are Helicobacter species and enterotoxigenic Bacteroides fragilis involved in inflammatory bowel disease? Dig Dis Sci 49:1425–1432

    Article  PubMed  Google Scholar 

  8. Toprak NU, Yagci A, Gulluoglu BM, Akin ML, Demirkalem P, Celenk T, Soyletir G (2006) A possible role of Bacteroides fragilis enterotoxin in the aetiology of colorectal cancer. Clin Microbiol Infect 12:782–786

    Article  CAS  PubMed  Google Scholar 

  9. Boleij A, Hechenbleikner EM, Goodwin AC, Badani R, Stein EM, Lazarev MG, Ellis B, Carroll KC, Albesiano E, Wick EC, Platz EA, Pardoll DM, Sears CL (2015) The Bacteroides fragilis toxin gene is prevalent in the colon mucosa of colorectal cancer patients. Clin Infect Dis 60:208–215

    Article  PubMed  Google Scholar 

  10. Sears CL (2009) Enterotoxigenic Bacteroides fragilis: a rogue among symbiotes. Clin Microbiol Rev 22:349–369

    PubMed Central  Article  CAS  PubMed  Google Scholar 

  11. Pantosti A, Malpeli M, Wilks M, Menozzi MG, D’Ambrosio F (1997) Detection of enterotoxigenic Bacteroides fragilis by PCR. J Clin Microbiol 35:2482–2486

    PubMed Central  CAS  PubMed  Google Scholar 

  12. Shetab R, Cohen SH, Prindiville T, Tang YJ, Cantrell M, Rahmani D, Silva J Jr (1998) Detection of Bacteroides fragilis enterotoxin gene by PCR. J Clin Microbiol 36:1729–1732

    PubMed Central  CAS  PubMed  Google Scholar 

  13. Zhang G, Weintraub A (1998) Rapid and sensitive assay for detection of enterotoxigenic Bacteroides fragilis. J Clin Microbiol 36:3545–3548

    PubMed Central  CAS  PubMed  Google Scholar 

  14. Rhee KJ, Wu S, Wu X, Huso DL, Karim B, Franco AA, Rabizadeh S, Golub JE, Mathews LE, Shin J, Sartor RB, Golenbock D, Hamad AR, Gan CM, Housseau F, Sears CL (2009) Induction of persistent colitis by a human commensal, enterotoxigenic Bacteroides fragilis, in wild-type C57BL/6 mice. Infect Immun 77:1708–1718

    PubMed Central  Article  CAS  PubMed  Google Scholar 

  15. Kato N, Liu CX, Kato H, Watanabe K, Tanaka Y, Yamamoto T, Suzuki K, Ueno K (2000) A new subtype of the metalloprotease toxin gene and the incidence of the three bft subtypes among Bacteroides fragilis isolates in Japan. FEMS Microbiol Lett 182:171–176

    Article  CAS  PubMed  Google Scholar 

  16. Apfalter P, Assadian O, Blasi F, Boman J, Gaydos CA, Kundi M, Makristathis A, Nehr M, Rotter ML, Hirschl AM (2002) Reliability of nested PCR for detection of Chlamydia pneumoniae DNA in atheromas: results from a multicenter study applying standardized protocols. J Clin Microbiol 40:4428–4434

    PubMed Central  Article  CAS  PubMed  Google Scholar 

  17. Perez IC, Le Guiner C, Ni W, Lyles J, Moullier P, Snyder RO (2013) PCR-based detection of gene transfer vectors: application to gene doping surveillance. Anal Bioanal Chem 405:9641–9653

    Article  CAS  PubMed  Google Scholar 

  18. Gould LH, Bopp C, Strockbine N, Atkinson R, Baselski V, Body B, Carey R, Crandall C, Hurd S, Kaplan R, Neill M, Shea S, Somsel P, Tobin-D’Angelo M, Griffin PM, Gerner-Smidt P; Centers for Disease Control and Prevention (CDC) (2009) Recommendations for diagnosis of shiga toxin-producing Escherichia coli infections by clinical laboratories. MMWR Recomm Rep 58:1–14

    PubMed  Google Scholar 

  19. Wu S, Rhee KJ, Albesiano E, Rabizadeh S, Wu X, Yen HR, Huso DL, Brancati FL, Wick E, McAllister F, Housseau F, Pardoll DM, Sears CL (2009) A human colonic commensal promotes colon tumorigenesis via activation of T helper type 17 T cell responses. Nat Med 15:1016–1022

    PubMed Central  Article  CAS  PubMed  Google Scholar 

  20. Wick EC, Rabizadeh S, Albesiano E, Wu X, Wu S, Chan J, Rhee KJ, Ortega G, Huso DL, Pardoll D, Housseau F, Sears CL (2014) Stat3 activation in murine colitis induced by enterotoxigenic Bacteroides fragilis. Inflamm Bowel Dis 20:821–834

    PubMed Central  Article  PubMed  Google Scholar 

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Acknowledgments

This work was supported by T32 DK007632-23 (National Institutes of Health) and a Clinical Research Award from the American College of Gastroenterology to LAC and R01CA151393 and R01CA151325 (National Institutes of Health) to CLS.

Conflict of interest

The authors declare that they have no conflict of interest.

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Affiliations

  1. Department of Medicine, Johns Hopkins School of Medicine, CRB2 Bldg, Suite 1M.05, 1550 Orleans Street, Baltimore, MD, 21231, USA

    L. A. Chen, S. Van Meerbeke, E. Albesiano, A. Goodwin, S. Wu & C. Sears

  2. Sidney Kimmel Comprehensive Cancer Center, Department of Oncology, Johns Hopkins School of Medicine, CRB1 1650 Orleans Street, Baltimore, MD, 21287, USA

    H. Yu

  3. Department of Pathology, Johns Hopkins School of Medicine, Meyer B1-193, 600 North Wolfe Street, Baltimore, MD, 21287, USA

    K. Carroll

Authors
  1. L. A. Chen
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  2. S. Van Meerbeke
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  3. E. Albesiano
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  4. A. Goodwin
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  5. S. Wu
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  6. H. Yu
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  7. K. Carroll
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  8. C. Sears
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Correspondence to C. Sears.

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Chen, L.A., Van Meerbeke, S., Albesiano, E. et al. Fecal detection of enterotoxigenic Bacteroides fragilis . Eur J Clin Microbiol Infect Dis 34, 1871–1877 (2015). https://doi.org/10.1007/s10096-015-2425-7

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  • DOI: https://doi.org/10.1007/s10096-015-2425-7

Keywords

  • Brain Heart Infusion
  • Touchdown Polymerase Chain Reaction
  • Peptone Yeast Glucose
  • Inflammatory Diarrhea
  • Multiple Intestinal Neoplasia