An overview of the pathogenesis & resistance of Bacteroides fragilis: an invader from the microbiota Professor Sheila Patrick BSc PhD DSc (Edin) Centre for Infection and Immunity Medicine, Dentistry & Biomedical Sciences Queen’s University Belfast European Congress for Clinical Microbiology & Infectious Disease, ESCMIDAmsterdam NL,eLibrary 9th April 2016 by author Outline Bacteroides fragilis – members of the normal human microbiota Mountains of Mourne Northern Ireland – opportunistic pathogens – virulence • Why we should be worried: MDR B. fragilis • Antibiotic resistance – overview – Metronidazole ESCMID• Horizontal gene transfer eLibrary by author Definitions MICROBIOTA = Microbes in an ecological niche Determined by sequencing taxonomic marker genes e.g. 16S rRNA genes (previously by culture) metataxonomics MICROBIOME = Collective microbial genomes in an ecological niche Determined by sequencing all the genetic material ESCMIDmetagenomics eLibrary by author MAJOR BACTERIA & ARCHAEA IN THE ADULT HUMAN FAECAL MICROBIOTA BYBY CULTURECULTURE Bacteria & Archaea Gram reaction Morphology Total viable counta (per g or faeces) Bacteroides - rod 109-1014 Eubacterium + rod 105-1013 Bifidobacterium + rod 105-1013 Clostridium + rod 103-1013 Lactobacillus + rod 104-1013 Peptostreptococcus + coccus 104-1013 Ruminococcus + coccus 105-1013 Streptococcus + coccus 107-1012 Methanobrevibacter + cocco-bacillus 107-1011 Desulphovibrio - rod 105-1011 Fusobacterium - rod 109 Enterococcus + coccus 107 Escherichia coli - rods 107 Prevotella/ - rods 104 Porphyromonas a Compiled from Willis 1991 and Macfarlane and Gibson 1994. Patrick,ESCMID S. 2002. Bacteroides. In: Molecular Medical Microbiology eds eLibraryM Sussman et al. Academic Press. by author Relative abundance -human faeces: metagenomic sequencing From: M Arumugam et al. Nature (2011) doi:10.1038/nature09944 S Patrick, RJ Ingram, T Schneiders & DC Fitzgerald 2016. Microbial regulation of gastrointestinal immunity in health and disease. Chapter 2 in: Neuro-Immuno-Gastroenterology. Constantinescu,ESCMID Cris S., Arsenescu, Razvan, Arsenescu, Violeta (Eds.) Springer, in press.eLibrary by author Outline Bacteroides fragilis – members of the normal human microbiota North Berwick – opportunistic pathogens Scotland – virulence • Why we should be worried: MDR B. fragilis • Antibiotic resistance – overview – Metronidazole • Horizontal gene transfer ESCMID eLibrary by author Bacteroides fragilis Veillon and Zuber 1897-98 Involvement of Gram negative Eiffel tower Paris: strictly anaerobic bacteria completed 1889 in appendicitis and peritonitis B. fragilis : Harry Houdini an ‘escaper’ from the normal microbiota ESCMID eLibrary by author Incidence of Bacteroides spp in faeces and clinical samples _______________________________________________________ aFaeces (%) aClinical samples (%) _______________________________________________________ B. vulgatus 43-45 2-3 B. thetaiotaomicron 15-29 13-17 Parabact. distasonis 9 3-6 B. fragilis 4-13 63-81 B. ovatus 4 0-7 ________________________________________________________ a Compiled from Namavar et al 1989, Willis 1991, Duerden 1980, Brook 1989 Patrick, S. 2002. Bacteroides Molecular Medical Microbiology M Sussman (Ed) Patrick 2015. Bacteroides Chapter 51 In: Molecular Medical Microbiology 2nd Edn. Tang, Sussman, Liu, PoxtonESCMID& Schwartzman (Eds) Academic Press. eLibrary by author Proportions of Bacteroides and related spp. from Wadsworth Anaerobe Collection database (>3000 isolates) P ESCMIDWexler H M Clin. Microbiol. Rev. 2007;20:593-621 eLibrary by author Trauma, Gut Normal microbiota Appendicitis Diverticulitis Peritoneal Normal microbiota - Cancer, Cavity Facultative + Anaerobes Surgery Lymphatic clearance Phagocytosis Host Early peritonitis – Abscess bacteraemia- Response mortality E. coli Streptococcus ‘milleri’ group Facultative e.g. E. coli Fibrin Abscess B. fragilis Formation Bacterial Containment B. fragilis ESCMIDBacteraemia eLibraryB. fragilis by author B. fragilis infections • Intra-abdominal infection B. fragilis abscess pus – Appendicitis, trauma, post- GI surgery • Gynaecological sepsis – post abortion (spontaneous or induced), post-partum, post-surgery, IUD associated • Soft tissue/ abscess Enterotoxicity/ diarrhoea: possible role in children – Diabetic osteomyelitis, brain, perianal abscess... BUT healthy carriers • Bacteraemia – mortality up to ≈30% Role of enterotoxin in infection unclear Non-sporing Gram Negative Anaerobes Patrick, S. & Duerden, B. I. 2006 Principles and Practice of Clinical Bacteriology. Gillespie, S. H. & Hawkey, P. M. (eds.). Wiley Blackwell, p. 541-556 Pantosti A, Malpeli M, Wilks M, Menozzi MG, D’Ambrosio F. Detection of enterotoxigenic Bacteroides fragilis by PCR. J ClinMicrobiol 1997;35:24826. Goldstein EJC 2002. Clinical Infectious Diseases 2002; 35(Suppl 1):S106–11. Wexler, HM Anaerobic Infections: A Clinical Overview Chapter 48 In: Molecular Medical Microbiology 2nd Edn. Tang, Sussman,ESCMID Liu, Poxton & Schwartzman (Eds) Academic Press. eLibrary by author Outline Bacteroides fragilis Birth place of the – members of the normal human microbiota Titanic – opportunistic pathogens – virulence • Why we should be worried: MDR B. fragilis • Antibiotic resistance – overview – Metronidazole •ESCMIDHorizontal gene transfer eLibrary by author Potential virulence determinants • Surface Structures/ Variation – phase and antigenic variation • Attachment • Host component/tissue destruction • Outer Membrane Vesicles • Avoidance/ perturbation of host defence Enterotoxin - not important in • Aerotolerance abscess context? Patrick S 2015. Bacteroides Chapter 51 In: Molecular Medical Microbiology 2nd Edn. Tang, Sussman, Liu, Poxton & Schwartzman (Eds) Academic Press. Wexler H M Clin. Microbiol. Rev. 2007;20:593-621 Patrick,ESCMID S. 2002. Bacteroides Molecular Medical Microbiology. M Sussman (Ed) eLibrary by author Surface structure variation: Phase Variation MICROCAPSULE SMALL CAPSULE LARGE CAPSULE (Reid, Patrick et al 1985, 1987; Lutton, Patrick et al 1991; Patrick et al 1995 PatrickESCMID et al 1999) eLibrary by author Surface structure variation: Antigenic Variation LARGE CAPSULE SMALL CAPSULE MICRO- CAPSULE 8 antigenically Lutton, Patrick et al 1991; Patrick et al 1995 different PatrickESCMID et al 1999 eLibrarymicrocapsules by author Antigenic variation: multiple antigenically different microcapsule polysaccharides PUS SAMPLE within B. fragilis culture AND DURING AN INFECTION BLOOD CULTURE ( LuttonESCMID, Patrick et al 1991; Patrick et al 1995; PatrickeLibrary et al 1999) by author ESCMID eLibrary Microcapsule by author First openly available Bacteroides fragilis complete genome sequence Bacteroides fragilis NCTC9343 Cerdeño-Tárraga, Patrick et al 2005. Extensive DNA inversions in the Bacteroides fragilis genome control variable gene expression Science 307, 1463-1465.ESCMID eLibrary by author Within strain surface antigen diversity generated by: • multiple synthesis loci • invertible promoters • different export mechanisms* • transcriptional anti-terminator inhibition** x8 *Patrick, S et al 2009, 'Mutational analysis of genes implicated in LPS and capsular polysaccharide biosynthesis in the opportunistic pathogen Bacteroides fragilis' Microbiology 155, pp. 1039-1049. **Chatzidaki-Livanis M et al 2009 A family of transcriptional antitermination factors necessary for synthesis of the capsular polysaccharides of Bacteroides fragilis. J Bacteriol 191:728895.ESCMID eLibrary by author Between strain surface antigen diversity generated by: different ‘sets’ of polysaccharide loci ESCMID eLibrary by author B. fragilis variation - summary • DNA inversion major mechanism controlling variation • Multiple variable surface and exported proteins • Multiple separate PS gene clusters generate – Within strain diversity and – Between strain diversity Extensive species pan-genome Evolved due to selective pressure in the GI tract Potential role in virulence ? ESCMID– Immune evasion eLibrary by author Potential virulence determinants • Surface Structures/ Variation – phase and antigenic variation • Attachment adhesion to host cells/tissues, biofilm formation • Host component/tissue destruction mucinase, hyaluronidase, fibrinolysin, DNAase, lipases, neuraminidase, glycosidases, proteases e.g. collagenase type IV, fibrinogenolytic activity Outer Membrane Vesicles Patrick S 2015. Bacteroides Chapter 51 In: Molecular Medical Microbiology 2nd Edn. Tang, Sussman, Liu, Poxton & Schwartzman (Eds) Academic Press. Wexler, HM Anaerobic Infections: A Clinical Overview Chapter 48 In: Molecular Medical Microbiology 2nd Edn. Tang, Sussman, Liu, Poxton & Schwartzman (Eds) Academic Press. Wexler H M Clin. Microbiol. Rev. 2007;20:593-621 ESCMIDPatrick, S. 2002. Bacteroides Molecular Medical Microbiology. M SussmaneLibrary (Ed) by author Trauma, Gut Normal microbiota Appendicitis Diverticulitis Peritoneal Normal microbiota - Cancer, Cavity Facultative + Anaerobes Surgery Lymphatic clearance Phagocytosis Host Early peritonitis – Abscess bacteraemia- Response mortality E. coli Streptococcus ‘milleri’ group Facultative e.g. E. coli Fibrin Abscess B. fragilis Formation Bacterial Containment B. fragilis ESCMIDBacteraemia eLibraryB. fragilis by author Fibrinogen interactions Fibrin clot formation Cleavage of A and B peptides ESCMID eLibrary by author B. fragilis: fibrinogenolysis: MM (kDa) M 0 24 48 M 0 24 48 M 0 24 48 M 0 24 48 64 - α 64 - α 64 - α 64 - α β β β 51 - 51 - β51 - 51 - γ γ γ γ 39 - 39 - 39 - 39 - CONTROL NCTC 9343 638R YCH46 Fibrinogen B. fragilis