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Enterococcus faecalis and friends

Enterococcus faecalis and friends. Primary endodontic cases Avg. 5 microbial species Primarily gram – rods Retreats Avg 1.3 species Usually gram + facultative cocci E. f aecalis 38% -90% of these cases 9x more likely

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Enterococcus faecalis and friends

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  1. Enterococcus faecalis and friends

  2. Primary endodontic cases • Avg. 5 microbial species • Primarily gram – rods • Retreats • Avg 1.3 species • Usually gram + facultative cocci • E. faecalis 38% -90% of these cases • 9x more likely • Higher % found with PCR detection (67-77% than with culturing methods

  3. Enterococcus faecalis • Facultative anaerobe • Gram-positive cocci • Widely distributed in nature, animals & humans • Endodontic • Oral – considered normal flora, but prob. not in every mouth • GI

  4. Why E. faecalis so prevalent in retreats? • Can adhere to collagen in the presence of serum - Love • Can invade dentinal tubules – Love • Proton pump – Evans • Nutrient deprived environment up to 1 year • Has ability to go semi-dormant – Sedgley • Can form biofilms – Distil

  5. Biofilms – a quick look • Unique ability to calcify in root canal environment – “shelter” • Biofiom infers 1000 times more resistance to phagocytosis, antibodies, and antimicrobials than non-biofilm producers

  6. BackgroundEnterococcus faecalis • Wide range of conditions • can grow at 10°C and 45°C (Sherman (1937)) • survive at 60°C for 30 minutes • High pH – can persist up to pH 11.5 • 6.5% NaCl (salt) broth • Following pre-exposure to sublethal stress conditions, can become less sensitive to normally lethal conditions • sodium dodecyl sulfate, bile salts, hyperosmolarity, heat, ethanol, hydrogen peroxide, acidity, and alkalinity (Flahautet al., 1996a,b,c, 1997) • Starving cells maintain their viability for extended periods and become resistant to: • UV irradiation, heat, sodium hypochlorite, hydrogen peroxide, ethanol, and acid (Giardet al., 1996; Hartkeet al., 1998)

  7. BackgroundEnterococcus faecalis • WhetherE. faecalis can ‘‘cause’’ periradicular infections has not been established. But we know it has virulence factors… *Sedgley et al. 2004, 2005 & 2006, Rocas et al 2004, Zehnder 2009 **Sedgley et al. 2005 & 2007 ***Reynaud et al. 2007, Sedgley et al. 2004

  8. E. faecalis virulence factors • Bacteriocin – anti-bacterial • Hemolysin • Gelatinase • extracellular enzyme capable of hydrolyzing gelatin, collagen and other peptides Bacteriocin Hemolysin

  9. E. faecalis virulence factors • Antibiotic resistance • LTA • Serine protease – cleave proteins • Collagen binding protein (Ace) which helps it bind to dentin • Clumping Response to pheromones • Secretion of Aggregation substance • Cell surface becomes sticky • Conjugation & DNA transfer easier

  10. BackgroundVirulence transfer in E. faecalis • Aggregation Substance is one of the keys to virulence of E. faecalis • Big part of conjugation and DNA transfer via plasmids • At the heart of why we care about E. faecalis …. • Biofilm Pheromones Aggregation substance conjugation virulence transfer *Dunny et al. 1979

  11. Gene Transfer • Types of gene transfer • Transformation • Uptake of naked DNA • Transduction • bacterophage • Conjugation • Cell to cell

  12. Agg. Substance & PMNs • AS promotes opsonin-independent binding to neutrophils (Vaneket al.) • Encouraging the neutrophil to attempt phagocytosis • Meanwhile AS slows phagocytosis (mechanism unknown) • E. faecalis bearing AS was shown to be resistant to killing by human neutrophils despite neutrophil activation (Rakitaet al., 1999).

  13. Figure. An endodontic disease model related to virulence factors of E. faecalis. Kayaoglu G , and Ørstavik D CROBM 2004;15:308-320

  14. How to treat it? • Full strength bleach is the fastest – Siquiera • Calcium hydroxide less effective • E. faecalis has a proton pump that can buffer it’s own cytoplasm in the presence of high alkalinity • E. faecalis killed in 30 seconds by 5.25% solution (in vitro)

  15. How bleach works? • Hypochlorous acid disrupts oxidative phosphorylation and other membrane-associated activities as well as DNA synthesis Oxidative phosphorylation machinery

  16. Bleach Mimics Neutrophils

  17. PMN’s & Myeloperoxidase • Phagocytosis in part …. • MPO (in neutrophil granulocytes) produces hypochlorous acid (HOCl) from hydrogen peroxide (H2O2) during the neutrophil's respiratory burst

  18. CRISPR-Cas • The recently discovered (~2007) Clustered Regularly Interspaced Short Palindromic Repeats (CRISPRs) and genes encoding CRISPR-associated (Cas) proteins, are a prokaryotic immune system widespread among archaea and bacteria, present in almost all archaea and 40% of bacteria. • The ‘‘health’’ of a microorganism may be related to the presence of its own protective ‘‘immune’’ system. • CRISPR-Cas confers resistance to mobile genetic elements, such as viruses (phages), plasmids and transposons carrying antibiotic resistance genes or virulence traits.

  19. Absence of a CRISPR-Casimmunity system might facilitate bacterial cell survival under certain conditions, e.g. by allowing uptake of antibiotic resistance genes in an antibiotic environment…. • …but could also render the cell more vulnerable to attack by other selfish genetic elements (e.g. phages).* *Takeuchi et al. 2012

  20. If it stops the bug from getting antibiotic resistance etc., then how does it help the bug? • Love-hate relationship between bacterial pathogens and their CRISPR-Cas systems. • On the one hand, it reduces the evolvability of the pathogen. • On the other, CRISPR-Cas systems can be repurposed to regulate gene expression and enhance pathogenesis. • Also, many viruses (phages) tell a bug to replicate and die. • For example, deletion of cas9 from the Type II CRISPR-Cas system in Nisseriameningitidis resulted in its reduced ability to adhere to, invade, and replicate within human epithelial cells

  21. CRISPR-Cas & E. faecalis • Not every strain has CRISPR-cas • Oral & endodontic strains found to be more likely to have CRISPR-cas than highly virulent hospital strains (pt’s with nosocomial infections) • For Example….

  22. CRISPR – Casin E. faecalis • V583 – (antibiotic-resistant E. faecalis strain from hospitalized pt) • More than a quarter of genome consists of mobile or foreign DNA • CRISPR-Cas is absent • OG1RF - oral strain • Almost no mobile genetic elements are found • CRISPR-Casis present

  23. CRISPR-Cas & E. faecalis • E. faecalisstrains with CRISPR – Cas … • Less likely to have… • antibiotic resistance • bacteriocin activity • clumping response to pheromones • hemolysin • gelatinase *Palmer and Gilmore 2010 ; **Sedgley 2013

  24. Conclusions…

  25. Thanks Mate!

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