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
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
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
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
Background Enterococcus faecalis Wide range of conditions can grow at 10°C and 45°C (Sherman (1937))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 (Flahaut et al., 1996a,b,c, 1997)Flahaut et al., 1996abc 1997 Starving cells maintain their viability for extended periods and become resistant to: UV irradiation, heat, sodium hypochlorite, hydrogen peroxide, ethanol, and acid (Giard et al., 1996; Hartke et al., 1998)Giard et al., 1996Hartke et al., 1998
Background Enterococcus faecalis Whether E. 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
E. faecalis virulence factors Bacteriocin – anti-bacterial Hemolysin Gelatinase extracellular enzyme capable of hydrolyzing gelatin, collagen and other peptides Bacteriocin Hemolysin
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
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 Background Virulence transfer in E. faecalis
Gene Transfer Types of gene transfer Transformation Uptake of naked DNA Transduction bacterophage Conjugation Cell to cell
Agg. Substance & PMNs AS promotes opsonin-independent binding to neutrophils (Vanek et 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 (Rakita et al., 1999).
Figure. An endodontic disease model related to virulence factors of E. faecalis. Kayaoglu G, and Ørstavik D CROBM 2004;15:308-320
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)
How bleach works? Hypochlorous acid disrupts oxidative phosphorylation and other membrane-associated activities as well as DNA synthesis Oxidative phosphorylation machinery
PMN’s & Myeloperoxidase Phagocytosis in part …. MPO (in neutrophil granulocytes) produces hypochlorous acid (HOCl) from hydrogen peroxide (H 2 O 2 ) during the neutrophil's respiratory bursthypochlorous acidhydrogen peroxiderespiratory burst
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.
Absence of a CRISPR-Cas immunity 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
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 Nisseria meningitidis resulted in its reduced ability to adhere to, invade, and replicate within human epithelial cells
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….
CRISPR – Cas in 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-Cas is present
CRISPR-Cas & E. faecalis E. faecalis strains with CRISPR – Cas … Less likely to have… antibiotic resistance bacteriocin activity clumping response to pheromones hemolysin gelatinase *Palmer and Gilmore 2010 ; **Sedgley 2013
Conclusions… E. faecalis is Hardy Treatment – HOCl – Like PMN’s Virulence Factors - make it a Likely cause of retreats CRISPR – Cas …lots to learn