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Enterococcus faecalis and friends
E. faecalis - Why we
care.
Treatment – How We
Copy Neutrophils
Virulence Factors
Prokaryotic immune system
Primary endodontic casesAvg. 5 microbial speciesPrimarily gram – rods
RetreatsAvg 1.3 speciesUsually gram + facultative cocciE. faecalis 38% -90% of these cases9x 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 yearHas 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)) 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)
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)
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
Gelatinaseextracellular enzyme capable of
hydrolyzing gelatin, collagen and other peptides
Bacteriocin Hemolysi
n
E. faecalis virulence factors
Antibiotic resistance
LTA
Serine protease – cleave proteins
Collagen binding protein (Ace) which helps it bind to dentin
Clumping Response to pheromonesSecretion of Aggregation substanceCell surface becomes stickyConjugation & 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 TransferTypes of gene transfer
TransformationUptake of naked DNA
Transductionbacterophage
ConjugationCell to cell
Agg. Substance & PMNsAS 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 effectiveE. faecalis has a proton pump that can buffer it’s own
cytoplasm in the presence of high alkalinityE. 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
Bleach Mimics Neutrophils
PMN’s & MyeloperoxidasePhagocytosis in part ….
MPO (in neutrophil granulocytes) produces hypochlorous acid (HOCl) from hydrogen peroxide (H2O2) during the neutrophil's respiratory 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
CRISPR – Cas …lots to learn
Treatment – HOCl – Like PMN’s
Virulence Factors - make it a Likely cause of retreats
Thanks Mate!