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!