Enterococcus faecalis -An Endodontic Challenge

REVIEW ARTICLE
Enterococcus faecalis -An Endodontic Challenge
1 Professor, 2 Lecturer
Department of Conservative Dentistry & Endodontics,
KSR Institute of Dental Science & Research,
KSR Kalvi Nagar, Thiruchengode, Tamil Nadu.
Pin: 637 215
Address for correspondence:
Sebeena Mathew, M.D.S.,
Professor,
Department of Conservative Dentistry &
Endodontics,
KSR Institute of Dental Science & Research,
KSR Kalvi Nagar, Thiruchengode, Tamil Nadu.
Pin: 637 215
Introduction
The objective of endodontic therapy is to
remove diseased tissue, elimination of bacteria
present in the canals and dentinal tubules and
to prevent post - endodontic recontamination
.Enterococcus faecalis has been mentioned with
increased frequency as it has been a challenge
to the very objective of endodontic therapy.
E.faecalis is one of the primary organisms in
patients with post treatment endodontic
infection 1 . The major cause of endodontic
failure is the survival of microorganisms in the
apical portion of root filled teeth. E.faecalis can
adhere to the root canal walls, accumulate, and
form communities organized in biofilm, which
helps it resist destruction by enabling the
bacteria to become 1000 times more resistant
to phagocytosis, antibodies, and antimicrobials
than non-biofilm producing organisms. 2 The
antimicrobial resistance of biofilm bacteria has
Sebeena Mathew 1 Boopathy T 2
Abstract:
Enterococcus faecalis is found in 4 to 40% of primary
endodontic infections. Failed root canal treatment
cases are nine times more likely to contain E.faecalis
than primary endodontic infections. This article
discusses the characteristics of E.faecalis, the factors
responsible for the virulence and survival of
E.faecalis , as well as the effect of disinfectants on
E.faecalis.
Key words:
E.faecalis , virulence factors, disinfectants.
been attributed to the protective barrier
provided by the extracellular polymeric matrix.
The purpose of this article is to discuss various
aspects of E.faecalis and treatment methods
that have been used to counteract this
organism.
Enterococcus faecalis
Enterococci were first placed under genus
streptococcus. In 1984, enterococci were given
a formal genus status after DNA-DNA and DNA-
RNA hybridization studies demonstrated a more
distant relationship with
streptococci. 3 Enterococci are gram positive
facultative anaerobic coccoid bacteria.
Enterococcal cells are ovoid and can occur
singly, in pairs or as short chains. Enterococci
grow at temperatures ranging from 10-45⁰ C, at
pH 9.6 and in 6.5 %NaCl. Enterococci survive at
60⁰C for 30 minutes.
E.faecalis belongs to the same group as
E.faecium, E.casseliflavus, E.mundtii and
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E.gallinarum. E.faecalis ferments mannitol,
sucrose, sorbitol and aesculin and grows on
tellurite bood agar producing black colonies.
E.faecalis posses a group D carbohydrate
cell wall antigen called Lancefield antigen,
which is an intracellular glycerol teichoic acid
associated with the cytoplasmic membrane. The
cell wall contains a large amount of
peptidoglycan and teichoic acid. Peptidoglycan,
a cross linked peptide sugar helps it to
counteract osmotic pressure of the cytoplasm
and provides cell wall strength and shape. It has
a polysaccharide backbone of alternating Nacetyl
glucosamine and N-acetylmuramic acids.
Virulence and survival factors of E.faecalis
E.faecalis possesses certain virulence factors
including lytic enzymes, Aggregation substance
(AS), pheromones and lipoteichoic acid. 4
Aggregation Substance. AS is a 37 KDa surface-
localized protein, a plasmid- encoded adhesin.
The adhesin mediates cell-cell contact which
facilitates the plasmid exchange between
recipient and donor strains. AS plays a role in
the dissemination of plasmid –encoded
virulence factors, protects against
polymorphonuclear leukocyte or macrophage
mediated killing and enhance the virulence by
activating the quorum –sensing mode of
cytolysin regulation.
Surface protein esp. It is a large chromosome
encoded surface protein which has multiple
repeat motifs.A study by Toledo-Arana et al
demonstrated that in the presence of the
surface protein esp, hydrophobicity,biofilm
formation and adherence to biotic surfaces
were increased.
Collagen -binding protein (Ace): It helps
E.faecalis bind to collagen in dentin.
Gelatinase and serine protease. E.faecalis
secretes proteases like gelatinase and serine
protease.Their secretion is autoregulated
through the fsr system.Gelatinase is a non
plasmid-encoded metalloendopeptidase.Gelatinase
contributes to bone
resorption and degradation of dentin organic
matrix,thus playing an important role in
pathogenesis of periapical inflammation.Serine
protease cleaves peptide bonds and was shown
to contribute to the binding of E.faecalis to
dentin.
Cytolysin: It is a plasmid encoded toxin that is
produced by beta-hemolytic E.faecalis isolates
.Cytolysin lyses erythrocytes,
polymorphonuclear neutrophils and
macrophages,kills bacterial cells and may lead
to reduced phagocytosis.
Lipoteichoic acid (LTA):Lipoteichoic acid can be
regarded as a molecule contributing to the
virulence of E.faecalis through the facilitation
of aggregate formation and plasmid
transfer.LTA of E.faecalis was reported to be
doubled in quantity during the viable but non
culturable (VBNC) state suggesting a role for
LTA during this period. 5
Hyaluronidase (spreading factor) is considered
to facilitate the spread of bacteria as well as
their toxins through host tissues. The presence
of microorganisms including E.faecalis in
periapical lesions may also be related to the
activity of a degrading bacterial enzyme such as
hyaluronidase.
Extracellular superoxide: It is associated with
enterococcal virulence, and it has been shown
that its production is significantly higher in
invasive strains than in commensal isolates.
Pheromones:Pheromones from E.faecalis are
chemotactic for human neutrophils and triggers
superoxide production. Antibiotic resistance
and other virulence traits, such as cytolysin
production can be disseminated among strains
of E.faecalis via sex pheromone system.
E.faecalis is a persister due to its ability to
survive under harsh environmental conditions.
It grows in high salt concentrations, wide
temperature range and tolerates a broad pH
range and starves until an adequate nutritional
supply becomes available. Studies have shown
that it might be serum delivered fluid from the
periapical tissues which sustain the microbial
flora. E.faecalis can enter the viable but non
culturable (VBNC) state, a survival mechanism
adopted by a group of bacteria when exposed
to environmental stress and resuccitate upon
returning to favourable conditions. It is small
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enough to invade the dentinal tubules and lives
in the dentinal tubules.Collagen binding protein
help it to bind to the dentin. E.faecalis has the
ability to establish mono-infections in
medicated root canals. The organism has the
ability to acquire, accumulate and share
extrachromosomal elements, encoding
virulence traits, which help to colonize,
compete with other bacteria, resist host
defense mechanisms and produce pathological
changes directly through the production of
toxins or indirectly through the induction of
inflammation.
E.faecalis has the advantage of forming
biofilms. Hence it has a certain degree of
protection and homeostasis. Biofilms grow in a
nutrient -deprived ecosystem as it concentrates
trace elements and nutrients by physical
trapping and electrostatic interaction. The
bacterial cells residing in a biofilm
communicate, exchange genetic materials and
acquire new traits. This communication takes
place by quorum sensing.
E.faecalis resists intra canal medicaments like
calcium hydroxide by maintaining pH
homeostasis.
E.faecalis as an endodontic biofilm.
E.faecalis forms intracanal biofilms,periapical
biofilms and biomaterial centered infection.
Invitro experiments have revealed distinct
stages of the development of E.faecalis biofilm
on root canal dentin. In stage 1, E.faecalis cells
adhered and formed microcolonies on the root
canal dentin surface. In stage 2, they induced
bacterial mediated dissolution of the mineral
fraction from dentine substrate. This localized
increase in calcium and phosphate ions
promotes mineralization (or calcification) of the
E.faecalis biofilm in stage 3. The mature biofilm
structure formed after 6 weeks of incubation
and showed signs of mineralization. The
mineralized E.faecalis biofilm showed a
carbonated –apatite structure as compared to
natural dentin which had carbonated-florapatite
structure. Dentin degradation occurred
in a nutrient deprived state and has been
observed to be a consequence of the
interaction of bacteria and their metabolic
products on dentine. E.faecalis biofilm has
been observed in root canal obturating
materials (biomaterial centered infection).
Disinfectants and E.faecalis
Studies have shown that 3% to full strength
sodium hypochlorite (NaOCl) is effective for all
presentations of E.faecalis including its
existence as a biofilm.
However, many of the studies about the
antibacterial effect of NaOCl against root canal
bacteria are in vitro studies, in either “neutral”
test tube conditions, in root canals of extracted
teeth, or in dentine blocks infected with a pure
culture of one organism at a time. EDTA
(Ethylene diamine tetraacetic acid) when used
along with NaOCl is effective in removing the
inorganic portion of the dentin. Smear layer
removal provides access of irrigants to the
dentinal tubules.
0.1% sodium benzoate solution added to 10%
citric acid solution will increase the chances of
killing E.faecalis.
A 2-min rinse of 2% chlorhexidine liquid can be
used to remove E.faecalis from the superficial
layers of dentinal tubules up to 100 µm. 2%
chlorhexidine gel is effective at completely
eliminating E.faecalis from dentinal tubules for
15 days.
Maria Teresa et al observed in their studies
that it was not possible to eradicate E.faecalis
biofilms using chlorhexidine alone. 6 They found
that the alternating use of chlorhexidine and
cetrimide (0.1% and 0.05%) killed 100% of
E.faecalis biofilm cultures. Cetrimide when
used destructures the EPS (Extra polymeric
substance) matrix. There after, chlorhexidine
was able to react more directly on E.faecalis,
possibly exerting its bactericidal potential to a
greater degree.
Orstavik D and Haapasalo M concluded from
their study that 2% / 4% Iodine, potassium
iodide (IKI) appeared as a more potent irrigant
than sodium hypochlorite and chlorhexidine.
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MTAD (a mixture of tetracycline isomer,acid
and detergent):MTAD is a formulation of
doxycycline, citric acid and Tween 80. Its
effectiveness is attributed to its anticollagenase
activity, low pH and ability to be gradually
released overtime. Mahmoud Torabinejad et al
observed that MTAD is effective against
E.faecalis . 7 MTAD is beneficial for retreatment
cases as well.
In one study a combination of calcium
hyrdroxide and camphorated
paramonochlorophenol completely eliminated
E.faecalis.Metapex (a silicone oil-based calcium
hydroxide paste containing 38% iodoform),
more effectively disinfected dentinal tubules
infected with E.faecalis than calcium hydroxide
alone. 2% chlorhexidine gel when combined
with calcium hydroxide achieves a pH of 12.8
and can completely eliminate E.faecalis within
dentinal tubules. Chlorhexidine and calcium
hydroxide when combined together have
shown better antimicrobial properties than
calcium hydroxide alone. 8
Nair, et al suggested the need of non antibiotic
chemomechanical measures to treat teeth with
infected and necrotic root canals so as to
disrupt the biofilm.
Er:Cr:YSGG laser employing radially emitting
laser tips demonstrated a considerable effect
on bacterial reduction within dentinal tubules
of root canal infected with E.faecalis. 9
Roth 811 sealer ,a zinc –oxide eugenol based
sealer has been shown to exert antibacterial
activity against E.faecalis.AH Plus (epoxy resin
based sealer) and Grossman’s sealer are
effective in killing E.faecalis within infected
dentinal tubules.
Steps that can be considered towards the
elimination and prevention of E.faecalis:
Treatment regimens should aim at prevention
and elimination of E.faecalis during treatment,
inbetween appointments and after completion
of treatment. We can prevent its re-entry by
following certain norms. That includes,
ensuring that the patient rinses with 0 .2%
chlorhexidine prior to the treatment,
disinfecting the tooth and rubber dam with
chlorhexidine or sodium hypochlorite and
disinfecting gutta-percha points with sodium
hypochlorite before insertion in the canal.
Besides the above norms the following can be
followed:
1. Adequate apical preparation
2. Use of canal irrigants such as 6 % sodium
hypochlorite, 17% EDTA and 2% chlorhexidine 4
3. Use of intracanal medicaments such as 2%
chlorhexidine gel or 2% chlorhexidine gel
+calcium hydroxide
4. Consider AH plus or Grossman’s sealer.
5. Proper coronal seal should be given.
Conclusion:
Numerous studies have been conducted on the
effects of various disinfectants on E.faecalis.
Most of them have been invitro. Clinically
E.faecalis still poses to be a challenge.
Endodontists studying root canal infections
need to include endodontic clinical isolates of
E.faecalis to analyze the properties of these
strains which includes putative virulence factors
or antibiotic resistance genes. 10 In order to
develop methods of eradicating E.faecalis from
persistent root canal infections, the mechanism
through which this organism maintains these
infections must be understood. 11
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