Enterococcus faecalis

Literature update
 Background
• Most treatment failures are caused by microorganisms
persisting in the apical parts of root canals of obturated teeth

• Microbiota of treated root canal different from untreated canal

• Enterococcus faecalis a prominent bacteria in treatment failed

root canal

• What are the factors leading to its persistence in root canal

 Evidence of E. faecalis in post treatment root
canals
Studies included for discussion are

1. Molander et al International Endodontic Journal (1998) 31, 1–7

2. Sundqvist et al Oral Surg Oral Med Oral Pathol Oral Radiol Endod
1998;85:86-93

3. Hancock et al Oral Surg Oral Med Oral Pathol Oral Radiol Endod
2001;91:579-86

4. Rôças et al J. Endod 2004; 30:7; 504

 Microbiological status of root-filled teeth with apical
periodontitis
Molander et al International Endodontic Journal (1998) 31, 1–7

Objective
Microbiological status of treatment failed root canals in Swedish population

Materials and methods

Selection of tooth
1. Hundred teeth with sign of apical pathosis (pathology group)
Root canal treated more than 4 years , No clinical symptoms indicating acute
apical periodontitis. Root-fillings end within 5 mm of the radiographic apex.
2. Twenty teeth without sign of apical pathosis (Technical group)
Microbiological sampling

• Root canal- apically enlarged to an ISO size 25 instrument

• Sampling fluid was introduced in the canal

• Canal content was then absorbed and transferred to a transport medium

using charcoal points

Bacteria identification

• By micromorphology, colony morphology, physical and biochemical tests

and by gas chromatography.

• Growth was semiquantified as very heavy, heavy, moderate, sparse and

very sparse on the agar plates, approximately corresponding to >10 000,
<10 000, <1 000, <100 and <10 colony forming units per 0.1ml VMGA III,
respectively.
 Result-pathology group

• Bacteria (microbiota) was detected in

68 of the 100 teeth

• Total number of strains isolated 117

• From most of the canals one or two

strains were isolated (85%)

• There was a predominance of Gram-

positive facultative anaerobes (69%).

• The most frequent - Enterococcus

group in 32 teeth.

• In 20 of these cases growth was

classed as very heavy.
Influence of various factors on microbial
growth
Result- Technical group
 Discussion
Enterococci- the most frequently isolated bacteria
• Engström (1964) reported 9% and Möller (1966) 17%
Difference in the antimicrobial dressing and number of appointments
Altered root canal ecology during treatment
• Use of an intracanal dressing directed specifically towards the anaerobic segment of the
microflora brought about a suitable environment for enterococcal growth (Molander 1990).
• Multiplication of E. faecalis in some canals following standard biomechanical treatment
procedures (Gomes et al. (1996)
Resistance of E. faecalis to calcium hydroxide
• Scandinavian countries- Ca(OH)2 is more often employed as the intracanal medicament
• E. faecalis is inherently resistant to Ca(OH)2 - iodine potassium-iodide more effective in
killing Enterococci?
 Discussion

Apical radiolucency

• Bacteria isolated from 9/20 teeth with absence of an apical radiolucency

• Lack of a visible periapical radiolucency does not necessarily imply the
absence of periapical pathosis
• Apical radiolucency could also be due to extra radicular infection
• Periapical cyst , foreign body reaction, or scar tissue formation, and by
extra radicular infections

Difference in microbiota of treated and untreated root canals

• More of facultative anaerobes in treated root canals

• Facultative anaerobic bacteria are less susceptible to antimicrobial

activities than are anaerobes.
 Conclusion

• All root filled tooth should be considered as potentially infected

• Care should be taken to avoid over instrumentation during retreatment

as it would give chance for the residual population to thrive
(based on clinical data on the association of development of
radiolucencies after retreatment due to over instrumentation)
Microbiologic analysis of teeth with failed endodontic
treatment and the outcome of conservative re-treatment
Sundqvist et al Oral Surg Oral Med Oral Pathol Oral Radiol Endod 1998;85:86-93

Objectives

• To determine the success rate of retreatment and identify factors that

might influence the prognosis.

• to obtain data on the composition of the microbial flora in previously

root-filled teeth with persisting periapical lesions

Materials and Method

Sampling
Fifty-four teeth were selected for re-treatment. Asymptomatic, had been
previously rootfilled, and showed radiographic evidence of periapical bone
lesions.
Crown remover to remove, post, core and crown
The surrounding surface was sterilized before microbiological sampling
First visit
• Sterile saline solution introduced into the canal and instrumented
• Bacteriologic sample taken using charcoaled paper points.
• The root canals were left empty to allow any surviving bacteria in the root canal
to multiply to a level that would be detectable at a subsequent appointment.
• The cavities were sealed with sterile foam pellets and zinc oxide-eugenol
cement at least 4 mm deep.
Second visit
• Second visit 7 days later, bacteriologic samples were taken after removal of the
temporary filling.
• The root canals were then instrumented by means of hand files and irrigation
with 0.5% sodium hypochlorite solution.
• After final irrigation the root canals were filled with calcium hydroxide paste
• The access cavities were sealed with zinc oxideeugenol cement at least 4 mm
thick.
Third visit
• Third appointment 7 to 14 days later, the dressing was rinsed out of the canals
with sterile saline solution.
• The canal was lightly filed to remove loose calcium hydroxide remnants, and a
postmedication sample was taken.

Subsequently the root canals were obturated and restoration was carried out
• The initial sample, taken at the first appointment after the previous root filling had
been removed, was taken in a liquid thioglycolate medium

• At the second appointment three samples were taken from each canal. Two
samples were taken in peptone yeast extract glucose broth (PYG) and one sample
in the agar-supplemented thioglycolate medium.

• All samples were introduced into an anaerobic box with an atmosphere of 10%
hydrogen and 5% carbon dioxide in nitrogen.

• The bacterial isolates were identified according to standard methods.

• The identity of some strains was confirmed by comparison of the mobility of their
soluble proteins in sodium dodecyl sulphate-polyacrylamide gel electrophoresis to
that of reference strains.
Follow-up examination

Patients were recalled yearly for clinical and radiographic examination. At

the recall appointments the type of restoration and any clinical signs or
symptoms associated with the teeth were recorded.

Strindberg's criteria were used to judge the success rate of the

Treatment was considered successful when either the contours, width, and
structure of the periodontal margin were normal or the periodontal contours
were widened mainly around an excess of filling material.
 Result- Microbiological examination

The bacterial species recovered from 24

of 54 canals after removal of the
previous root filling

In 20 cases there was growth from both

the sample taken at the initial
appointment and the sample taken at the
second appointment.

In all nine cases in which Enterococcus

faecalis was isolated, it was the only
microorganism present in the canal.
 Result- success of the re-treatment

• Fifty of the 54 treated cases (93%) were available for recall.

• Thirty-seven of the lesions healed completely
• Thirteen cases were judged as failures
• Success rate 74%

Successful re-treatment Unsuccessful re-treatment

(a) Initial,
(b) 15 months after treatment
(c) 5 years after treatment
 Association between bacterial presence and success of the
treatment
• Eighteen of 24 teeth (75%) from which bacteria were isolated after removal of the
root filling healed completely

• Nineteen of the 26 teeth from which there were no cultivable microorganisms

healed--a success rate of 73%. (bacteria were present or not…cultivation
technique?)
• The difference between the outcomes for the two groups was not statistically
significant.

• The success rate for the teeth from which E. faecalis was isolated after removal of
the earlier root filling was somewhat lower (66%) than the average for the whole
material.

• At the time of root filling, microorganisms were recovered from six root canals.
Four of the lesions associated with these teeth did not heal. (3- E. faecalis, 1- A.
israelii).

• Two factors that were shown to have a negative influence on the prognosis are the
presence of infection at the time of root filling and the size of the periapical lesion.
 Discussion

Bacterial composition differs from

untreated canal

E. faecalis was isolated in 38% of teeth

that had recoverable microorganisms-
suggests that it is an important agent
in endodontic failure.

Survival in re-treated root canal

The capacity to withstand antibacterial
measures, nutrient deprivation and
minimal cooperative relationships
with other bacteria
 Discussion
• E. faecalis enter during the time of treatment?

• Higher proportion of E. faecalis in teeth whose canals lacked an

adequate seal for a period during the treatment or were treated over 10
or more visits (Siren et al 1997)

• When E. faecalis is present in low numbers initially, it can usually be

eliminated; however, once established in the root canal system it is a
difficult organism to eradicate.

• The presence of infection at the time of re-obturation significantly

increased the success rate of teeth undergoing re-treatment. (typo?)

• Teeth that had positive bacteriologic samples at the time of root filling
had a success rate of just 33%, whereas those that had negative samples
had a success rate of 80%.
Bacteria isolated after unsuccessful endodontic treatment in a
North American population
Hancock et al 2001 Oral Surg Oral Med Oral Pathol Oral Radiol Endod 91:579-86)

Objective.
Determine the composition of the microbial flora present in teeth after the failure of
root canal therapy in a North American population.

Molander et al suggested that use of an intracanal dressing other than Ca (OH)2

would reduce the incidence of E. faecalis

Materials and Methods

Fifty-four teeth were selected for retreatment from referrals made to the University
of North Carolina Department of Endodontics. All the re-treated teeth
demonstrated radiographic signs of chronic apical periodontitis and had
completed root canal therapy more than 3 years earlier.
The obturation was removed without using any solvent
The canal content was transferred to liquid dental transport medium uisng paper
point, or by incubating the tip of the K file used for instrumentation of the apical
portion.
The bacteriological samples were plated and incubated anaerobically

E faecalis was detected through use of a bacteroides bile esculin plate.

The growth on agar plates was subcultured for identification on the basis of
anaerobic, facultative anaerobic, or aerobic growth. Gram-stain, micromorphology,
colony morphology, physical and biochemical tests, and selective media were used
to identify both the anaerobic and the aerobic strains.
This study indicated that the occurrence of E faecalis in a North American
population is not significantly different from those reported in the
Scandinavian studies.

Thus, the use of Ca(OH)2 as an intracanal medicament does not appear to

increase the likelihood of cultivating this bacteria in obturated teeth with
persistent apical periodontitis.
 Evidence of E. faecalis in persistent infection in
south Korean population
Rôças et al JOURNAL OF ENDODONTICS, 2004; 30:7; 504

Material and methods

• Fourteen root-filled teeth with persistent periradicular diseases
were selected for retreatment.

• After removal of the root canal filling, the canals were sampled,
and a PCR assay (using taxon-specific oligonucleotide primers)
• Bacteria were present in all cases, as revealed by amplification
using ubiquitous 16S rDNA primers.

Result
• Enterococcus faecalis (64%), Streptococcus spp. (21%) and
Tannerella forsythensis (14%).
E. faecalis in treated root canal

Biofilm
formation

Immune Virulence
modulation factors
The presence of E. faecalis in root canal

Did it enter the root canal during the treatment

or

Did it with stand the treatment

Source of Enterococcus faecalis
• Enterococcal root canal infections could be food-borne
(Razavi et al. Oral Microbiology And Immunology 22 (4): 248-251 Aug 2007).
 E. faecalis in peridontitis
Souto and Colombo. Archives of oral biology, 2007. sep issue)

The role of the oral cavity as a reservoir for this species is unclear, particularly in
the presence of oral infection.

Method
Samples were obtained from 56 periodontally healthy and 169 chronic
periodontitis subjects.

Result
In general, E. faecalis was detected in 34.9% of all samples evaluated.
No significant difference in the prevalence of this species between subgingival
biofilm (34.6%) and saliva (35.1%).
E. faecalis was detected significantly more often in saliva and subgingival
samples of periodontitis patients (40.5% and 47.8%, respectively) compared to
controls (14.6% and 17.1%, respectively; p<0.05).

Conclusion
E. faecalis is frequently detected in the oral microbiota of periodontitis patients
suggesting that periodontal infection may favour the colonization by this
species.
 Microbiological findings and clinical treatment
procedures in endodontic cases selected for
microbiological investigation
Siren et (Haapasalo group) International Endodontic Journal (1997) 30, 91–95
Objective

Study the relationship between clinical treatment procedures and the

occurrence of facultative enteric bacteria in root canal infections.

Microbiological sampling

Microbial samples were sent to the Oral Microbiological Service

Laboratory at the Institute of Dentistry in Helsinki.

The first group consisted of teeth where Enterococcus faecalis and/or

other facultative enteric bacteria or Pseudomonas sp.

The second group consisted of teeth where only non-enteric bacteria

were found.
• The samples taken with sterile forceps using sterile paper points inserted
into the canal at the apical portion of the root canal.
• After sampling the paper points were immediately placed into the transport
medium, VMGA III gel
• Incubated at 37°C in 5% CO2 and anaerobically
• Bacterial colony types were enumerated, isolated and identified.
 Result
Enterococcus faecalis was the most common
finding in the enteric bacteria group.
In 33% of the cases where E. faecalis was
isolated, it appeared as a monoinfection.

Other species most often found together with

enteric bacteria were Streptococcus sp.,
Fusobacterium nucleatum and Prevotella
intermedia

Enteric bacteria were more frequently isolated

in cases with a high number of visits before
sampling

In the cases with 10 or more visits enteric

bacteria were found in 12 out of 13 samples.

If the root canals had been unsealed at some

time during the treatment, enteric bacteria were
found
 Result

35% of the infections caused by

enteric bacteria were monoinfections.

Calcium hydroxide is the most

commonly used root canal
dressing in Scandinavia.

In an earlier study, of the 27 strains

tested the most resistant species to
calcium hydroxide was Enterococcus
faecalis
 Discussion

• The observations of the present study that both a high number of

visits and lack of an adequate seal significantly increased the
probability of finding enteric bacteria in the root canal, indicate that
these bacteria enter the root canal during the treatment.

• In theory, the high number of appointments could also be a sequel to

the presence of enteric bacteria in the root canal and not vice versa.
However, the present study cannot rule out either possibility.

In conclusion
Root canals should not be left open during endodontic therapy and
emphasize the importance of maintaining a high level of asepsis
throughout the treatment.
Antimicrobial susceptibility and
virulence factors
 Effect of endodontic procedures on enterococci,
enteric bacteria and yeasts in primary endodontic
infections
Ferrari et al International Endodontic Journal, 38, 372–380, 2005

Objective
To detect enterococci, enteric bacteria and yeast species in teeth with
primary endodontic infections before and after root canal preparation.

Materials and methods

Twenty-five systemically healthy patients 23–49 years of age
Single roots, intact pulp chambers and showed an asymptomatic apical
periodontitis without communication to the mouth through fistula or
otherwise
Using paper points root bacterial samples were taken from the root canal
before and after the root canal treatment
Sampling was repeated at the second and third visit after removing the
temporary restoration
Plate cultured bacteria were identified using biochemcial assays
 Result

The first sample at the time of intracoronal

access, microorganisms were isolated
from 23/25 (92%) root canals and 22% of
them were infected by enterococci, enteric
bacteria or yeasts and another (4%) by
yeasts.

After 7 days without intracanal dressing,

microorganisms were isolated from 25/25
(100%) root canals (sample 3).
Nine (36%) of these were infected by
enterococci only

After 7 days of intracanal dressing,

microorganisms were recovered (sample
4) from nine (36%) root canals
These strains were identified as E. faecalis
and E. faecium.
 Discussion

• Despite mechanical instrumentation and disinfection of the root canal

system, microorganisms were recovered in five (20%) canals (sample 2)

• clearly showing that root canal preparation is unable to eliminate all

bacteria from the root canal system

• These microorganisms were probably present from the outset in low

numbers that could not be isolated

• It is believed that environmental change due the biomechanical preparation

which eliminated the most sensitive microorganisms, provided better
growth conditions for enterococci, in addition to the fact that enterococci
have the ability to penetrate dentinal tubules
 Discussion

As mechanical instrumentation and irrigation may not eliminate all

microorganisms, it has been emphasized that antimicrobial agents
should be used between appointments

The results support the hypothesis that enterococci do not need any
other interactions tosurvive
 Antimicrobial susceptibility of enterococci
isolated from the root canal
Dahlen et al Oral Microbiol Immunol 2000: 15: 309–312

Objective
Study examined the occurrence of different enterococci in root canal
samples and tested the in vitro antimicrobial susceptibility.

Materials and methods

A total of 29 strains were isolated from 29 endodontic samples at the
Laboratory for Oral Microbiology, Faculty of Odontology, Go¨ teborg
University.

Minimal inhibitory concentrations (MICs) were determined by placing

the E-test strips containing the continous gradient of antibiotics were
placed radially on the surface of the agar plated with bacteria.

Antibiotics tested include:benzylpenicillin, ampicillin, clindamycin,

erythromycin, metronidazole, tetracycline and vancomycin.
 Result

All strains were susceptible to vancomycin (®8 mg/ml)

Four strains were highly resistant to tetracycline (.96 mg/ml), while all
strains were susceptible to erythromycin

All strains were resistant to metronidazole (.256 mg/ml) and most

strains also to clindamycin (25 strains had MIC of >12 mg/ml).
 Discussion
• The lack of sufficient nutrition and the presence of antimicrobial
agents may reduce or eliminate sensitive strains, while more
resistant species such as enterococci may survive
• The multiresistance also includes vancomycin, which has created
treatment problems due to lack of therapeutic alternatives.
• All of the examined strains in this study were susceptible to
vancomycin.
• Does not exclude the possibility that such strains may occur in
endodontic infections in the future.
• It is therefore recommended that susceptibility testing be
performed on enterococcal strains isolated from persisting
endodontic infections.
• The known pathological potential of enterococci recognized from
other body sites and the increasing multiresistance to antibiotics
indicate that the need for microbiological diagnosis on root canal
infections should focus on treatment-resistant bacteria such as
enterococci.
Virulence factors- significant in
enterococcal infection
 Virulence factors

An endodontic disease model

related to virulence factors of E.
faecalis.

Virulence factors help the persistence of E.

faecalis in root canal (gelatinase, Ace, etc)

The virulence factors of the bacterium inside

the dentinal tubules and the root canal are
released to the periradicular area, where they
elicit leukocyte attraction or stimulate
leukocytes to produce inflammatory mediators
or lytic enzymes (AS and gelatinase)
Virulence, phenotype and genotype characteristics of
endodontic Enterococcus spp.
Sedgley et al Oral Microbiology Immunology 2005: 20: 10–19

Objective
Investigated the virulence, phenotype and genotype of 33 endodontic enterococcal
isolates

Materials and method

Thirty-three enterococci recovered from the root canals of patients.

Strains were rehydrated in Todd Hewitt Broth aliquot was plated on THB
agar supplemented with 4% defibrinated horse blood
E. faecalis identified by 16S rRNA analysis
Hemolysin, gelatinase production, bacteriocin production, aggregation
substance and antimicrobial susceptibility were assayed
Virulence genes were detected by PCR
Pulsed-field gel electrophoresis (PFGE) was conducted to assay for plasmid
DNA
 Result

• Thirty-one isolates were identified as E. faecalis

• Two isolates were identified as E. faecium (GS11 and GS20)

• Twenty-three strains (70%) expressed gelatinase activity

• Bacteriocin production was evident in 14 strains (42%)

• None of the isolates expressed hemolysin activity on horse, rabbit, sheep or

bovine blood agar

• All were susceptible to ampicillin, benzylpenicillin, chloramphenicol,

erythromycin, fusidic acid, kanamycin, rifampin, streptomycin, and
vancomycin.
 Result
• An esp gene was present in 20 isolates
(61%), including both E. faecium isolates. A
cylA gene was present in six isolates (18%).
• An aggregation substance gene, asa, was
present in 100% of the isolates.

• PFGE evaluation of genomic DNA showed

genetic polymorphism with evidence of
genetically related E. faecalis isolates (clonal Pulsed field gel electrophoresis of
groups) SmaI-digested genomic DNA from
endodontic E. faecalis strains.
• Plasmid DNA preparations showed that at
least 25 strains contained plasmid DNA,
with one to four plasmids per strain.
• Based on size and restriction pattern, a
similar small (5.1 kb) plasmid occurred in 16
of the isolates.
 Discussion
• Phenotypic tests showed that 23 strains (70%) produced gelatinase.

• Gelatinase activity was expressed in 13/14 (93%) of strains recovered from primary endodontic
infections compared to 2/8 (25%) from retreatment cases.

• Gelatinase are produced by a large proportion of E. faecalis isolated from hospitalized patients
and patients with endocarditis and may contribute to increased severity of endocarditis in
animal models

• Phenotypic testing revealed apparent ‘silent’ genes where the presence of the determinant
using PCR did not correlate with its phenotypic expression

• Expression of gelatinase is regulated by a quorum sensing system encoded by the fsr gene
cluster

• Genes for the adherence factors EfaA protein, and Ace (adhesin of collagen from enterococci)
protein were present in all endodontic strains

• A PCR product representative of aggregation substance,Asa, was found in all endodontic

strains.
 Discussion
• PFGE of endodontic isolates demonstrated genotypic polymorphism with
evidence of several clonal groups, defined as genetically related isolates

• Plasmid DNA was recovered from 25 of the 33 endodontic isolates

• Sixteen strains responded (‘clumped’) to a culture filtrate known to

contain numerous pheromones, suggesting the potential to transfer
plasmid DNA between strains

• Despite differences in geographic location (Sweden vs. United States),

sampling years (1994/1995 vs. 2002/2003) and sample size and
processing, oral and endodontic enterococci demonstrated susceptiblility
to most antibiotics ( are they testing the true mode of bacterial growth ?).

• However, differences included a greater incidence of gelatinase activity

and pheromone response in endodontic strains from Sweden (76% and
42%, respectively) compared to oral rinse strains from the United States
(36% and 9%, respectively)
 Influence of growth phase, environment, and isolate on
the expression of virulence factors
(Hew et al. Systematic and applied microbiology 30 (4): 257-267 2007)

Virulence factors studied

gelE -a metalloproteasethat targets biomolecules
agg -an aggregation protein involved in conjugation
cylBMALlLsI -an operon of genes involved in the production of cytolysin
efaA-a cell surface protein associated with endocarditis isolates
gls24- a general stress protein related to virulence in a rabbit model
esp -an enterococcal surface protein involved in adhesion
Different strains of E. faecalis - virulence factors
associated
 Real time PCR based study

Conditions tested

LB broth – basic environmental condition

Brain Heart Infusion (BHI) - infections of the heart (endocarditis).
Porcine fecal extract - lower intestine (colon)
The fecal sample
Supernatants from E. faecalis strains TMW 2.63 and TMW 2.622 grown
in LB broth to late exponential and stationary phases - presence of
molecules involved in quorum sensing.
A liquid meat – represent conditions in sausage
pH and salt- influence virulence gene expression.
 Result

Virulence gene relative

expression ratios for E.
faecalis TMW 2.63 cells in
’exponential phase and
stationary phase
Virulence gene relative
expression ratios for E.
faecalis TMW 2.622 cells
in exponential phase and
stationary phase
 Discussion

Importance of virulent gene with respect to E. faecalis mediated

infections
• Genes involved in stress response, clpP, clpX and gls24, - higher level of
expression in exponential phase cells compared to stationary phase cells for all
environments examined

• agg and efaA, both produce surface proteins involved in adhesion- Increased
binding to renal tubular cells and fibrin , increased binding and invasion of
enterocytes, as well as the internalization and survival within neutrophils to cause
infection beyond the digestive tract

• efaA- expression is increased upon exposure to serum due to the absence of free
manganese (Mn2+), which is an important micronutrient for E. faecalis
 Discussion

• The absence or limited availability of Mn2+ -environmental signal

for the initialization of genes involved in virulence

• gelE is important for translocation of E. faecalis across a cell

monolayer (Zeng et al)

• Upregulation of gelE in E.faecalis cells exposed to serum (Shepard

and Gilmore )

• Upregulation of virulence factors - result of stress (starvation stress)

 Biofilm Formation in Medicated Root Canals
Distel et al 2002 Journal of endodontics 28 (10) 689-693

The hypothesis that Enterococcus faecalis resists common intracanal medications

by forming biofilms was tested in this study

Biofilm
Biofilm is a microbially derived sessile community characterized by cells that
are irreversibly attached to a substratum or interface or to each other, are
embedded in a matrix of extracellular polymeric substances that they have
produced, and exhibit an altered phenotype with respect to growth rate and
gene transcription. (Donlan and Costerton. Clinical Microbiology Reviews 2002)

Question-can E. faecalis biofilm resist common intracanal medicament and cause chronic
infections
Materials and methods
Prepared teeth (n=46) was filled with Ca(OH)2 paste, inserted with
Ca(OH)2 points or kept without any medicament (control)

A cotton pledget was placed in the reservoir of the test roots in groups
and positive control with 50uL of a pure culture of E. faecalis
Incubation was continued till the bacteria penetrated through the root
canal in to the fresh reservoir below (the assembly)
SEM and SCLM
Protein extration and SDS-PAGE (bacteria that reached the reservoir
chamber to give turbidity)
 Result

In some samples of Ca(OH)2 paste,

fewer colonies were observed than in
Ca(OH)2 points.

Calcium hydroxide paste medication;

Fig. 1B) showed total colonization of all
samples after an average of 77 days of
incubation.
 Result

One root, inoculated for 86 days showed

colonization of both the dentin surface
and the surface of calcium hydroxide.

Root, inoculated for 160 days (Fig.3),

showed colonies forming a mushroom-
shape with apparent water channels

SDS-PAGE showed no notable

difference between bacteria from biofilm
and control.
No difference between bacteria from
medicated and nonmedicated root
canal…?
Discussion
This study demonstrated that in both short-term and long-term
incubation periods, E. faecalis colonized medicated root canals with
possible biofilm formation in the long-term experiments.

the depths of the biofilms measured 28 to 30 m after 160 days of

inoculation and 21 m after 86 days.

If E. faecalis can form biofilms in root canals, this might explain its
ability to persist in that environment.

Compared with planktonic cells, biofilm bacteria are up to 1000-fold

more resistant to phagocytosis, antibodies, and many antibiotics

This study presented evidence of E. faecalis colonization and biofilm

formation in medicated root canals of human teeth.
Aggregation substance- relevance
and working

Potential recipient cells secrete small peptide

pheromones that elicit a specific mating
response by plasmid-carrying donor cells.

Upon exposure to pheromone, donor cells

exhibit a 10,000-fold or greater increase in
plasmid transfer frequency .

This mating response is induced within 20 to

30 min of exposure to pheromone and is
characterized by the synthesis of a surface
adhesin that facilitates the formation of mating
aggregates.

This protein has been termed aggregation

substance
 Plasmid-Associated Hemolysin and Aggregation
Substance Production Contribute to Virulence in
Experimental Enterococcal Endocarditis
Chow et al 1993. Antimicrobial Agents And Chemotherapy 37 2474-2477

The expression of hemolysin-bacteriocin and aggregation activity conferred

by the enterococcal plasmid pAD1 was altered by the insertion of the
erythromycin resistance transposon Tn917 (creating mutants lacking the
As and cytolysin activity)

These cells were used to determine the effects of these properties on

virulence in
enterococcal endocarditis.

Experimental endocarditis, was induced in female New Zealand White

rabbits
 Result

FA2-2 (hemolysin negative, aggregation negative)

FA2-2 containing pAD1 (hemolysin positive, aggregation positive)
pAM9058 (hemolysin negative, aggregation positive)
pAM944 (hemolysin positive, aggregation negative)
pAM947 (hemolysin positive, aggregation negative).
The FA2-2(pAD1) strain was associated with significantly higher mortality
than were all of the other strains tested.

This strain and the hemolysin-negative derivative FA2-2(pAM9058) were

associated with a significantly higher vegetation weight compared with the
aggregation-negative strains.

Showed that aggregation substance and cytolysin have synergistic actions,

which enhance the virulence by activating the quorum-sensing mode of
cytolysin regulation.
 Gelatinase activity and type of obturation on E. faecalis
Survival in root canal
(Sedgley CM. Journal Of Endodontics 33 (5): 561-566 May 2007)

• Tooth obturated with Gele +ve or –ve E. faecalis

• 8 months of incubation at 37 degrees C

• Viable E. faecalis was recovered from more teeth sealed with RoekoSeal (95%)
compared with AH-Plus (40%) (p=0.0004, Fisher's exact test) and Roth's sealer
(45%) (p=0.0012, Fisher's exact test).

• In the RoekoSeal groups, viable counts of E. faecalis OG1RF were higher than E.
faecalis TX5128 (p=0.03, Mann-Whitney U test)
• Suggested that gelatinase activity plays a role in long-term survival of E. faecalis in
obturated root canals.
Response of bacteria to altered root
canal environment
 Starvation-Induced Multiresistance in Enterococcus
faecalis JH2-2
Giard et al Current Microbiology 1996;32; 264–271

Objective

The ability of E. faecalis to develop cross-resistance against five

disparate stresses (heat, ethanol, acid pH, H2O2, and UV
irradiation) during 24 h of glucose starvation.

Material and Methods

Control cells (exponential growth phase cells) and starved cells were
resuspended in fresh semisynthetic medium; 10 ml of each culture
received one of the following treatments:
62°C, (2) 20 mM H2O2, (3) pH 3.7 (adjusted with lactic acid), (4) 17%
(vol/vol) ethanol.
Protein was analysed using 2D gel electrophoresis
Starved bacteria are more
resistant to ethanol, acid, heat,
and oxidative challenge but not
to UV irradiation (Fig. 1)
2-D analysis of starvation-induced proteins.

About 100 of the approximately 312 proteins observed in control cells

showed reduction or even complete inhibition after 24 h of glucose
starvation. On the other hand, at least 22

Control group 24 hours of nutrient

deprivation
 Discussion

The increase of acid resistance in the stationary phase was blocked at any
time of starvation by the addition of chloramphenicol.

This shows that protein synthesis is crucial for the development of acid
tolerance in E. faecalis.

Starvation-induced proteins identified by 2-D gel analysis and their

temporal appearance could be involved in the increased resistance.

Study showed that early stationaryphase protein synthesis is important

for the acquisition of resistance against heat, acid, and oxidative stresses
 Effects of prolonged exposure to alkaline pH on
Enterococcus faecalis survival and specific gene transcripts
Appelbe and Sedgley. Oral Microbiology Immunology 2007: 22: 169–174

Objective

Examine the survival and targeted gene expression of E. faecalis

maintained
in media at pH 7, 10, 11 and 12 for 1 week.

Materials and Methods

E. faecalis JH2-2 cells were suspended in buffered BHI broth at pH

7, 10,
11 or 12 and incubated at either 25C or 37C.

RNA was extracted and RT PCR was conducted at defined time

intervals
 Result

The approximate percentage

survival of cells compared to time
zero after 1 week in alkaline media
was 1% (pH 10), 0.001% (pH 11)
and 0.00001% (pH 12).
At 25C all genes showed a decrease in
transcripts at all time intervals.

At 37C when E. faecalis was grown in

media at pH 7 and pH 10, all genes
except tuf showed an increased level of
transcripts at one or more time intervals
but never at 168 h.
The greatest increase in transcription level involved ftsZ at pH 10 at 37C,
which increased by 37-fold after 120 h.

FtsZ gene involved in cell division increased despite a decrease in log number
of bacteria.
Could be due to the cytoskeleton functional homology between FtsZ and the
eukaryotic protein tubulin the increase in gene trascript could account for
increased cytoskeletal function in the remaining viable cells.

Interestingly, at 25C, or room temperature, all the genes showed a decrease in

level of transcripts at every time interval…probably due to the cell death.

At 37C …Levels of the Na+/H+ antiporter encoding NapA only increased at

120 h in pH 10 medium, suggesting that the internal pH of the cells might have
stabilized, but once the cells adapted, the increased expression of napA may
have no longer been necessary.
 Adhesion to medical device materials and biofilm
formation capability of some species of enterococci in
different physiological states
Lleo et al. FEMS Microbiol Lett 2007 274: 232–237

Most of the studies on cells growing and dividing normally but most of the time
enterococci encountered adverse environments where they may survive by
activating stress-responding mechanisms

Human body would hardly be in an exponentially growing state but would

mostly be in physiological states, such as starvation or the viable but
nonculturable (VBNC) state

The described survival mechanisms allow cells to maintain viability,

pathogenicity and other biological characteristics such as metabolic activity
and gene expression even if they are blocked in their dividing capability.

This study analyzed the ability to adhere and form biofilms in different
enterococcal species and in enterococcal exponentially growing and
nondividing cells responding to adverse environmental conditions.
Material and methods
Twelve enterococcal species and some E. faecalis laboratory and clinical
strains were used in this study.
VBNC state was induced
Known OD of E. faecalis cells was introduced in lake water and was kept
under direct light at 40Cto induce VBNC

Adherence study
Cells were harvested from the microcosms and added at the same
concentration to the biomaterial supports.
After incubation for 15 h at 37 1C, the Petri dishes and slides were washed
three times with PBS. Cells were fixed with Bouin solution (Sigma) and
Gram stained.
Adherent bacterial cells were observed by oil immersion microscopy, and the
mean count was determined in 15 microscopic fields. Each experiment was
repeated three times.

Biofilm assay
These cells are not in VBNC but on the process of induction into VBNC
 Discussion
• Studying pathogenic characteristics in stressed and nonculturable cells
would be of help fordefinitively establishing the possible role of VBNC
bacteria as potential infectious agents capable of causing disease.

• E. faecalis which had reached the VBNC state and lost its culturability is
capable of adhering, although with a reduced efficiency, but is unable to
form a measurable biofilm on the same polystyrene plates.

• Enterococci of medical interest, when in adverse environments, maintain

the ability to form biofilms, for a given period of time before complete
loss of culturability, indicating their capacity to respond to stressing
conditions and protect themselves by creating a biofilm matrix.
 Discussion

The ability to form a biofilm disappears well in advance with respect

to the loss of culturability might be due to changes in the metabolic
activity and gene expression profile of the cells subjected to
environmental conditions leading to the VBNC state, and not to the
loss of their culturability.

This characteristic, support to the hypothesis that enterococcal stressed

cells and even nonculturable forms could play a role as infectious
agents and consequently could represent a risk to human health.
Interaction with immune system
 Enterococcus faecalis Constitutes an Unusual
Bacterial Model in Lysozyme Resistance
Hebert et al Infection and immunity 2007; 75 (11) 5390–5398

• Lysozyme resistance to survive and colonize the host

• Lysozyme is a muramidase that cleaves peptidoglycan (PG) between the

glycosidic beta-1,4-linked residues of N-acetylmuramic acid (NAM) and N-
acetylglucosamine

• Enterococci are resistant to lysozymes

• Two main mechanisms involved in lysozyme resistance have been

characterized for different species of bacteria

• The first mechanism is related to the modification of PG backbone structure

via either O acetylation occurring on the C-6 hydroxyl moiety of the NAM
residues or the deacetylation of the N-acetylglucosamine residues

• The second mechanism is based on the production of lysozyme inhibitors

which have a protective function.
Objective

The causes of the extraordinary resistance to lysozyme of E.

faecalis was investigated.

Two proteins were studied for the lysozyme resistance and

survivla in mouse peritoneal macrophage , the EF_0783 and
EF_1843 proteins that display homology with OatA and PgdA,
respectively.

The role of EF_0783 and EF_1843 on the PG structure of E.

faecalis was also studied
Materials and Methods

• E. faecalis strain JH2-2 (49) and its derivatives were used in the study

• E. faecalis mutants of EF_0783, EF_1843, and EF_0783 EF_1843 were

constructed

• Lysozyme sensitivity assays were performed on 3 ml of Mueller-Hinton

agar (pH 7.4) medium containing different lysozyme concentrations (0, 5,
20, 40, and 60 mg/ml) and potassium tellurite at 0.8 g/ml disposed into
five-by-five comparmented square culture plates

• Survival assays in mouse peritoneal macrophages. Survival of E.

faecalis in mouse peritoneal macrophages was tested by using an in
vivo/in vitro infection

• PG structure analysis. E. faecalis PG structure was analyzed by reverse-

phase high-performance liquid chromatography (RP-HPLC) and matrix-
assisted laser desorption ionization–time of flight (MALDI-TOF) mass
spectrometry
 Result
• The in silico analysis of the genome sequence of E. faecalis V583 strain (GenBank
accession no. AE016830) revealed an open reading frame, EF_0783, showing
significant similarity with an acetyltransferase gene of Staphylococcus aureus

• EF_1843 protein product (301 amino acids) of E. faecalis shares 35% identity
(53% similarity) with PgdA over 204 amino acids.

EF_0783 mutant and the EF_0783 EF_1843

double mutant are more sensitive to lysozyme
than the parental JH2-2
(is there an increased activity of these
enzymes once E. faecalis is subjected to
nutrient deprivation ?)
Time course of intracellular survival of the
E. faecalis and E. coli DH5 strains within
murine peritoneal macrophages.

Mutants were cleared faster than wild

strain

The peptide analysis of PG of bacteria

from wild strain and mutant showed that
EF_0783 gene encodes the E. faecalis PG
O-acetyltransferase. ■E. faecalis JH2-2; ▲ EF_0783 mutant; F,
EF_1843 mutant● EF_0783 ▼EF_1843 double
mutant; □ E. coli DH5.
The peptide analysis of PG in a simulated
macrophage condition also showed the
modification (O-acetylation)
 Discussion

O acetylation of NAM plays a role in controlling the activity of

enterococcal cell wall-hydrolyzing enzymes.

The sensitivity of the non-O-acetylated strains remains relative because

their growths are only slightly inhibited under 20 mg/ml of lysozyme.

Data suggest that EF_0783 is involved in lysozyme resistance but also

that PG O acetylation and de-N-acetylation are not major determinants
for lysozyme resistance of E. faecalis unlike it is the case with other
bacteria

E. faecalis may possesses mechanisms other than PG modification that

inhibit the nonenzymic and/or nonlytic mode of action of lysozyme.