E.

faecalis also have a spesific protein that could be detected; attributed to its
presence in a sample. SDS PAGE method may be used to see materials protein profile.
Application of calsium hydroxide and PDT laser will change the protein profile and show
the sensitivity of E.faecalis towards irrigation material which is combined with laser beam.
To eliminate E.faecalis that becomes persistent bacteria in the root canal, we should
know its genotype, phenotype i.e protein profile changes, polysachharide capsule profile,
and bacterial sensitivity to PDT laser and calcium hydroxide.

Kerangka Teori
Persistent Intra Radicular
Bacterium : 5.7.132
E. faecalis, Candida albicans
Dental morphology:70

Accessory canal, curved canal,

iregular canal wall, lateral canal,
UNIVERSITAS
INDONESIA
dead-end
canal, isthmus
dan
ramification

2
Primary and secondary
Intra Radicular Bacterium:
128,131

Successful root canal

care

Pulp and Periapical Disease 128,132

Root canal care: 134,135

Endodontic triad:37,40,134,136

5,128,129

Sources of irritant:
mechanical, termal, chemical,
bacterial

Unsuccessful root canal

care

E. faecalis : 6,16,56,130
Persistent intra radicular infection

E. faecalis
Genotype:118
MLEE, RFLD,RAPD,
PFGE, MLST, CPS.
Genotype profile:
cps 1, cps 2, cps 5

Extra Radicular
Bacterium:7,130,132 Actinomyces
sp , P. propionicum
E. faecalis Phenotype:6,14,16,22,56,130
AS, Esp, LTA gelatinase, sitolisin
hyaluronidase, AS-48, extracellular
superxide, protein profile, capsule
carbohydrate, sensitivity to
irigation solution, sensitivity to
laser beam

Irigation solution: 171-173

Ca(OH)2 Ca2+ + OH-

Access, cleaning and shaping

(instrumentation, irigation,
medicamentosa), Filling

Failure factor:56,70,132
Bacterial
Non bacterial :
- Iatrogenic
- Exogen :
cellulose
- Endogen :
Cholesterol crystal

LAI cavitation100,101,104,107,109

PDT ROS 65,66,72,98

Figure 1
Theoretical Framework

UNIVERSITAS INDONESIA

Conceptual Framework

Isolated E. faecalis:
Primary Intra Radicular Infection
Persistent Intra Radicular Infection

Amount of cps 1
Amount of cps 2
Amount of cps 5

Persistent Intra
Radicular E. faecalis
Infection:

PDT Laser

Genotyping :
Cps type

LED 630 nm
Laser 810 nm

50% Calcium
Hydroxide solution

Genotype Profile:
cps 1
cps 2
cps 5
Protein Profile
Capsule Polysaccharide Profile

E. faecalis after intervention

Phenotype :

Protein profile
Capsule polysaccharide profile
Sensitivity to PDT Laser and
50% Calcium Hydroxide
Solution

Figure 2
Conceptual Framework

General Hypothesis
Cps genotype are different between isolated E.faecalis from root canal with
primary intra radicular infection and persistent intraradicular infection. There are also
differences in phenotype characteristic on persistent intra radicular infection towards
photodynamic laser therapy and 50% calcium hydroxide solution.
Spesific Hypothesis
1. There is difference in amount of E.faecalis which is isolated from root canal
with primary intra radicular infection and persistent intra radicular infection.
2. There are cps genotype variations of E.faecalis which is isolated from root canal
with primary intra radicular infection and persistent intra radicular infection.

UNIVERSITAS INDONESIA

3. There is difference in sensitivity of E.faecalis which is isolated from root canal

with primary intra radicular infection before and after intervention using
photodynamic laser therapy and 50% calcium hydroxide solution.
4. There is difference in protein profiles between E.faecalis which is isolated from
root canal with persistent intra radicular infection before and after intervention
using photodynamic laser therapy and 50% calcium hydroxide solution.
5. There is difference in polysaccharide capsule profiles between E.faecalis which
is isolated from root canal with persistent intra radicular infection before and
after intervention using photodynamic laser therapy and 50% calcium hydroxide
solution.
6. There is difference in sensitivity of E.faecalis which is isolated from root canal
with persistent intra radicular infection before and after intervention using
photodynamic laser therapy and 50% calcium hydroxide solution among
viability, protein profile and polysaccharide capsule profile.
Study Design
This study begins with molecular epidemiological survey that compares the
amount of E.faecalis in primary and persistent intra radicular infections. Quantification
of E.faecalis was performed using q-PCR. Study subjects was choosed randomly based
on clinical diagnosis, primary and persistent intra radicular infection. E.faecalis clinical
sample was taken from root canal during endodontic routine care, but the sample was
taken at the beginning before the endodontic measure was continued. Cps type was
determined using conventional PCR method. Firstly, E.faecalis was confirmed by doing
culture in selective chromogenic agar medium. The greenish blue colonies then cultured
in BHI broth for further made to the stock of bacteria. After that, DNA was extracted
and confirmation using PCR techniques was done. Later, determination of the type of
capsule polysaccharide (cps) was done to these colonies that had been confirmed as
E.faecalis before. This cps was subsequently being sample in next step.
The next step was experimental laboratory test, which analyzed phenotype
including protein profile and capsule polysaccharide profile of E.faecalis that has been
isolated from root canal with persistent intra radicular infection using SDS PAGE

UNIVERSITAS INDONESIA

method to analyze protein profile and Stain All method to analyze capsule
polysaccharide profile, also analyzed sensitivity sensitivity of E.faecalis towards PDT
laser and irrigation materials i.e 50% calcium hydroxide solution. Measurement of
E.faecalis sensitivity to PDT laser and calcium hydroxide was done through bacterial
viability test counting growing colonies in agar medium after intervention, analyzed
protein profile and capsule polysaccharide profile.

UNIVERSITAS INDONESIA

Study Design
Subjects (114)

Primary Intra Radicular Infection

(61)

RT q-PCR

Persistent Intra Radicular Infection (53)

Chromogenic Agar Culture

Amount E. faecalis

BHI Broth

Protein profile: SDS PAGE

Capsular profile : All Stain

PCR

Cps Genotype

Persistent Intra
RadicularInfection

Genotype cps 1.2 dan 5

Sensitivity Test :
Viability Test towards Photodynamic Laser Therapy
Viability test towards 50% Calcium Hydroxide solution
(see sensitivity test plot)
Protein profile: SDS PAGE
Capsule Polysaccharide profile : All Stain

E. faecalis phenotype

Figure 3
Schema of Study Design

UNIVERSITAS INDONESIA

Sensitivity Test
146 single-root premolar

Root canal access, preparation

Autoclav sterilization
2 teeth was excluded
for control (-)

Insert isolated enterococcus faecalis from root

canal: persistent intra radicular infection
2 weeks incubation
128 teeth divided into 5 groups

32 teeth
Group cps 1

32 teeth
Group cps 2

32 teeth
Group cps 5

16 teeth
Group cps 1+2

16 dikeluarkan
sebagai kontrol (+)
Masing-masing cps
dan cps gabungan

16 teeth
Group cps 1+5

A : 2 teeth : Fotosan 60
B : 2 teeth : Fotosan + BT 30
C : 2 teeth : Fotosan + BT 60
D : 2 teeth : ARC Laser 60
E : 2 teeth : ARC Laser+Emundo 60
F : : 2 teeth : CH 50% 60
G : : 2 teeth CH 50% 60
H : : 2 teeth : CH 50% 24 hour

Spreading colonies on the BHI agar

surfaces (CFU/mL)

Figure 4
Schema of Sensitivity Test

UNIVERSITAS INDONESIA

Place and Time of Study

Determination of subjects and preparation of clinical test material took place in
Bunda Margonda Hospital,

Hermina Hospital Bogor, and Pondok Indah Hospital.

While the genotype and phenotype bacterial analysis based on the examination of
clinical sample was conducted at Biologi Oral laboratory, Faculty of Dentistry,
University of Indonesia.
This study was conducted over 18 months from July 2014 until December 2015.
Ethical Clearance
This study has been approved by the Ethical Commitee for medical research,
Faculty of Dentistry, University of Indonesia with letter number 99/ethical
clearance/FKGUI/X/2014
RESULTS
Molecular Epidemiological Analysis
Molecular epidemiological survey using real time q-PCR showed that the
average ammount of E.faecalis in persistent intra radicular infection group was
approximately two times more higher than primary intra radicular infection group
(Figure 5). Meanwhile, the average of relative amount of E.faecalis in persistent intra
radicular infection group was approximately seven times much more than in primary
intra radicular infection group. Nonetheless, both of these differences was not
significant statistically, p>0,05 (Figure 5). These results indicate that E.faecalis can be
isolated from root canal with primary and persistent intra radicular infection, and more
common found in root canal with persistent intra radicular infection. Compared with all
microbacterium in the root canal, relative amount of E.faecalis in the case of persistent
intra-radicular infection is higher than in case of primary intraradicular infection.

UNIVERSITAS INDONESIA

450.0000

424.3510

400.0000
350.0000
300.0000
250.0000

CFU/mL

amount of E.faecalis

208.7600

relative amount of
E.faecalis

200.0000
150.0000
100.0000
50.0000
3.7145
0.5377
0.0000
Primary infection (n=61)

Gambar 5
Difference of average amount and average relative amount of E.faecalis
in case of primary and persistent intra radicular infection

Cps Genotype Analysis

Overall, proportion of cps1 was more dominant (76,3%) compared with the
combination of cps 1-2 (9,7%) and cps 1-5 (14%) in both primary and persistent intra
radicular infection (Figure 6).

11.50%
8.20%

17.00%

14.00%

11.30%

9.70%

71.70%

76.30%

Persistent Infection

Total

80.30%

Primary Infection

CPS-1

CPS-1-2

CPS-1-5

Figure 6
Diagram of E.Faecalis cps Genotype Proportion (cps-1, cps-1-2, and
cps-1-5) isolated from primary and persistent intra-radicular
infection
UNIVERSITAS INDONESIA

10

E. faecalis and cps type confirmed by PCR

1

200 bp
150
100 bp

Figure 7
Electrophoresis of E. faecalis sample:
Column 1= first cps 1 pertama; column 2= second cps 1; Column 3= first
cps 2; Column 4= second cps 2; Column 5= first cps 5; Column 6=
second cps 5, Column M=marker DNA Ladder up to 500 bp; Column 7=
negative control; Column 8= positive control ATCC 29212 around 138
bp. All the sample showed that E. faecalis was positive.

UNIVERSITAS INDONESIA