Diversity of Enterococcus Faecalis Clonal types in Endodontic Failures

© Ramaiah University of Applied Sciences
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Faculty of Dental Sciences
Diversity of Enterococcus faecalis Genotypes
from Multiple Oral Sites Associated with
Endodontic Failure Using Repetitive
Sequence-based Polymerase Chain Reaction and
Arbitrarily Primed Polymerase Chain Reaction
Maraısa G. Delboni,Brenda P.F.A. Gomes, Priscila A. Francisco, Fabrıcio B. Teixeira David Drake
JOE2017 Mar;43(3):377-382
Presented : Dr. Arbiya Anjum S
Modulater : Dr. B V Srinivas Murthy

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Conventional endodontic therapy failure - treatment is done inadequately,
Procedures under highest standards showed nonhealing of apical periodontitis
Ability of some microorganisms to survive after current treatment
protocols
Coronal microleakage
Introduction

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Nonmotile, gram-positive,
spherical bacterium. Singly, in
pairs/in short chains in large
intestine of humans
First to the 3rd leading cause of
Nosocomial infections
UTI, Bacteremia, Endocarditis,
Meningitis, Foodborne disease,
Wound infections and Intra-
abdominal abscesses
Root canals with apical
periodontitis , major
endodontic pathogen in
secondary infections
Enterococcus faecalis

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Polymerase chain reaction (PCR
Polymerase chain reaction
Primers bind to specific regions of DNA in proper
orientation and within an optimum distance - generation of
species/strain-specific amplification products
Primers i.e REP1R and REP2, derived from repetitive
extragenic palindromic sequences used in technique
known as repetitive sequence-based PCR
Arbitrarily primed PCR - any form of PCR that uses
primers of arbitrary sequence

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the goals of this study were
1. To identify and locate E. faecalis in each clinical case using in vivo
sampling protocols and culture techniques;
2. To analyze DNA fingerprints from the saliva, pulp chambers, and
root canals of endodontically treated teeth with apical periodontitis
using 2 molecular methods; and
3. To determine the diversity and similarity of genotypes present within
the oral cavity.
Persistent root canal infection – endodontic failure
Although unsatisfactory coronal restorations
are associated with lower rates of complete apical healing ,the biological aspects of
coronal microleakage have not been clearly investigated
It is critical to evaluate this species occurrence in the patient’s saliva and under the
restorative materials
Overall success rate of nonsurgical retreatment of teeth with apical periodontitis is
76.7%
Poor prognosis -difficulties in the elimination of the particular resistant microbiota(e.
Faecalis)
It would be of interest to know in vivo the pathways of the root canal reinfection,
starting from the saliva and then entering the internal surface of the pulp chamber
and spreading through the root-filled canal
system

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the goals of this study were
1. To identify and locate E. faecalis in each clinical case using in vivo
sampling protocols and culture techniques;
2. To analyze DNA fingerprints from the saliva, pulp chambers, and
root canals of endodontically treated teeth with apical periodontitis
using 2 molecular methods; and
3. To determine the diversity and similarity of genotypes present within
the oral cavity.
Aim Of Study
To identify and locate E. faecalis in
each clinical case using in vivo
sampling protocols and culture
techniques
To analyze DNA fingerprints from the
saliva, pulp chambers, and root canals
of endodontically treated teeth with
apical periodontitis using 2 molecular
methods
To determine the diversity and
similarity of genotypes present within
the oral cavity

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All the protocols and the specimen collection methods for this
investigation were approved by the institutional review board of the Piracicaba
Dental School, State University of Campinas, Piracicaba, S~ao
Paulo, Brazil. Informed consent forms were provided and signed by
the patients involved in the study. Forty-two teeth of 20 patients were
included. The patients were screened and scheduled to receive nonsurgical
endodontic retreatment because of radiographic evidence of apical
periodontitis. Medical history and dental records were obtained.
The inclusion criteria used to select teeth and patients were as follows:
1. Previously endodontically treated teeth with radiographic evidence
of apical periodontitis
2. Previously endodontically treated teeth with persistence of
symptoms
3. No evidence of longitudinal fractures
4. No antibiotic therapy provided 3 months before the consultation
visits
5. Absence of systemic disease and periodontal disease
Sampling Procedure
Microbial sampling of the saliva followed by the pulp chamber and
the root canals was collected during the first dental appointment.
Aseptic techniques were used throughout the nonsurgical root canal retreatment
and before sample collection. The sampling protocols used in
this study were previously detailed by Gomes et al (4, 23) and were
adapted for this study as described.
Materials and Methods
Inclusion criteria
42 teeth of 20
patients
(informed
consent )
Previously
endodontically
treated teeth
with
radiographic
evidence
of apical
periodontitis
Previously
endodontically
treated teeth
with persistence
of
symptoms
No evidence of
longitudinal
fractures
No antibiotic
therapy
provided 3
months before
the consultation
visits
Absence of
systemic
disease and
periodontal
disease

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42 saliva samples - 20
patients
Patients were informed
to not brush teeth or eat
anything 2 hrs before
appointment
1 mm of whole saliva
was collected from the
patients and placed in a
sterile plastic receptacle
External surfaces of
crowns were then
disinfected with 30%
h2o2 followed by 2.5%
naocl
Solutions were
inactivated with 5%
sodium thiosulfate to
avoid interference with
bacteriological sampling
Internal surfaces of the
coronal restorations or
posts were collected
using a sterile swab
Root canal filling materials
were then removed with
gates-glidden drills and k-
files without the use of
endodontic solvents
Sampling was
performed during all
retreatment procedures
Gp & debris sample
collected using paper
points till WL for 60 secs
then taken out
126 microbial samples,
42 from each of 3 sites
during the retreatment
procedures

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Culture Procedure
All sample- transferred to
Viability Medium
G€otemborg Agar
transport medium and
promptly taken to
laboratory for processing
for 4 hrs
Inside the chamber, each
transport medium was
shaken thoroughly
in a mixer for 60 seconds
Samples were plated
using sterile plastic
spreaders onto m-
Enterococcus agar and
incubated at 35C for 2
days in duplicate plates

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The amplification was performed in a thermocycler with an initial denaturation of 95C for 7mins
followed by 30 cycles of denaturation at 90C for 30 secs, annealing at 52C for 1 min, and extension at
65C for 8 mins. A final extension was performed at 65C for 16 minutes.
DNA Fingerprinting
• E. faecalis isolates extracted and DNA fingerprints of 74 strains were developed using Rep PCR (primers
ERIC1 and ERIC2) and AP-PCR (primer RW3A)
• Amplifications were performed in an ultraviolet sterile biohood
• The amplification was performed in a thermocycler with an initial denaturation of 95C for 7mins followed by
30 cycles of denaturation at 90C for 30 secs, annealing at 52C for 1 min, and extension at 65C for 8 mins..
• PCR products were separated by gel electrophoresis
• Gel Compar II software was used to determine clonal types

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The data collected from each case were entered into a spreadsheet
and analyzed statistically using SPSS for Windows
Statistical Analysis
Data collected - entered into a spreadsheet and analyzed
statistically using SPSS for Windows

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using SPSS for Windows
Results
• E. faecalis was isolated from 10 clinical cases involving 8
patients
• 74 E. faecalis strains - biochemically identified; saliva -10, pulp
chamber-17, and root canal-47
• API identifications of strains- confirmed via PCR with E. faecalis
16S ribosomal RNA probes, all of them were identified as E
faecalis
• DNA fingerprints of 74 strains using Rep-PCR (primers ERIC1
and ERIC2) and AP-PCR (primer RW3A) showed different results
• 7 genotypes groups were identified among the 74 isolates
• By comparing techniques, most isolates classified as identical,
related, or different

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Results

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using SPSS for Windows
E. faecalis was more
frequently found in
the root canal than
in the saliva of the
same patients
E. faecalis
transiently colonizes
the oral cavity via
foodborne infection
, which is an
exogenous route of
infection
Some research
indicates that E.
faecalis, mostly
present in the
human intestine,
is not a permanent
colonizer of the oral
cavity
The recovery of E.
faecalis from
previously treated
root canals
with apical
periodontitis is still
the subject of
research
Therefore,
microorganisms from
saliva are present in
higher or lower
quantities depending
on several factors,
which will differ from
subject to subject
Furthermore, the
host’s systemic
condition (eg, obesity)
is reportedly
associated with the
microbiota structure
in saliva
The salivary
microbiome, although
stable , can suffer
from the influence of
human genetic
makeup, diet, age,
surroundings,
smoking, and personal
oral hygiene

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The data collected from each case were entered into a spreadsheet
and analyzed statistically using SPSS for Windows
Finding the same microorganism in different sites using the culture technique could be only a coincidence, but
molecular methods used in this work showed the similarity of the strain
Additionally, multiple colonies isolated from each sampling site showed homogeneous genotypes.Root-filled canal
infections appear to be attributed to a large number of different E. faecalis genotypes that share a large degree of
homology
In conclusion, E. faecalis genotypes isolated from saliva, pulp chamber, and root canal were similar using Rep-PCR
and AP-PCR
methods.
These findings suggest that coronal microleakage is a conceivable cause of endodontic failure.

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Thank you

Diversity of Enterococcus Faecalis Clonal types in Endodontic Failures

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