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57]

Original Article

Comparing the antimicrobial efficacy of pediocin with

chlorhexidine and calcium hydroxide as intracanal
medicaments against persistent root canal infections
Hui Ying Ooi, Wan Yi Tee, Fabian Davamani1, Venkateshbabu Nagendrababu2
School of Dentistry, International Medical University, 1Division of Applied Biomedical Science and Biotechnology, School of Health
Sciences, International Medical University, 2Division of Clinical Dentistry, School of Dentistry, International Medical University, Kuala
Lumpur, Malaysia

Abstract
Introduction: The aim of this study is to compare the antimicrobial activity of pediocin with chlorhexidine (CHX) and calcium
hydroxide (Ca(OH)2) against Enterococcus faecalis and Staphylococcus epidermidis biofilms.
Materials and Methods: The prepared root canals of 80 teeth were contaminated with E. faecalis (n  = 40) and
S. epidermidis (n = 40) for 21 days to create biofilms. The samples in each group were allocated randomly into the following
four subgroups (n = 10) according to the decontamination protocol: Group 1: 1% Pediocin, Group 2: 2% CHX, Group 3:
Ca(OH)2, and Group 4: saline (negative control). At 5 days, the antimicrobial efficacy of the medicaments against E. faecalis
and S. epidermidis was assessed by collecting dentin shavings from the canal walls created using Gates Glidden drill sizes
4 and 5, corresponding to a depth into the root canal walls of 200 µm and 400 µm, respectively. The total number of
colony‑forming units (CFUs) was counted. The Wilcoxon signed‑rank test was used to compare the difference in CFUs between
the two depths (P > 0.05).
Results: There was no bacterial growth in samples treated with pediocin, CHX, or Ca(OH)2 at either depth.
Conclusion: In this laboratory experimental model, pediocin exhibited the same antimicrobial properties against E. faecalis
and S. epidermidis as CHX and Ca(OH)2.
Keywords: Calcium hydroxide; chlorhexidine; Enterococcus faecalis; Staphylococcus epidermidis

INTRODUCTION
Bacteria are the cause of pulpal and periapical disease.[1] periodontitis.[2] Thus, for a successful outcome following root
Persistent bacteria inside dentinal tubules have been reported canal treatment, it is necessary to eliminate the bacteria from
as the most common reason for the resurgence of apical the root canal system to promote healing of the periradicular
tissues.[3] During root canal preparation, it has been reported
Address for correspondence: that 35% of the root canal walls remains untouched by the
Dr. Fabian Davamani, Division of Applied Biomedical Science and instruments[4] due to the anatomical complexity of the
Biotechnology, School of Health Sciences, International Medical root canal system. Further difficulties in removing bacteria
University, Kuala Lumpur, Malaysia. are associated with the presence of biofilms and the
E‑mail: fabian_davamani@imu.edu.my structure and composition of dentin.[5] Hence, intracanal
Dr. Venkateshbabu Nagendrababu, Division of Clinical Dentistry, medicaments play a key role in reducing bacteria, which
School of Dentistry, International Medical University, Bukit Jalil remain after mechanical debridement.[6] In endodontics,
57000, Kuala Lumpur, Malaysia.
E-mail: venkateshbabu@imu.edu.my This is an open access journal, and articles are distributed under the terms
of the Creative Commons Attribution‑NonCommercial‑ShareAlike 4.0
Date of submission : 22.11.2018
Review completed : 28.01.2019 License, which allows others to remix, tweak, and build upon the work
Date of acceptance : 23.05.2019 non‑commercially, as long as appropriate credit is given and the new
creations are licensed under the identical terms.
Access this article online For reprints contact: reprints@medknow.com
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Website:
How to cite this article: Ooi HY, Tee WY, Davamani F,
www.jcd.org.in
Nagendrababu V. Comparing the antimicrobial efficacy of
pediocin with chlorhexidine and calcium hydroxide as intracanal
DOI: medicaments against persistent root canal infections. J Conserv
10.4103/JCD.JCD_521_18
Dent 2019;22:241-4.

© 2019 Journal of Conservative Dentistry | Published by Wolters Kluwer - Medknow 241

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Ooi, et al.: Antimicrobial efficacy of pediocin
chlorhexidine  (CHX)[7] and calcium hydroxide (Ca(OH)2)[8]
have been used widely as an intracanal medicament.
Enterococcus faecalis has been reported to be the most
common persistent intraradicular microorganism associated
with posttreatment endodontic disease following root canal
treatment.[9,10] E. faecalis can penetrate into dentinal tubules and
to adapt to harsh environmental conditions, and therefore, it is
difficult to eradicate from the root canal system.[9] Staphylococci
are nonmotile, nonspore‑forming, and facultative anaerobic
cocci. Staphylococcus epidermidis is a common organism
present associated with persistent root canal infections when
investigated by checkerboard DNA–DNA hybridization.[11]
Bacteriocins are ribosomally encoded proteinaceous
molecules produced by bacteria of all genera to kill or inhibit
the growth of other bacteria.[12,13] Pediocins are cationic
peptides that belong to class IIa bacteriocins.[14] All class IIa
bacteriocins are active against Listeria and other Gram‑positive
pathogenic bacteria.[14] It has been reported that bacteriocins
have antimicrobial efficacy against periodontal pathogens.[15]
The application of pediocins for root canal disinfection as
intracanal medicaments has not been studied. The hypothesis
for the study was that bacteriocins have better antimicrobial
efficacy than CHX or Ca(OH)2 against E. faecalis and S.
epidermidis biofilms. The null hypothesis was stated as that
all the tested medicaments had similar antimicrobial efficacy.
Hence, the aim of the current laboratory study was to
compare the antimicrobial efficacy of pediocin with CHX and
Ca(OH)2 against E. faecalis and S. epidermidis biofilms within
prepared root canals of extracted human teeth.
MATERIALS AND METHODS
The International Medical University Joint Committee on
Research and Ethics (BDS I‑01/14 (05) 2017) approved the
study protocol. Eighty single‑rooted human teeth were
selected. Teeth with open apex, previous root canal filling,
and crack/fracture in root surface were excluded for this
study. The teeth were then sectioned horizontally below
the cementoenamel junction using rotary diamond discs
and again at the apical part of the root to leave the middle
portion (6 mm) of the root for the study. The cementum was
removed from the root cylinders, and the canal diameter
was standardized with size 3 Gates Glidden drills (Mani
Inc., Tachigi‑ken, Japan). Dentinal debris was removed by
placing the dentin cylinders in an ultrasonic bath (Fisher
scientific FB 15061)‑containing 3% sodium hypochlorite
and followed by 17% ethylenediamine tetraacetic acid
for 5 min each. To remove traces of chemicals from the
canals, they were rinsed in sterile water for 1 min. The
samples were then sterilized in an autoclave for 121°C
for two cycles. To ensure no microbial contamination, the
specimens were incubated in brain heart infusion broth at
37°C for 24 h.
242 Journal of Conservative Dentistry | Volume 22 | Issue 3 | May-June 2019
E. faecalis (ATCC 29212) and S. epidermidis (ATCC 14990)
were obtained from the American Type Culture Collection.
The bacteria were grown using tryptic soy agar/broth
(TSA/TSB, Thermo Fisher). The bacterial suspension was
adjusted to 0.5 McFarland units 1.5 × 108 CFU/ml before
the microbiological experiments. Fifty microliters of
the bacterial inoculum were transferred to presterilized
individual microcentrifuge tubes containing the dentin
cylinders and immersed in 1 mL of the TSB. The dentin
cylinders were then transferred to the fresh broth‑containing
E. faecalis (n = 40) and S. epidermidis (n = 40) every 2nd day.
All the procedures were carried out under laminar flow
conditions (biological safety cabinet Thermo scientific). The
purity of the culture was checked by subculturing 50 μL of
broth from the incubated specimens in TSB on TSA plates.
The dentin cylinders were contaminated for 21 days.
Eighty specimens were divided into two groups contaminated
with E. faecalis (n  = 40) and S. epidermidis (n  = 40). After
21 days, the specimens in each group were irrigated
using 10 mL of sterile saline, and the specimens in each
group were randomly allocated into the following four
subgroups (n = 10): group 1‑1% Pediocin (Sigma, St. Louis,
MO, USA); Group 2‑2% CHX (Consepsis® V, Ultradent Products,
Inc.); Group 3 ‑ (Ca(OH)2) (Calasept® Plus, Directa); and
Group 4 ‑ saline (negative control). The medicaments used in
Groups 1 and 4 were converted into intracanal medicament
form using methylcellulose as a thickening agent. Paraffin
wax was used to seal both ends of the dentin cylinders after
placing the medicaments inside the root canals. They were
incubated in an anaerobic environment at 37°C for 5 days.
At the end of 5 days, an assessment of microbial cells was
carried out. Dentin chips were collected at a depth of 200
µm and 400 µm by running the Gates Glidden drills size 4
and 5, respectively, along the root canal walls. The Gates
Gliden drills sequentially remove the dentin chips from the
inner surface of the tooth.[16] The collected dentin chips were
transferred into 100 µL of sterile saline and the contents
serially diluted four times. A volume of 10 µL of the dilution
were then plated on TSA plates and incubated for 24 h. The
colonies were counted and readings were tabulated.
Statistical analysis was not possible between groups
because no bacterial growth occurred. The Wilcoxon
signed‑ranks test was used to check the difference in CFUs
between the 200 and 400 µm depths (P > 0.05).
RESULTS
The mean and standard deviation of CFU observed for
the four groups are shown in Table  1. In CHX, pediocin,
and Ca(OH)2 groups, no bacterial growth was observed at
both the 200 µm and 400 µm depths. Hence, there was a
100% reduction of E. faecalis and S. epidermidis when CHX,
pediocin, and Ca(OH)2 were used as intracanal medicament
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Table 1: Mean and standard deviation of the colony‑forming units after treating with various medicaments
Groups
Enterococcus faecalis
200 µm 400 µm
Pediocin 0 0
Chlorhexidine 0 0
Calcium hydroxide 0 0
Saline a
15.54×105±27.32×105 a
17.83×105±29.53×105
a
Groups with same superscript alphabets have no significant difference. SD: Standard deviation
for 5 days. E. faecalis and S. epidermidis were present in
the groups where saline was used. However, there was
no significant difference between the depths of 200 and
400 µm.
DISCUSSION
Endodontic diseases are biofilm‑mediated infections and
the removal or reduction of persistent bacteria from the
root canal system is challenging. Hence, it is important
to develop and introduce new antimicrobial agents
for effective root canal disinfection. The evolution of
antibiotic resistance to microbes is of concern to the global
community and is a growing issue worldwide. To reduce
the rapid emergence of antibiotic‑resistant strains of
bacteria, bacteriocins are now considered as antimicrobials
compounds to treat human and animal infections. Hence,
in this study, we explored the application of pediocin as an
intracanal medicament.
The prevalence of E. faecalis in persistent root canal
infections ranges from 67% to 77% when using polymerase
chain reaction.[9] A meta‑analysis reported that E. faecalis
was the most common microorganism in persistent
intraradicular infections compared with primary chronic
periapical periodontitis.[17] S. epidermidis was one of the most
prevalent species in persistent apical periodontitis.[11] It has
been reported that S. epidermidis and Staphylococcus xylosus
were associated with secondary root canal infections,
which might be responsible for the persistent symptoms.[18]
Hence, in the present study, E. faecalis and S. epidermidis
were selected as the test organisms. The age of biofilm
plays a key role in studying the effectiveness of disinfection
agents. The matured biofilm (21 days) will be difficult in
eradicating compared to young biofilm (2–14  days).
Bacteria in mature biofilms are more resistant to CHX in
killing than in young biofilms.[1] Hence, in our study, we
standardized the age of biofilm to 21 days (3 weeks).
No growth of E. faecalis and S. epidermidis was observed
after treating root canals with CHX, Pediocin, and Ca(OH)2.
Pediocin is a ribosomal synthesized antimicrobial peptide
produced by bacteria to serve as their defense against
microorganisms.[14] In the present study, 1% pediocin was
associated with complete inhibition of E. faecalis and
S. epidermidis at both canal wall depths after 5 days. The
Journal of Conservative Dentistry | Volume 22 | Issue 3 | May-June 2019
Ooi, et al.: Antimicrobial efficacy of pediocin
Mean±SD
Staphylococcus epidermidis
200 µm 400 µm
0 0
0 0
0 0
a
19.55×105±26.18×105 a
23.12×105±30.75×105
bactericidal mode of action of pediocin involves its binding
to the cytoplasmic membrane and insertion of C‑terminal
regions of pediocin molecules into the membranes of
target cells causing pore formation.[15] Target cells lose
their permeability barrier and membrane potential which
leads to cell death.[14] Peptide‑based antibiotics are largely
considered as a replacement to overcome the increasing
resistance to conventional antibiotics due to their
antibacterial efficacy against Listeria and Clostridium spp.
and Enterococcus spp.[12,14] It has also been revealed that
peptides are active against bacteria that are resistant to
conventional antibiotics and that they could kill a broad
range of bacteria in vitro at concentrations ranging from
0.25 to 4 µg/mL.[19] Due to the antimicrobial property of
bacteriocins, they have been used as food preservatives
and in the field of bio‑preservation, pharmaceutical, and
aquaculture.[20]
The antimicrobial efficacy of CHX and Ca(OH)2 has been
studied extensively in endodontics. The possible reasons
of complete inhibition in CHX could be due to its high
bactericidal concentration of 2% and also increased the
diffusion of the medicament into the dentinal tubules.
CHX is a positively charged molecule that interacts with
cell walls of bacteria which are negatively charged.[21,22] This
interaction alters the osmotic equilibrium of the bacteria
leading to increased permeability of cell membranes, which
allows the penetration of CHX molecules into the bacteria
and causes leakage of low‑molecular‑weight compounds
such as potassium ions.[23] At higher concentration (2%),
CHX exerts its bactericidal effect by causing precipitation
of the cytoplasmic contents, which results in cell death.[22]
In this study, Ca(OH)2 completely inhibited E. faecalis and
S. epidermidis at depths of 200 µm and 400 µm. Plausible
reasons could be the high concentration of Ca(OH)2
and its high alkalinity. The Ca(OH)2 used in the present
study contained a very high concentration of >41%
of Ca(OH)2, which allowed it to generate an effective
and long‑lasting antimicrobial effect. The antimicrobial
effect of Ca(OH)2 is due to hydroxyl ions release in an
aqueous environment. The high alkalinity of Ca(OH)2 has
the potential to change the integrity of the cytoplasmic
membrane by the destruction of phospholipids.[24,25] The
use of Ca(OH)2 at intervals of at least 7 days can reduce the
total number of bacteria remaining even after biochemical
243
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Ooi, et al.: Antimicrobial efficacy of pediocin
preparation.[25] Disinfection ability of intracanal
medicaments will be studied at 1, 3, and 5 days. In this
study, we assessed the efficacy of pediocin only at the
end of day 5 and found it to be highly effective. However,
a shorter duration for about 1–3  days could have given
similar or lowered effect, which will be evaluated in our
future projects. This could be considered as the limitation
of the present study.
CONCLUSION
Within the limitation of the present study, pediocin
exhibited antimicrobial activity against persistence root
canal infections. Further laboratory tooth model studies
are needed to confirm the viability of bacteria after treating
with pediocin followed by in vivo studies to validate its
application as an intracanal medicament.
Financial support and sponsorship
This work was financially supported by the International
Medical University under Grant no: BDS I‑01/14 (05) 2017.
Conflicts of interest
There are no conflicts of interest.
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