ORIGINAL ARTICLE

Year : 2019 | Volume : 22 | Issue : 3 | Page : 241--244

Comparing the antimicrobial efficacy of pediocin with chlorhexidine and calcium hydroxide as intracanal medicaments
against persistent root canal infections
Hui Ying Ooi1, Wan Yi Tee1, Fabian Davamani2, Venkateshbabu Nagendrababu3,
1 School of Dentistry, International Medical University, Kuala Lumpur, Malaysia
2 Division of Applied Biomedical Science and Biotechnology, School of Health Sciences, International Medical University, Kuala Lumpur, Malaysia
3 Division of Clinical Dentistry, School of Dentistry, International Medical University, Kuala Lumpur, Malaysia

Correspondence Address:
Dr. Fabian Davamani
Division of Applied Biomedical Science and Biotechnology, School of Health Sciences, 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.

How to cite this article:

Ooi HY, Tee WY, Davamani F, Nagendrababu V. Comparing the antimicrobial efficacy of pediocin with chlorhexidine and calcium hydroxide as intracanal medicaments against persistent
root canal infections.J Conserv Dent 2019;22:241-244

How to cite this URL:

Ooi HY, Tee WY, Davamani F, Nagendrababu V. Comparing the antimicrobial efficacy of pediocin with chlorhexidine and calcium hydroxide as intracanal medicaments against persistent
root canal infections. J Conserv Dent [serial online] 2019 [cited 2019 Aug 20 ];22:241-244
Available from: http://www.jcd.org.in/text.asp?2019/22/3/241/262022

Full Text

Introduction

Bacteria are the cause of pulpal and periapical disease.[1] Persistent bacteria inside dentinal tubules have been reported as the most common reason for the resurgence of apical
periodontitis.[2] Thus, for a successful outcome following root canal treatment, it is necessary to eliminate the bacteria from the root canal system to promote healing of the periradicular
tissues.[3] During root canal preparation, it has been reported that 35% of the root canal walls remains untouched by the instruments [4] due to the anatomical complexity of the root
canal system. Further difficulties in removing bacteria are associated with the presence of biofilms and the structure and composition of dentin.[5] Hence, intracanal medicaments play a
key role in reducing bacteria, which remain after mechanical debridement.[6] In endodontics, 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.

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 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.{Table 1}

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 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
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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Tuesday, August 20, 2019

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