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 Table of Contents  
ORIGINAL RESEARCH
Year : 2017  |  Volume : 8  |  Issue : 4  |  Page : 94-99

Antimicrobial efficacy of silver nanoparticles with and without different antimicrobial agents against Enterococcus faecalis and Candida albicans


1 Department of Conservative Dentistry Endodontics, King George’s Medical University, Lucknow, Uttar Pradesh, India
2 Department of Molecular Bioprospection, Council of Scientific Industrial Research, Central Institute of Medicinal Aromatic Plants (CSIR-CIMAP), Lucknow, Uttar Pradesh, India

Date of Web Publication29-Nov-2017

Correspondence Address:
Simith Yadav
Department of Conservative Dentistry and Endodontics, King George’s Medical University, Lucknow, Uttar Pradesh
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/denthyp.denthyp_17_17

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  Abstract 

Introduction: The aim of this ex vivo study was to check the antimicrobial efficacy of silver nanoparticles with and without different antimicrobials against Enterococcus faecalis and Candida albicans. Materials and Methods: Two-hundred and fifty two freshly extracted single-rooted human teeth were contaminated with E. faecalis and C. albicans. The teeth were randomly divided into five experimental (n = 21) and one control group (n = 21). Each subgroup was then exposed to different antimicrobials, namely calcium hydroxide (Ca(OH)2) (Group 1), 2% chlorhexidine (CHX) (Group 2), silver nanoparticles (SNP) (Group 3), SNP with Ca(OH)2 (Group 4), SNP with 2% CHX (Group 5), and saline as control group (Group 6). Cultures were made from each group after 24 h, 7 days, and 14 days, and colony forming units were counted. The Kruskal–Wallis test was used to compare the study parameters among the groups at 24 h, 7 days, and 14 days. Results: Significant difference was found in the antimicrobial efficacy of different intracanal medicaments against E. faecalis and C. albicans after 24 h, 7 days, and 14 days. 2% CHX was found to be the most effective medicament at 24 h, 7 days, and 14 days against E. faecalis and C. albicans. Combination of SNP with 2% CHX and Ca(OH)2 and SNP alone ranked second in their antimicrobial efficacy against E. faecalis and C. albicans at 24 h, 7 days, and 14 days, respectively. Conclusion: 2% CHX was more effective as intracanal medicament against E. faecalis and C. albicans biofilm in both short as well as long-term duration, i.e., at 24 h, 7 days, and 14 days.

Keywords: Calcium hydroxide, Candida albicans, chlorhexidine, Enterococcus faecalis, nanoparticles


How to cite this article:
Chandra A, Yadav RK, Shakya VK, Luqman S, Yadav S. Antimicrobial efficacy of silver nanoparticles with and without different antimicrobial agents against Enterococcus faecalis and Candida albicans. Dent Hypotheses 2017;8:94-9

How to cite this URL:
Chandra A, Yadav RK, Shakya VK, Luqman S, Yadav S. Antimicrobial efficacy of silver nanoparticles with and without different antimicrobial agents against Enterococcus faecalis and Candida albicans. Dent Hypotheses [serial online] 2017 [cited 2017 Dec 11];8:94-9. Available from: http://www.dentalhypotheses.com/text.asp?2017/8/4/94/219444


  Introduction Top


Successful endodontic treatment depends on various factors such as chemo-mechanical debridement, optimum irrigation, appropriate and specific intracanal medicaments, and three-dimensional obturation in order to completely seal the root canal system.[1] Mechanical debridement with the help of endodontic files has its own limitations. Past studies have suggested that biomechanical preparation to its maximum efficacy still leaves around 30 to 35% of the root canal untouched, and thereby raising concerns about complete root canal debridement.[2] Chemical disinfection through various irrigants and intracanal medicaments, such as sodium hypochlorite, chlorhexidine (CHX), and calcium hydroxide, have also been reported to be insufficient as far as their action against the resistant microbes that cause secondary infection is concerned.[2],[3]

The most commonly isolated species from the secondary infection of the root canal system is Enterococcus faecalis and is reported to be present in more than 60% of the re-infection cases.[4] Studies have suggested that resistance incorporated in E. faecalis against commonly used intracanal medicaments is because of its ability to form biofilm.[5] Biofilm is a complex extracellular polymeric matrix that protects the bacteria against nutrient deprived and other unfavorable conditions, such as high alkaline and salt concentrations, created by intracanal medicaments.[5],[6]

Culture studies have indicated the presence of multiple flora in secondary infection of the root canal system rather than single microorganism. Apart from E. faecalis, the other commonly isolated microorganism is Candida species.[7],[8] Among the Candida species, Candida albicans is the most commonly isolated species in secondary root canal infection.[8] Because of the increasing evidence suggestive of biofilm-mediated resistance of the microflora toward the commonly used intracanal medicaments, a greater emphasis is now being laid in developing materials that can disrupt the biofilm and eliminate the microorganisms from the root canal system.

Nanoparticles are class of newer medicaments which are hypothesized to have antibacterial effect by causing disruption of the biofilm because of their smaller nanosize structure.[9] The nanosize provides increased surface area which can adsorb other medicaments and exert antimicrobial effect.[9],[10] They have charged polycationic structure that provides greater interaction with the negatively charged bacterial cell wall and causes more and more of antibiotic interaction with the microorganism.[9]

Various nanoparticles have been reported to have antibacterial effect such as Ag, Mg, Zn, etc. Silver nanoparticles (SNP) prevent adhesion of microorganism and biofilm formation and have antimicrobial effect against majority of the organisms such as Streptococci, fungi, viruses, etc.[11] Because of the promising antimicrobial effect of SNPs against various microorganisms, This study was conducted with the aim to check the antimicrobial efficacy of SNPs with and without different antimicrobial agents such as calcium hydroxide and CHX against E. faecalis and C. albicans. Till now, there is no study which has evaluated the antimicrobial efficacy of 10 nm-sized SNPs in combination with 2% CHX against E. faecalis and C. albicans. The null hypothesis tested was that there will be no difference among the tested medicaments in their antimicrobial action against E. faecalis and C. albicans in short and long-term therapy.


  Materials and Methods Top


Preparation of teeth specimen

The study was conducted after getting ethical clearance from the Research Cell, King George’s Medical University, Lucknow, India in collaboration with Council of Scientific and Industrial Research (CSIR) and Central Institute of Medicinal and Aromatic Plants (CIMAP), Lucknow, India.

The study was conducted on 252 freshly extracted single-rooted human teeth. The teeth were radiographically confirmed to have single canal. The teeth were decoronated at 14 mm from the root apex using diamond disc. The working length was established 1 mm short of the apex (13 mm) and it was confirmed by inserting 15K file (Dentsply Maillefer, Ballaigues, Switzerland) into the canal till the point it became visible from the apex and then withdrawing it 1 mm short of the apex. Biomechanical preparation was done using a series of ProTaper universal files (Dentsply Maillefer, Ballaigues, Switzerland) to an F3 master apical file. Glyde™ (Dentsply Maillefer, Ballaigues, Switzerland) was used as lubricant for assisting the file motion inside the canal, and in between the mechanical preparation, 3% NaOCl was used an irrigant. 3 ml of 17% ethylene diamine tetra-acetic acid (EDTA) [Prevest Denpro Limited, Jammu, India] and 3 ml of 3% NaOCl were used as irrigants at the end of the biomechanical preparation for 3 min to remove the smear layer. Final irrigation was performed with 3 ml physiological saline solution. Outer surface of the teeth specimen was coated with two layers of nail polish and apex was sealed with self-cure glass ionomer cement. The teeth were then transferred into 2 ml microtubes and autoclaved at 121°C under 15 lbs pressure for 15 min.[12]

Microbiological procedures

Clinical isolates of E. faecalis and C. albicans were used as the test microorganisms. Bacterial and fungal colonies isolated for 24 h were suspended in 5 ml of brain/heart infusion (BHI) broth and Sabouraud Dextrose Broth (SDB), respectively, and were then incubated at 37°C for 4 h.

The 252 root specimens were transferred into sterile 11 cell culture well plates (each plate having 24 wells) under sterile conditions. Roots were mounted in the well plates containing 2% sterile agar media which was allowed to solidify so that root specimens can be stabilized.

10 µl of 0.5 McFarland solution of the bacterial and fungal suspension was transferred into each canal of laminar flow hood under sterile conditions using micro pipettes. After injection, each specimen corresponding to the bacterial group was entirely submerged in BHI broth and specimens corresponding to the fungal group were submerged in SDB and the tubes were incubated anaerobically at 37°C for 24 h. Then the cell culture plates were recapped and sealed using three layers of paraffin, and were incubated at 37°C for 21 days. Every alternate day, 10 µl of fresh BHI/SDB broth was added to ensure the viability of bacteria and fungi.

Antimicrobial activity of the medicaments

At the end of the incubation period, the medium in the well plates was aspirated aseptically and the canals were dried using sterile paper points. Then the teeth were randomly divided into five experimental groups (n = 21) and one control group (n = 21) subjected to the following intracanal dressings: Group 1–0.1 g Ca(OH)2 per 1 ml of sterile water, Group 2–2% CHX (Prevest Denpro Limited, Jammu, India), Group 3–100 ppm of preformed SNP (Reinste Nano ventures Pvt. Ltd, New Delhi, India) of 10 nm size, Group 4–100 ppm of 10 nm SNP + Ca(OH)2 in 1:1 ratio, Group 5–100 ppm of 10 nm SNP + 2% CHX in 1:1 ratio, and Group 6 (control, i.e., only sterile saline).

The medicaments were carried into the canal using 3 ml syringe and 27 gauge needle under aseptic conditions into all experimental group until the canals were totally full. The canal orifices were covered with sterile aluminum foil. The teeth were then put in sterile well plates and they were sealed with several layers of paraffin. After loading the various medicaments, all groups were subdivided randomly into three equal subgroups and then incubated for different time periods of 24 h, 7 days, and 14 days at 37°C temperature and 100% humidity.

Microbiological sampling

Each root canal specimen was flushed with 5 ml of sterile saline, irrigated with 1 ml of 0.5% citric acid to neutralize Ca(OH)2. The specimens medicated with 2% CHX solution were neutralized with 0.5% Tween 80 in 0.07% lecithin, and again irrigated with 5 ml sterile saline.

Microbiological samples were obtained by inserting a sterile # 30 paper point and placing it into the canal for 60 s and then, transferring this paper point into the micro test tube containing 1 ml of physiological saline solution and shaking it for 30 s on a vortex. Then, a 0.1 ml aliquot of the microbial suspension was seeded on a BHI/SDB agar plate respectively. All the samplings were carried out two times for each group and the average was analyzed. After 48 h of incubation, the number of colony forming units (CFUs) were recorded. Bacterial purity was verified using gram staining and colony morphology.

Data analysis

The results are presented in mean ± SD. The Kruskal–Wallis test was used to compare the study parameters among the groups at 24 h, 7 days, and 14 days. The Wilcoxon rank-sum test was used to compare the mean change in the study parameters from 24 h to 7 days and 14 days within the group. The P-value <0.05 was considered significant. All the analyses were carried out on Statistical Package for the Social Sciences, version 16 (SPSS Inc., Released 2007, SPSS for Windows, Version 16.0, Chicago, SPSS Inc.).


  Results Top


Comparison of medicaments after 24 h

A significant difference (P = 0.0001) was found in the antimicrobial efficacy of different intracanal medicaments against E. faecalis and C. albicans after 24 h. Significantly reduced number of microbiological colonies were recovered on the experimental plates when compared with the control plate. In the E. faecalis group, minimum number of CFUs was found in 2% CHX followed by SNP + 2% CHX group [[Table 1]; [Figure 1]]. In the C. albicans group, minimum number of CFUs was noted in 2% CHX followed by SNP [[Table 2]; [Figure 2]].
Table 1: Comparison of mean change in E. Faecalis within the group from 24 hours to 7 and 14 days

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Figure 1: Comparison of mean change in E. Faecalis within the group from 24 hours to 7 and 14 days. There was significant (p=0.0001) in E. Faecalis in all the groups from 24 hours to 7 and 14 days.

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Table 2: Comparison of mean change in Candida Albicans within the group from 24 hours to 7 and 14 days

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Figure 2: Comparison of mean change in Candida albicans within the group from 24 hours to 7 and 14 days. There was significant (p=0.0001) in Candida Albicans in all the groups from 24 hours to 7 and 14 days.

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Comparison of medicaments after 7 days

A significant difference (P = 0.0001) was found in the antimicrobial efficacy of different intracanal medicaments against E. faecalis and C. albicans after 7 days. Significantly reduced number of microbiological colonies was recovered on the experimental plates when compared with the control plate. In the E. faecalis group, minimum number of CFUs was found in 2% CHX followed by SNP + Ca(OH)2 group [[Table 1]; [Figure 1]]. In the C. albicans group, minimum number of CFUs was noted in 2% CHX followed by Ca(OH)2 [[Table 2]; [Figure 2]].

Comparison of medicaments after 14 days

A significant difference (P = 0.0001) was found in the antimicrobial efficacy of different intracanal medicaments against E. faecalis and C. albicans after 14 days. Significantly reduced number of microbiological colonies was recovered on the experimental plates when compared with the control plate. In the E. faecalis group, minimum number of CFUs was found in 2% CHX followed by SNP group [[Table 1]; [Figure 1]]. In the C. albicans group, minimum number of CFUs was noted in Ca(OH)2, followed by 2% CHX group [[Table 2]; [Figure 2]].


  Discussion Top


Complete root canal disinfection is the mainstay of successful endodontic treatment.[13] Major challenges involved in achieving the above-mentioned goal are the microorganism-induced biofilms and the limitations associated with the currently used intracanal medicaments and irrigants in eliminating the bacteria located inside the dentinal tubules and other root canal ramifications such as delta, isthmuses, etc.[14] Biofilms belonging to E. faecalis group are specifically very resistant to elimination because of its ability to tolerate high pH, stress, starvation, and high salt concentration.[15] Moreover, presently there is no definite way to ensure complete root canal disinfection in single root canal session. Hence, an effective root canal medicament is required for definite period of time to obtain more predictable results.[16] Especially, microorganisms recovered from the persistent periradicular infection or the secondary infection are found to be associated with biofilms that have high resistance against the commonly used intracanal medicaments.[9],[17] Studies have suggested that the most common bacteria isolated from the secondary root canal infection is E. faecalis.[4] In an earlier reported study, the most common fungal species isolated from the infection of root canal system was C. albicans.[18] Hence the present study was conducted with the objective of checking the antimicrobial efficacy of SNPs with and without different antimicrobials against E. faecalis and C. albicans.

Ca(OH)2 has been used historically because of its proven antimicrobial property. But its action against E. faecalis is very limited. Ca(OH)2 acts by releasing hydroxyl ions and creating alkaline environment.[19] E. faecalis because of its resistant nature is able to survive in alkaline environment and easily skip the action of Ca(OH)2.[20] Hence, the search for a novel intracanal medicament propelled researchers to introduce nanoparticles in the field of endodontics.

CHX has unique property of substantivity[21] and has been proven to have efficient antimicrobial action against E. faecalis and C. albicans either alone, or in combination with other antimicrobials.[21],[22],[23] But recently, biofilm formation by the microorganisms is a major obstacle for the action of CHX.[24]

SNPs of 10 nm size have been used in the current study because its smaller size provides more surface area and hence, more amount of antimicrobial activity can be anticipated.[25],[26] In earlier studies, it was found that nanoparticles of 10 nm or lesser size are likely to have more antimicrobial effect.[26] Although various other nanoparticles are also reported to have antimicrobial activity, in the present study, specifically, SNPs were used because of its ability to react with the thiol group of DNA molecule of bacteria, causing its condensation and cell death.[27]

In the present study, SNPs were used as vehicle for calcium hydroxide and CHX, and the antimicrobial efficacy of the combinations was compared with respect to the individual antimicrobials after 24 h, 7 days, and 14 days. 24-h evaluation showed CHX alone to be significantly better in comparison with the other medicaments in both E. faecalis group and C. albicans group followed by combination of SNP and 2% CHX. The above result can be explained by the fact that antimicrobial efficacy of CHX is reduced when combined with the other antimicrobials.[28] 2% CHX alone showed significantly better results in comparison to SNP alone. To the best of the authors’ knowledge, when this study was conducted, there was no other study that evaluated the effect of combination of SNPs with CHX against E. faecalis and C. albicans. Combination of SNP with Ca(OH) 2 was found to be better in comparison to SNP and Ca(OH)2 alone. This finding was in agreement with the results of the previous study.[29] Possible reasons for this can be: (i) Inherent capacity of E. faecalis to tolerate alkaline environment created by Ca(OH)2 and (ii) creation of pits by SNP in the cell wall of the microorganism leading to disruption of biofilm and increased amount of Ca(OH)2 delivery. No significant difference was found between Ca(OH)2 and combination of SNP with Ca(OH)2 against C. albicans at 24 h. In the present study, SNP alone was found to be significantly better in comparison to the combination of Ca(OH)2 and SNP against C. albicans. The reason for this can be attributed to high surfactant activity of SNP, which disrupts the cell wall of yeast and increases its sensitivity to SNP.

In the present study, evaluation after 7 days showed 2% CHX to be more effective followed by combination of SNP and Ca(OH)2 against E. faecalis. These results are in agreement with the findings of the similar studies reported earlier.[29],[30] In those studies, authors found significantly less CFUs in the group having combination of SNP and Ca(OH)2 in comparison to Ca(OH)2 alone group. In the present study, antifungal activity of the 2% CHX and Ca(OH)2 was found to be significantly better in comparison to their combination with SNP. These findings corroborate with the findings of the other study[29] where CHX and Ca(OH)2 alone and in combination with each other have shown better antifungal activity. In the present study, the concentration and viscosity of SNP used might have been one of the reasons for the decreased antimicrobial efficacy of the combinations against C. albicans because earlier studies have shown that dissociation of Ca(OH)2 into hydroxyl and calcium ions depends on the concentration of vehicle used.[31],[32]

After 14 days, evaluation showed 2% CHX and Ca(OH)2 to be better against both E. faecalis and C. albicans rather than their combination with SNP. Similar results were reported in an earlier study where the authors noticed combination of CHX and Ca(OH)2 to be less effective in comparison to the individual medicaments.[33] Very limited studies exist which have evaluated the effect of combination of medicaments against C. albicans. The results of the present study can be attributed to the lesser availability of free Ca(OH)2 and CHX when combined with SNP over 14 days of time. Therefore, from the results of the present study, we can reject the null hypothesis that there will be no difference among tested antimicrobial agents in short- and long-term therapy.

Present study is possibly the first study that has evaluated the antimicrobial efficacy of SNPs in combination with CHX against E. faecalis and C. albicans. Size and concentration of SNPs used in the present study was very small, i.e., 10 nm and 100 ppm, respectively, which has not been reported in dentistry till now. Although concerns regarding the cytotoxicity of SNPs have been raised, recent study by Gomes Filhalo et al.[22] reported that 47 ppm of SNPs were biocompatible in comparison to 2.5% NaOCl against fibrous connective tissue of rat.


  Conclusion Top


2% CHX alone was significantly better in its antimicrobial efficacy against E. faecalis and C. albicans over the period of 24 h, 7 days, and 14 days. SNPs show great potential in their ability to be used as a vehicle for Ca(OH)2 and 2% CHX against the resistant microbes of root canal infection. Although 2% CHX was found to have significantly better results in comparison to the SNPs combination in the present ex vivo setup, more studies with in vivo conditions are required to validate the findings of the present study.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.

 
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