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 Table of Contents  
ORIGINAL ARTICLE
Year : 2019  |  Volume : 7  |  Issue : 1  |  Page : 23-27

Comparative evaluation of silver nanoparticles and 5.25% sodium hypochlorite for rapid chairside decontamination of artificially infected gutta-percha with Escherichia coli: An In vitro Study


1 Department of Conservative Dentistry and Endodontics, Purvanchal Institute of Dental Sciences, Gorakhpur, Uttar Pradesh, India
2 Department of Conservative Dentistry and Endodontics, People's Dental Academy, Bhopal, Madhya Pradesh, India
3 Department of Orthodontics and Dentofacial Orthopaedics, Babu Banarasi Das College of Dental Sciences, Lucknow, Uttar Pradesh, India

Date of Web Publication12-Apr-2019

Correspondence Address:
Priyesh Mishra
S/o Dr. S.C. Mishra, Opposite Gramin Bank, New Civil Lines, Meerabhawan, Pratapgarh - 230 001, Uttar Pradesh
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/dmr.dmr_37_18

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  Abstract 


Introduction: The aim of this in vitro study was to evaluate the efficacy of 50 ug/ml silver nanoparticle (AgNPs), 70 ug/ml AgNPs and 5.25% sodium hypochlorite (NaOCl) as a final irrigant for rapid chair-side decontamination of artificially infected gutta-percha (GP) with Escherichia coli. Materials and Methods: A series 120 GP cones size 40 were taken from new batch. The GP cones were divided into six experimental group: Group I - Evaluation of the contamination of GP cones in manufacturer's Box-(20 GP cones), Group II - Evaluation of GP artificially contaminated with E. coli(100 GP cones), Group III - Evaluation of the effectiveness of 50 ug/ml of AgNPs for decontamination of the cones contaminated with E. coli(n = 20 GP infected cones from Group II), Group IV - Evaluation of the effectiveness of 70 ug/ml of AgNPs for decontamination of the cones contaminated with E. coli(n = 20 GP infected cones from Group II), Group V - Evaluation of the effectiveness of 5.25% NaOCl for decontamination of the cones contaminated with E. coli(n = 20 GP cones infected from Group II). Aliquots from the experimental GP cones were plated on brain heart infusion agar (HiMedia Lab, Mumbai, India), and the colony-forming units were evaluated under colonimeter. Results: 70 ug/ml of AgNPs, 5.25% NaOCl exhibited similar antimicrobial effect (P = 1). Highly significant difference were found when 50 ug/ml compared with 70 ug/ml and 5.25% NaOCl (P < 0.0001). Conclusion: 70 ug/ml of AgNPs (0.007%) proved to be highly effective against E. coli and showed similar antimicrobial efficacy as 5.25% NaOCl at 750 times lesser concentration.

Keywords: Decontamination, Gram-negative bacteria, gutta-percha, silver nanoparticle, sodium hypochlorite


How to cite this article:
Mishra P, Tyagi S, Tripathi D. Comparative evaluation of silver nanoparticles and 5.25% sodium hypochlorite for rapid chairside decontamination of artificially infected gutta-percha with Escherichia coli: An In vitro Study. Dent Med Res 2019;7:23-7

How to cite this URL:
Mishra P, Tyagi S, Tripathi D. Comparative evaluation of silver nanoparticles and 5.25% sodium hypochlorite for rapid chairside decontamination of artificially infected gutta-percha with Escherichia coli: An In vitro Study. Dent Med Res [serial online] 2019 [cited 2019 Jun 15];7:23-7. Available from: http://www.dmrjournal.org/text.asp?2019/7/1/23/256023




  Introduction Top


Microorganisms play a fundamental role in the etiology of pulpal and periapical diseases, and their elimination from the root canal by chemical and mechanical procedures is one of the essential steps to secure successful treatment.[1],[2],[3],[4],[5],[6] In clinical practice, the dentist is occasionally faced with the problem of re-infection after endodontic treatment. Moreover, penetration of bacteria into the blood stream of systemically compromised patients should be prevented because of the risk of infective endocarditis.[1] One possible explanation for this phenomenon may be the introduction of contaminated gutta-percha (GP) cones into the root canal.[2] Therefore, much care must be taken during this procedure to prevent contamination of filling materials.[3],[7]

GP cones are the most commonly used material for filling the root canal. They are biocompatible, dimensionally stable, radiopaque, thermoplastic and easily removed from the root canal. Even though GP cones are produced under aseptic conditions and have potentially antimicrobial properties, especially owing to their zinc oxide component, they can be contaminated by handling and also by aerosols and physical sources during the storage process.[2],[3],[8],[9] Therefore, the handling of GP cones must follow the basic principles of infection control. In addition, GP cones that have been in contact with patients should be discarded1. Owing to the thermoplastic characteristic of GP cones, they may not be sterilized by the conventional process in which moist or dry heat is used, because this may cause alteration to the GP structure.[2],[3],[4],[6],[8],[10] For this reason, previous studies suggested that several different chemical agents could be used for the rapid decontamination of GP cones in dentistry.

Montgomery[11] showed that commercially available GP cones may be contaminated, even if carefully removed from factory-sealed containers. Contamination of GP cones may also occur during treatment, prior to their use. A GP master cone which does not fit well in one prepared root canal could still be used in another if effectively sterilized. Since the use of standard high temperature sterilization would deform GP cones, a rapid, chairside decontamination method is needed. Several studies have examined cold sterilizing agents for GP.[11],[12],[13],[14] One of the earlier reports regarding sterilization of GP cones, by Buchbinder,[15] recommended exposing the cones to paraformaldehyde vapour for 3 h. Senia et al.[16] found that formocresol vapour sterilized GP cones within 16 h. Such methods may be satisfactory for long-term storage of GP cones, but it is not suitable for rapid decontamination of GP during treatment.

Several chemical solutions for cold disinfection have been evaluated, such as polyvinylpyrrolidone-iodine, ethyl alcohol, sodium hypochlorite (NaOCl), hydrogen peroxide, quaternary of ammonium, glutaraldehyde, chlorhexidine liquid, Deconex 53plus. The time for these substances to kill microorganisms ranges from a few seconds to substantial periods of time.[12],[14],[17],[18],[19] NaOCl is one of the most widely used endodontic solutions, either as an irrigant or for rubber dam and cone decontamination. Its concentration ranges from 0.5% (Dakin solution) to 5.25%.[17]

Various nano-particles have gained popularity as antimicrobial agents as a result of their broad spectrum of activity and biocompatibility.[20] Recent studies have focused on using nano-particulate materials to disinfect root canals.[21],[22] Nanoparticulates exhibit higher antibacterial activity as a result of their polycationic/polyanionic nature with higher surface area and charge density, resulting in greater degree of interaction with the bacterial cell.[21] It has been observed that the size of nanoparticulates plays an important role in their antibacterial activity, with smaller particles showing higher antibacterial activity than the macro-scaled ones.[22],[23],[24]

Silver nanoparticle (AgNPs) shows antibacterial effect; it also exhibits novel physicochemical and biological activities.[21] In the medical field, there are wound dressings, contraceptive devices, surgical instruments and bone prostheses that are all coated or embedded with AgNPs.[25],[26],[27] Previous studies have investigated the antibacterial effect of AgNPs on both Gram-positive and negative bacteria such as Staphylococcus aureus,  Escherichia More Details coli, Pseudomonas aeruginosa and Staphylococcus epidermidis.[28],[29],[30]

Though antimicrobial activity of AgNPs has already been investigated but rapid decontamination of GP by AgNPs has never been studied.

The main objective of this paper is to evaluate and compare two different concentration of AgNPs and NaOCl when used as a chair side decontaminant after artificially infecting GP cones with E. coli.


  Materials and Methods Top


Gutta-percha cones

A series of 120 GP cones 6% size 40 (Dentsply, Maillefer, Ballaigues, Switzerland) were used throughout the experiment. The GP cones were divided into 5 groups:

  • Group I-Evaluation of the contamination of GP cones in manufacturer's Boxes-(20 GP cones)
  • Group II-Evaluation of GP artificially contaminated with E. coli (100 GP cones)
  • Group III-Evaluation of the effectiveness of 50 ug/ml of AgNPs for decontamination of the cones contaminated with E. coli (n = 20 GP infected cones from Group II)
  • Group IV-Evaluation of the effectiveness of 70 ug/ml of AgNPs for decontamination of the cones contaminated with E. coli (n = 20 GP infected cones from Group II)
  • Group V-Evaluation of the effectiveness of 5.25% NaOCl for decontamination of the cones contaminated with E. coli (n = 20 GP cones infected from Group II).


Microorganism and media

The microorganism used in the study was procured from ATCC (American type culture collection), representing the one of the bacteria that may act as contaminants in dental surgery E. coil. (ATCC 10536, Gram-negative). The culture was maintained on desired media agar slants at optimum temperature forin vitro growth, dated and labeled properly, stored at 4°C and sub-cultured periodically. Successive sub-culturing of the microbial strains was done with particular attention. Their purity was examined at regular intervals by staining and biochemical test. The growth media used in the current study were Brain Heart Infusion (BHI) media, for bacteria.

Contamination of gutta-percha cones

The GP cones were artificially contaminated with E. coli. A 24-h old pure culture of E. coli was grown in BHI agar plates. The plates were then incubated at 37°C for bacteria for 24–48 h, aerobically in a bacteriological incubator. Microbial suspensions of these cultures were prepared in sterile saline solution (0.85% w/v of NaCl), with the density of 0.5 McFarland corresponding to 1.5 CFU/ml × 108 CFU/ml (CFU = colony forming units) for bacteria adjusted spectrophotometrically.

BHI broth (for bacteria) was prepared by dissolving appropriate amount of media in distilled water and sterilized by autoclaving at 121°C (15 lb/in2) for 15 min. Under aseptic conditions, the GP cones were contaminated by immersion in broth previously inoculated with 0.1 ml of standardized microbial suspensions of the microorganism. In order to promote surface contamination, the cones were incubated at 37°C for bacteria for 24–48 h, aerobically in bacteriological incubator. Thereafter, the cones were transferred to sterile paper filter pads in petridish, briefly dried at room temperature under sterile condition and then disinfected.

Decontamination of gutta-percha cones

The artificially contaminated GP cones were subsequently subjected to decontamination using 50 ug/ml AgNPs, 70 ug/ml of AgNPs and 5.25% NaOCl and their disinfectant property were evaluated and compared. Two different concentrations of Ag-NP were used i. e. 50 μg/ml and 70 μg/ml while NaOCl were used with 5.25%. The testing times to disinfectant the cones was: 1 min. The contaminated cones were immersed individually in micro-centrifuge tubes containing 1.5 ml of the respective disinfectant to be tested and were vortexed for 1 min. After that, the cones were transferred to tubes containing saline solution (0.85% NaCl) and shaken for 30 s to detach any remaining microbial cells. These saline suspensions were then used as an inoculum for further procedure.

The pour-plate method (for bacteria) was employed to observe the presence of microbial colonies. For this, the BHI agar medium was prepared and sterilized by autoclaving at 121°C (15 lb/in2) for 15 min. The petriplates were washed thoroughly, sterilized in autoclave and dried in hot air oven at 160°C for 2 h for complete removal of moisture.

Pour-plate method (for bacteria)

Under aseptic condition, 0.1 ml of each saline suspension (bacterial inoculum) was added to the respective sterile, labeled and empty petridishes and approximately 15–18 ml of sterilized molten BHI agar was added to it. Prior to pouring, the agar media was cooled between 45°C and 50°C to avoid killing of bacterial cells and to minimize the amount of condensation that forms. The plates were then gently tilt and swirl to ensure proper mixing of inoculum with the media and were allowed to completely solidify at room temperature. These solidified plates were then incubated at 37°C for 24 h and observed for (CFU/ml).

Control group

The possibility of initial contamination of GP cones as well as the effectiveness of the contamination procedure employed was examined by cultivating decontaminated GP cones (negative-control) and contaminated GP cones (positive-control) individually into sterile BHI broth. All control groups were inoculated in selected agar media and incubated at appropriate temperature and for 24–48 h.

Statistical analyses

Statistically analyses used for result were unpaired t-test and one-way analyses of variance (ANOVA) was used for the investigation of difference in CFU of E. coli.


  Results Top


  • Group I – Evaluation of the contamination of GP cones in manufacturer's Boxes: None of the cones removed from new boxes were culture positive
  • Group II – Evaluation of GP artificially contaminated with E. coli: 100 presterilized GP cones from were artificially contaminated with E. coli since all the culture came out to be positive. 1476 CFU/ml were observed in colonimeter
  • Comparison between Group III and Group IV – Evaluation of the effectiveness of 50 ug/ml of AgNPs and 70 ug/ml of AgNPs for decontamination of the cones contaminated in E. coli: Group III–CFU/ml in 50 ug/ml of AgNPs came out to be 81 where as no CFU/ml in 70 ug/ml of AgNPs was observed. Stastically significant difference was observed when unpaired t-test was applied (P ≤ 0.0001) [Table 1]
  • Comparison between Group III and Group V – Evaluation of the effectiveness of 50 ug/ml of AgNPs and 5.25% NaOCl for decontamination of the cones contaminated in E. coli:CFU/ml in 50 ug/ml of AgNPs came out to be 81 where as no CFU/ml in 70 ug/ml of AgNPs was observed. Stastically significant difference was observed when unpaired t-test was applied (P ≤ 0.0001) [Table 2]
  • Comparison between Group IV and GroupV – Evaluation of the effectiveness of 70 ug/ml of AgNPs and 5.25% NaOCl for decontamination of the cones contaminated in E. coli: No CFU/ml were found when contaminated GP were disinfected by 70 ug/ml of AgNPs and 5.25% of NaOCl. No significant difference was observed when unpaired t-test was applied (P = 1) [Table 3]
  • Comparison between Group III, Group IV and Group V – Evaluation of the effectiveness of 50 ug/ml of AgNPs, 70 ug/ml of AgNPs and 5.25% NaOCl for decontamination of the cones contaminated in E. coli: ANOVA test was applied for intergroup analyses. Statistically highly significant difference was observed (P ≤ 0.0001) [Figure 1] and [Table 4].
Table 1: Comparison of disinfection efficacy between two concentration of Silver Nanoparticles (AgNPs)

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Table 2: Comparison of disinfection efficacy between 50ug/ml of AgNPs and 5.25% NaOCl

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Table 3: Comparison of disinfection efficacy between 70ug/ml of AgNPs and 5.25% NaOCl

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Figure 1: Graphical representation of disinfectants and Escherichia coli colonies

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Table 4: Comparison of disinfection efficacy between 50ug/ml of AgNPs, 70ug/ml AgNPs and 5.25% NaOCl

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  Discussion Top


In the present study all the GP from the recently opened pack did not contain any bacteria still other studies have shown that GP cones can be easily contaminated when exposed to the dental operatory environment, during storage and when manipulated incorrectly by the operator.[8] Other studies have shown that 5%–8% of GP cones from sealed packages can be contaminated with bacteria.[8],[11]

The results of this study showed that none of the brand new GP cones that were cultured showed bacterial contamination as evaluated by culture. This corroborates others that also found no contamination in sealed new GP cones.[31],[32] However, our study is in contradiction to several studies[8],[11] which did find contamination on GP cones from new packaging.

The use of 5.25% of NaOCl in this study showed that it was effective after 1 min which was in accordance with previous studies which confirmed the efficiency of 5.25% of NaOCl.[6],[8]

AgNPs was another disinfectant used in the present study have shown excellent antimicrobial property. The antibacterial activity of 70 ug/ml AgNPs against E. coli was found to be higher than that of 50 ug/ml [Table 1]. The present study was in accordance with the study conducted by Kim et al.[33] who reported that the minimum inhibitory concentration of AgNPs for E. coli, Gram-negative bacteria(0.33 nM) was lower than that for S. aureus, Gram-positive bacteria (3.3 nM), which was a 10-fold difference. This may be attributed to the fact that Gram-negative bacteria have relatively thin cell walls and thus are more prone to physical destruction.

In the present study the size of the AgNPs was 21.3–2 9.1 nm. Shape of AgNPs used in the present study was truncated triangular which was found to provide better bactericidal action which corroborates with earlier studies.[33],[34] Truncated triangular silver nanoplates with a (111) lattice plane as the basal plane displayed the strongest biocidal action, compared with spherical and rod-shaped nanoparticles and with Ag (in the form of AgNO3). It is proposed that nanoscale size and the presence of a (111) plane combine to promote this biocidal property.

Study conducted by Hwan et al.[35] showed that Minimum Inhibitory concentration for inhibiting S. aureus is 100 ug/ml and 150 ug/ml for E. coli. On contrary in the present study 70 ug/ml completely eliminated E. coli which has bettered the previous studies. Study conducted by Raffi et al.[36] found that complete elimination of E. coli at 60 ug/ml which was analogous to present study result.

Some researchers have reported that the antimicrobial effect of the AgNPs on Gram-negative bacteria was dependent on the concentration of Ag in the nanoparticles and was closely related to the formation of “pits” in the cell walls.[37] Further, negatively charged AgNPs accumulated in the bacterial membrane increased the permeability of the membrane.

Miura and Shinohara[38] determined the biologic effects of AgNPs exposure to mammalian cells. In their paper, Hela cells were evaluated by being exposed to different concentrations of AgNPs. They concluded that 120 μg/ml concentration could be harmful for Hela cells.

By considering this study, a therapeutic window can be provided to use AgNPs in 0.005% and 0.007% concentration as a novel intracanal irrigant. On the other hand, because of broad spectrum antibacterial action of AgNPs and its influence on Gram-positive and negative bacteria, more investigations are needed to consider AgNPs as an irrigant in primary polymicrobial endodontic infections.


  Conclusion Top


In the present study it has been clearly observed that there was huge reduction of microcolonies of E. coli by both AgNPs and NaOCl. Lesser concentration of AgNPs has been able to reduce the colony growth but was unable to completely eliminate the growth. However, 70 ug/ml (0.007%) of AgNPs completely annihilates the bacterial growth so does 5.25% NaOCl. So it can be inferred that AgNPs has same antimicrobial efficacy at 750 times lesser concentration.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.



 
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    Figures

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    Tables

  [Table 1], [Table 2], [Table 3], [Table 4]



 

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