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ORIGINAL ARTICLE
Year : 2017  |  Volume : 7  |  Issue : 1  |  Page : 29-35

Antibacterial effect of silver nanoparticles against Enterococcus faecalis


Department of Restorative Dental Sciences, Division of Endodontics, King Saud University College of Dentistry, Riyadh, Saudi Arabia

Date of Web Publication10-Jan-2017

Correspondence Address:
Abdelhamied Yousef Saad
King Saud University College of Dentistry, Riyadh
Saudi Arabia
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/1658-5984.197989

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  Abstract 

Objective: This study aimed to investigate the bactericidal effect of silver nanoparticles (AgNPs) in reducing bacterial infection in root canal when used as intracanal medicament alone or in addition to the conventionally used calcium hydroxide (Ca(OH)2).
Materials and Methods: The root canals of 110 single-rooted teeth were cleaned, shaped, and sterilized. All groups, except for negative control, were inoculated with Enterococcus faecalis for 48 h. Then, teeth were divided into five groups according to the intracanal medicaments to be used. Group I (n = 30): Ca(OH)2. Group II (n = 30): AgNP. Group III (n = 30): AgNP + Ca(OH)2. Group IV (n = 10) was used as a positive control where the root canals were inoculated with E. faecalis and left without treatment. Group V (n = 10) was used as a negative control where the root canals were checked for the absence of bacterial growth. Specimens were incubated for 1 and 2 weeks. Pre- and post-medication samples were obtained by paper points, and the colony-forming units were counted.
Results: Ca(OH)2 resulted in a higher percentage of bacterial reduction in both 1 and 2 weeks of application (81.5% and 98%, respectively). AgNP was ineffective against E. faecalis with 32.9% bacterial reduction in 1 week and 56.5% after 2 weeks.
Conclusion: It was concluded that the antibacterial effect of AgNP was lower than Ca(OH)2 or combination of both materials.

Keywords: Antibacterial, calcium hydroxide, Enterococcus faecalis, silver nanoparticles


How to cite this article:
Alabdulmohsen ZA, Saad AY. Antibacterial effect of silver nanoparticles against Enterococcus faecalis. Saudi Endod J 2017;7:29-35

How to cite this URL:
Alabdulmohsen ZA, Saad AY. Antibacterial effect of silver nanoparticles against Enterococcus faecalis. Saudi Endod J [serial online] 2017 [cited 2019 Dec 9];7:29-35. Available from: http://www.saudiendodj.com/text.asp?2017/7/1/29/197989


  Introduction Top


The role of bacteria and their by-products in the initiation, propagation, and persistence of pulpal and periapical disease has been confirmed via several investigators.[1],[2] Elimination of microorganisms from infected root canal system to create a favorable healing environment is the major goal of root canal treatment. Most infecting bacteria and necrotic pulp debris may be removed by routine intracanal procedures such as mechanical cleaning and shaping and chemical irrigation of the pulp space.[3],[4] Augmentation of the antibacterial protocol can be achieved by utilizing intracanal interappointment medicaments, which are capable of further reducing the bacterial load.[5],[6]

Calcium hydroxide Ca(OH)2 is considered the most commonly used intracanal medicament. The role of Ca(OH)2 in endodontics as an intracanal medicament is based on its ability to induce hard tissue formation during apexogenesis or pulp capping,[7] its antibacterial actions,[8] and tissue-dissolving capability.[9] The antibacterial effect is thought to be mainly due to the nonspecific bactericidal action of high alkalinity [10] and its ability to eliminate apical exudates.[11] At a pH level of 9.5-12, most of the microorganisms can be eliminated and few can survive at a level of 11 and above.[11],[12]

Enterococcus faecalis has been considered the main etiological pathogen associated with posttreatment disease due to its prominence in root-filled teeth with apical periodontitis, where it has also been detected as monocultures.[13] Several studies have found that E. faecalis to be highly resistant to routine randomized controlled Trial.[14],[15],[16],[17]E. faecalis overcomes the challenges of survival within the root canal system in several ways. It has an intrinsic resistance to irrigant solutions, intracanal medicaments, several antibiotics, and high alkaline pH. Moreover, one advantage of E. faecalis over other species is its ability to tolerate harsh environmental conditions, formation of biofilm and invading deeply into the dentinal tubule. Despite its excellent properties, Ca(OH)2 is not equally effective against all microorganisms within root canals.

Nanotechnology is considered to be a breakthrough in the field of medicine. It can be useful in producing advanced biomaterials with unique physical, chemical, and biological properties.[18],[19] This is mainly approached by enhancing surface-to-volume ratio. To control various biological processes, we can use nanomaterials with predefined geometries, surface characteristics, and mechanical strength.[20]

Silver nanoparticles (AgNP) have been used in various medical applications because of its antibacterial and anti-inflammatory effects. AgNPs having a size range of 10–100 nm have showed strong bactericidal potential against Gram-positive, Gram-negative, and multidrug-resistance bacteria.[21],[22],[23],[24] Silver antimicrobial property is defined by the release rate of silver ion. Silver in bulk status is considered an inert material. However, when it gets ionized by moisture, it will result with highly reactive state. At this stage, silver can interact with bacterial cell wall leading to structural changes due to its binding to tissue protein.[25] Previous studies have shown that AgNP was more effective than Ca(OH)2 in the removal of E. faecalis.[26],[27],[28] However, few studies have evaluated the antibacterial effect of combined AgNP and Ca(OH)2 as an intracanal medicament.[26]

The aim of this investigation was to determine the effect of AgNP in reducing bacterial infection in root canal when used as intracanal medicament alone or in combination with Ca(OH)2.


  Materials and Methods Top


Materials used in this investigation were Ca(OH)2 paste (Calasept ® Plus, Nordiska Dental, Sweden) and AgNP gel (Huzheng ® Nano Technology Co, Ltd, Shanghai, China).

Teeth selection and preparation

One hundred and ten single-rooted, freshly extracted human teeth were collected and stored in saline. Teeth were embedded in Ortho-resin, and a standardized length of 15 mm was obtained by low-speed, precision saw (Isomet ® 2000 Precision, Saw, Buehler Ltd, Lake Bluff, IL, USA). After removal of pulp tissue, a #10 K file was used to check the patency of the canals, and standard root canal cleaning and shaping were performed using rotary Ni–Ti ProTaper to size F3 file (Dentsply Tulsa Dental; Tulsa, Oklahoma). Glyde (MD-ChelCream, Meta ® Biomed Co., Ltd, Korea) was used as a lubricant. After each instrument change, irrigation with 5 mL of 2.5% sodium hypochlorite was done and 3 mL of 17% EDTA was applied for 1 min as the final irrigation. The external surface of the teeth was dried with sterile gauze, apices were sealed with resin composite (Megafill MH, Megadenta Dentalprodukte, Germany), and two layers of nail polish were applied over the entire external surface of all specimens except the canal orifice. Specimens were sterilized under 25 kGy gamma radiation. To confirm the efficacy of the sterilization process, random samples were selected and placed in microcentrifuge tube containing brain heart infusion (BHI), incubated for 24 h, and then checked for turbidity.

Bacterial inoculation of specimens

A 24-h pure culture suspension of E. faecalis (ATCC 29212) was collected by centrifugation (1000 ×g for 10 min), and the pellet was suspended in sterile BHI broth at 37°C. Teeth were inoculated with 15 μL of 1 × 108 colony-forming unit (CFU)/mL suspension of E. faecalis (determined by serial dilution and plating) and incubated at 37°C and 95% humidity for 48 h to grow in a planktonic form. After incubation, the teeth were removed from the tubes aseptically and gently rinsed with sterile phosphate-buffered saline (PBS) to remove the culture medium and nonadherent bacteria. Then, 15 μl of saline was injected in each specimen, and premedication sample (S1) was obtained by sterilized #25 paper point applied in root canal for 1 min. This procedure was performed twice for each specimen. Subsequently, root canals were dried by paper points before the application of the medication.

Antibacterial activity of intracanal medicament

In this pilot study, different combinations of AgNP + Ca(OH)2 were tested and the ratio of 1:2 was selected for better handling of the materials. In addition, 0.1 ml of these materials was found to completely fill the root canal system, and this amount was used in this experiment.

Teeth were labeled and divided into five groups as follows:

  • Group I (n = 30): Ca(OH)2 only – 0.1 mL of Ca(OH)2 paste was inserted into each root canal.
  • Group II (n = 30): AgNP only – 0.1 mL of AgNP gel was inserted into each root canal.
  • Group III (n = 30): Ca(OH)2 + AgNP – 1.0 mL of AgNP gel was mixed with 2 mL of Ca(OH)2, and 0.1 mL of that mixture was inserted into the root canal.


The medicaments were placed into the canals by using size 27-gauge needle and 3 mL of sterilized syringe.

Group IV (n = 10): Positive control - The specimens were inoculated with E. faecalis and left untreated to confirm the presence of infection.

Group V (n = 10): Negative control - The specimens with no bacterial contamination or medication were applied in BHI and checked for sterility.

The pulp chamber of all the specimens was sealed with sterile dry cotton pellet and Cavit (MD Temp, Meta Biomed Co, Ltd, Korea) to prevent any external contamination. Teeth were then incubated at 37°C anaerobic jar for 1 and 2 weeks. At each time period, specimens from each experimental group (n = 15) were irrigated with 5 ml of sterile saline to remove the medicament with the slight instrumentation of root canal wall with #20 K file. In addition, five teeth from each control group were examined after 1 and 2 weeks.

Assessment of antibacterial activity

Bacteriological sampling was obtained using sterile paper points (S2). Two paper points were placed individually inside each root canal for 1 min and placed individually in 1.5 mL Eppendorf tubes containing 1 ml PBS and vortexed at the highest speed for 30 s each. Twenty-five microliters of the suspension was plated on BHI agar plate. All the plates were incubated at 37°C in anaerobic jar for 24 h, after which the colonies were enumerated by using a colony counter (Reichert-Jung). The process was carried out twice for each group of all the samples, and the average was reported. After confirming the purity of the positive cultures using Gram-staining, colony morphology, and biochemical identification, the number of CFUs of each specimen was determined.

Statistical analysis

Data were collected and analyzed using SPSS software computer program (Statistical Package for Social Sciences) version 21 (SPSS Inc., Chicago, USA). Descriptive statistics (mean, standard deviation, and standard error) was used to describe the quantitative study variables. Student's paired t-test and independent t-test were used to compare the mean values of CFU before and after the application of drugs and between two time points (1 week and 2 week). P < 0.05 was used to report the statistical significance of the results.


  Results Top


One-week exposure to intracanal medicament

No bacterial growth was detected in the negative group at this incubation period or any other periods. The percentage of bacterial reduction was higher in Ca(OH)2 group (81.5%) followed by Ca(OH)2 + AgNP (56.3%), and (32.9%) in AgNP group [Figure 1],[Figure 2],[Figure 3].
Figure 1: E.faecalis plate for sample obtained after incubation for 1 week with Ca(OH)2

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Figure 2: E.faecalis plate for sample obtained after incubation for 1 week with AgNP

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Figure 3: E.faecalis plate for sample obtained after incubation for 1 week with AgNP + Ca(OH)2

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Two-week exposure to intracanal medicament

The percentage of bacterial reduction was higher in Ca(OH)2 group (98.5%) followed by Ca(OH)2 + AgNP (85.4%), and (56.5%) in AgNP group [Figure 4],[Figure 5],[Figure 6].
Figure 4: E.faecalis plate for sample obtained after incubation for 2 weeks with Ca(OH)2

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Figure 5: E.faecalis plate for sample obtained after incubation for 2 weeks with AgNP

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Figure 6: E.faecalis plate for sample obtained after incubation for 2 weeks with AgNP + Ca(OH)2

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Comparison between one and two weeks of medication

There was a statistically significant difference in the mean value of CFU between 1 and 2 weeks after the application of medicaments in Ca(OH)2 group. No statistically significant difference was observed between the values of the other three groups (AgNP, Ca(OH)2 + AgNP, and positive) after 1 or 2 weeks [Table 1] and [Table 2].
Table 1: Comparison of the mean values of colony-forming units between 1 and 2 weeks duration, in each of the four study groups

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Table 2: Comparison between before and after the application of medications in both time periods and also the percentage of bacterial reduction for each group

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  Discussion Top


To gain a successful endodontic treatment of teeth with apical periodontitis, an effective control of the root canal infection must be achieved.[3],[4] Chemomechanical cleaning and shaping of the infected root canal results in the reduction of microorganism counts, but cannot eliminate it completely. This is mainly because of anatomical complexity and access limitations to the root canal system by instruments and irrigants.[5],[29] In some cases where microorganisms are resistant to regular therapy and present with pain and exudation, the need for intracanal medication is increased.[30] Although Ca(OH)2 is widely used, it cannot be considered a universal intracanal medicament, since it is not equally effective against all bacteria found in the root canal. Indeed, several studies have reported the failure of Ca(OH)2 to eliminate Enterococci effectively, as they tolerate high pH values, varying from 9 to 11.[31],[32],[33]

Our results revealed a significant difference in the antibacterial effect of Ca(OH)2 and Ca(OH)2 + AgNP groups in both 1 and 2 weeks of application compared with AgNP group. The effect of Ca(OH)2 group is in accordance with the results showed in the previous studies where Ca(OH)2 was highly effective against E. faecalis.[8],[34],[35] The results of this experiment were in contrast with several previous studies where Ca(OH)2 was appeared ineffective against E. faecalis.[31],[32],[33] The difference may be attributed to the variation in Ca(OH)2 vehicle, experimental model, use of bacteria in biofilm, time of application, and use of different analytical methods.

AgNP group was not effective against E. faecalis after 1 week of exposure. However, a significant reduction of bacterial count was observed after 2 weeks. Although there was an antibacterial effect with AgNP, this effect is lower than the one with Ca(OH)2 alone. This is in contrast with numerous studies where a strong antibacterial effect of AgNP against different bacterial strains was detected.[27],[36],[37] This variation in results may be explained due to the differences in the mode of application, concentration, and particle diameter used.

The present results showed a signifi cant reduction of CFU in Ca(OH)2 + AgNP group compared to AgNP alone. However, when compared with Ca(OH)2 group, the latter resulted in a lower CFU with no significant difference between them. In the current work, the addition of AgNP to Ca(OH)2 reduced the efficacy of the latter. This may be due to an antagonistic action between both. No previous research explained this antagonistic action so far. In contrast, Javidi et al.[26] have found that the number of CFU was significantly less in a combination of Ca(OH)2 + AgNP than Ca(OH)2 alone. This may be due to the difference in the time of drug application which was 1 and 7 days. In addition, they used AgNP with a concentration of 200 ppm and an average diameter of 70 nm in suspension. In our study, we used a concentration of 20 ppm with an average diameter of 2 nm in the form of colorless gel. Our results were in contrast with the results obtained by Afkhami et al.[28] In our study, the specimens were incubated for 1 and 2 weeks, and the combination of Ca(OH)2 + AgNP was found to be mildly effective against E. faecalis. In their study, the incubation of the medicament was for 1 week and 1 month. The combination of Ca(OH)2 + AgNP was most effective in 1-week samples comparing to Ca(OH)2, but after 1 month, it was similar to the other groups. This difference in results may be attributed to the differences in concentration (20 ppm vs. 100 ppm) and in particle diameter (2 nm vs. 20 nm) used in our study and their study, respectively.

The fact that E. faecalis is proved to be resistant to variable intracanal medications and it is frequently associated with persistent disease after endodontic treatment led to its use for the inoculums in this study.[9],[38] In addition, E. faecalis is relatively easy to culture, and it has been used successfully in most of the studies.[26],[27],[28],[39] One of the mechanisms that enabled E. faecalis to resist treatment and to survive is its ability to form biofilm and become more resistant to phagocytosis, antibodies, and antimicrobial agents than nonbiofilm-producing organisms.[40]E. faecalis in planktonic form was used in the present study to evaluate the response of bacteria without being in biofilm structure.


  Conclusion Top


From the scope of this investigation, we can conclude the following:

  • The AgNP is less effective against E. faecalis than the Ca(OH)2 alone or combination of both materials
  • Although the combination of Ca(OH)2 and AgNP has reduced the bacterial count in both time periods, this reduction is considered lower than the one obtained with Ca(OH)2 alone.


Acknowledgment

The authors would like to thank Microbiological laboratory in the College of Dentistry- King Saud University for their continuous assistance during this project. The authors would like also to acknowledge the contribution of Dr. Shaik Shaffi who assisted with the statistical analysis.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.

 
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    Figures

  [Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5], [Figure 6]
 
 
    Tables

  [Table 1], [Table 2]


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