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ORIGINAL ARTICLE
Year : 2012  |  Volume : 4  |  Issue : 1  |  Page : 26-31

An in vitro comparative evaluation of the antibacterial efficacy of 10% metronidazole gel, 2% chlorhexidine gel, and a combination of calcium hydroxide and 2% chlorhexidine gel against Enterococcus faecalis


1 Department of Conservative Dentistry and Endodontics, Kamineni Institute of Dental Sciences, Narketpally, India
2 Department of Conservative Dentistry and Endodontics, Bangalore Institute of Dental Sciences, Lakkasandra, Bangalore, Karnataka, India
3 Department of Conservative Dentistry and Endodontics, SIBAR Institute of Dental Sciences, Guntur, Andhra Pradesh, India
4 Department of Oral Pathology, Kamineni Institute of Dental Sciences, Narketpally, India

Date of Web Publication10-Sep-2012

Correspondence Address:
Jaya Nagendra Krishna
Department of Conservative Dentistry and Endodontics, SIBAR Institute of Dental Sciences, Takellapadu, Guntur, Andhra Pradesh
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/0975-8844.99886

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  Abstract 

Background and Objectives: A major objective in endodontic therapy is to disinfect the root canal system prior to obturation. This is because the residual root canal infection can sustain persistent or recurrent periapical disease. Hence, the use of an intracanal medicament between appointments helps in the elimination of bacteria that remain even after cleaning and shaping. The objective of this in vitro study is to compare the antibacterial efficacy of 10% metronidazole gel, 2% chlorhexidine (CHX) gel, and a combination of calcium hydroxide and 2% CHX gel against Enterococcus faecalis (E. faecalis) using a culture technique. Materials and Methods: The study included 40 single-rooted, human permanent teeth, ­extracted for periodontal or other reasons. Conventional access to the root canals was obtained using access preparation burs in a high speed handpiece. The working length was determined using the Ingle's radiographic method and the canals were instrumented using a Step-back technique with K-files, up to size 40, at the apex and irrigated with 1 ml of 2.5% Sodium hypochlorite and Ethylenediaminetetraacetic acid gel. The root canals were filled with a sterile Casein Soya meal peptone solution (CSL) and autoclaved twice for 30 minutes at 121°C. An inoculum of E. faecalis was injected into the canals using a sterile syringe and it was incubated aerobically at 37°C for nine days. The specimens were then randomly divided into three experimental groups (10% metronidazole gel, 2% CHX gel, and a combination of calcium hydroxide and 2% CHX gel) and one control group, each containing 10 samples. Following this, the canals were cleaned using an ultrasonically activated No.15 K-file, along with sodium ­hypochlorite irrigation. After medicament removal, each root canal was prepared manually with a new size 40 hedstrom file. The colony forming units per millimeter were determined by the standard laboratory methods. The obtained data was subjected to statistical analysis using the Mann-Whitney test and the χ2 test. Results: In our study, Group II (2% CHX gel) showed significant reduction of E. faecalis. Group I (10% metronidazole gel) showed minimal ­reduction, and Group III (2% CHX gel with calcium hydroxide) showed moderate ­reduction. There was no ­reduction in observed in Group IV (control group) patients. Conclusion: Our study ­signifies that 2% CHX gel showed substantial antimicrobial activity against E. faecalis. The ­combination of calcium hydroxide and 2% CHX gel also showed good ­antimicrobial activity. Hence, the efficacy of 2% CHX gel was greater than its ­combination with ­calcium hydroxide. The least effective drug against the microbes was metronidazole.

Keywords: Colony forming units, CSL, E. faecalis, ultrasonic #15 K-file


How to cite this article:
Krishna JN, Raghu R, Bolla N, Muddanna K. An in vitro comparative evaluation of the antibacterial efficacy of 10% metronidazole gel, 2% chlorhexidine gel, and a combination of calcium hydroxide and 2% chlorhexidine gel against Enterococcus faecalis. J Orofac Sci 2012;4:26-31

How to cite this URL:
Krishna JN, Raghu R, Bolla N, Muddanna K. An in vitro comparative evaluation of the antibacterial efficacy of 10% metronidazole gel, 2% chlorhexidine gel, and a combination of calcium hydroxide and 2% chlorhexidine gel against Enterococcus faecalis. J Orofac Sci [serial online] 2012 [cited 2020 Dec 3];4:26-31. Available from: https://www.jofs.in/text.asp?2012/4/1/26/99886


  Introduction Top


Pulpal infection due to caries or trauma can result in the microbial colonization of the root canal system including ramifications, isthmuses, apical deltas, and dentinal tubules. [1],[2],[3] Root canal infections are usually polymicrobial in nature and are dominated by anaerobic bacteria. These microorganisms and their toxic metabolic products are responsible for the development and persistence of apical periodontitis of endodontic origin. [1] The microbial elimination from the endodontium, is therefore, crucial in the treatment of the infected root canal. [1]

Root canal disinfection is usually accomplished by mechanical instrumentation and various irrigating solutions, which eliminate bacteria in approximately 50% of the root canals. [4] Residual bacteria within the root canals grow and multiply, causing persistent or recurrent periapical disease. [5] Hence, the use of an intracanal medicament between appointments helps in bacterial elimination. [6]

Various intracanal medicaments have been used in the past, such as, essential oils, phenolic compounds, halogens, and antibiotics. [7] However, calcium hydroxide has remained the intra-canal medicament of choice due to its antibacterial activity. Although effective against a majority of endodontic pathogens, E. faecalis and Candida albicans survive and are resistant to calcium hydroxide. [1],[5] E. faecalis have frequently been associated with persistent endodontic infections and failed root canal therapy. [8]

The search for a better alternative to calcium hydroxide has led to the introduction of newer antimicrobial agents like metronidazole, chlorhexidine (CHX), and so on. [6] Metronidazole is bactericidal against most anaerobic bacteria, due to its radical mediated mechanism. Of late, 2% metronidazole gel has been reported to exhibit good antibacterial activity against E. faecalis, and increasing its concentration may completely eliminate E. faecalis. [6]

CHX gluconate is a cationic biguanides, which has bacteriostatic and bactericidal effects at low and high concentrations, respectively. [9],[10] In endodontics, the application of CHX in gel form in concentrations of 0.2 or 2% has been suggested as an intracanal medicament. [6],[11]

Recent studies have suggested that a combination of calcium hydroxide and CHX has improved antimicrobial efficacy against calcium hydroxide−resistant microbes, as adding CHX could increase the antimicrobial activity of calcium hydroxide. [12]

Hence, this in vitro study was done to compare the antibacterial efficacy of 10% metronidazole gel, 2% CHX gel, and a combination of calcium hydroxide and 2% CHX gel, against E. faecalis.


  Materials and Methods Top


Forty single-rooted, human permanent teeth extracted for periodontal or other reasons were selected for the study and stored in physiological saline, to prevent dehydration before use.

Conventional accesses to the root canals were obtained using access preparation burs in a high speed handpiece. The working length was determined using the Ingle's radiographic method and instrumented using a Step-back technique with K-files up to size 40 at the apex. With each change in file size, the canal was irrigated with 1 ml of 2.5% sodium hypochlorite and Ethylenediaminetetraacetic acid (EDTA) gel. The final irrigation was done for one minute using 17% EDTA solution to eliminate the smear layer produced during the initial preparation.

All teeth were dried with filter paper and paper points and weighed. Thereafter, the canals were filled with water, and the teeth were weighed again, to calculate the volume of the canals in microliters. The apical foramina of all teeth were sealed with epoxy resin to prevent microleakage. All teeth were stored in physiological saline to prevent dehydration.

Before microbiological investigations, the surfaces were dried with filter paper and with paper points in the root canal. The root canals were filled with a sterile Casein Soya meal peptone solution (CSL) and the specimens were placed in test tubes containing CSL and autoclaved twice for 30 minutes at 121° C. Finally, the CSL was removed with a sterile syringe, and the roots were blotted dry with filter paper.

The E. faecalis organism was cultured for 24 hours at 37° C in CSL. An inoculum of E. faecalis was injected into the canals using a sterile syringe. The inoculated specimens were placed in test tubes filled with agar, covered with 200 μl of physiological saline, to prevent them from drying. The test tubes were covered with a glass lid and incubated aerobically at 37° C, for nine days.

After the last inoculation, the contaminated CSL was removed with a sterile syringe and the canals were blotted dry with sterile paper points. The specimens were then randomly divided into three experimental groups and one control group [Table 1], each containing 10 samples.
Table 1: Distribution of samples

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After placement of the intracanal medicaments in the teeth of all the groups, the specimens were incubated at 37°C, for seven days. Following this, the canals were cleaned using an ultrasonically activated No.15 K-file, along with water irrigation, for complete removal of the medicament.

After the medicament removal, each root canal was prepared manually with a new Hedstrom file, size 40, with sterile gloves worn in each case. Each file, together with the dentin removed from the canal wall, was transferred to a test tube containing 10 ml CSL with inactivating substances. The instrument was shaken up in the test-tube for 10 seconds with a vibrator. The colony forming units (CFU) per millimeter in the shaken solution were determined by standard laboratory methods.

On account of the differences in the volumes of the root canals, the determined CFU values were converted to 10 μl, as follows, to obtain a standardized volume for all root canals:

V 1 * CFU / V 2 = CFU cor

Where V 1 = Standardized volume of 10 μl

V 2 = Actual volume of the root canal

CFU = CFU in 1 ml of shaken solution

CFU cor = CFU corrected per 1 ml of shaken solution

A Mann-Whitney test was used to evaluate the differences between the log reduction factors of different disinfectants. Additionally, an χ2 test was performed on the number of teeth with positive cultures, after treatment with one of the three medicaments [Figure 1] and [Figure 2].
Figure 1: Colony forming units (CFU) of Medicaments and Control

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Figure 2: Reduction factor of different medicaments used

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  Results Top


Group I with 10% metronidazole gel showed minimal reduction. The results of the present study indicate that the Group I samples had 7.72 × 105 CFU/ml [Table 2], [Table 3] and [Table 4] of E. faecalis with 10% Metronidazole gel, in our study. [Figure 3]. However this was not very effective against E. faecalis, as seen in the results.
Table 2: Colony forming units of dentin layers of control teeth (n = 10)

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Table 3: Log reduction of colony forming units of dentin layers of control teeth (n = 10)

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Table 4: Pairwise significance by Mann Whitney U test – Log reduction of CFU / ml

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Figure 3: Colony forming units of different groups. Group – I: 10% metronidazole gel

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In our study, Group II samples with 2% CHX gel, showed significant reduction of E. faecalis. Group II samples had excellent efficacy against E. Faecalis, because in a total of 10 canals incubated with the bacterial strain and treated with CHX, only 2.80 × 105 CFU / ml were found [Figure 4]. Samples in Group III, medicated with a combination of calcium hydroxide and 2% CHX gel, showed a moderate reduction which demonstrated 5.08 × 105 CFU /ml of E. Faecalis [Figure 5]. This was significantly inferior to the performance of 2% CHX alone [Table 2], [Table 3] and [Table 4].
Figure 4: Colony forming units of different groups Group – II: (2% chlorhexidine gel)

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Figure 5: Colony forming units of different groups Group – III: Combination of calcium

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There was no reduction observed in Group IV (control group) patients. Group IV (Control Group) did not show any antibacterial activity as no intracanal medicament was placed and it had 9.77 × 105 CFU / ml [Table 2], [Table 3] and [Table 4] E. Faecalis [Figure 6]. This reiterates the importance of intracanal medicaments as interappointment dressings.
Figure 6: Colony forming units of different groups hydroxide and 2% chlorhexidine gel) Group – IV: Control group

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  Discussion Top


The success of endodontic therapy can be greatly enhanced if bacteria are eliminated during obturation. [9] Treatment strategies involving chemomechanical means eliminate 40 - 60% of the cultivable bacteria in the root canal. [9] One reason for the persistent endodontic infection could be the microbial retention within the dentinal tubules. [2] Therefore, placement of intracanal medicaments is often recommended. [5],[9]

Calcium hydroxide, the most popular intracanal medicament is antibacterial due to its high pH 12. [1],[4],[13],[14],[ 15] Its other advantages are, osteoclastic activity inhibition, bacterial cell product detoxification, and radiopacity. [8],[16],[17] Although effective against most root canal microorganisms, several studies have reported its ineffectiveness against E. faecalis. [1],[4],[9],[14],[18]

E. faecalis is one of the most resistant microorganisms found in root canal infection. [1],[14] It can infect the dentinal tubules rapidly, migrating up to 250 μm, due to the presence of Ace, a bacterial adhesion. [1],[6] It can persist for at least 10 days without nutrient supply and also has a proton pump activity resisting the high pH of calcium hydroxide. [6] E. faecalis is chosen as the test organism, as it is easy to culture and identify, rapidly invades the dentinal tubules, and has been used in previous in vitro studie (Haapasalo and Orstavik, Schafer and Bossmann). [1],[5],[14],[19]

For removal of the intracanal medicament, passive ultrasonic activation, with a size 15 K-file and water as the irrigant, has been done in several studies. Lumley, Walmsley have reported this to be a superior method. [20] CFU have been used in the evaluation method, as they allow quantification of bacteria per milligram of dentin. [18]

Two percent CHX gel, 10% Metronidazole gel, and a combination of Calcium hydroxide and 2% CHX gel were selected as the intracanal medicaments, as previous studies have reported these agents to be more effective against E. faecalis than Calcium hydroxide alone. [1],[6],[9],[11],[21],[22]

CHX has been used as an antibacterial agent in dentistry since 1962. [14] It is a cationic bis-biguanide, which is active against gram-positive and gram-negative bacteria, bacterial spores, lipophillic virus, yeast, and dermatophytes, being bacteriostatic at low concentrations and bactericidal at high concentrations. [23] Several advantages for the clinical use of CHX as a root canal disinfectant include, its low toxicity, substantivity, more tolerable odor than sodium hypochlorite, better taste, and non-bleaching effects. [9]

As an irrigant, various concentrations of CHX solution, ranging from 0.002 to 2%, have been employed, but the 2% solution instilled greater and longer lasting antimicrobial activity. [1] As an intracanal medicament, the use of 2% CHX gel is beneficial over the use of 2% CHX solution, because the viscosity of the gel provides better retention within the root canal, promoting a better cleansing effect. [9] In addition, its antimicrobial activity is sustained and powerful. [9] Various authors have reported that 2% CHX gel is particularly effective against E. faecalis. The 2% CHX gel used in the present study contains 1% natrosol, 17% EDTA, and 2% CHX, similar to that used by Ferraz et al. [9]

Several studies have reported the improved antimicrobial efficacy of the combination of Calcium hydroxide and 2% CHX gel. The improved physical properties could be a possible explanation for the improved antibacterial efficacy of this combination. [9],[12],[21],[22],[24] The rationale behind the combined use of Calcium hydroxide and 2% CHX gel could be explained as follows:

  1. Calcium hydroxide is a well-established, long-term, intracanal medicament [22]
  2. CHX has broad spectrum antimicrobial activity, reduced cytotoxicity, and the beneficial property of substantivity [21],[22]
  3. Their combination affords a synergistic effect of their beneficial properties without any adverse effects. [22]


Group III samples medicated with a combination of Calcium hydroxide and 2% CHX gel demonstrated a significantly inferior performance than that of 2% CHX alone. Association of calcium hydroxide with CHX could reduce the antimicrobial activity of CHX, while increasing the antimicrobial activity of calcium hydroxide, resulting in CHX precipitation, which happens at high pH. [1],[9],[21]

Metronidazole is bactericidal against most anaerobes that contain electron transport components, such as ferrodoxin, which donate electrons to metronidazole, forming highly reactive nitro radical anions that kill susceptible organisms by a radical-mediated mechanism. [6],[25] Two percent Metronidazole gel has demonstrated 86.5% reduction of E. faecalis, while increasing the concentration of metronidazole results in complete elimination. [6] Ten percent Metronidazole gel in this study has not been very effective against E. faecalis, as it is active against strict anaerobes, but is ineffective against facultative anaerobes. [6]

Group IV (Control Group) did not show any antibacterial activity, as no intracanal medicament was placed.

The model used in vitro produces different conditions than that found in the infected root canal in vivo.[14] In addition, the medications were tested against only one species of microorganisms, E. faecalis, but the infected root canal usually contains more than one species of pathogen. [14] Also, in clinical situations, the root canal contains necrotic and / or viable tissues and tissue fluids, which may reduce the activity of the medications. [14] Hence, further research should be done in vivo to assess the clinical efficiency of the medicaments tested.


  Conclusion Top


Within the limitations of this study, 2% CHX gel showed substantial antimicrobial activity against E. faecalis. The combination of Calcium hydroxide and 2% CHX gel also showed good antimicrobial activity, while 10% Metronidazole gel was least effective.

 
  References Top

1.Schafer E, Bossmann K. Antimicrobial efficacy of CHX and two Calcium hydroxide formulations against Enterococcus faecalis. J Endod 2005;31:53-6.  Back to cited text no. 1
    
2.Stephen JC, Belanger M. Dentinal tubule disinfection using three Calcium hydroxide formulations. J Endod 2005;31:50-2.   Back to cited text no. 2
    
3.Barbosa CA, Gonclaves RB. Evaluation of the antibacterial activities of Calcium hydroxide, Chlorhexidine and Camphorated paramonochlorophenol as Intracanal medicaments. A Clinical and Laboratory study. J Endod 1997;23:297-301.   Back to cited text no. 3
    
4.Behnen MJ, West LA. Antimicrobial activity of several Calcium hydroxide preparations in root canal dentin. J Endod 2001;27:765-7.  Back to cited text no. 4
    
5.Basrani B, Tjaderhane L, Santos JM. Efficacy of Chlorhexidine and Calcium hydroxide containing medicaments against Enterococcus faecalis in vitro. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 2003;96:618-24.   Back to cited text no. 5
    
6.Krithikadatta J, Indira R, Dorothykalyani AL. Disinfection of dentinal tubules with 2% chlorhexidine, 2% metronidazole, bioactive Glass when compared with calcium hydroxide as intracanal medicaments. J Endod 2007;33:1473-6.   Back to cited text no. 6
    
7.Ingle JI, Bakland, Baumgartner. Text book of Endodontics. 6 h ed. Philadelphia: BC Decker Publications; 2008. p. 258-61.  Back to cited text no. 7
    
8.Christopher PM, Zhang P. pH required to kill Enterococcus faecalis in vitro. J Endod 2004;30:218-9.   Back to cited text no. 8
    
9.Wang CS, Arnold RR, Trope M. Clinical efficiency of 2% Chlorhexidine gel in reducing intracanal bacteria. J Endod 2007;33:1283-9.   Back to cited text no. 9
    
10.Yu-heng Lin, Mickel AK, Chogle S. Effectiveness of selected materials against Enterococcus faecalis: Part 3. The antibacterial effect on Calcium hydroxide and Chlorhexidine on Enterococcus Faecalis. J Endod 2003;29:565-6.   Back to cited text no. 10
    
11.Behnen MJ, West LA. Antimicrobial activity of several calcium hydroxide preparations in root canal dentin. J Endod 2001;27:765-7.  Back to cited text no. 11
    
12.Basrani B, Ghanem A, Tjaderhane L. Physical and chemical properties of Calcium hydroxide containing medication. J Endod 2004;30:413-7.   Back to cited text no. 12
    
13.Heling I, Steinberg D, Kenig S. Efficacy of a sustained release device containing chlorhexidine and Ca(OH) 2 in preventing secondary infection of dentinal tubules. Int Endod J 1992;25:20-4.   Back to cited text no. 13
    
14.Almyroudi A, Mackenzie D. The effectiveness of various disinfectants used as endodontic intracanal medications: An in vitro study. J Endod 2002;283:163- 7.  Back to cited text no. 14
    
15.Sukawat C, Srisuwan T. A comparison of the antimicrobial efficacy of three calcium hydroxide formulations on human dentin infected within Enterococcus faecalis. J Endod 2002;28:102-4.   Back to cited text no. 15
    
16.Stephen Cohen, Kenneth M. Hargreaves. Pathways of the Pulp. 9 th ed. United States; Elsevier Mosby; 2006. p. 1080-97.   Back to cited text no. 16
    
17.Estrela C, Pimenta FC. Antimicrobial evaluation of Calcium hydroxide in infected dentinal tubules. J Endod 1999;25:416-8.  Back to cited text no. 17
    
18.Evans.MD, Baumgartner JC Efficacy of calcium hydroxide: chlorhexidine paste as an intracanal medication in bovine dentin. J Endod 2003;29:338-9.  Back to cited text no. 18
    
19.Siren EK, Markus PP, Haapasalo. In vitro antibacterial effect of calcium hydroxide combined with chlorhexidine or Iodine potassium iodide on Enterococcus faecalis. Eur J Oral Sci 2004;112:326-31.  Back to cited text no. 19
    
20.Lumley PJ, Walmsley AD, Lard WR. Ultrasonic instruments in Dentistry: 2. Endosonics. Dent Update 1988;15:362-9.   Back to cited text no. 20
    
21.Gomes BP, Vianna ME, Sena NT. In vitro evaluation of the antimicrobial activity of Calcium hydroxide combined with Chlorhexidine gel used as intracanal medicament. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 2006;102:544-50.  Back to cited text no. 21
    
22.Podbielski A, Spahr A. Additive antimicrobial activity of calcium hydroxide and chlorhexidine on common endodontic bacterial pathogens. J Endod 2003;29:340-5.   Back to cited text no. 22
    
23.Leonardo MR, Filho MT. In vivo antimicrobial activity of 2% chlorhexidine used as a root canal irrigating solution. J Endod 1999;25:167-71.   Back to cited text no. 23
    
24.Yeung SY, Huang CS, Chan CP. Antioxidant and pro-oxidant properties of chlorhexidine and its interaction with calcium hydroxide solutions . Int Endod J 2007;40:837-44.  Back to cited text no. 24
    
25.Siqueria JF, Uzeda MD. Intracanal Medicaments: Evaluation of the antibacterial effects of chlorhexidine, metronidazole and calcium hydroxide associated with three vehicles. J Endod 1997;23:167-9.  Back to cited text no. 25
    


    Figures

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

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



 

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