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ORIGINAL ARTICLE
Year : 2011  |  Volume : 3  |  Issue : 1  |  Page : 21-24

An in vitro investigation of a newer intracanal medicament Nisin on Enterococcus faecalis in comparison with chlorhexidine and calcium hydroxide


1 Department of Conservative Dentistry and Endodontics, Indira Gandhi Institute of Dental Sciences, Puducherry, India
2 Department of Conservative Dentistry and Endodontics,Ragas Dental College and Hospital, Chennai, India
3 Department of Conservative Dentistry and Endodontics,Sri Venkateswara Dental College and Hospital, Chennai, India

Date of Web Publication29-Jul-2013

Correspondence Address:
Suneel Kumar Chinni
Indira Gandhi Institute of Dental Sciences, Pillyarkuppam, Puducherry
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/2231-0754.115770

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   Abstract 

Aim: To determine the antibacterial efficacy of Nisin in comparison with Calcium hydroxide and Chlorhexidine. Materials and Methods: Fifty extracted lower premolar single-rooted teeth were collected and were instrumented with K3 rotary files. Then, root canals were inoculated with a bacterial solution of Enterococcus faecalis. After 21 days, the canals were inoculated with Saline, Nisin, Vancomycin, Calcium hydroxide, and Chlorhexidine. The roots were left for 7 days and on the 8 th day, to investigate the degree of infection of the radicular dentin, specimens of the dentin chips from the full length of the root canal were harvested using a sterile rotary K3 instrument (size 25 6% taper). Results: The results of the present study showed that Nisin and Chlorhexidine showed none of Colony Forming Units (CFU) in their respective group. Conclusion: Within the limits of the study, Nisin was effective in eradicating E. faecalis cells in pure culture and root canal dentin.

Keywords: Antibacterial efficacy, calcium hydroxide, chlorhexidine, Enterococcus faecalis


How to cite this article:
Chinni SK, Veni AB, Srinivasan MR, Rajamani I. An in vitro investigation of a newer intracanal medicament Nisin on Enterococcus faecalis in comparison with chlorhexidine and calcium hydroxide. J Int Clin Dent Res Organ 2011;3:21-4

How to cite this URL:
Chinni SK, Veni AB, Srinivasan MR, Rajamani I. An in vitro investigation of a newer intracanal medicament Nisin on Enterococcus faecalis in comparison with chlorhexidine and calcium hydroxide. J Int Clin Dent Res Organ [serial online] 2011 [cited 2019 Nov 11];3:21-4. Available from: http://www.jicdro.org/text.asp?2011/3/1/21/115770


   Introduction Top


Successful treatment of apical periodontitis depends on elimination of the infective microflora from the necrotic root canal system and favors the healing of the periapex. Apical periodontitis is an inflammatory process involving the periradicular tissues, causing pain and resorption of periradicular structures. It has been shown that anaerobes are the primary etiological agents of apical periodontitis. [1],[2] One of the factors that contribute to a persistent periradicular infection following root canal therapy is the survival of microorganism in the apical portion of root-filled tooth. Unlike primary endodontic infections, which are polymicrobial in nature and dominated by Gram-negative anaerobic rods, the microorganisms involved in secondary infections are composed of one or a few bacterial species.

Enterococcus faecalis (E. faecalis) is a persistent organism that, despite making up a small portion of the microflora in untreated canals, plays a major role in the etiology of persistent periradicular lesions after root canal treatment. It is commonly found in a high percentage of root canal failures and it is able to survive in the root canal as a single organism or as a major component of the flora due to its ability to survive a period of starvation up to 12 months. [3],[4],[5] Thus, it is an important microorganism in cases of endodontic retreatments than in primary root canal treatments.

However, the aberrations in the pulp space morphology contribute to the complexities in the root canal system, such as ramifications, webs, fins, etc., reveal colonization of bacteria in the dentinal tubules other than the main canal which are inaccessible to routine cleaning and shaping. [6]

Although chemomechanical preparation of root canal is able to reduce the number of bacteria, an intracanal medicament with antibacterial action is required to maximize the disinfection of the root canal system in infected cases. [7] The need for intracanal medication increases, especially in those cases where the infection is resistant to regular treatment and the therapy cannot be successfully completed due to the presence of pain or continuing exudates. For this reason, a wide variety of antibacterial intracanal medicaments have been used, such as Phenolic derivatives, aldehydes, halides, antibiotics, calcium hydroxide pastes, and chlorhexidine gels. [8]

Calcium hydroxide plays an important role in endodontics through its versatile action like inducing tissue formation, exerting antibacterial action, and interrupting the nutrient supply to remaining bacteria. [9]

Chlorhexidine gluconate use in endodontics has been proposed both as irrigant and intracanal medicament. Chlorhexidine [10],[11],[12],[13] has inhibitory effects on bacteria commonly found in endodontic infections [14] acting against Gram-positive and Gram-negative microorganisms. [15]

A newer intracanal medicament, Nisin which is a naturally occurring antimicrobial peptide (discovered in 1928) is found to be effective against the E. faecalis. Nisin is produced by Lactococcus lactis and which is a class I bacteriocin. It is used as food preservative and found to be safe in human beings. [16]

The objectives of the present study is to determine the efficacy of Nisin against E. faecalis and its efficiency is compared with other intracanal medicaments like Calcium hydroxide, Chlorhexidine in human radicular dentin.


   Materials and methods Top


Preparation of teeth

Fifty extracted lower premolar single-rooted teeth were collected and stored in distilled water until required. The teeth were decoronated using a rotary diamond saw with water irrigation (Gillings-Hamco, Rochester, NY, USA) and only roots with a Type-I root canal configuration [17] and a round root canal at the cut surface were chosen. The roots were divided into five groups of 10 roots. The root lengths were measured and the working length was determined as 1 mm less than the file length when the tip was visible at the apical foramen. Coronal root structure was removed perpendicular to the long axis of the root to produce roots with a working length of 10 mm.

The canals were instrumented with engine-driven nickel-titanium K3 rotary files (SYBRON ENDO USA) used in a sequential crown down technique to a standardized apical size of 25.4% taper instrument. 2.5% sodium hypochlorite was used as an irrigant during instrumentation with a sterile endodontic needle (Monoject; Sherwood Medical, St Louis, MO, USA) and 5 ml Luer lock syringe (Monoject; Sherwood Medical, St Louis, MO, USA). The roots were then rinsed in water for 30 minutes, then rinsed in 17% ethylenediaminetetraacetic acid (EDTA) (Vishal biotech, Mumbai, India) for 5 minutes in an ultrasonic bath to remove the smear layer and rinsed in water for a further 30 minutes. Then, the teeth were stored in sterile water until used.

Each root was dried and three layers of clear nail varnish were placed over all external root surfaces with care not to occlude the root canal entrance and teeth were allowed to dry. Then, the roots were autoclaved at 121°C for 15 minutes.

Root canal infection

Bacterial samples of E. faecalis of American Testing Cell Culture of 29212 (ATCC 29212) were taken for this study. Each root canal was inoculated with a bacterial solution up to the canal entrance using a sterile endodontic needle and 5 ml Luer lock syringe. Each canal was sealed with a dental wax and all samples were incubated in a closed container at 37°C for a period of 21 days. The canals were reinoculated with fresh bacterial samples every 3 days. To check for cell viability and purity of culture, samples were taken from each canal with a sterile paper point and inoculated onto Muller Hilton agar plate and incubated aerobically for 24 hours at 37°C.

Root canal medication

After 21 days, the canal contents were aspirated and each canal was rinsed with 5 ml saline using a sterile endodontic needle and 5 ml Luer lock syringe, then dried with sterile paper points. The test medicament was applied to the corresponding groups. In Group I, 5 μl saline (Claris life sciences, India); Group II, 5 μl Nisin (Vasta Biotech, Chennai); Group III, 5 μl Vancomycin; Group IV, 5 μl Calcium Hydroxide (Sigma-Aldrich Co., USA); Group V, 5 μl Chlorhexidine (Sigma-Aldrich Co., USA) was placed using Tuberculin syringe. In this study, saline group (group I) was taken as negative control and Vancomycin (group V) which is the drug of choice for E. faecalis was taken as positive control. The roots were left for 7 days at 37°C after sealing with paraffin wax. On the 8 th day, to investigate the degree of infection of the radicular dentin, specimens of the dentin chips from the full length of the root canal were harvested using a sterile rotary Nickel titanium K3 instrument (size 25 6% taper).


   Results Top


The results of the present study showed that Nisin and Chlorhexidine showed none of Colony Forming Units (CFU) in their respective group as shown in [Table 1]. Saline group which served as negative control showed 120 CFU (10 5 /ml). Vancomycin group which served as positive control showed no growth of E. faecalis. Calcium hydroxide group showed 84 CFU (10 5 /ml). Statistical analysis was carried out using Students Unpaired 't' test using SPSS windows 12.0 version. P value is considered significant when P value is less than 0.05. Nisin group showed very high significant difference in eradicating E. faecalis than Calcium hydroxide and Saline. Chlorhexidine group also showed very high significant difference in eradicating E. faecalis than Calcium hydroxide and Saline. Nisin and Chlorhexidine are equally effective with positive control Vancomycin in eradicating E. faecalis.
Table 1: Number of colony forming units for each group

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   Discussion Top


The results of the present study have showed that calcium hydroxide group has 84 CFU. E. faecalis are able to survive in a highly alkaline environment and are resistant to its effects. [18] The ability of Enterococci to invade dentinal tubules, even under stressed environmental conditions, is thought to play a role in its ability to resist chemomechanical instrumentation and reinfect the root canal system. [19] The presence of proton pump has been illustrated as a primary resistance mechanism of E. faecalis to resist the alkaline effects of calcium hydroxide in vitro even in an alkaline environment up to pH 11.1. [18] The buffering capacity of dentine provides a decreasing pH gradient from inner to peripheral root dentine. [20],[21] A pH of 11.5 is required to effectively kill E. faecalis[18] and this is not commonly achieved within radicular dentine by a Ca(OH) 2 medicament, with an alkalinity of only pH 10.3 being reported in vitro. [21],[22]

Chlorhexidine gluconate is a cationic bisbiguanide that acts mainly by adsorbing onto the cell wall of the microorganism and causing leakage of intracellular components. [23] At low concentrations of chlorhexidine, small molecular weight substances will leak out, resulting in a bacteriostatic effect. At higher concentrations, chlorhexidine has a bactericidal effect due to precipitation and coagulation of the cytoplasm probably caused by protein cross linking. [24] One of the mechanisms that explain its efficacy is based on the interaction between the positive charge of the molecule and negatively charged phosphate groups on the bacterial cell wall, which allows the chlorhexidine molecule to penetrate into the bacteria with toxic effects. [25],[26] The results of the present study were in accordance with the previous studies of Gomes et al.

The effect of Nisin as an antibacterial agent is recorded in several studies. Nisin-based mouth wash was tested on beagle dogs and was found to significantly decrease the number of bleeding sites compared with placebo. [27] An in vitro investigation of the antibacterial effect of Nisin in root canals and canal wall radicular dentin found that Nisin is effective against E. faecalis. [16]

The most commonly used intracanal medicament Calcium hydroxide is not much effective in eradicating the E. faecalis. This may be due to the resistance and survival techniques of E. faecalis previously mentioned. However, the mode of action of Nisin differs to that of calcium hydroxide and is not reliant on a highly alkaline environment for effective killing.

Severina E et al. have demonstrated that nisin had a wide and powerful bactericidal effect often accompanied by rapid lysis against a large panel of bacterial pathogens. [28]

From previous studies, the mode of action of nisin have shown that this basic polypeptide acts by inserting into the plasma membrane and triggering the activity of bacterial murein hydrolases resulting in damage or degradation of the peptidoglycan and lysis of the cells. [29]

Nisin was able to effectively kill all bacteria within the intraradicular canal space. The present study of investigation Nisin as an Intracanal medicament has provided some encouraging results. The ability of nisin to effectively kill E. faecalis by a mechanism that is not reliant on achieving a high pH may provide a means to eliminate this species by a method to which it has no defense mechanism. Nisin is reported to be active against a broad range of Gram-positive bacteria. However, infection of radicular dentinal tubules is dominated by Gram-positive species namely, Streptococci and Enterococci. Given the association of E. faecalis in cases of chronic failure in endodontically treated teeth, a medication aimed specifically at this species may be of value. This may be especially pertinent in cases of conventional endodontic retreatment where E. faecalis is the most commonly recovered species.


   Conclusions Top


Within the limits of the study, Nisin was effective at eradicating E. faecalis cells in pure culture and was comparable with chlorhexidine, positive control Vancomycin in elimination of E. faecalis from within the root canal system.

 
   References Top

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8.Gomes BP, Souza SF, Ferraz CC, Teixeira FB, Zaia AA, Valdrighi L, et al. Effectiveness of 2% chlorhexidine gel and calcium hydroxide against Enterococcus faecalis in bovine root dentine in vitro. Int Endod J 2003;36:267-75.  Back to cited text no. 8
    
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17.Vertucci FJ. Root canal anatomy of the human permanent teeth. Oral Surg Oral Med Oral Pathol Oral Radiol and Endod 1984;58:589-99.  Back to cited text no. 17
    
18.Evans M, Davies JK, Sundqvist G, Figdor D. Mechanisms involved in the resistance of Enterococcus faecalis to calcium hydroxide. Int Endod J 2002;35:221-8.  Back to cited text no. 18
    
19.Love RM. Enterococcus faecalis- a mechanism for its role in endodontic failure. Int Endod J 2001;34:399-405.  Back to cited text no. 19
    
20.Wang J, Hume W. Diffusion of hydrogen ion and hydroxyl ion from various sources through dentin. Int Endod J 1988;21:17-26.  Back to cited text no. 20
    
21.Nerwich A, Figdor D, Endo D, Messer HH. pH changes in root dentin over a 4-week period following root canal dressing with calcium hydroxide. J Endod 1993;96:302-6.  Back to cited text no. 21
    
22.Minana M, Carnes D, Walker WI. pH changes at the surface of root dentin after intracanal dressing with calcium hydroxide. J Endod 2001;27:43-5.  Back to cited text no. 22
    
23.Greenstein G, Berman C, Jaffin R. Chlorhexidine an adjunct to periodontal therapy. J Periodontol 1986;57:370-6.  Back to cited text no. 23
    
24.Kontakiotis E, Nakov M, Georgopoulou M. Invitro study of the indirect action of calcium hydroxide on the anaerobic flora of the root canal. Int Endod J 1995; 28:285-9.  Back to cited text no. 24
    
25.Lindskog S, Pierce AM, Blomlof L. Chlorhexidine as a root canal medicament for treating inflammatory lesions in the periodontal space. Endod Dent Traumatol 1998;14:186-90.  Back to cited text no. 25
    
26.Hugo WB, Longworth AR. Some aspects of the mode of action of chlorhexidine. J Pharm Pharmacol 1964;16:665-72.  Back to cited text no. 26
    
27.Howell TH, Fiorellini JP, Blackburn P, Projan SJ, De la Harpe J. Williams RC. The effect of a mouthrinse based on nisin, a bacterocin, on developing plaque and gingivitis in beagle dogs. J Clin Periodontol 1993;20:335-9.  Back to cited text no. 27
    
28.Severina E, Severin A, Tomasz A. Antibacterial efficacy of nisin against multidrug-resistant Gram-positive pathogens. J Antimicrob Chemother 1998;41:341-7.  Back to cited text no. 28
    
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    Tables

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