| Abstract|| |
Aim: The present study evaluated the antimicrobial efficacy of Chlorhexidine, Nisin and Linezolid and a control group (Normal saline ) against Enterococcus faecalis (EF).
Methods: Human single rooted premolars with type I canal anatomy were instrumented with ProTaper using NaOCl as an irrigant. Supension of EF was inoculated into each root specimen and incubated. The medicaments were syringed into each root and incubated. After 24 hours, 8 samples per group (among the 4 groups) were retrieved. A hole was drilled on each root, and the dentinal shavings obtained were allowed to fall in brain-heart infusion (BHI) broth. Dilutions from the broth were plated and spread over blood agar. Colony-forming units (CFU) of EF was counted. The procedure was repeated after 72 hrs and 1 week.
Results: In group Nisin, the mean CFU was 10.6250 at 24 hrs, 6.6250 at 72 hrs and 6.2500 after 1 week respectively (statistically significant). In group Chlorhexidine, mean CFU was found to be the lowest of 10.5000 at 24 hrs, with further gradual increase to 13.7500 at 72 hrs and further increase to 15.8750 by 1 week. Similarly, in group linezolid , the mean CFU was found to decrease from 49.0000 at 24 hrs to 29.8750 at 72hrs and then increase to 34.8750 in 1 week
Keywords: Chlorhexidine, Enterococcus faecalis, intracanal medicaments, linezolid, nisin
|How to cite this article:|
Somanath G, Samant PS, Gautam V, Singh Birring OJ. To comparatively evaluate the antimicrobial efficacy of chlorhexidine, nisin and linezolid as an intracanal medicament on Enterococcus faecalis: An in vitro study. Indian J Dent Res 2015;26:613-8
Microbiology plays a detrimental role in endodontic practice, as without the presence of microbes or their products, the living pulp and the periapex cannot become toxic. A critical objective in endodontic therapy is the complete elimination of microorganism from the root canal system. 
|How to cite this URL:|
Somanath G, Samant PS, Gautam V, Singh Birring OJ. To comparatively evaluate the antimicrobial efficacy of chlorhexidine, nisin and linezolid as an intracanal medicament on Enterococcus faecalis: An in vitro study. Indian J Dent Res [serial online] 2015 [cited 2020 Jun 6];26:613-8. Available from: http://www.ijdr.in/text.asp?2015/26/6/613/176926
Enterococcus faecalis is a Gram-positive bacterium often isolated in persistent root canal infections. It has been detected in 77% of failed endodontic cases and 50% of cases with chronic apical periodontitis. ,
Although cleaning and shaping procedures effectively reduce microbiota, these procedures do not completely eliminate the bacteria in the lateral and accessory canals, isthmus and apical deltas.  Thus, intracanal medication between appointments is recommended to further reduce bacteria in the root canal system, and multiple visits may be required even in cases in which biological concerns are not an issue. 
Chlorhexidine (CHX) has the ability to maintain its antibacterial action for a prolonged duration, i.e. its substantivity and its optimal microbial action at pH 5.5-7 has been recommended for root canal irrigation and as an intracanal medicament. 
Nisin (NI) is a naturally occurring antimicrobial peptide produced by strains of Lactococcus lactis and was discovered in 1928. The use of NI in dentistry has been limited. Studies have, however, shown the effectiveness of the use of NI against Enterococcus faecalis (EF) and Streptococcus mutans. ,
Linezolid (LZ) has gained popularity on the basis of its wide spectrum of activity against Gram-positive organisms, including vancomycin-resistant EF. It is an oxazolidine agent that acts by inhibiting initiation of bacterial protein synthesis.  A recent study has shown LZ to be very effective against EF, faring better than calcium hydroxide. 
The purpose of this study was to compare the efficacy of 2% CHX, LZ, and NI in reducing the colony forming unit (CFU) of EF and to compare the rapidity with which these medications act at intervals of 24 h, 72 h, and 1 week.
| Subjects and Methods|| |
Ninety-six extracted single-rooted premolars were collected and stored in distilled water; the teeth were cleaned to remove any attached debris and calculus. All the samples were decolonized using a diamond disc at 18 mm to standardized root length. Working length was established using no 10 K file until it was visible thru apical foramen. The canals were shaped with nickel-titanium rotary instruments (Protaper, Dentsply Maillefer, Switzerland) following the usual protocol up to size F3 as the last file used at the working length. 5.25% sodium hypochlorite was used for irrigation after each instrument use. Final irrigation was done with 5 mL of 17% ethylenediaminetetraacetic acid (EDTA) for 3 min for each specimen. The root apices were then sealed with glass ionomer cement, mounted on acrylic blocks, and the specimens were then sterilized in an autoclave at 121°C for 40 min at 15 psi pressure. The complete eradication of bacteria and their spores was confirmed by conducting a pilot study prior to the experiment by placing the samples in culture medium. No growth confirmed the effectiveness of the procedure.
Isolated 24 h colony of pure culture of EF (MTCC 439) grown in blood agar was collected with the help of wired loop and suspended in brain heart infusion (BHI) broth. The cell suspension was incubated at 36.5°C for 24 h. The turbidity was verified by using the McFarland Turbidity Scale and adjusted to 0.5, corresponding to 10 8 organisms/ml.
Fifteen microliters of this bacterial suspension were inoculated into each canal using a micropipette. The teeth were sealed using modeling wax molded in flame. The specimens were then incubated at 36.5°C for 7 days to ensure penetration of EF into dentinal tubules. Asepsis was maintained throughout the procedures using standard precautions with two flames in a biosafety cabinet. After incubation for 7 days, the inoculated specimens were divided into four study groups containing 24 specimens each.
The wax was removed, and the canals were dried using ProTaper Universal paper points of size F3 (Dentsply Maillefer) before placement of medicaments. NI (2.5%) was prepared by dissolving in sterile distilled water to a concentration of 100 mg/ml.  2.5 g of NS was electronically weighed and dissolved in 100 ml of sterile distilled water in a sterile beaker. LZ intravenous suspension (2 mg/ml) (Cipla Pharmaceuticals) and CHX (2%) (RC-Chlor, DEOR dental, and oral care) were used.
Standard volume of each medicament (0.5 ml) was syringed into each respective canal. The teeth were again sealed with 3 mm of modeling wax and incubated at 36.5°C until evaluation.
After 24 h, randomly 8 samples per group were retrieved from the incubator, dried with sterile paper points, and the swab taken with the help of sterile paper point which is placed in a test tube filled with 2 ml of BHI broth. Along with this, using a number 2 sterile round bur, 1 mm in diameter (SS White Burs, Inc., Lakewood, NJ), was used to drill a hole on each root 3 mm short of the apex. The dentinal shavings obtained were allowed to fall in 2 ml of the same BHI broth. This was then vortexed for maximum leaching of bacteria from the samples.
Five hundred microliters of the solution was then used to obtain decimal series of dilutions up to 10 − 8 . One hundred microliters of each dilution was plated and spread over a 6-inch nutrient agar plate. The agar plates were then incubated at 36.5°C for 24 h [Figure 1]. CFU was then counted using a colony counter. The above procedures were repeated at time intervals of 72 h and 1 week in eight samples each, respectively.
|Figure 1: Chlorhexidine group at (a) 24 h, (b) 72 h, and (c) 1 week, linezolid group at (d) 24 h, (e) 72 h, and (f) 1 week, NI group at (g) 24 h, (h) 72 h, and (i) 1 week|
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Growth of organism that occurred in nutrient agar was confirmed to be EF by the following tests: (a) Gram-staining: Gram-positive organism. (b) Catalase test: Found to be negative for enterococci.
The data were analyzed using "SPSS" version 18.0 (SPSS 18 developed by IBM, Chicago, USA). One-way ANOVA was done by post hoc analysis was done for multiple comparisons between the different groups by Tukey's test. The level of significance was established at P < 0.05.
| Results|| |
The CFU count indicating the number of viable bacterial colonies was found to be the highest in Group NI at all-time intervals of 24 h, 72 h, and after 1 week [Figure 2]. In Group LZ, the mean CFU was found to decrease at 72 h and then increase in 1 week. In Group CHX, the mean CFU was found to be lowest at 24 h with a significant increase after 72 h and 1 week. In Group NI, the mean CFU was found to reduce significantly with time at all three-time intervals. The number of viable colonies was found to be the minimum in Group CHX at 24 h, Group NI at 72 h and after 1 week.
|Figure 2: Mean colony forming units plotted for all the groups at 24 h, 72 h, and 1 week interval|
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Multiple comparisons among the different groups done by post hoc analysis [Table 1], revealed that Group LZ had significantly higher growth when compared to Group CHX and NI (P = 0.00) and significantly lower growth when compared to Group NS (P = 0.00).
|Table 1: Multiple comparisons between all the groups by using post hoc analysis by Tukey's test |
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Group CHX showed a significantly lower CFU when compared to Group NS and Group LZ (P = 0.00), but no significant difference was found between Groups CHX and NI (P = 0.481).
In Group NI, the mean CFU was found to be significantly lower in comparison to Groups NS and LZ (P = 0.00). However, there was no significant difference between Groups NI and CHX (P = 0.481).
| Discussion|| |
In literature, three different techniques have been used to determine the effectiveness of any antimicrobial agent, namely dilution test, agar diffusion, and direct exposure methods.
The dilution method provides quantitative information about the amount of antimicrobial agent required but has the disadvantage of being able to evaluate only substances that are soluble in the culture media. 
The agar diffusion method is the most commonly used method. However, it does not distinguish between bacteriostatic and bactericidal properties of dental medicaments nor provide information about microbial viability after the test.  Besides, rather than its actual efficacy against the organism, the test results depend on the medicament's solubility and diffusibility in agar.
The direct exposure method provides qualitative information about the material because of its direct contact with the microorganism being considered. The bacterial sampling method followed in this study is as recommended by Pavaskar et al., 2012.
Bacterial samples were taken with sterile paper points as well as in the form of dentinal shavings. The use of sterile paper point has the advantage that it can be used in vitro and in vivo and is the methodology recommended by Mercade et al. to closely mimic a clinical setup.  Dentinal shavings were obtained with a punch hole drilled using a number 2 round bur 3 mm from the apex.  Both the methods were employed to minimize error and attain more accurate results.
In this study, the protocol for instrumentation was the same for all the groups. The canals were uniformly instrumented 1 mm beyond the apex to ensure that the apical third was adequately cleaned. EDTA was used to remove the smear layer that might have remained in the canal after instrumentation.
Sodium hypochlorite is an effective root canal irrigant which causes irreversible oxidation of hydrosulfuric groups of essential enzymes, disturbing metabolic functions in the microorganism.  The use of 5.25% sodium hypochlorite preceded with autoclaving helped in making the root canal sterile prior to inoculation with EF.
EF was chosen as the test organism in this study because it is one of the most common microorganism recovered from root canals of previously root-filled teeth with persistent periapical pathology. The implication of EF in endodontic treatment failure due to its resistance to the highly alkaline environment produced by calcium hydroxide and the ability to invade dentinal tubules under stressed conditions are indicative that any method of eradicating this species would be beneficial to endodontic treatment. ,,,,,,
Though calcium hydroxide is the most commonly used intracanal medicament, EF appears to be highly resistant to the medicaments used during treatment and is known to resist the antibacterial effect of dressing with calcium hydroxide. ,,
Several studies have compared the efficacy of LZ, CHX, and NI in comparison with calcium hydroxide ,,, against EF. This study, however, compares these medicaments among themselves to evaluate the agent with the best antimicrobial efficacy.
After data analysis, all the three medicaments exhibited antimicrobial effect and significantly reduced the mean CFU of EF. However, none of the three medicaments could completely eliminate EF in 1 week.
LZ acts by preventing the formation of 70S ribosome complex that is responsible for the initiation of protein synthesis. , In Group LZ, the mean CFU was found to decrease from 49.0000 at 24 h to 29.8750 at 72 h and then increase to 34.8750 in 1 week. The decrease in CFU at 24-72 h is similar to a previous study,  however, the further increase in CFU could be attributed to the development of resistance to the drug which occurs as a result of the mutation of ribosomal binding site. 
CHX, a cationic bisbiguanide, crosses the cell wall, presumably by passive diffusion, and subsequently attacks the cytoplasmic membrane. , It is found to be a potent antimicrobial agent that is particularly effective against EF.  In Group CHX, mean CFU was found to be the lowest of 10.5000 at 24 h, with a further gradual increase to 13.7500 at 72 h, and further increase to 15.8750 by 1 week. The property of substantivity exhibited by CHX is what imparts its long-term antimicrobial effect. According to reports, the antimicrobial effect lasts for up to 12 weeks, which directly corresponds to the CHX concentration. Hence, the concentration of CHX retained in the root canal gradually decreased which could be the reason, the mean CFU values increased gradually at 72 h.
NI was found to effectively kill Streptococcus gordonii and EF both in solution and in vitro intracanal environment. , In this study, NI was prepared in the concentration of 100 mg/ml which was in accordance to the concentration used by Turner et al.  Group NI exhibited the best antimicrobial effect with mean CFU of 10.6250 at 24 h, 6.6250 at 72 h, and 6.2500 after 1 week. On multiple comparisons, the action was found to be comparable with CHX. The medicament was found to be effective even after 1 week. Similar effect was seen in the study conducted by Hemadri et al., 2011 according to which NI was found to exhibit the best results against EF in comparison to calcium hydroxide at 1 week.  However, the exact mechanism of action of NI on EF needs further investigation at the molecular level.
Though every effort was made, this in vitro study could not simulate the intraoral environment of the infected root canal. In this study, the medicaments were dispensed in the liquid medium and were standardized by volume. Further investigation is needed for a suitable base, which can dispense these medicaments more effectively for a longer period.
Certain variables such as the quantity of dentinal shavings analyzed per sample, the amount of time required for drilling a punch hole and the amount of heat generated during the procedure could not be standardized.
From this study, NI is found to be a promising agent in eliminating EF in comparison with the other medicaments tested. However, the systemic effect of this medicament, its biocompatibility, allergic potential, and bacterial resistance needs further investigation. Hence, this study can serve as a baseline for future in vivo studies to evaluate the effects of NI and its combinations on biofilm organisms.
| Conclusions|| |
Nisin was found to be the most effective in reducing the bacterial count of Enterococcus faecalis e week. It's action was found to be comparable with Chlorhexidine. Linezolid was found to be short acting with gradual decrease in antimicrobial action after 72 hours.
We thank Dr. Shivarajan and the team of Department of Microbiology, Universal College of Medical Sciences, Bhairahawa, Nepal, for their help and support in conducting the study.
Financial support and sponsorship
Conflicts of interest
There are no conflicts of interest.
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Dr. Geethu Somanath
Department of Conservative Dentistry and Endodontics, College of Dental Surgery, Universal College of Medical Sciences, Bhairahawa
Source of Support: None, Conflict of Interest: None
[Figure 1], [Figure 2]