The quest for the best intra canal medicament for root canals has remained a struggle ever since endodontic literature has been written. Although, various medicaments exist, each has its advantages and disadvantages. Enterococcus faecalis (EF) has been found in asymptomatic and persistant root canal infections [1,2]. It has also been found in 77% of failed endodontic cases [2], and in 50% cases with chronic apical periodontitis [3]. Chlorhexidine (CX) has been known for its broad spectrum of action against gram positive and negative organisms. It has a unique ability to adsorb on to dental tissues and mucous membrane (Substantivity), while releasing itself gradually [4]. Calcium hydroxide (CH) has been popular as an intra canal medicament due to its additional action on gram negative bacteria [5,6]. Some authors have discussed its effectiveness in eliminating EF [7,8]. Combinations of CH with iodoform have been previously believed to enhance periapical bone regeneration simultaneous with resorption of excess material, with a success rate of 84% to 100% [9]. Vitapex® (VP), a combination of CH and iodoform, resorbs from apical tissues within a time span of a week to two months in primary teeth. It is radio-opaque, non-setting, easily inserted and retrieved [10].
Linezolid (LZ) has gained popularity due to its activity against gram positive organisms, including vancomycin resistant EF. It is an oxazolidine agent that acts by inhibiting the initiation of bacterial protein synthesis. It has a half life of four to six hours, gets 31% protein bound and has good CSF penetration [11]. The effectiveness of CH on EF, with or without iodoform, has been controversial. Moreover, the effectiveness of LZ, or a combination of LZ with CH (LC), on root canal related EF has not yet been reported. Hence, this study was conducted to evaluate the efficacy of CX, CH, VP, LZ and LC on EF at time intervals of 24 hours and 72 hours using the agar diffusion method. The null hypothesis assumed was that there would be no difference amongst the medicaments in their action against EF.
Materials and Methods
The study was conducted in the Department of Conservative Dentistry and Endodontics, in association with the Departments of Microbiology, Pharmacology and Pharmaceutics. Agar plates were prepared on sterilized glass petri dishes and were left overnight at 37°C. EF strains were mixed with peptone water and the turbidity was adjusted to the McFarland’s turbidity standard tube No: 0.5. The inoculum obtained was used to make lawn cultures on the agar plates using sterile cotton swabs. Following this, wells that were three millimeters in diameter and four millimeters in depth were punched on the agar plates. A total of 30 agar plates were prepared, such that each plate had five wells, into which each of the five medicaments were placed in no particular order. However, the medicament name was labeled under each well after each medicament was placed to enable a blind evaluation. The five medicament groups were:
Group I (CX) – 2% Chlorhexidine
Group II (CH) - Calcium hydroxide based intra canal medicament (Ultracal XS®, Ultradent, South Jordan, UT).
Group III (VP) - Vitapex® (Diadent® Group International Inc., Burnaby, B.C.,Canada).
Group IV (LZ) - Linezolid based intra canal medicament (0.3%)
Group V (LC) - Calcium hydroxide and Linezolid based intra canal medicament (3% LZ, 30% CH)
The plates were then incubated overnight at 37°C, after which, the samples were evaluated for zones of inhibition after intervals of 24 hours and 72 hours. The zone of inhibition was measured with a boley guage. The readings corresponding to each medicament were statistically evaluated by paired t-test, ANOVA and Post-hoc analysis using Tukey’s HSD.
Results
The difference between values of the zones of inhibition around various medicaments after 24 hours and 72 hours was found to be statistically significant [Table/Fig-1]. The maximum mean value of the zones of inhibition after 24 hours was shown by group LC (26.31 ± 0.597), while the minimum was shown by group CH (0.68 ± 0.545). After 72 hours, the maximum value was again shown by group LC (27.13 ± 0.540), while the least was shown by group CH (0.23 ± 0.264). Groups LC, LZ and CHX showed an increase in their values after 72 hours compared to those after 24 hours. Among these the greatest increase was shown by group CHX, followed by LC and LZ. However, groups VP and CH showed a decline in their values after 72 hours compared to those after 24 hours, out of which the greatest decline was observed for group VP, followed by CH. A comparison between the five groups after 24 hours [Table/Fig-2] (Post-hoc analysis using Tukey’s HSD) showed that each group differed significantly from the rest of the groups. A comparison between the five groups after 72 hours [Table/Fig-3] Post-hoc analysis using Tukey’s HSD) also showed that each group differed significantly from the rest of the groups.
Comparison between groups after 24 and 72 hours using the paired t-test
| n | At 24 hrs | At 72 hrs | t-value | p-value |
|---|
| LC | 30 | 26.31 ± 0.597 | 27.13 ± 0.540 | 13.025 | <0.001*** |
| LZ | 30 | 22.55 ± 0.560 | 22.78 ± 0.592 | 4.455 | <0.001*** |
| CX | 30 | 15.37 ± 0.574 | 16.36 ± 0.545 | 56.512 | <0.001*** |
| VP | 30 | 5.50 ± 0.426 | 1.57 ± 0.356 | 57.00 | <0.001*** |
| CH | 30 | 0.68 ± 0.545 | 0.23 ± 0.264 | 7.072 | <0.001*** |
*** p < 0.001; Highly significant
Comparison between five groups at 24 hours using ANOVA
| n | Range | Mean | SD | SEm | ANOVA |
|---|
| LC | 30 | 25.20-27.20 | 26.310 | 0.597 | 0.109 | p < 0.0001 Very Highly Significant |
| LZ | 30 | 21.50-23.90 | 22.547 | 0.560 | 0.102 |
| CX | 30 | 14.30-16.20 | 15.370 | 0.574 | 0.105 |
| VP | 30 | 4.80-6.20 | 5.480 | 0.433 | 0.079 |
| CH | 30 | 0.00-1.90 | 0.680 | 0.545 | 0.099 |
Comparison between five groups at 72 hours using ANOVA
| n | Range | Mean | SD | SEm | ANOVA |
|---|
| LC | 30 | 26.20-27.90 | 27.130 | 0.539 | 0.098 | p < 0.0001 Very Highly Significant |
| LZ | 30 | 21.70-23.90 | 22.780 | 0.592 | 0.108 |
| CX | 30 | 15.30-17.20 | 16.360 | 0.545 | 0.099 |
| VP | 29 | 1.10-2.20 | 1.569 | 0.356 | 0.066 |
| CH | 30 | 0.00-0.80 | 0.227 | 0.264 | 0.048 |
Discussion
Three different methods have been used to determine the effectiveness of any medicament: Dilution, 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 direct exposure method provides qualitative information about the substance due to its direct contact with the microorganism being considered. The agar diffusion method presents a zone of inhibition around the wells containing the medicament [12]. It is by far the most commonly used method.
CH releases OH ions that are responsible for the creation of a highly alkaline environment. High pH has a destructive effect on bacterial cell membrane and protein structure [12]. A pH of 10.5 to 11 delays the growth of EF, while, a pH of 11 or more eliminates EF [13,14]. However, CH was the least effective against EF in our study. This is in agreement with a few previous studies [14–16]. The mean zone of inhibition was (0.68 ± 0.545) after 24 hours of incubation, which increased to 0.23 ± 0.264 after 72 hours. When CH is placed in agar, its high pH starts to precipitate it, preventing its diffusion [17]. Moreover, the release of Ca and OH ions decrease the pH of the media, enhancing growth of the organisms being tested [15]. These factors may have been responsible for its lack of effectiveness against EF in blood agar. Moreover, the proton pump of EF carries protons to the interior of the cell, acidifying its cytoplasm in situations of increased alkalinity when subjected to CH [14]. All these factors might have contributed to the pH decline of CH.
Two percent CX has been proven to be more effective than lesser concentrations [18]. That is why it was decided to use this concentration for the study. CX was found to be effective against EF after 24 hours (15.37 ± 0.574) and 72 hours (16.36 ± 0.545) of incubation in agar. This is in accordance with the results obtained by other authors [14,19–21]. However, it is in disagreement with Estrela et al., [22]. It was found to be more effective than CH, which is in agreement with results obtained by Ballal et al., and Gomes et al., [21,23]. However, it was not as effective as LC or LZ.
VP showed effectiveness against EF after 24 hours (5.50 ± 0.426), however, the effect declined after 72 hours (1.57 ± 0.356). The ineffectiveness of VP on organisms has been previously reported [15,24]. In a study by Amorim et al., VP produced no zone of inhibition in the agar diffusion method, however, was found to be effective against EF through direct exposure test [12]. This example confirms the unreliability of the agar diffusion method in assessing VP.
LZ acts by preventing the formation of 70S ribosome complex that is responsible for the initiation of protein synthesis, by binding to the 23S subunit of the 50S subunit [25]. However, enterococcal resistance to the drug occurs due to mutation of the ribosomal binding site [26]. LZ showed good results against EF with the mean zone of inhibition being 22.55 ± 0.560 after 24 hours, and 22.78 ± 0.592 after 72 hours. However, the best results were shown by LC with a mean values of 26.31 ± 0.597 and 27.13 ± 0.540 after 24 hours and 72 hours, respectively. LZ is known to cause adverse effects on systemic administration, like nausea, diarrhea, tongue discoloration, oral moniliasis, taste perversion, headache and myelosupression [27]. However, there has not been an in-vivo study yet evaluating the effectiveness of LZ as an intra canal medicament against EF or any other organism. This in vitro study could not simulate the intra-oral environment inside an infected root canal.
It has been shown that EF rarely appears in primary endodontic lesions [28]. However, some have documented its occurrence in primary as well as secondary endodontic lesions [1,29]. EF can survive inside dentin tubules for atleast 10 days without nutrient supply [16,30]. It may also survive in smear layer and debris, and be extremely difficult to eliminate during instrumentation and irrigation [22]. Moreover, serine protease and ACE aid in the adhesion of EF with dentin [31]. EF has virulence factors (aggregation substance, enterococcal surface proteins (Esp), gelatinase, cytolysin toxin, extracellular superoxide production, capsular polysaccharides, antibiotic resistance determinant) that can facilitate its adherence with host cells and extracellular matrix; help in its invasion into tissues; immunomodulation effect and cause toxin mediated damage [32]. These factors make EF resistant to most intra canal medications.
From this study, we found LC to be promising in eliminating EF in comparison to the other medicaments tested. Hydroxyl ions liberated by CH act on enzymes in the cytoplasmic membrane. The membrane is similar, irrespective of the organisms morphological, tinctorial and respiratory characteristics. This means that CH will have similar effects on aerobic, anaerobic, gram positive and gram negative organisms [12]. Therefore, we recommend the LC combination, because CH could broaden the spectrum of activity beyond the sole gram positive spectrum of action of LZ. But, it might need periodic replacement to ensure absence of any dehydration or reduction in pH of CH, that might invariably affect the potency of LZ.
Endodontic infections are polymicrobial, therefore, a medicament effective against EF need not be as effective in the root canal because the root canal is a habitat for various organisms. Moreover, the agar diffusion method does not distinguish between microbiostatic and microbicidal properties of dental medicaments, neither does it provide information about microbial viability after the test [33]. Also, the test results depend on the medicament’s solubility and diffusability in agar, rather than its actual efficacy against the organism. Therefore, we recommend future studies evaluating the effectiveness of the above medicaments using in vivo methods.
Conclusion
Within the confines of our study, it was found that LC has the greatest effectiveness against EF, followed by LZ, CX, VP and CH, as evaluated by the agar diffusion method.
*** p < 0.001; Highly significant