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An In Vitro Study Comparing the Intracanal Effectiveness of Calcium Hydroxide– and Linezolid-based Medicaments against Enterococcus faecalis
Basic Research—Technology
An In Vitro Study Comparing the Intracanal Effectiveness of Calcium Hydroxide– and Linezolid-based Medicaments against Enterococcus faecalis Rajdeep Pavaskar, MDS,* Ida de Noronha de Ataide, MDS,* Paul Chalakkal, MDS,† Maria J. Pinto, MD,‡ Kristlee Sabrin Fernandes, MDS,* Ramachandra V. Keny, MPharma,§ and Anagha Kamath, MD, DNBk Abstract Introduction: This study evaluated the efficacy of calcium hydroxide (CH), Vitapex (VP), linezolid (LZ), a combination of LZ with CH (LC), and a control group (N, no medicament) against Enterococcus faecalis (EF). Methods: Human single-rooted premolars were instrumented up to ProTaper size F3 files. EF suspension was inoculated into each root specimen and incubated. The medicaments were syringed into each root by weight and incubated. After 72 hours, 6 samples per group (among the 5 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 BHI agar and blood agar. Colony-forming units (CFU) of EF were measured from BHI agar. The procedure was repeated after 8 days and 14 days. Results: In group CH, the mean CFU (log 10 values) after 72 hours, 8 days, and 14 days were 1.17 1.16, 3.33 1.97, and 4.17 1.17, respectively (statistically significant). In group VP, the mean CFU were 0.83 0.75, 4.00 1.67, and 4.83 1.72. In group LZ, the mean CFU at 72 hours and after 8 days was 0.17 0.41, and no CFU were found on the fourteenth day. Similarly, in group LC, the mean CFU at 72 hours and after 8 days was 0.50 0.84, which increased to 1.33 1.51 on the fourteenth day (not significant). Conclusions: LZ was found to be most effective on EF, followed by LC, CH, and VP. (J Endod 2012;38:95–100)
From the *Department of Conservative Dentistry and Endodontics, Goa Dental College and Hospital, Bambolim, Goa; †Department of Pedodontics and Preventive Dentistry, Goa Dental College and Hospital, Bambolim, Goa; ‡Department of Microbiology, Goa Medical College and Hospital, Rajiv Gandhi Medical Complex, Bambolim, Goa; §Department of Pharmaceutics, Goa College of Pharmacy, Panaji, Goa; and k Department of Gynecology and Obstetrics, Kasturba Medical College, Mangalore, India. Address requests for reprints to Paul Chalakkal, Lecturer, Department of Pedodontics and Preventive Dentistry, Goa Dental College and Hospital, Bambolim 403202, Goa, India. E-mail address: [email protected] 0099-2399/$ - see front matter Copyright ª 2012 American Association of Endodontists. doi:10.1016/j.joen.2011.09.031
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Key Words Calcium hydroxide, Enterococcus faecalis, linezolid, vitapex
T
horough irrigation of root canals with any antimicrobial solution might not be sufficient to eliminate all microorganisms from the root canal (1). Enterococcus faecalis (EF) has been detected in asymptomatic and persistent root canal infections (2, 3). It has also been detected in 77% of failed endodontic cases (3) and in 50% of cases with chronic apical periodontitis (4). Calcium hydroxide (CH) has been widely accepted as an intracanal medicament because of its antimicrobial properties, especially because of its action on gram-negative bacteria (5, 6). Some authors have discussed its effective role in eliminating EF (7, 8). A combination of CH and iodoform has been documented to enhance periapical bone regeneration with resorption of excess material, with a success rate of 84%–100% (9). Vitapex (VP), a combination of CH and iodoform, resorbs from the apical tissues from 1 week to 2 months in primary teeth. It is radiopaque, does not set to a hard mass, and is easily inserted and retrieved (10). A recent therapeutic, 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 (11). The effectiveness of CH on EF, with or without iodoform, has been speculated by authors. Moreover, the effectiveness of LZ, or a combination of LZ with CH (LC), on root canal related EF has not previously been reported. Hence this study was conducted to evaluate the efficacy of CH, VP, LZ, and LC on EF at various time intervals by using the bacterial sampling method. The null hypothesis assumed was that there would be no difference among 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. One hundred fifty single-rooted human premolar teeth with completed apices and straight roots were collected for 10 days from the Department of Oral and Maxillofacial Surgery. The teeth were gently scrapped externally with 5.25% sodium hypochlorite by using sterile gauze to remove debris. The teeth were then washed with distilled water and stored in saline. The crowns of all the teeth were sectioned with a diamond disk (Carbodent; Gysi S.A, Buenos Aires, Argentina), and the canal lengths were standardized to 18 mm. Access preparation was made on each tooth by using a high-speed Endo access bur (Dentsply Maillefer, Ballaigues, Switzerland) with water spray. A size 10 K-file (Mani Inc, Tochigi, Japan) was placed into the canal until it was visible at the apical foramen to ensure that the canal was patent. Root canals were instrumented 0.5 mm beyond the apex up to size 25 instruments. Roots with apical diameter greater than 20 K-file size or a canal anatomy other than Type 1 were discarded. Only 95 teeth fulfilled the inclusion criteria, and these were then instrumented 1 mm short of their apices up to ProTaper size F3 files (Dentsply Maillefer) by using the crown-down technique. Five milliliters of 5.25% sodium hypochloride
Intracanal Effectiveness of Calcium Hydroxide– and Linezolid-based Medicaments against E. faecalis
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Basic Research—Technology TABLE 1. Mean CFU (log 10 values) in All Groups at 72 Hours Kruskal-Wallis test Group
N
Range
Mean
SD
SEM
Mean rank
c2
P value
N CH VP LZ LC
6 6 6 6 6
5–8 0–3 0–2 0–1 0–2
6.67 1.17 0.83 0.17 0.50
1.211 1.169 0.753 0.408 0.837
0.494 0.477 0.307 0.167 0.342
27.50 15.67 14.50 8.67 11.17
18.003
.001*
SD, standard deviation; SEM, standard error of mean. *P < .01, significant at 1% significance level.
(Vensons, Malleshwaram, India) was used for irrigation after each instrument use. Final irrigation was done with 5 mL of 17% ethylenediaminetetraacetic acid (EDTA) (Dent Wash; Prime Dental, Chicago, IL) for 3 minutes for each specimen. The teeth were then introduced into an ultrasonic bath containing 17% EDTA for 1 minute. This was followed by irrigation with physiologic saline solution to eliminate any residual EDTA. The root apices were then sealed with nail varnish, and the specimens were sterilized in an autoclave at 121 C for 20 minutes at 20 psi pressure. An autoclave label (Signaloc; Johnson & Johnson Ltd, Mumbai, India) was used to ensure adequate sterilization. A turbid suspension of EF (ATCC 29212; Microbiologics, Medimark Europe) was obtained by growing the cells in brain-heart infusion (BHI) broth for 24 hours at 36.5 C. The turbidity was verified by using the Mac Farland Turbidity Scale and adjusted to 0.5, corresponding to 108 organisms per milliliter. Fifteen microliters of this bacterial suspension was inoculated into each canal by using a micropipette (Kasablanka, digital variable micropipette, Mumbai, India). The specimens were then incubated at 36.5 C for 6 hours to ensure penetration of EF into dentinal tubules. Two autoclaved foams with 90 punch holes were used to hold the prepared specimens. Asepsis was maintained throughout the procedures by using standard precautions with 2 flames in a biosafety cabinet. The specimens were divided into 5 groups, each comprising 19 specimens. One specimen per group (chosen blindly) was sectioned 3 mm from the apex to obtain a 0.8-mm-thick cross section. The section was observed under a scanning electron microscope to verify EF penetration into the dentinal tubules. The remaining 18 specimens per group were then used to evaluate the effectiveness of the medicaments being considered. The 5 groups were the following: group I (N): negative control, no intracanal medicament; group II (CH), calcium hydroxide based intracanal (Ultracal XS; Ultradent, South Jordan, UT); group III (VP), Vitapex (Diadent Group International Inc, Burnaby, BC, Canada); group IV (LZ), linezolid based intracanal medicament (0.3%); and group V (LC), calcium hydroxide and linezolid based intracanal medicament (3% LZ, 30% CH).
The canals were dried by using ProTaper Universal paper points of size F3 (Dentsply Maillefer) before placement of medicaments. To standardize the amount of intracanal medicament that was to be loaded into each root, 0.8 mg of each medicament was weighed on an electronic scale (Essae, India) and syringed into each canal. The teeth were then sealed with 3 mm of intermittent restorative material MD-Temp (Meta Biomed, Korea). The specimens were then incubated at 36.5 C in an incubator until evaluation. After 72 hours, 6 samples per group were retrieved from the incubator and analyzed. A no. 2 sterile round bur, 1 mm in diameter (SS White Burs, Inc, Lakewood, NJ), was used to drill a hole on each root, 1 mm deep and 3 mm short of the apex. The dentinal shavings obtained by doing this were allowed to fall in 200 mL of BHI broth. By using sterile pipette tips and micropipettes (0–200 mL), decimal series of dilutions were made up to 104. One hundred microliters of each dilution was plated and spread over a 6-inch BHI agar plate and blood agar by using a spreader to rule out the presence of organisms other than EF. Both BHI and blood agar plates were incubated at 36.5 C for 24 hours. Colony count of EF was done from BHI agar by using a stereomicroscope and expressed as colony-forming units (CFU)/mL. Growth of the organism that occurred on BHI agar was confirmed to be that of EF by the following tests: (1) Gram staining, (2) catalase test (negative for enterococci), (3) growth on Pfizer selective enterococcus agar (Liofilchem, Roseto degli Abruzzi, Italy), and (4) growth on nutrient agar. The above procedures were repeated at time intervals of 8 days and 14 days. Statistical analysis of CFU (log 10 values) from the various groups was done by using Kruskal-Wallis test, Kolmogorov-Smirnov Z test, and the Wilcoxon signed rank test.
Results The mean values of CFU (log 10) differed significantly among the 5 groups (Tables 1 through 3) after 72 hours (P = .001), 8 days (P < .001), and 14 days (P < .001). By multiple comparisons after 72 hours by using the Kolmogorov-Smirnov test (Table 4), it was found that CFU were significantly greater in the control group than in the study groups
TABLE 2. Mean CFU (log 10 values) in All Groups after 8 Days Kruskal-Wallis test Group
N
Range
Mean
SD
SEM
Mean rank
c2
P value
N CH VP LZ LC
6 6 6 6 6
4–7 1–6 2–6 0–1 0–2
5.33 3.33 4.00 0.17 0.50
1.211 1.966 1.673 0.408 0.837
0.494 0.803 0.683 0.167 0.342
24.58 18.58 20.67 6.00 7.67
21.771
<.001*
SD, standard deviation; SEM, standard error of mean. *P < .001, highly significant.
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Basic Research—Technology TABLE 3. Mean CFU (log 10 values) in All Groups after 14 Days Kruskal-Wallis test Group
N
Range
Mean
SD
SEM
Mean rank
c2
P value
N CH VP LZ LC
6 6 6 6 6
5–7 3–6 2–7 0–0 0–3
6.33 4.17 4.83 0.00 1.33
0.816 1.169 1.722 0.000 1.506
0.333 0.477 0.703 0.000 0.615
25.83 17.67 20.25 5.00 8.75
23.295
<.001*
SD, standard deviation; SEM, standard error of mean. *P < .001, highly significant.
(P < .01). However, no significant difference was found between the 4 study groups at 72 hours (P > .05). By multiple comparisons after 8 days and 14 days (Tables 5 and 6), it was found that CFU were significantly greater in the control group as compared with LZ and LC groups (P < .01). CH and VP groups showed no significant difference in mean CFU as compared with N group (P > .05). Also, there were no significant differences between CH, VP, and LC groups (P > .05). VP group showed significantly more CFU than LZ (P < .01) and LC (P < .05) groups. No significant difference was found between LZ and LC groups (P > .05). Group CH showed significantly more CFU than LZ after 8 days (P < .05; Table 5) and 14 days (P < .01; Table 6). On the eighth day, least number of CFU were seen in group LZ, followed by LC (Table 5). However, after 14 days, no CFU were seen in group LZ, whereas group LC showed 1.33 1.506 CFU (Table 6). The Wilcoxon signed rank test determined the intragroup variation in CFU of all groups at 72 hours, 8 days, and 14 days (Fig. 1 and Table 7). The maximum increase in CFU among the 4 study groups was found in group VP, followed by groups CH and LC. Group LZ showed the best results, with no CFU after 14 days.
Discussion The canals were instrumented 1 mm beyond the apex up to size 25 K-file and 1 mm short of the apex up to ProTaper size F3 file (Dentsply Maillefer). This was done to ensure that the apical third was adequately cleaned. In this study, teeth were introduced in an ultrasonic bath containing 17% EDTA for 1 minute to remove the smear layer that might have remained in the canal after instrumentation. Because of the demineralizing effect of EDTA, smear layer formation is reduced during instrumentation, thereby augmenting NaOCl penetration into tubules (12, 13). NaOCl has gained popularity with regard to root canal irrigation because of various virtues. It brings about lavage of debris, tissue lubrication, and dissolution (12, 14). When it couples with TABLE 4. Multiple Comparisons by Using Kolmogorov-Smirnov Z Test (at 72 hours) Comparison N vs CH N vs VP N vs LZ N vs LC CH vs VP CH vs LZ CH vs LC VP vs LZ VP vs LC LZ vs LC
Mean difference
Z value
P value
5.500 5.833 6.500 6.167 0.333 1.000 0.667 0.667 0.333 0.333
1.732 1.732 1.732 1.732 0.289 0.866 0.577 0.866 0.577 0.289
.005* .005* .005* .005* 1.000† .441† .893† .441† .893† 1.000†
*P < .01, significant at 1% significance level. † P > .05 (not significant).
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water molecules, there is formation of hypochlorite acid that contains active chlorine. Chlorine, a powerful oxidizing agent, causes irreversible oxidation of hydrosulfuric groups of essential enzymes, disturbing metabolic functions in the microorganism. It also adheres to bacterial cytoplasmic components, forming toxic N-chloro compounds (14). Its high pH interferes with bacterial cytoplasmic membrane integrity, causing biosynthetic alteration in cell metabolism and phospholipid destruction (15). The ineffectiveness of using lower concentrations of NaOCl against EF has been reported (16, 17). Hence, the highest concentration recommended, 5.25% NaOCl, was used in this study. Although EF has been reported to be sensitive to 5.25% NaOCl, the irrigant is unable to completely eliminate the viability of enterococci (7, 17). Dentinal shavings from the infected specimens were allowed to fall in BHI broth from a 1-mm-deep punch hole drilled by using a no. 2 round bur 3 mm from the apex. This distance was chosen because the apical 3 mm is the most critical zone during instrumentation. Moreover, this was the closest distance from where a 1-mm-deep hole could be comfortably drilled without perforating into the root canal. Perforation of the canal would have meant contamination of the dentinal shavings with the medicament that could have interfered with bacterial counts. Three different techniques have been used to determine the effectiveness of any antimicrobial agent: 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 because of its direct contact with the microorganism being considered. The agar diffusion method presents with a zone of inhibition around the wells containing the medicament (18). It is by far the most commonly used method. However, it does not distinguish between microbiostatic and microbicidal properties TABLE 5. Multiple Comparisons by Using Kolmogorov-Smirnov Z Test (after 8 days) Comparison N vs CH N vs VP N vs LZ N vs LC CH vs VP CH vs LZ CH vs LC VP vs LZ VP vs LC LZ vs LC
Mean difference
Z value
P value
2.000 1.333 5.167 4.833 0.667 3.167 2.833 3.833 3.500 0.333
0.866 0.577 1.732 1.732 0.289 1.443 1.155 1.732 1.443 0.289
.441* .893* .005† .005† 1.000* .031‡ .139* .005† .031‡ 1.000*
*P > .05 (not significant). † P < .01, significant at 1% significance level. ‡ P < .05, significant at 5% significance level.
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Basic Research—Technology TABLE 6. Multiple Comparisons by Using Kolmogorov-Smirnov Z Test (after 14 days) Comparison
Mean difference
Z value
P value
2.167 1.500 6.333 5.000 0.667 4.167 2.833 4.833 3.500 1.333
1.155 0.866 1.732 1.732 0.577 1.732 1.155 1.732 1.443 0.866
.139* .441* .005† .005† .893* .005† .139* .005† .031‡ .441*
N vs CH N vs VP N vs LZ N vs LC CH vs VP CH vs LZ CH vs LC VP vs LZ VP vs LC LZ vs LC
*P > .05 (not significant). † P < .01, significant at 1% significance level. ‡ P < .05, significant at 5% significance level.
Figure 1. Comparison of mean CFU (log 10 values) in all groups at different time intervals.
of dental medicaments, and it does not provide information about microbial viability after the test (19). Also, the test results depend on the medicament’s solubility and diffusibility in agar, rather than its actual efficacy against the organism. When CH is placed in agar, its high pH starts to precipitate it, preventing its diffusion (20). Moreover, disassociation of Ca and OH ions decreases the pH of the medium, enhancing growth of the organisms being tested (21). Therefore, previous studies that have tested the efficacy of CH against EF by using the agar diffusion method might have documented imperfect results. However, in this study, the bacterial sampling method was used, which does not have any of the disadvantages other methods have. Sampling was done in 3 stages at 72 hours, 8 days, and 14 days. CH releases OH ions that are responsible for the creation of high alkalinity. Increased pH has a destructive effect on bacterial cell membrane and protein structure (18). In our study, the mean CFU of the CH group at 72 hours was 1.17 1.16, which rose to 3.33 1.97 (P > .05) on the eighth day and 4.17 1.17 (P < .05) after 14 days (Table 7). The change in CFU from 72 hours to 14 days was statistically significant. The reason for decline in its effect might be due to the decrease in pH with time. A pH of 10.5–11 delays the growth of EF, whereas a pH of 11 or more eliminates EF (22, 23). Although many authors have stated little effect of CH on EF (1, 21, 23), our study has proved a time-related decline in its effect on EF. It has been shown that CH requires 60 days to exert any action against EF (24); however, we found it to be most effective soon after placement. These differences among results could be due to the agar diffusion method that was used in most of these studies, which could have yielded imperfect results. Recently, it has been shown that CH attenuates the inflammatory action of EF lipoteichoic acid through deacylation of the lipid moiety (25). Dentin has a buffering potential, whereby the proton donor in the hydroxyapatite hydrated layer can reduce pH and thus the effect of calcium hydroxide within tubules. The pH of CH might reduce to 10.3 because of the dentin buffer effect (23, 26). The low solubility and diffusibility of CH might further make it difficult to penetrate
into dentinal tubules to exert any action (27). Moreover, the proton pump of EF carries protons to the interior of the cell, acidifying its cytoplasm, in situations of increased alkalinity in its environment (when subjected to CH) (23). All these factors might have contributed to the pH decline of CH. In the VP group (Table 7), the mean CFU after 72 hours was 0.83 0.75, which rose significantly to 4.00 1.67 after 8 days (P < .05) and 4.83 1.72 after 14 days (P < .05). The ineffectiveness of VP on organisms has been previously reported (21, 28). In a study by Amorim et al (18), VP produced no zone of inhibition in the agar diffusion method; however, it was found to be effective against EF through the direct exposure test. This example further confirms the unreliability in using the agar diffusion method. 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 (29). However, enterococcal resistance to the drug occurs as a result of mutation of the ribosomal binding site (30). In group LZ, the mean CFU at 72 hours and after 8 days was 0.17 0.41, with no CFU after 14 days (Table 7). In group LC, the mean CFU at 72 hours and after 8 days was 0.50 0.84, which increased to 1.33 1.51 after 14 days (Table 7). However, this increase was not statistically significant (P > .05). This might have occurred as a result of dehydration of CH, which could have also affected the penetrability of LZ, resulting in increased CFU. The drug is known to cause adverse effects on systemic administration, such as nausea, diarrhea, tongue discoloration, oral moniliasis, taste perversion, headache, and myelosuppression (31). However, these effects were found on systemic administration. There has not been a study yet evaluating the effectiveness of LZ as an intracanal medicament against EF or any other organism. This in vitro study could not simulate the intraoral environment inside an infected root canal. Although every effort was made to deliver quantified amounts of microorganisms and intracanal medicaments, it was not possible to standardize certain variables such as the quantity of dentinal shavings analyzed per sample, the time used for drilling a punch
TABLE 7. Intragroup Variation in CFU after 72 Hours, 8 Days, and 14 Days (Wilcoxon signed rank test) Time
Group N
P value
Group CH
P value
Group VP
P value
Group LZ
P value
Group LC
P value
72 hours 8 days 14 days
6.67 1.21 5.33 1.21 6.33 0.82
– .063* .581*
1.17 1.16 3.33 1.97 4.17 1.17
– .104* .024†
0.83 0.75 4.00 1.67 4.83 1.72
– .039† .027†
0.17 0.41 0.17 0.41 0.00 0.00
– – .317*
0.50 0.84 0.50 0.84 1.33 1.51
– – .276*
*P > .05, not significant. † P < .05, significant at 5%.
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Basic Research—Technology hole, and amount of heat generated during the procedure. It was necessary to standardize the medicaments by weight rather than volume because the medicaments used (except LZ and LC) were available commercially in terms of weight. Therefore, it was convenient to standardize all the medicaments by weight. It has been shown that EF rarely appears in primary endodontic lesions (32). However, some authors have documented its occurrence in primary as well as secondary endodontic lesions (2, 33). Primary colonization by EF might be explained as a consequence of coronal colonization after contaminated food ingestion. EF can survive inside dentin tubules for at least 10 days without nutrient supply (1, 34). It can also survive in smear layer and debris and might be extremely difficult to eliminate during instrumentation and irrigation (35). Moreover, serine protease and Ace aid in the adhesion of EF with dentin (36). It has been recently documented that the tolerance of EF to CH can be attributed to its association with collagen (37). These factors make EF resistant to most intracanal medications. However, certain studies have documented positive effects of propolis against EF (38, 39). From this study, we found linezolid to be promising in eliminating EF in comparison with the other medicaments tested. Hydroxyl ions liberated by CH act on the enzymes in the cytoplasmic membrane of the organism. The membrane is similar, irrespective of the organisms’ morphologic, tinctorial, and respiratory characteristics. This means that CH will have similar effects on aerobic, anaerobic, gram-positive, and gram-negative organisms (18). Therefore, we recommend the LC combination rather than LZ alone, because CH could widen the spectrum of activity against a variety of organisms, compared with the sole gram-positive spectrum of action of LZ. However, it might need replacement every 8 days to ensure absence of any dehydration or reduction in pH of CH, which might invariably affect the potency of LZ. This study used planktonic EF cells, which are different in characteristics from those found in intracanal biofilms. However, this study serves as a baseline for future in vivo studies that could evaluate the effects of LZ and its combinations on biofilm organisms. The periapical effects of LZ such as sensitization, allergy, and bacterial resistance also need to be investigated.
Conclusion From the present study it might be concluded that calcium hydroxide effectively inhibits EF within dentinal tubules up to 72 hours, and the effect gradually reduces in time. The addition of iodoform to calcium hydroxide, as in Vitapex, did not increase its activity against EF. Linezolid was found to be most effective against EF in comparison with all tested medicaments, followed by a combination of linezolid with calcium hydroxide.
Acknowledgments The authors deny any conflicts of interest related to this study. However, the authors have financial interests in the linezolid based intracanal medicaments that were formulated for this study and hold a patent application for the same (2619/MUM/2011).
References 1. Bystrom A, Claesson R, Sundqvist G. The antibacterial effect of camphorated paramonochlorophenol, camphorated phenol and calcium hydroxide in the treatment of infected root canals. Endod Dent Traumatol 1985;1:170–5. 2. Pirani C, Bertacci A, Cavrini F, et al. Recovery of Enterococcus faecalis in root canal lumen of patients with primary and secondary endodontic lesions. New Microbiol 2008;31:235–40.
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3. Siqueira JF Jr, R^oc¸as IN. Polymerase chain reaction-based analysis of microorganisms associated with failed endodontic treatment. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 2004;97:85–94. 4. Peciuliene V, Reynaud AH, Balciuniene I, Haapasalo M. Isolation of yeasts and enteric bacteria in root-filled teeth with chronic apical periodontitis. Int Endod J 2001;34:429–34. 5. Athanassiadis B, Abbot PV, Walsh LJ. The use of calcium hydroxide, antibiotics and biocides as antimicrobial medicaments in endodontics. Aust Dent J 2007;52: 64–82. 6. Menezes MM, Valera MC, Jorge AO, Koga-Ito CY, Camargo CH, Mancini MN. In vitro evaluation of the effectiveness of irrigants and intracanal medicaments on microorganisms within root canals. Int Endod J 2004;37:311–9. 7. Lana PEP, Scelza MFZ, Silva LE, Mattos-Guaraldi AL, Junior RH. Antimicrobial activity of calcium hydroxide pastes on enterococcus faecalis cultivated in root canal systems. Braz Dent J 2009;20:32–6. 8. Chai WL, Hamimah H, Cheng SC, Sallam AA, Abdullah M. Susceptability of enterococcus faecalis biofilm to antibiotics and calcium hydroxide. J Oral Sci 2007;49: 161–6. 9. Reddy VV, Fernandes. Clinical and radiological evaluation of zinc-oxide eugenol and Maisto’s paste as obturating materials in infected primary teeth: nine months study. J Indian Soc Pedod Prev Dent 1996;14:39–44. 10. Nurku C, Garcia-Godoy F. Evaluation of a calcium hydroxide iodoform paste (Vitapex) in root canal therapy for primary teeth. J Clin Ped Dent 1999;23: 289–94. 11. Narang M, Gomber S. Linezolid. Indian Pediatr 2004;41:1129–32. 12. Berutti E, Marini R, Angeretti A. Penetration ability of different irrigants into dentinal tubules. J Endod 1997;23:725–7. 13. Goldberg F, Abramovich A. Analysis of the effect of EDTAC on the dentinal walls of the root canal. J Endod 1977;3:101–5. 14. Siqueira JF Jr, R^oc¸as IN, Favieri A, Lima KC. Chemomechanical reduction of the bacterial population in the root canal after instrumentation and irrigation with 1%, 2.5% and 5.25% sodium hypochlorite. J Endod 2000;26: 331–4. 15. Estrela C, Ribeiro RG, Estrela CR, Pecora JD, Souza-Neto MD. Antimicrobial effect of 2% sodium hypochlorite and 2% chlorhexidine tested by different methods. Braz Dent J 2003;14:58–62. 16. Gomes BP, Ferraz CC, Vianna ME, Berber VB, Teixeira FB, Souza–Filho FJ. In vitro antimicrobial activity of several concentrations of sodium hypochlorite and chlorhexidine gluconate in the elimination of Enterococcus faecalis. Int Endod J 2001;34:424–8. 17. Fidalgo TKS, Barcelos R, Portela MB, Soares RMA, Gleiser R, Silva-Filho FC. Inhibitory activity of root canal irrigants against Candida albicans, Enterococcus faecalis and Staphylococcus aureus. Braz Oral Res 2010;24:406–12. 18. Amorim LFG, Toledo OA, Estrela CRA, Decurcio DA, Estrela C. Antimicrobial analysis of different root canal filling pastes used in pediatric dentistry by two experimental methods. Braz Dent J 2006;17:317–22. 19. Estrela C, Estrela CRA, Bammann LL, Pecora JD. Two methods to evaluate the antimicrobial action of calcium hydroxide paste. J Endod 2001;27:720–3. 20. Souza-Filho FJ, Soares AJ, Vianna ME, Zaia AA, Ferraz CCR, Gomes BPFA. Antimicrobial effect and pH of chlorhexidine gel and calcium hydroxide alone and associated with other materials. Braz Dent J 2008;19:28–33. 21. Gangwar A. Antimicrobial effectiveness of different preparations of calcium hydroxide. Indian J Dent Res 2011;22:66–70. 22. McHugh CP, Zhang P, Michalek S, Eleazer PD. pH required to kill Enterococcus faecalis in vitro. J Endod 2004;30:218–9. 23. 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. 24. Estrela C, Cynthia RA, Pecora JD. A study of the time necessary for calcium hydroxide to eliminate microorganisms in infected canals. J Appl Oral Sci 2003;11:133–7. 25. Baik JE, Jang KS, Kang SS, et al. Calcium hydroxide inactivates lipoteichoic acid from Enterococcus faecalis through deacylation of the lipid moiety. J Endod 2011;37:191–6. 26. Mi~nana M, Carnes DL Jr, Walker WA. PH changes at the surface of root dentin after intracanal dressing with calcium oxide and calcium hydroxide. J Endod 2001;27: 43–5. 27. Gomes BPFA, Souza SFC, Ferraz CCR, et al. Effectiveness of 2% chlorhexidine gel and calcium hydroxide against Enterococcus faecalis in bovine root dentin in vitro. Int Endod J 2003;36:267–75. 28. Blanscet ML, Tordik PA, Goodell GG. An agar diffusion comparison of the antimicrobial effect of calcium hydroxide at five different concentrations with three different vehicles. J Endod 2008;34:1246–8. 29. Bozdogan B, Esel D, Whitener C, Browne FA, Appelbaum PC. Antimicrobial susceptibility of a vancomycin-resistant Staphylococcus aureus strain isolated at the Hershey Medical Center. J Antimicrob Chemother 2003;52:864–8.
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Basic Research—Technology 30. Hamel JC, Stapert D, Moerman JK, Ford CW. Linezolid, critical characteristics. Infection 2000;28:60–4. 31. Ba BB, Arpin C, Nso BBB, Dubois V, Saux MC, Quentin C. Activity of linezolid in an in vitro pharmacokinetic-pharmacodynamic model using different dosages and staphylococcus aureus and enterococcus faecalis strains with and without a hypermutator phenotype. Antimicrob Agents Chemother 2010; 54:1443–52. 32. Siren EK, Haapasalo MP, Ranta K, Salmi P, Kerosuo ENJ. Microbiological findings and clinical treatment procedures in endodontic cases selected for microbiological investigation. Int Endod J 1997;30:91–5. 33. Sakamoto M, Rocas IN, Siqueira JF, Benno Y. Molecular analysis of bacteria in asymptomatic and symptomatic endodontic infections. Oral Microbiol Immunol 2006;21:112–22. 34. Siqueira JF Jr. Aetiology of root canal treatment failure: why well treated teeth can fail. Int Endod J 2001;34:1–10.
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35. Estrela C, Estrela CRA, Decurcio DA, Hollanda ACB, Silva JA. Antimicrobial efficacy of ozonated water, gaseous ozone, sodium hypochlorite and chlorhexidine in infected human root canals. Int Endod J 2007;40:85–93. 36. Hubble TS, Hatton JF, Nallapareddy SR, Murray BE, Gillespie MJ. Influence of Enterococcus faecalis proteases and the collagen-binding protein, Ace, on adhesion to dentin. Oral Microbiol Immunol 2003;18:21–6. 37. Kayaoglu G, Erten H, Bodrumlu E, Ørstavik D. The resistance of collagenassociated, planktonic cells of Enterococcus faecalis to calcium hydroxide. J Endod 2009;35:46–9. 38. Kayaoglu G, Omurlu H, Akca G, et al. Antibacterial activity of propolis versus conventional endodontic disinfectants against Enterococcus faecalis in infected dentinal tubules. J Endod 2011;37:376–81. 39. Madhubala MM, Srinivasan N, Ahamed S. Comparative evaluation of propolis and triantibiotic mixture as an intracanal medicament against Enterococcus faecalis. J Endod 2011;37:1287–9.
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An In Vitro Study Comparing the Intracanal Effectiveness of Calcium Hydroxide– and Linezolid-based Medicaments against Enterococcus faecalis Rajdeep Pavaskar, MDS,* Ida de Noronha de Ataide, MDS,* Paul Chalakkal, MDS,† Maria J. Pinto, MD,‡ Kristlee Sabrin Fernandes, MDS,* Ramachandra V. Keny, MPharma,§ and Anagha Kamath, MD, DNBk Abstract Introduction: This study evaluated the efficacy of calcium hydroxide (CH), Vitapex (VP), linezolid (LZ), a combination of LZ with CH (LC), and a control group (N, no medicament) against Enterococcus faecalis (EF). Methods: Human single-rooted premolars were instrumented up to ProTaper size F3 files. EF suspension was inoculated into each root specimen and incubated. The medicaments were syringed into each root by weight and incubated. After 72 hours, 6 samples per group (among the 5 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 BHI agar and blood agar. Colony-forming units (CFU) of EF were measured from BHI agar. The procedure was repeated after 8 days and 14 days. Results: In group CH, the mean CFU (log 10 values) after 72 hours, 8 days, and 14 days were 1.17 1.16, 3.33 1.97, and 4.17 1.17, respectively (statistically significant). In group VP, the mean CFU were 0.83 0.75, 4.00 1.67, and 4.83 1.72. In group LZ, the mean CFU at 72 hours and after 8 days was 0.17 0.41, and no CFU were found on the fourteenth day. Similarly, in group LC, the mean CFU at 72 hours and after 8 days was 0.50 0.84, which increased to 1.33 1.51 on the fourteenth day (not significant). Conclusions: LZ was found to be most effective on EF, followed by LC, CH, and VP. (J Endod 2012;38:95–100)
From the *Department of Conservative Dentistry and Endodontics, Goa Dental College and Hospital, Bambolim, Goa; †Department of Pedodontics and Preventive Dentistry, Goa Dental College and Hospital, Bambolim, Goa; ‡Department of Microbiology, Goa Medical College and Hospital, Rajiv Gandhi Medical Complex, Bambolim, Goa; §Department of Pharmaceutics, Goa College of Pharmacy, Panaji, Goa; and k Department of Gynecology and Obstetrics, Kasturba Medical College, Mangalore, India. Address requests for reprints to Paul Chalakkal, Lecturer, Department of Pedodontics and Preventive Dentistry, Goa Dental College and Hospital, Bambolim 403202, Goa, India. E-mail address: [email protected] 0099-2399/$ - see front matter Copyright ª 2012 American Association of Endodontists. doi:10.1016/j.joen.2011.09.031
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Key Words Calcium hydroxide, Enterococcus faecalis, linezolid, vitapex
T
horough irrigation of root canals with any antimicrobial solution might not be sufficient to eliminate all microorganisms from the root canal (1). Enterococcus faecalis (EF) has been detected in asymptomatic and persistent root canal infections (2, 3). It has also been detected in 77% of failed endodontic cases (3) and in 50% of cases with chronic apical periodontitis (4). Calcium hydroxide (CH) has been widely accepted as an intracanal medicament because of its antimicrobial properties, especially because of its action on gram-negative bacteria (5, 6). Some authors have discussed its effective role in eliminating EF (7, 8). A combination of CH and iodoform has been documented to enhance periapical bone regeneration with resorption of excess material, with a success rate of 84%–100% (9). Vitapex (VP), a combination of CH and iodoform, resorbs from the apical tissues from 1 week to 2 months in primary teeth. It is radiopaque, does not set to a hard mass, and is easily inserted and retrieved (10). A recent therapeutic, 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 (11). The effectiveness of CH on EF, with or without iodoform, has been speculated by authors. Moreover, the effectiveness of LZ, or a combination of LZ with CH (LC), on root canal related EF has not previously been reported. Hence this study was conducted to evaluate the efficacy of CH, VP, LZ, and LC on EF at various time intervals by using the bacterial sampling method. The null hypothesis assumed was that there would be no difference among 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. One hundred fifty single-rooted human premolar teeth with completed apices and straight roots were collected for 10 days from the Department of Oral and Maxillofacial Surgery. The teeth were gently scrapped externally with 5.25% sodium hypochlorite by using sterile gauze to remove debris. The teeth were then washed with distilled water and stored in saline. The crowns of all the teeth were sectioned with a diamond disk (Carbodent; Gysi S.A, Buenos Aires, Argentina), and the canal lengths were standardized to 18 mm. Access preparation was made on each tooth by using a high-speed Endo access bur (Dentsply Maillefer, Ballaigues, Switzerland) with water spray. A size 10 K-file (Mani Inc, Tochigi, Japan) was placed into the canal until it was visible at the apical foramen to ensure that the canal was patent. Root canals were instrumented 0.5 mm beyond the apex up to size 25 instruments. Roots with apical diameter greater than 20 K-file size or a canal anatomy other than Type 1 were discarded. Only 95 teeth fulfilled the inclusion criteria, and these were then instrumented 1 mm short of their apices up to ProTaper size F3 files (Dentsply Maillefer) by using the crown-down technique. Five milliliters of 5.25% sodium hypochloride
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Basic Research—Technology TABLE 1. Mean CFU (log 10 values) in All Groups at 72 Hours Kruskal-Wallis test Group
N
Range
Mean
SD
SEM
Mean rank
c2
P value
N CH VP LZ LC
6 6 6 6 6
5–8 0–3 0–2 0–1 0–2
6.67 1.17 0.83 0.17 0.50
1.211 1.169 0.753 0.408 0.837
0.494 0.477 0.307 0.167 0.342
27.50 15.67 14.50 8.67 11.17
18.003
.001*
SD, standard deviation; SEM, standard error of mean. *P < .01, significant at 1% significance level.
(Vensons, Malleshwaram, India) was used for irrigation after each instrument use. Final irrigation was done with 5 mL of 17% ethylenediaminetetraacetic acid (EDTA) (Dent Wash; Prime Dental, Chicago, IL) for 3 minutes for each specimen. The teeth were then introduced into an ultrasonic bath containing 17% EDTA for 1 minute. This was followed by irrigation with physiologic saline solution to eliminate any residual EDTA. The root apices were then sealed with nail varnish, and the specimens were sterilized in an autoclave at 121 C for 20 minutes at 20 psi pressure. An autoclave label (Signaloc; Johnson & Johnson Ltd, Mumbai, India) was used to ensure adequate sterilization. A turbid suspension of EF (ATCC 29212; Microbiologics, Medimark Europe) was obtained by growing the cells in brain-heart infusion (BHI) broth for 24 hours at 36.5 C. The turbidity was verified by using the Mac Farland Turbidity Scale and adjusted to 0.5, corresponding to 108 organisms per milliliter. Fifteen microliters of this bacterial suspension was inoculated into each canal by using a micropipette (Kasablanka, digital variable micropipette, Mumbai, India). The specimens were then incubated at 36.5 C for 6 hours to ensure penetration of EF into dentinal tubules. Two autoclaved foams with 90 punch holes were used to hold the prepared specimens. Asepsis was maintained throughout the procedures by using standard precautions with 2 flames in a biosafety cabinet. The specimens were divided into 5 groups, each comprising 19 specimens. One specimen per group (chosen blindly) was sectioned 3 mm from the apex to obtain a 0.8-mm-thick cross section. The section was observed under a scanning electron microscope to verify EF penetration into the dentinal tubules. The remaining 18 specimens per group were then used to evaluate the effectiveness of the medicaments being considered. The 5 groups were the following: group I (N): negative control, no intracanal medicament; group II (CH), calcium hydroxide based intracanal (Ultracal XS; Ultradent, South Jordan, UT); group III (VP), Vitapex (Diadent Group International Inc, Burnaby, BC, Canada); group IV (LZ), linezolid based intracanal medicament (0.3%); and group V (LC), calcium hydroxide and linezolid based intracanal medicament (3% LZ, 30% CH).
The canals were dried by using ProTaper Universal paper points of size F3 (Dentsply Maillefer) before placement of medicaments. To standardize the amount of intracanal medicament that was to be loaded into each root, 0.8 mg of each medicament was weighed on an electronic scale (Essae, India) and syringed into each canal. The teeth were then sealed with 3 mm of intermittent restorative material MD-Temp (Meta Biomed, Korea). The specimens were then incubated at 36.5 C in an incubator until evaluation. After 72 hours, 6 samples per group were retrieved from the incubator and analyzed. A no. 2 sterile round bur, 1 mm in diameter (SS White Burs, Inc, Lakewood, NJ), was used to drill a hole on each root, 1 mm deep and 3 mm short of the apex. The dentinal shavings obtained by doing this were allowed to fall in 200 mL of BHI broth. By using sterile pipette tips and micropipettes (0–200 mL), decimal series of dilutions were made up to 104. One hundred microliters of each dilution was plated and spread over a 6-inch BHI agar plate and blood agar by using a spreader to rule out the presence of organisms other than EF. Both BHI and blood agar plates were incubated at 36.5 C for 24 hours. Colony count of EF was done from BHI agar by using a stereomicroscope and expressed as colony-forming units (CFU)/mL. Growth of the organism that occurred on BHI agar was confirmed to be that of EF by the following tests: (1) Gram staining, (2) catalase test (negative for enterococci), (3) growth on Pfizer selective enterococcus agar (Liofilchem, Roseto degli Abruzzi, Italy), and (4) growth on nutrient agar. The above procedures were repeated at time intervals of 8 days and 14 days. Statistical analysis of CFU (log 10 values) from the various groups was done by using Kruskal-Wallis test, Kolmogorov-Smirnov Z test, and the Wilcoxon signed rank test.
Results The mean values of CFU (log 10) differed significantly among the 5 groups (Tables 1 through 3) after 72 hours (P = .001), 8 days (P < .001), and 14 days (P < .001). By multiple comparisons after 72 hours by using the Kolmogorov-Smirnov test (Table 4), it was found that CFU were significantly greater in the control group than in the study groups
TABLE 2. Mean CFU (log 10 values) in All Groups after 8 Days Kruskal-Wallis test Group
N
Range
Mean
SD
SEM
Mean rank
c2
P value
N CH VP LZ LC
6 6 6 6 6
4–7 1–6 2–6 0–1 0–2
5.33 3.33 4.00 0.17 0.50
1.211 1.966 1.673 0.408 0.837
0.494 0.803 0.683 0.167 0.342
24.58 18.58 20.67 6.00 7.67
21.771
<.001*
SD, standard deviation; SEM, standard error of mean. *P < .001, highly significant.
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Basic Research—Technology TABLE 3. Mean CFU (log 10 values) in All Groups after 14 Days Kruskal-Wallis test Group
N
Range
Mean
SD
SEM
Mean rank
c2
P value
N CH VP LZ LC
6 6 6 6 6
5–7 3–6 2–7 0–0 0–3
6.33 4.17 4.83 0.00 1.33
0.816 1.169 1.722 0.000 1.506
0.333 0.477 0.703 0.000 0.615
25.83 17.67 20.25 5.00 8.75
23.295
<.001*
SD, standard deviation; SEM, standard error of mean. *P < .001, highly significant.
(P < .01). However, no significant difference was found between the 4 study groups at 72 hours (P > .05). By multiple comparisons after 8 days and 14 days (Tables 5 and 6), it was found that CFU were significantly greater in the control group as compared with LZ and LC groups (P < .01). CH and VP groups showed no significant difference in mean CFU as compared with N group (P > .05). Also, there were no significant differences between CH, VP, and LC groups (P > .05). VP group showed significantly more CFU than LZ (P < .01) and LC (P < .05) groups. No significant difference was found between LZ and LC groups (P > .05). Group CH showed significantly more CFU than LZ after 8 days (P < .05; Table 5) and 14 days (P < .01; Table 6). On the eighth day, least number of CFU were seen in group LZ, followed by LC (Table 5). However, after 14 days, no CFU were seen in group LZ, whereas group LC showed 1.33 1.506 CFU (Table 6). The Wilcoxon signed rank test determined the intragroup variation in CFU of all groups at 72 hours, 8 days, and 14 days (Fig. 1 and Table 7). The maximum increase in CFU among the 4 study groups was found in group VP, followed by groups CH and LC. Group LZ showed the best results, with no CFU after 14 days.
Discussion The canals were instrumented 1 mm beyond the apex up to size 25 K-file and 1 mm short of the apex up to ProTaper size F3 file (Dentsply Maillefer). This was done to ensure that the apical third was adequately cleaned. In this study, teeth were introduced in an ultrasonic bath containing 17% EDTA for 1 minute to remove the smear layer that might have remained in the canal after instrumentation. Because of the demineralizing effect of EDTA, smear layer formation is reduced during instrumentation, thereby augmenting NaOCl penetration into tubules (12, 13). NaOCl has gained popularity with regard to root canal irrigation because of various virtues. It brings about lavage of debris, tissue lubrication, and dissolution (12, 14). When it couples with TABLE 4. Multiple Comparisons by Using Kolmogorov-Smirnov Z Test (at 72 hours) Comparison N vs CH N vs VP N vs LZ N vs LC CH vs VP CH vs LZ CH vs LC VP vs LZ VP vs LC LZ vs LC
Mean difference
Z value
P value
5.500 5.833 6.500 6.167 0.333 1.000 0.667 0.667 0.333 0.333
1.732 1.732 1.732 1.732 0.289 0.866 0.577 0.866 0.577 0.289
.005* .005* .005* .005* 1.000† .441† .893† .441† .893† 1.000†
*P < .01, significant at 1% significance level. † P > .05 (not significant).
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water molecules, there is formation of hypochlorite acid that contains active chlorine. Chlorine, a powerful oxidizing agent, causes irreversible oxidation of hydrosulfuric groups of essential enzymes, disturbing metabolic functions in the microorganism. It also adheres to bacterial cytoplasmic components, forming toxic N-chloro compounds (14). Its high pH interferes with bacterial cytoplasmic membrane integrity, causing biosynthetic alteration in cell metabolism and phospholipid destruction (15). The ineffectiveness of using lower concentrations of NaOCl against EF has been reported (16, 17). Hence, the highest concentration recommended, 5.25% NaOCl, was used in this study. Although EF has been reported to be sensitive to 5.25% NaOCl, the irrigant is unable to completely eliminate the viability of enterococci (7, 17). Dentinal shavings from the infected specimens were allowed to fall in BHI broth from a 1-mm-deep punch hole drilled by using a no. 2 round bur 3 mm from the apex. This distance was chosen because the apical 3 mm is the most critical zone during instrumentation. Moreover, this was the closest distance from where a 1-mm-deep hole could be comfortably drilled without perforating into the root canal. Perforation of the canal would have meant contamination of the dentinal shavings with the medicament that could have interfered with bacterial counts. Three different techniques have been used to determine the effectiveness of any antimicrobial agent: 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 because of its direct contact with the microorganism being considered. The agar diffusion method presents with a zone of inhibition around the wells containing the medicament (18). It is by far the most commonly used method. However, it does not distinguish between microbiostatic and microbicidal properties TABLE 5. Multiple Comparisons by Using Kolmogorov-Smirnov Z Test (after 8 days) Comparison N vs CH N vs VP N vs LZ N vs LC CH vs VP CH vs LZ CH vs LC VP vs LZ VP vs LC LZ vs LC
Mean difference
Z value
P value
2.000 1.333 5.167 4.833 0.667 3.167 2.833 3.833 3.500 0.333
0.866 0.577 1.732 1.732 0.289 1.443 1.155 1.732 1.443 0.289
.441* .893* .005† .005† 1.000* .031‡ .139* .005† .031‡ 1.000*
*P > .05 (not significant). † P < .01, significant at 1% significance level. ‡ P < .05, significant at 5% significance level.
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Basic Research—Technology TABLE 6. Multiple Comparisons by Using Kolmogorov-Smirnov Z Test (after 14 days) Comparison
Mean difference
Z value
P value
2.167 1.500 6.333 5.000 0.667 4.167 2.833 4.833 3.500 1.333
1.155 0.866 1.732 1.732 0.577 1.732 1.155 1.732 1.443 0.866
.139* .441* .005† .005† .893* .005† .139* .005† .031‡ .441*
N vs CH N vs VP N vs LZ N vs LC CH vs VP CH vs LZ CH vs LC VP vs LZ VP vs LC LZ vs LC
*P > .05 (not significant). † P < .01, significant at 1% significance level. ‡ P < .05, significant at 5% significance level.
Figure 1. Comparison of mean CFU (log 10 values) in all groups at different time intervals.
of dental medicaments, and it does not provide information about microbial viability after the test (19). Also, the test results depend on the medicament’s solubility and diffusibility in agar, rather than its actual efficacy against the organism. When CH is placed in agar, its high pH starts to precipitate it, preventing its diffusion (20). Moreover, disassociation of Ca and OH ions decreases the pH of the medium, enhancing growth of the organisms being tested (21). Therefore, previous studies that have tested the efficacy of CH against EF by using the agar diffusion method might have documented imperfect results. However, in this study, the bacterial sampling method was used, which does not have any of the disadvantages other methods have. Sampling was done in 3 stages at 72 hours, 8 days, and 14 days. CH releases OH ions that are responsible for the creation of high alkalinity. Increased pH has a destructive effect on bacterial cell membrane and protein structure (18). In our study, the mean CFU of the CH group at 72 hours was 1.17 1.16, which rose to 3.33 1.97 (P > .05) on the eighth day and 4.17 1.17 (P < .05) after 14 days (Table 7). The change in CFU from 72 hours to 14 days was statistically significant. The reason for decline in its effect might be due to the decrease in pH with time. A pH of 10.5–11 delays the growth of EF, whereas a pH of 11 or more eliminates EF (22, 23). Although many authors have stated little effect of CH on EF (1, 21, 23), our study has proved a time-related decline in its effect on EF. It has been shown that CH requires 60 days to exert any action against EF (24); however, we found it to be most effective soon after placement. These differences among results could be due to the agar diffusion method that was used in most of these studies, which could have yielded imperfect results. Recently, it has been shown that CH attenuates the inflammatory action of EF lipoteichoic acid through deacylation of the lipid moiety (25). Dentin has a buffering potential, whereby the proton donor in the hydroxyapatite hydrated layer can reduce pH and thus the effect of calcium hydroxide within tubules. The pH of CH might reduce to 10.3 because of the dentin buffer effect (23, 26). The low solubility and diffusibility of CH might further make it difficult to penetrate
into dentinal tubules to exert any action (27). Moreover, the proton pump of EF carries protons to the interior of the cell, acidifying its cytoplasm, in situations of increased alkalinity in its environment (when subjected to CH) (23). All these factors might have contributed to the pH decline of CH. In the VP group (Table 7), the mean CFU after 72 hours was 0.83 0.75, which rose significantly to 4.00 1.67 after 8 days (P < .05) and 4.83 1.72 after 14 days (P < .05). The ineffectiveness of VP on organisms has been previously reported (21, 28). In a study by Amorim et al (18), VP produced no zone of inhibition in the agar diffusion method; however, it was found to be effective against EF through the direct exposure test. This example further confirms the unreliability in using the agar diffusion method. 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 (29). However, enterococcal resistance to the drug occurs as a result of mutation of the ribosomal binding site (30). In group LZ, the mean CFU at 72 hours and after 8 days was 0.17 0.41, with no CFU after 14 days (Table 7). In group LC, the mean CFU at 72 hours and after 8 days was 0.50 0.84, which increased to 1.33 1.51 after 14 days (Table 7). However, this increase was not statistically significant (P > .05). This might have occurred as a result of dehydration of CH, which could have also affected the penetrability of LZ, resulting in increased CFU. The drug is known to cause adverse effects on systemic administration, such as nausea, diarrhea, tongue discoloration, oral moniliasis, taste perversion, headache, and myelosuppression (31). However, these effects were found on systemic administration. There has not been a study yet evaluating the effectiveness of LZ as an intracanal medicament against EF or any other organism. This in vitro study could not simulate the intraoral environment inside an infected root canal. Although every effort was made to deliver quantified amounts of microorganisms and intracanal medicaments, it was not possible to standardize certain variables such as the quantity of dentinal shavings analyzed per sample, the time used for drilling a punch
TABLE 7. Intragroup Variation in CFU after 72 Hours, 8 Days, and 14 Days (Wilcoxon signed rank test) Time
Group N
P value
Group CH
P value
Group VP
P value
Group LZ
P value
Group LC
P value
72 hours 8 days 14 days
6.67 1.21 5.33 1.21 6.33 0.82
– .063* .581*
1.17 1.16 3.33 1.97 4.17 1.17
– .104* .024†
0.83 0.75 4.00 1.67 4.83 1.72
– .039† .027†
0.17 0.41 0.17 0.41 0.00 0.00
– – .317*
0.50 0.84 0.50 0.84 1.33 1.51
– – .276*
*P > .05, not significant. † P < .05, significant at 5%.
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Basic Research—Technology hole, and amount of heat generated during the procedure. It was necessary to standardize the medicaments by weight rather than volume because the medicaments used (except LZ and LC) were available commercially in terms of weight. Therefore, it was convenient to standardize all the medicaments by weight. It has been shown that EF rarely appears in primary endodontic lesions (32). However, some authors have documented its occurrence in primary as well as secondary endodontic lesions (2, 33). Primary colonization by EF might be explained as a consequence of coronal colonization after contaminated food ingestion. EF can survive inside dentin tubules for at least 10 days without nutrient supply (1, 34). It can also survive in smear layer and debris and might be extremely difficult to eliminate during instrumentation and irrigation (35). Moreover, serine protease and Ace aid in the adhesion of EF with dentin (36). It has been recently documented that the tolerance of EF to CH can be attributed to its association with collagen (37). These factors make EF resistant to most intracanal medications. However, certain studies have documented positive effects of propolis against EF (38, 39). From this study, we found linezolid to be promising in eliminating EF in comparison with the other medicaments tested. Hydroxyl ions liberated by CH act on the enzymes in the cytoplasmic membrane of the organism. The membrane is similar, irrespective of the organisms’ morphologic, tinctorial, and respiratory characteristics. This means that CH will have similar effects on aerobic, anaerobic, gram-positive, and gram-negative organisms (18). Therefore, we recommend the LC combination rather than LZ alone, because CH could widen the spectrum of activity against a variety of organisms, compared with the sole gram-positive spectrum of action of LZ. However, it might need replacement every 8 days to ensure absence of any dehydration or reduction in pH of CH, which might invariably affect the potency of LZ. This study used planktonic EF cells, which are different in characteristics from those found in intracanal biofilms. However, this study serves as a baseline for future in vivo studies that could evaluate the effects of LZ and its combinations on biofilm organisms. The periapical effects of LZ such as sensitization, allergy, and bacterial resistance also need to be investigated.
Conclusion From the present study it might be concluded that calcium hydroxide effectively inhibits EF within dentinal tubules up to 72 hours, and the effect gradually reduces in time. The addition of iodoform to calcium hydroxide, as in Vitapex, did not increase its activity against EF. Linezolid was found to be most effective against EF in comparison with all tested medicaments, followed by a combination of linezolid with calcium hydroxide.
Acknowledgments The authors deny any conflicts of interest related to this study. However, the authors have financial interests in the linezolid based intracanal medicaments that were formulated for this study and hold a patent application for the same (2619/MUM/2011).
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