Antimicrobial efficacy of triple antibiotic paste and calcium hydroxide mixed 2% chlorhexidine or saline against enterococcus faecalis: an in vitro study.
Abstract
Aim: The aim of this study was to evaluate antimicrobial efficacy of triple antibiotic paste and calcium hydroxide when mixed with either 2% chlorhexidine or saline against Enterococcus faecalis (E. Faecalis).
Material and Methods:An agar well diffusion assay method was used to determine the efficacy of the experimental medicaments against E. faecalis(ATCC 292122). Medicaments were divided into 4 groups; triple antibiotic powder with saline or chlorhexidine, and Ca(OH)2 (plus normal saline or 2% chlorhexidine).The diameters of the growth inhibition zones against E. faecalis for each group were recorded and compared. The differences between groups were analyzed by one-way ANOVA and Tukey’s post hoc tests for intergroup analysis.
Results: The triple antibiotic paste groups exhibited significantly higher zones of inhibition. The highest was exhibited by the combination of TAP with 2% chlorhexidene followed by saline (p<0.001). The Ca(OH)2combined with saline exhibited the least zone of inhibition in all the groups (p>0.0001).
Conclusion: The combination of triple antibiotic paste in combination with 2% chlorhexidiene exhibits better antibacterial action against E. faecalis.
Keywords: Enterococcus faecalis, Agar diffusion test , Triple antibiotic paste ‘
Introduction
The most active role in the initiation and maintenance of pulp and periapical diseases is of microorganisms; hence it is essential during endodontic treatment to totally eliminate them from the root canal system1.For long-term success of endodontic treatment ,complete debridement and subsidising of the bacterial infection from the root canal space is utmost priority2,3. The eradication of microorganisms from the infected root canal system is a complicated task including instrumentation, irrigation and application of intracanal medicaments. Many researchers have observed that after mechanical instrumentation, significant portions of the root canal walls remain untouched4. However in addition to mechanical debridement, chemical irrigators and intracanal medicaments seem necessary for eradication of infected tissues and microorganisms5. The most common factor for the failure of treatment is persistence of microorganisms in apical third of the root canals4.
E. faecalis is an enteric facultative gram positive bacterium with a special feature of growing independently in the root canal without the assistance of other bacteria6-14. The ability to tolerate the rough environmental changes which is believed to be due to its high alkali tolerance15 and tubular invasion ability of this cocci which protects it from intracanal endodontic medicaments, has made E. Faecalis a treatment-resistant microorganism16.
Calcium hydroxide (CH) is the most common intracanal medicament that has the ability to kill the bacteria because of its high alkalinity. The high pH of CH destroys the bacterial cell membrane and protein structures17. However, there are some evidences which shows that due to E. faecalis ability to penetrate into deeper layers of dentinal tubules, where its pH is neutralized by the buffering action of dentin, it is resistant to CH18-19. So considering the shortcomings of CH, finding an alternative intracanal medicament would be beneficial.
Recently, triple antibiotic paste (a mixture of metronidazole, ciprofloxacin and minocycline) has been used as an intracanal medicament for disinfecting the root canal during tissue regeneration20. A retrospective study showed that, because of the antibacterial properties of Ca(OH) and triple antibiotic paste,when used as an intracanal medicament in immature necrotic teeth,it aids in development of the pulp dentin complex21. Sato et al. showed triple antibiotic paste can destroy the bacteria in deep areas of the root canal system22. In a study on dogs, the results indicated the effectiveness of triple antibiotic paste in the disinfection of immature teeth with apical periodontitis23.
To date, there is insufficient documentation regarding the antimicrobial effect of triple antibiotic paste in eliminating E. faecalis. For this purpose, this investigation compared the in vitro antimicrobial efficacy of triple antibiotic paste and calcium hydroxide in combination with either 2% chlorhexidine or saline against E. Faecalis.
Materials and Method:
In the current study the medicaments tested were in powdered form. They were as follows: the antibiotic mixture, the individual components of the triple antibiotic mixture-metronidazole (Flagyl, Winthrop Pharmaceuticals, UK), ciprofloxacin(Bayer plc, UK) and minocycline (Expanscience Laboratories,Paris, France), Ca(OH)2 powder. The triple antibiotic mixture and Ca(OH)2 powder were either mixed normal saline (NaCl 0.9%, Darupakhsh, Tehran,Iran) or 2% chlorhexidine (Consepsis, Ultradent Inc., South Jordan, UT. USA). Ca(OH)2 powder mixed with normal saline served as the control group. The triple antibiotic paste was prepared for each group with identical amount of the three antibiotic powders (mg) and then mixed with 1mL normal saline or 2% chlorhexidine.
Preparation of the Medium for Enterococcus faecalis:-
The strains of microorganisms used for the study were standard strains of Enterococcus faecalis ATCC 29212(Department of Bacteriology, Haffkine Institute, Mumbai, India) and were subcultured in blood agar plate and were incubated at 37”C for 24 hours. A pure, single Enterococcus faecalis colony was isolated from the same cultured plate and Gram’s staining was done to confirm its growth, which was observed under oil immersion microscope and then inoculated with a brain heart infusion broth (BHI- Broth). The BHI- broth was incubated at 37”C for 24 hours period and checked for bacterial growth by changes in turbidity.
A drop of BHI(brain heart infusion) broth containing Enterococcus faecalis was placed into saline solution and checked for correct bacterial concentration with a spectrophotometer.The density of the bacterial suspension is standardized by comparing the broth at a density equivalent to the barium sulfate standard of 0.5 McFarland units, which was equivalent to 1.5 x 108 colony forming units per milliliter (CFU/ml).
Antimicrobial activity by Agar Well-Diffusion Method:-
Plates were prepared with Mueller Hinton Agar. The sterility of the plates were checked and the fresh inoculums of Enterococcus faecalis of 0.5 McFarland standard suspensions were prepared. A sterile non-toxic cotton swab was dipped on a wooden applicator into standardized inoculums (turbidity so adjusted, as to obtain confluent growth on the Petri plate) and the soaked swab was rotated firmly against the upper inside wall of the tube to express excess fluid. The entire agar surface of the plate was streaked with the swab three times, and the plate was turned at 600angles between each streaking. The inoculums was allowed to dry for 5-15 minutes with lid in place.
Four wells were created using 8mm sterile cork borer. The desired amount of the medicament to be tested were mixed and were placed in the well against E.Faecalis. 10-15 minutes were allowed for diffussion of the medicament in agar and then were incubated immediately at 35” 20C for 24 hours. The whole experiment was carried out under aseptic conditions and was be repeated twenty times to ensure reproducibility.
Measurement of Inhibition Zones
Zones of bacterial growth inhibition will be measured at the end of 24 hours. The zone of inhibition will be measured in milimeters using inhibition zone measuring scale.
Statistical Analysis
After data collection, data entry was done in Microsoft Excel and analyzed using the SPSS software v20 . Descriptive statistics were shown as Mean and standard deviation. To evaluate the differences between the antimicrobial efficacy of triple antibiotic paste and calcium hydroxide in combination with either normal saline or 2% chlorhexidine one-way ANOVA test was used and for comparison within the groups Tukey’s multiple comparison test(table 2) was used. P values less than 0.05 were considered as significant.
Results
The means of the diameters of the growth inhibition zones for each group of medicament are presented in (Figure 1).The range of inhibitory values between experimental groups varied broadly.One way ANOVA was used to calculate P-value and showed significant differences (P<0.0001)(Table 1).Overall, triple antibiotic paste/saline, triple antibiotic paste/2% chlorhexidine (groups 1, 2) had thelargest zones of growth inhibition in the well diffusion assay (figure 1).Ca(OH)/2% chlorhexidine showed minimal range of inhibitory effect against E.faecalis. Ca(OH)2/normal saline showed least antimicrobial efficacy against E. Faecalis.
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Figure 1. The zones of inhibition against E. faecalis, for the groups.
Figure 2. Meanwise presentation of the antibacterial efficacy by means of zones of inhibition.
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Source Sum of square Degree of freedom Mean square MS F Statistics P
VALUE
TREATMENT 3157.6854 3 1052.5618 117.08 <.0001
Error 683.2545 76 8.9902
Total 3840.9399 79
Table 1:Statistical analysis (ANOVA) against Enterococcus Faecalis.
Group P value Conclusion
TAP + CHX vs. TAP + Saline < 0.05 Significant
TAP + CHX vs. Ca(OH) + CHX and Ca(OH) + Saline < 0.01 Significant
TAP + Saline vs. Ca(OH) + CHX and Ca(OH) + Saline < 0.01 Significant
Ca(OH) + CHX vs. Ca(OH) + Saline < 0.01 Significant
Table 2: Statistical analysis Tukey’s post hoc multiple comparisons against Enterococcus Faecalis
Discussion
Traditionally, agar diffusion method and agar dilution method are commonly employed for detecting antimicrobial susceptibility. In our study, Kirby-Bauer method (Agar Disc Diffusion method) was chosen instead of the Agar Dilution method. The agar diffusion test we used in this study is useful for evaluating and comparing the in vitro antimicrobial activities of medicaments; many studies have used this method for evaluations of antibacterial effects of various endodontic materials.. The disadvantage of the Agar Dilution method is that this technique can alter some of the properties of the material being tested.
Hence, we chose the Agar Disc Diffusion method, as in this method the chemical properties of the medicaments are not changed and the antimicrobial resistance can be detected by challenging bacterial isolates with antimicrobial discs. Moreover, this is an easy and less technique sensitive method.
The bacterial species E. Faecalis was selected as representing an organism. There have been several studies to evaluate the effects of antibacterial agents which applied E. Faecalis as a target microorganism24-26.In fact, previously treated failed teeth are nine times more likely to be infected with E.faecalis than primary endodontic infections27.When a high pH is not maintained, E. Faecalis is also resistant to calcium hydroxide, a commonly used intracanal medicament28-30. Some studies have shown desirable effects of chlorhexidine in a 2% gel or liquid form to reduce or completely eliminate E. faecalis from the root canal space and dentinal tubules31-33. Turk et al. stated that calcium hydroxide mixed with 2% chlorhexidine digluconate was more effective against E. faecalis than calcium hydroxide mixed with other vehicles34.Therefore we utilised chlorhexidine as one of the vehicles for this study. To evaluate which substance had the highest antibacterial effect,each component was independently mixed with normal saline. The results showed the triple antibiotic powder, either mixed with normal saline or 2% chlorhexidine, produced the largest zone of inhibition against E. faecalis. Antibacterial activity of chlorhexidine may be attributed to its chemical characteristics.
In the present study, the antimicrobial effect of the triple antibiotic paste against E. faecalis were compared to calcium hydroxide, either mixed with normal saline or 2% chlorhexidine. Considering the low antibacterial capability of Ca(OH)2/chlorhexidine and the high ability of triple antibiotic powder/normal saline in this study, we conclude the ability of the former is related to its mixed antibiotic contents, not to the vehicle’s properties.
The zones of inhibition generated by groups containing calcium hydroxide were much smaller than the zones ofthe antibiotic-containing groups, concurring with previous literature that calcium hydroxide is not an effective intracanal medicament when root canals are infected by E. Faecalis. E. faecalis is the most common species in failed root canal therapy cases11-13; therefore the findings of the present study support the use of triple antibiotic paste as a potential intracanal medicament in the treatment of endodontic failures. It is noteworthy to mention that triple antibiotic paste is utilised in primary infections of open apex teeth, in which E. faecalis is not a prevalent bacteria35-38.
Conclusion
Under the limitations of this study, the triple antibiotic paste is very effective against E. faecalis and can be considered as a more powerful root canal medicament compared to calcium hydroxide pastes.
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