|Year : 2012 | Volume
| Issue : 2 | Page : 70-74
Comparative evaluation of the antimicrobial efficacy of MTAD, oxytetracycline, sodium hypochlorite and chlorhexidine against Enterococcus faecalis: An ex-vivo study
Rakesh Mittal, Meenu G Singla, Ashima Garg, Sumit Gupta, Vandana Dahiya
Department of Conservative Dentistry and Endodontics, Sudha Rustagi College of Dental Sciences and Research, Kheri More, Village Bhopani, Faridabad, Haryana, India
|Date of Web Publication||6-Mar-2013|
Department of Conservative Dentistry and Endodontics, Sudha Rustagi College of Dental Sciences and Research, Kheri More, Village Bhopani, Faridabad, Haryana - 121 002
Source of Support: None, Conflict of Interest: None
Objectives: To evaluate and compare the antibacterial efficiency of MTAD, Oxytetracycline, 5% NaOCl, and 2% chlorhexidine when used as root canal irrigants against Enterococcus faecalis. Materials and Methods: Fifty extracted human single rooted anterior teeth were selected. The decoronated sterilized root samples were infected with 10μl of 24 hours pure culture suspension of E. faecalis for 48 hours except for 10 teeth in negative control group (Group V). The test samples were divided into four groups (n = 10) as: Group I- 5% Sodium Hypochlorite, Group II- MTAD, Group III- Oxytetracycline and Group IV- 2% Chlorhexidine. The root canals were instrumented while using respective root canal irrigant solution. The bacterial cultures were taken from each root canal and colony forming units were counted on agar plates. The data was statistically analyzed. Results: It was observed that Group-III (Oxytetracycline) showed the maximum antibacterial efficacy against E. faecalis followed by Group II (MTAD), Group IV (2% Chlorhexidine), Group I (5% Sodium hypochlorite). Conclusion: Oxytetracycline has a great potential as a root canal irrigating agent because of its superior antimicrobial efficacy against E. faecalis, easy availability and cost effectiveness.
Keywords: Antimicrobial activity, chlorhexidine, MTAD, oxytetracycline, sodium hypochlorite
|How to cite this article:|
Mittal R, Singla MG, Garg A, Gupta S, Dahiya V. Comparative evaluation of the antimicrobial efficacy of MTAD, oxytetracycline, sodium hypochlorite and chlorhexidine against Enterococcus faecalis: An ex-vivo study. Saudi Endod J 2012;2:70-4
|How to cite this URL:|
Mittal R, Singla MG, Garg A, Gupta S, Dahiya V. Comparative evaluation of the antimicrobial efficacy of MTAD, oxytetracycline, sodium hypochlorite and chlorhexidine against Enterococcus faecalis: An ex-vivo study. Saudi Endod J [serial online] 2012 [cited 2019 Dec 8];2:70-4. Available from: http://www.saudiendodj.com/text.asp?2012/2/2/70/108152
| Introduction|| |
Irrigation plays a crucial role in determining the outcome of root canal treatment. An irrigant serves to flush out debris from within the instrumented root canals, dissolve organic tissue remnants, disinfect the root canal space and provide lubrication during instrumentation, without causing irritation to biological tissues. 
The most popular irrigating solution is sodium hypochlorite (NaOCl). It is an effective antimicrobial agent, good lubricant and an excellent organic solvent. , However, it is highly irritating to the periapical tissues, especially at high concentrations.  Demineralizing agents such as ethylenediamine tetraacetic acid (EDTA) show high efficiency in removing smear layer. In addition to their cleaning ability, chelators may detach biofilms adhering to root canal walls despite its limited antibacterial capacity.  Chlorhexidine gluconate (CHX) is a potent antimicrobial agent,  holds substantivity and has a low grade toxicity.  CHX is bacteriostatic at low concentrations (0.2%), bactericidal at high concentrations (2%), and adsorbs to dental tissue resulting in its prolonged gradual release at therapeutic levels.  However, chlorhexidine is unable to dissolve pulp tissue and debris may remain on canal walls, obstructing the dentinal tubules. 
Torabinejad introduced MTAD [a mixture of a tetracycline isomer, citric acid, and a detergent (Tween 80)] as an endodontic irrigant for disinfection of the root canal system.  It represents an innovative approach in simultaneous removal of endodontic smear layer and complete disinfection of root canals. It has been shown to be clinically effective and biocompatible, with potential antibacterial substantivity. ,
Tetracyclines are a group of broad spectrum antibiotics which are effective against a wide variety of microorganisms. They readily attach to dentine and are subsequently released without losing their antibacterial activity.  Oxytetracycline, a derivative of tetracycline, is produced by Streptomyces rimosus and like other tetracyclines, act by inhibiting bacterial protein synthesis by preventing the association of aminoacyl-tRNA with the bacterial ribosome. Tetracyclines, besides acting as antibiotics, may also affect inflammation, immunomodulation, cell proliferation, and angiogenesis. , These properties may be of importance when tetracyclines are used as root canal irrigants. The literature contains only one report on antimicrobial efficacy of oxytetracycline as root canal irrigant. 
The present study was conducted to evaluate and compare the antimicrobial efficacy of 5% sodium hypochlorite, 2% chlorhexidine gluconate, MTAD and Oxytetracycline against E. faecalis in an ex-vivo model.
| Materials and Methods|| |
Fifty freshly extracted intact non-carious human single rooted teeth with fully formed apices were collected, cleaned and stored in normal saline until use. Each tooth was examined radiographically for the presence of a single canal. Teeth were handled for the entire procedure inside laminar air flow (Toshiba, Delhi, India) under fully aseptic conditions. Teeth were decoronated with a diamond disc using a low speed straight hand piece under water cooling up to 12 mm length. Working length for each tooth was recorded radiographically using a size 10 K-file up to 0.5 mm short of the apical foramen. The teeth were numbered, placed in glass Petri dish More Details and sterilized in an autoclave at a temperature of 121°C under 15 psi for 20 minutes.
Inoculation with E. faecalis
E. faecalis [MTCC439; Microbial Type Culture Collection and Gene Bank (MTCC), Institute of Microbial Technology, Chandigarh, India] was grown in Trypticase Soy Broth (HIMEDIA, Mumbai, India) for 24 hours at 37°C. The turbidity of the suspension was standardized by comparison with a 0.5 McFarland Standard to give an approximate count of 10  colony-forming units (CFU)/ml. An inoculum of 10 μl of this culture was suspended into each root canal of forty teeth by using sterile micropipettes. The remaining ten teeth were left un-inoculated and served as negative controls. Each tooth was transferred into a test tube containing 4ml of sterile physiological saline and incubated (Navyug, Ambala, India) for 48 hours at 37°C at 100% relative humidity. After incubation, the teeth were removed, rinsed thoroughly with sterile physiological saline and mounted in resin blocks. Forty inoculated teeth were randomly divided into 4 study groups of 10 teeth each as per the irrigating solution used:
Apical enlargement of all the samples was done up to size 40 and the root canals were filed with step back technique using hand K-files (Dentsply Maillefer). During the process of instrumentation, canals were irrigated with 2ml of respective irrigant solution using 26 gauge stainless steel needles with disposable syringe. A final rinse of each root canal was carried out with 3ml of the respective irrigant solution for 5 minutes followed by irrigation with 4ml of sterile saline, with the excess irrigant being removed continuously through suction.
- Group I- 5% NaOC (Ammdent Pvt. Ltd. India)
- Group II- MTAD (Dentsply Tulsa, OK, USA)
- Group III- Oxytetracycline (Oxytetracycline injection I.P. 50 mg/ml, Piramal Healthcare Ltd. India)
- Group IV- 2% Chlorhexidine (Ammdent Pvt. Ltd. India)
- Group V- Normal saline (un-inoculated teeth- negative control)
Irrigant efficacy by bacteriological culture
After removal of excess moisture from the canals, dry sterile standardized paper points (one per tooth) of size 40 were kept in the canals of all teeth to the full working length for 45 seconds. Later the paper points were taken out and transferred serially into test tubes containing 2ml SPS (Sterile Physiological Saline) solution and vortexed for 1 minute. 100 μl of this suspension was placed on the surface of dried Trypticase Soy Agar (TSA) plate and spread using sterilized glass rod L-spreader and plates were incubated for 48 hours at 37°C under aerobic conditions. The bacterial colonies that appeared were counted by keeping the plates over the illuminator grid of digital colony counter (LA660-HIMEDIA, Mumbai, India). The data for each sample was recorded and tabulated.
A Kruskal-Wallis test was conducted on mean number of colony forming units to evaluate differences among the four irrigants. Follow-up tests were conducted to evaluate pairwise differences among the four groups by using the Mann Whitney U test. The significance level was set as P > 0.05.
| Results|| |
The mean CFU counts for the groups were as follows (in ascending order): Oxytetracycline group 60 + 88.57 < MTAD group 263.40 + 206.2 < CHX group 546.00 + 253.64 < NaOCl group 880 + 288.29. As expected the teeth in group V (negative control) did not show any microbial growth. A Kruskal-Wallis test was conducted to evaluate differences among the four irrigants on mean number of CFU. The test was significant (P = 0.000). Follow-up tests were conducted to evaluate pair wise differences among groups using Mann Whitney U test. The results of these tests indicated a significant difference between all the groups.
| Discussion|| |
The investigation model adopted in the present study was counting CFUs in infected human teeth ex vivo to evaluate the antimicrobial efficacy of different irrigating agents. This model has been used earlier , and allows to study the possible effects of root canal morphology and dentin components on the antimicrobial properties of the test irrigants. 
Enterococci have been implicated in endodontic infections and especially endodontic treatment failures. Enterococcus faecalis is a non-spore-forming, fermentative, facultatively anaerobic, Gram-positive coccus. Its inherent antimicrobial resistance, ability to adapt to harsh environmental changes, and its growth potential in root canal walls as biofilm makes it responsible for endodontic failures. The rapid emergence of antimicrobial resistance among enterococci makes it difficult to treat the chronic infections.  Hence, this microorganism was used to assess the antimicrobial activity of various root canal irrigants in this study.
In group I, NaOCl failed to inhibit the growth of E. faecalis completely in any of the ten samples. The antimicrobial efficacy of NaOCl group was significantly lower as compared to other groups. The result of the present study is consistent with those observed by Shabahang S et al.  They had concluded that NaOCl was not an effective antimicrobial agent. In contrast, some in vitro studies ,, showed excellent results with NaOCl. The difference in the results as compared to ours could be due to the difference in the study design as the present study was an ex-vivo model. Irrigating agents show better results in in-vitro model than ex-vivo as root canal morphology and dentin components affect negatively on their antimicrobial properties.
In group II MTAD was able to inhibit the growth of E. faecalis completely in two of the samples. The antimicrobial efficacy of MTAD group was significantly greater as compared to NaOCl and CHX groups but lesser than oxytetracycline group. The result of the present study is consistent with the previous authors. ,, In an in vitro study by Torabinejad et al.,  MTAD was effective in killing E. faecalis up to 200X dilution; NaOCl ceased to exert its antibacterial activity beyond 32X dilution, whereas EDTA did not exhibit any antibacterial activity. Only MTAD was able to kill E. faecalis after an exposure of 2 or 5 minutes. Shabahang et al.,  showed that the combination of 1.3% NaOCl as a root canal irrigant and 5-minute application of MTAD as a final rinse was significantly more effective than other regimens because of its greater penetrating ability into the dentinal tubules due to the presence of detergent in MTAD (Tween 80) which lowers the surface tension. Newberry et al.,  also showed that MTAD inhibited most strains of E. faecalis when diluted 1:8192 times and killed most strains of E. faecalis when diluted 1:512 times. However, Dunavant et al.  reported that MTAD was significantly ineffective in killing biofilm bacteria. Giardino et al.,  found that only 5.25% NaOCl could disaggregate and remove the biofilm after only 5 minutes, whereas the same effect was reached by Tetraclean after 60 minutes. Tetraclean removed 90% of the bacteria present in biofilm after 5 minutes of contact and more than 99% after 30 minutes whereas MTAD was unable to reach this goal at every considered time. The difference in result of Dunavant et al.  and Giardino et al.  from the present investigation can be explained by the fact that the present investigation was done on planktonic bacteria whereas their studies were on bacterial biofilms.
In this ex-vivo study the antimicrobial efficacy of injectable form of oxytetracycline as root canal irrigant was evaluated to avoid the problem of insolubility of antibiotics in water.  Oxytetracyline was able to inhibit the growth of E. faecalis better than rest of the groups. Similar findings were reported by Chai et al.  who investigated in an in vitro model the antimicrobial efficacy of antibiotics and Ca(OH) 2 against E. faecalis biofilm and found that oxytetracycline, Ca(OH) 2 and erythromycin were 100% effective. However, the results of the present study can be considered more favorable as the experimental setup simulated the clinical conditions.
In group IV (2% CHX) all the samples showed growth of E. faecalis. 2% CHX showed significantly less antimicrobial efficacy as compared to MTAD and oxytetracycline groups but significantly more than NaOCl group. In another agar diffusion study by Ferraz et al.  largest inhibition zones were produced when the test bacteria were in contact with 2% CHX gluconate gel (11.79 mm), being significantly different from those produced by all NaOCl concentrations (9.54 mm). Ercan et al.  observed that CHX gluconate was significantly more effective against E. faecalis. Whereas Clegg et al.  showed 6% and 3% NaOCl were significantly more effective in disrupting and removing the biofilm; 1% NaOCl and 1% NaOCl with MTAD were capable of disrupting the biofilm, but not eliminating bacteria; 2% CHX was not capable of disrupting the biofilm. The better antibacterial efficacy of NaOCl observed in comparison to CHX by Clegg et al.  and Arias-Moliz et al.  could be because of anti-biofilm effects of NaOCl. The NaOCl acts as organic tissue solvent thus eliminating the bacterial attachment to dentin and other organisms.
From the results of our study, it can be summarized that oxytetracycline has superior antimicrobial efficacy against E. faecalis. The results in this ex-vivo tooth model, which may more closely simulate in vivo conditions, emphasize the fact that oxytetracycline has great potential as irrigating agent specially in retreatment cases because of its antimicrobial efficacy against E. faecalis, easy availability and cost effectiveness.
Further studies are required that focus on other aspects of an ideal root canal irrigant like smear layer removal, pulp tissue dissolution, activity against bacterial biofilm to establish the use of oxytetracycline as an ideal irrigant when used alone or in combination.
| References|| |
|1.||Cheung GS, Stock CJ. In vitro cleaning ability of root canal irrigants with and without endosonics. Int Endod J 1993;26:334-43. |
|2.||Estrela C, Silva JA, Gonçalves De Alencar AH, Leles CR, Decurcio DA. Efficacy of sodium hypochlorite and chlorhexidine against Enterococcus Faecalis - A systematic review. J Appl Oral Sci 2008;16:364-8. |
|3.||Vianna ME, Gomes BP, Berber VB, Zaia AA, Ferraz CC, Jose' de Souza-Filho F. In vitro evaluation of the antimicrobial activity of chlorhexidine and sodium hypochlorite. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 2004;97:79-84. |
|4.||Ercan E, Ozekinci T, Atakul F, Kadri G. Antibacterial activity of 2% chlorhexidine gluconate and 5.25% sodium hypochlorite in infected root canal: In-vivo study. J Endod 2004;30:84-7. |
|5.||Zehnder M. Root Canal Irrigants. J Endod 2006;32389-92. |
|6.||Jeansonne MJ, White RR. A comparison of 2% chlorhexidine gluconate and 5.25% sodium hypochlorite as anti-microbial endodontic irrigants. J Endod 1994;20:276-8. |
|7.||Filho MT, Leonardo MR, Silva LA, Anibal FF, Faccioli LH. Inflammatory response to different endodontic irrigation solutions. Int Endod J 2002;35:735-9. |
|8.||White RR, Hays GL, Janer LR. Residual antimicrobial activity after canal irrigation with chlorhexidine. J Endod 1997;23:229-31. |
|9.||Kuruvilla JR, Kamath MP. Antimicrobial activity of 2.5% sodium hypochlorite 0.2% chlorhexidine gluconate separately and combined as endodontic irrigants. J Endod 1998;24:472-6. |
|10.||Torabinejad M, Khademi AA, Babagoli J, Johnson YW, Bozhilov K, Kim J, et al. A new solution for the removal of the smear layer. J Endod 2003;29:170-5. |
|11.||Torabinejad M, Shabahang S, Aprecio R, Kettering JD. The antimicrobial effect of MTAD: An in vitro investigation. J Endod 2003;29:400-3. |
|12.||Shabahang S, Pouresmail M, Torabinejad M. In vitro antibacterial efficacy of MTAD and sodium hypochorite. J Endod 2003;29:450-2. |
|13.||Khademi AA, Mohammadi Z, Havaee A. Evaluation of the antibacterial substantivity of several intra-canal agents. Aust Endod J 2006;32:112-5. |
|14.||Shapira LL, Soskolne WA, Houri Y, Barak V, Halabi A, Stabholz A. Protection against endotoxic shock and lipopolysaccharideinduced local inflammation by tetracycline: Correlation with inhibition of cytokine secretion. Infect Immun 1996;64:825-8. |
|15.||Golub LM, Lee HM, Ryan ME, Giannobile WV, Payne J, Sorsa T. Tetracyclines inhibit connective tissue breakdown by multiple non-antimicrobial mechanisms. Adv Dent Res 1998;12:12-26. |
|16.||Chai WL, Hamimah H, Cheng SC, Sallam AA, Abdullah M. Susceptibility of enterococcus faecalis biofilm to antiboitics and calcium hydroxide. J Oral Sci 2007;49:161-6. |
|17.||Srikumar GP, Varma KR, Shetty HK, Vidya. Comparison of the antibacterial efficiency of MTAD, 2.5% Sodium Hypochlorite and 2% Chlorhexidine against Enterococcus faecalis- an ex vivo study. Endodontology 2009;21:42-7. |
|18.||Singamaneni V, Shekhar MG, Himagiri S. In vitro effectiveness of different endodontic irrigants on the reduction of Enterococcus faecalis in root canals. J Clin Exp Dent 2010;2:e169-72. |
|19.||Smith JJ, Wayman BE. An evaluation of the antimicrobial effectiveness of citric acid as a root canal irrigant. J Endod 1986;12:54-8. |
|20.||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 E. Faecalis. Int Endod J 2001;34:424-8. |
|21.||Shabahang S, Torabinejad M. Effect of MTAD on Enterococcus faecalis- Contaminated Root Canals of Extracted Human Teeth. J Endod 2003;29:576-9. |
|22.||Ahangari Z, Samiee M, Yolmeh MA, Eslami G. Antimicrobial activity of three root canal irrigants on Enterococcus Faecalis: An in vitro study. Iran Endod J 2008;3:33-7. |
|23.||Newberry BM, Shabahang S, Johnson N, Aprecio RM, Torabinejad M. The antimicrobial effect of Biopure MTAD on eight strains of Enterococcus faecalis: An in vitro investigation. J Endod 2007;33:1352-4. |
|24.||Dunavant TR, Regan JD, Glickman GN, Solomon ES, Honeyman AL. Comparative evaluation of endodontic irrigants against Enterococcus faecalis biofilms. J Endod 2006;32:527-31. |
|25.||Giardino L, Ambu E, Savoldi E, Rimondini R, Cassanelli C, Debbia EA. Comparative evaluation of antimicrobial efficacy of sodium hypochlorite, MTAD, and Tetraclean against Enterococcus faecalis biofilm. J Endod 2007;33:852-5. |
|26.||Ferraz CC, Gomes BP, Zaia AA, Teixeira FB, Souza-Filho FJ. Comparative study of the antimicrobial efficacy of chlorhexidine gel, chlorhexidine solution and sodium hypochlorite as endodontic irrigants. Braz Dent J 2007;18:294-8. |
|27.||Clegg MS, Vertucci FJ, Walker C, Belanger M, Britto LR. The effect of exposure to irrigant solutions on apical dentin biofilms in vitro. J Endod 2006;32:434-7. |
|28.||Arias-Moliz MT, Ferrer-Luque CM, Espigares-Garcýa M, Baca P. Enterococcus faecalis biofilms eradication by root canal irrigants. J Endod 2009;35:711-4. |