|Year : 2016 | Volume
| Issue : 1 | Page : 7-10
Comparative evaluation of the effect of proton pump inhibitors associated with calcium hydroxide on Enterococcus faecalis: An in vitro study
Chitharanjan Ganesh, Preethi Ann Koshy, Lekshmy S Devi, Sreeja Sreedhar, Lakshmi Aravind, Siddharth Nair, George Chacko, SL Satheesh
Department of Conservative Dentistry and Endodontics, Sri Sankara Dental College, Trivandrum, Kerala, India
|Date of Web Publication||21-Jun-2016|
Preethi Ann Koshy
Department of Conservative Dentistry and Endodontics, Sri Sankara Dental College, Vennecode P O, Varkala, Trivandrum - 695 318, Kerala
Source of Support: None, Conflict of Interest: None
Introduction: The purpose of this in vitro study was to evaluate the concentration dependent effect of proton pump inhibitors on the action of calcium hydroxide against E. faecalis using a broth dilution study.
Methods: Calcium hydroxide at a concentration of 16 mg/ml, pantoprazole and Lansoprazole at a concentration of 1 mg/ml made into dilutions of 6.25 and 25 μg/ml was incorporated into a broth containing E. faecalis and spectrophotometrically analysed at 630 nm at two time parameters of 12 hours and 24 hours.
Results: At 12 hours there was statistically significant growth inhibition by group 5 (calcium hydroxide and lansoprazole 6.25 μg/ml). At 24 hours,the maximum Inhibition of E. faecalis was demonstrated by group 2 (calcium hydroxide and chlorhexidine 2%) and group 5 (calcium hydroxide and lansoprazole 6.25 μg/ml).
Conclusion: Concentration dependent usage of proton pump inhibitors along with calcium hydroxide have potential in the eradication of persistent apical periodontitis.
Keywords: Calcium hydroxide; Enterococcus faecalis; proton pump inhibitors; root canal.
|How to cite this article:|
Ganesh C, Koshy PA, Devi LS, Sreedhar S, Aravind L, Nair S, Chacko G, Satheesh S L. Comparative evaluation of the effect of proton pump inhibitors associated with calcium hydroxide on Enterococcus faecalis: An in vitro study. Endodontology 2016;28:7-10
|How to cite this URL:|
Ganesh C, Koshy PA, Devi LS, Sreedhar S, Aravind L, Nair S, Chacko G, Satheesh S L. Comparative evaluation of the effect of proton pump inhibitors associated with calcium hydroxide on Enterococcus faecalis: An in vitro study. Endodontology [serial online] 2016 [cited 2019 Jan 19];28:7-10. Available from: http://www.endodontologyonweb.org/text.asp?2016/28/1/7/184322
| Introduction|| |
The complete chemomechanical disinfection of the root canal system is essential for endodontic treatment success. Complete bacterial eradication with conventional instrumentation and irrigation is difficult to achieve in the apical third due to natural anatomical complexities.  It is a well-established fact that persistent apical periodontitis is due to the presence of microorganisms in the root canal system.  Enterococcus faecalis is a normal inhabitant of the oral cavity, but it is associated with both primary and persistent periradicular infections.  The frequency of E. faecalis association in primary endodontic infections ranges from 4 to 40%  and 24-77% in persistent infections. 
Cleaning and shaping procedures and irrigation are not able to adequately eliminate endodontic microbiota because of less contact time. Intracanal medicaments have been used extensively in root canal disinfection due to their virtue of longer contact time with the endodontic pathogens. Ever since its introduction into endodontics by Hermann in 1920,  calcium hydroxide has been considered the gold standard of intracanal medicaments. However, E. faecalis can resist calcium hydroxide dressing even up to 10 days due to an inherent proton pump. 
Proton pump inhibitors (PPIs) have been used extensively in the treatment of peptic ulcer of microbial origin along with antibiotics. The effect of incorporating PPI omeprazole with calcium hydroxide has been evaluated by an in vivo study,  with promising results. However, newer PPIs such as pantoprazole and lansoprazole have not been investigated in this aspect.
The in vitro comparison of the effect of using different concentrations of PPIs pantoprazole and lansoprazole associated with calcium hydroxide on E. faecalis.
The PPIs pantoprazole and lansoprazole in association with calcium hydroxide have no concentration-dependent effect on the inhibition of E. faecalis.
| Methodology|| |
Calcium hydroxide was mixed with deionized water at a concentration of 16 mg/ml  and mixed every 15 min for 1 h and supernatant was taken after vortexing. The pH of the prepared calcium hydroxide was determined using a pH meter (Oakton Pvt. Ltd., Hyderabad, Telangana, India) as 12.3. PPIs pantoprazole and lansoprazole powder (Prevacid Ltd.) was mixed with deionized water at a concentration of 1 mg/ml and dilutions made at 6.25 and 25 μg/ml.  Chlorhexidine 2% solution (Asep RC) was used as the positive control.
E. faecalis strain (NCTC 12697 Himedia) was incubated in Luria-Bertani broth. Bacterial growth of this master broth was confirmed by the presence of turbidity. Two milliliters of the sterilized Luria broth was inoculated with 50 μl of E. faecalis from the master broth and incubated at 39°C for 5 h. The growth of bacteria was confirmed by the turbidity of the broth. The tubes containing broth were divided into two time parameters for evaluation after 12 h and 24 h. Broth and organism (BO) and broth and calcium hydroxide (BC) served as controls for optical density (OD) evaluation. Tubes were divided into six groups each for evaluation at 12 and 24 h.
- Group 1 - calcium hydroxide alone
- Group 2 - calcium hydroxide + chlorhexidine 2%
- Group 3 - calcium hydroxide + pantoprazole 6.25 μg/ml
- Group 4 - calcium hydroxide + pantoprazole 25 μg/ml
- Group 5 - calcium hydroxide + lansoprazole 6.25 μg/ml
- Group 6 - calcium hydroxide + lansoprazole 25 μg/ml.
Fifty microliters of calcium hydroxide, 50 μl of calcium hydroxide with 50 μl of chlorhexidine, and 50 μl calcium hydroxide with PPIs pantoprazole and lansoprazole in the two dilutions as described were added to the test tubes and incubated at 39°C. The tubes were then evaluated for OD at 630 nm at 12 and 24 h after addition of test solutions, and the mean of five readings of each dilution was taken. The percentage inhibition of growth was calculated as per Gomes et al. 
Statistical analysis was done using SPSS 16.0 software [Figure 1] (SPSS, Chicago, IL, USA) using one-way anova for comparison between groups and post hoc Tukey test for multiple comparisons. The level of significance (P) was set at 0.05 [Table 1].
|Figure 1: Bar diagram depicting inhibition percentage of different groups at 12 and 24 h (X-axis groups, Y-axis-inhibition percentage)|
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|Table 1: Mean percentage inhibition and standard deviation of the groups at 12 and 24 h|
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| Results|| |
The null hypothesis was rejected as there was definite concentration-dependent effect of the PPIs on the growth inhibition of E. faecalis. At 12 h, there was statistically significant E. faecalis growth inhibition by Group 5 (calcium hydroxide and lansoprazole 6.25 μg/ml) with P = 0. At 24 h, the maximum E. faecalis growth inhibition was demonstrated by Group 2 (calcium hydroxide and chlorhexidine 2%) and Group 5 (calcium hydroxide and lansoprazole 6.25 μg/ml) with P < 0.001
| Discussion|| |
Root canal treatment success entirely depends on the complete elimination of microorganisms from the root canal anatomy. However, this is not always possible with traditional cleaning and shaping and irrigation procedures. Intracanal medicaments by virtue of their longer contact time with the microorganisms can effectively eliminate these pathogens from the root canal system.
Calcium hydroxide is the most commonly used intracanal medicament in clinical dentistry. The antimicrobial effect of calcium hydroxide is related to the release of hydroxyl ions which diffuse into the dentinal tubules, a highly reactive oxidative species in an aqueous environment.  The pH of calcium hydroxide has been determined as 12.5, and the most bacterial species found in the root canal environment are eliminated when in contact with calcium hydroxide for a sufficient period of time.  However, inside the root canal, this pH is never achieved. It was found by previous studies that the intracanal pH achieved was 12.2, in the circumadjacent dentin this pH reduced to 8, and in the most peripheral dentin of the root the pH was 7.4.  This can be due to the buffering effect of dentin and the protective effect of biofilms covering the root canal wall. Dentin has buffering capacity due to the presence of proton donors such as H 2 PO 4− , H 2 CO3 , and HCO3− from hydroxyapatite.  Further, the formation of endodontic biofilms, essentially seen in endodontic infections, prevents the full microbial eradication by calcium hydroxide. 
E. faecalis is a persistent biofilm forming microorganism which possesses extraordinary ability to withstand adverse conditions and live inside dentinal tubules. It possesses widespread genetic polymorphism, secretes serine protease, gelatinase, and collagen binding protein which all help in dentin binding.  It is also able to endure prolonged periods of starvation.  It can utilize serum as a nutritional source which also helps it in dentin binding.  It can resist intracanal dressing of calcium hydroxide for over 10 days.  Many reasons have been proposed for the resistance of E. faecalis with calcium hydroxide, out of which the most important is the presence of a proton pump, which helps to maintain cytoplasmic pH  by pumping protons into the cell to lower the pH. However, at a pH of 11.5 or greater, E. faecalis is unable to survive.  To positively determine whether it is the presence of a proton pump which imparts resistance to calcium hydroxide, Evans et al.  used a PPI carbonyl cyanide-m-chlorophenylhydrazone along with calcium hydroxide and found a 20-fold more reduction in microbial load compared to plain calcium hydroxide after 30 min.
Omeprazole was the first PPI to be developed; it is a weak base, highly lipophilic, and easily crosses the cell membrane.  Omeprazole has been used to eradicate Helicobacter pylori which is implicated as the main causative agent of peptic ulcer by affecting its proton pump.  In dental literature, the first attempt to eradicate E. faecalis using calcium hydroxide supplemented with omeprazole was by Wagner et al.,  who used a rat model of periapical lesion and compared it to conventional calcium hydroxide intracanal dressing. They obtained superior healing rates with calcium hydroxide supplemented with omeprazole than conventional calcium hydroxide dressing. The effect of intracanal medications is commonly investigated using the agar well-diffusion study model. In our preliminary pilot study, when we attempted to do the same, it was found that the calcium hydroxide precipitated at the bottom of the agar well. According to Al-Nazhan,  a possible cause for failure is the limited solubility of calcium hydroxide and the inability to diffuse through agar. Thus, the broth dilution study model was selected as evidenced by Baron et al.,  which yielded satisfactory results.
Pantoprazole and lansoprazole represent the new generation of PPIs used extensively in medicine for gastric disorders. In the present study, at 12 h, maximum E. faecalis growth inhibition was shown by Group 5 (calcium hydroxide and lansoprazole 6.25 μg/ml). At 24 h, the maximum E. faecalis growth inhibition was demonstrated by Group 2 (calcium hydroxide and chlorhexidine 2%) and Group 5 (calcium hydroxide and lansoprazole 6.25 μg/ml); the difference between the groups was not statistically significant. The inter-time comparison was statistically significant for Group 2 but was not significant for Group 5 which could be related to the substantivity of chlorhexidine.  Pantoprazole did not enhance the bactericidal effect of calcium hydroxide on E. faecalis compared to lansoprazole which was statistically significant. Similarly, Suresh and Abraham  observed no effect of pantoprazole associated with calcium hydroxide on E. faecalis.  This is the first study where the concentration of PPIs to be incorporated with calcium hydroxide as intracanal medicament was evaluated. The exact mechanism of concentration-dependent effect of PPIs on calcium hydroxide is not clearly understood. Further studies regarding the exact mechanism need to be conducted.
Pretreatment with regular irrigants such as sodium hypochlorite and chlorhexidine has a significant role in reduction of E. faecalis count by virtue of their increased activity at higher temperature  and substantivity,  respectively. Use of increased taper allows more irrigation dynamics in clinical setting.  Although such measures reduce the microbial count significantly, it is often increased in the inter-appointment time. This is where the intracanal medicaments supplemented with PPIs come into play. In conclusion, PPIs have a promising future in the elimination of E. faecalis in primary and persistent periradicular infections. Further in vivo studies need to be conducted to evaluate the effectiveness of these medicaments in the clinical setup.
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Conflicts of interest
There are no conflicts of interest.
| References|| |
Nair PN. Pathogenesis of apical periodontitis and the causes of endodontic failures. Crit Rev Oral Biol Med 2004;15:348-81.
Siqueira JF Jr. Aetiology of root canal treatment failure: Why well-treated teeth can fail. Int Endod J 2001;34:1-10.
Molander A, Reit C, Dahlén G, Kvist T. Microbiological status of root-filled teeth with apical periodontitis. Int Endod J 1998;31:1-7.
Rôças IN, Siqueira JF Jr., Santos KR. Association of Enterococcus faecalis
with different forms of periradicular diseases. J Endod 2004;30:315-20.
Pinheiro ET, Gomes BP, Ferraz CC, Sousa EL, Teixeira FB, Souza-Filho FJ. Microorganisms from canals of root-filled teeth with periapical lesions. Int Endod J 2003;36:1-11.
Hermann BW. Calcium hydroxyd Als Mittel Zum Behandein und fullen Von Zahnwurzelkanalen (Dissertation). Wurzburg, Med Diss V; 29 September.
Stuart CH, Schwartz SA, Beeson TJ, Owatz CB. Enterococcus faecalis
: Its role in root canal treatment failure and current concepts in retreatment. J Endod 2006;32:93-8.
Wagner C, Barth VC Jr., de Oliveira SD, Campos MM. Effectiveness of the proton pump inhibitor omeprazole associated with calcium hydroxide as intracanal medication: An in vivo
study. J Endod 2011;37:1253-7.
Dean JA. Lange′s Handbook of Chemistry. New York: McGraw-Hill; 1979.
Ni W, Cai X, Liang B, Cai Y, Cui J, Wang R. Effect of proton pump inhibitors on in vitro
activity of tigecycline against several common clinical pathogens. PLoS One 2014;9:e86715.
Gomes BP, Drucker DB, Lilley JD. Associations of specific bacteria with some endodontic signs and symptoms. Int Endod J 1994;27:291-8.
Freeman BA, Crapo JD. Biology of disease: Free radicals and tissue injury. Lab Invest 1982;47:412-26.
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.
Tronstad L, Andreasen JO, Hasselgren G, Kristerson L, Riis I. pH changes in dental tissues after root canal filling with calcium hydroxide. J Endod 1981;7:17-21.
Wang JD, Hume WR. Diffusion of hydrogen ion and hydroxyl ion from various sources through dentine. Int Endod J 1988;21:17-26.
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.
McHugh CP, Zhang P, Michalek S, Eleazer PD. pH required to kill Enterococcus faecalis in vitro
. J Endod 2004;30:218-9.
Kendall MJ. Review article: Esomeprazole - The first proton pump inhibitor to be developed as an isomer. Aliment Pharmacol Ther 2003;17 Suppl 1:1-4.
Andersen LP, Colding H, Kristiansen JE. Potentiation of the action of metronidazole on Helicobacter pylori
by omeprazole and bismuth subcitrate. Int J Antimicrob Agents 2000;14:231-4.
Al-Nazhan S. Antimicrobial activity of extracts of calcium hydroxide points. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 2002;93:593-5.
Baron EJ, Finegold SM, Peterson LR. Bailey and Scott′s Diagnostic Microbiology. 9 th
ed. Mosby, St. Louis: Mosby Inc.; 1994.
Mohammadi Z, Abbott PV. The properties and applications of chlorhexidine in endodontics. Int Endod J 2009;42:288-302.
Suresh M, Abraham TA. Pantoprazole-does it enhance the antibacterial efficacy of calcium hydroxide against Enterococcus faecalis
? Int J Pharm Bio Sci 2015;6:734-9.
Sirtes G, Waltimo T, Schaetzle M, Zehnder M. The effects of temperature on sodium hypochlorite short-term stability, pulp dissolution capacity, and antimicrobial efficacy. J Endod 2005;31:669-71.
Böttcher DE, Sehnem NT, Montagner F, Fatturi Parolo CC, Grecca FS. Evaluation of the effect of Enterococcus faecalis
biofilm on the 2% chlorhexidine substantivity: An in vitro
study. J Endod 2015;41:1364-70.
Brunson M, Heilborn C, Johnson DJ, Cohenca N. Effect of apical preparation size and preparation taper on irrigant volume delivered by using negative pressure irrigation system. J Endod 2010;36:721-4.