Efficacy of various proton-pump inhibitors and calcium hydroxide as intracanal medicaments against Enterococcus Faecalis: An in vitro study

Tejas Koparkar; Sai Kalyan; Lalitagauri Mandke; Mansi Vandekar

doi:10.4103/endo.endo_102_22

ORIGINAL ARTICLE

Year : 2022 | Volume : 34 | Issue : 4 | Page : 259-264

Efficacy of various proton-pump inhibitors and calcium hydroxide as intracanal medicaments against Enterococcus Faecalis: An in vitro study

Tejas Koparkar¹, Sai Kalyan², Lalitagauri Mandke¹, Mansi Vandekar¹
¹ Department of Conservative Dentistry and Endodontics, D. Y. Patil School of Dentistry, Navi Mumbai, Maharashtra, India
² Department of Conservative Dentistry and Endodontics, Terna Dental College, Navi Mumbai, Maharashtra, India

Date of Submission	14-Apr-2022
Date of Decision	29-May-2022
Date of Acceptance	31-May-2022
Date of Web Publication	28-Dec-2022

Correspondence Address:
Dr. Tejas Koparkar
501, Shri Chintamani Apts., Sahakar Colony, Behind Deodhar Hospital, Naupada, Thane (W), Maharashtra
India

Source of Support: None, Conflict of Interest: None

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DOI: 10.4103/endo.endo_102_22

Abstract

Aim: The purpose of this study was to compare the efficacy of four proton-pump inhibitors (PPIs) (pantoprazole, omeprazole, lansoprazole, and rabeprazole) when combined with calcium hydroxide and used as intracanal medicaments against Enterococcus faecalis.
Methods: A total of 550 microliters of E. faecalis strain was inoculated into two mixtures 6.25 μg/ml and 25 μg/ml containing calcium hydroxide (concentration 16 mg/ml) and PPIs (concentration 1 mg/ml). The efficacy was evaluated by comparing the optical density of the mixtures at 630 nm with two time parameters of 18 h and 24 h.
Results: At 18 h, O6 (calcium hydroxide and omeprazole 6.25 μg/ml) showed the maximum percentage inhibition of E. faecalis strain and R25 (calcium hydroxide and rabeprazole 25 μg/ml) showed the least inhibition. At 24-h time interval, O6 showed the maximum inhibition, while R6 (calcium hydroxide and rabeprazole 6.25 μg/ml) showed the least inhibition of E. faecalis strain.
Conclusion: The use of PPIs with calcium hydroxide did show promising results and the combination could be used successfully as an intracanal medicament.

Keywords: Calcium hydroxide, Enterococcus faecalis, intracanal medicament, proton-pump inhibitors, recurrent endodontic infection

How to cite this article:
Koparkar T, Kalyan S, Mandke L, Vandekar M. Efficacy of various proton-pump inhibitors and calcium hydroxide as intracanal medicaments against Enterococcus Faecalis: An in vitro study. Endodontology 2022;34:259-64

How to cite this URL:
Koparkar T, Kalyan S, Mandke L, Vandekar M. Efficacy of various proton-pump inhibitors and calcium hydroxide as intracanal medicaments against Enterococcus Faecalis: An in vitro study. Endodontology [serial online] 2022 [cited 2023 Jan 28];34:259-64. Available from: https://www.endodontologyonweb.org/text.asp?2022/34/4/259/365804

Introduction

Endodontic retreatment is a challenge for endodontists and practitioners all over the world. While there are many reasons for failure of an endodontic therapy, one of the most common and underestimated reasons is the failure to achieve adequate disinfection.

Enterococcus faecalis is native to oral cavity and is analogous to both primary and persistent periradicular infections.^[1] Its association in primary endodontic infections ranges from 4% to 40%, while that for persistent infections ranges from 24% to 77%.^[2] E. faecalis is one of the most tenacious microorganisms present in the root canal system and is one of the major culprits in endodontic treatment failure. It is capable of surviving alkaline environment and can go dormant for extensive periods during the phases of decreased nutrition within the smear layer. It is capable of surviving intracanal calcium hydroxide dressings for longer periods.^[3]

Calcium hydroxide is considered a gold standard as an intracanal medicament. It has antimicrobial property, as well as a pH of 12 within the canal that can destroy a majority of microorganisms. The inherent presence of proton-pump mechanism within the plasma membrane of E. faecalis facilitates constant cytoplasmic pH maintenance. This consequently provides E. faecalis an upper hand in surviving calcium hydroxide medication for up to 10 days.^[3],[4] In order for calcium hydroxide to be effective, its contact time with the canal walls needs to be prolonged. Proton-pump inhibitors (PPIs) are a class of drugs that inhibit activation of proton-pump channel, thereby preventing acid production. PPIs, such as pantoprazole and omeprazole, have already showed promising results when used with calcium hydroxide against E. faecalis colonies.^[5],[6]

Aim

The aim of our in vitro study was to compare and evaluate the effectiveness of different concentrations of PPIs when used along with calcium hydroxide to inhibit the growth of E. faecalis.

Materials and Methods

A mixture of calcium hydroxide powder and deionized water was prepared at a concentration of 16 mg/ml, after mixing the solution every 15 min for 1 h and collecting the supernatant from the vortex. A pH meter was used to determine the pH of the solution, which was established at 12.3.

A mixture of 4 PPIs each with deionized water was prepared at a concentration of 1 mg/ml dilutions. From the following, mixtures were prepared at 6.25 μg/ml and 25 μg/ml for each PPI. Incubation of E. faecalis strain (ATCC 29212 Himedia, Mumbai, India) was done in Luria Bertani broth. Bacterial growth in the broth was verified by the presence of turbidity. 2 ml of sterile Luria Bertani broth was inoculated with 550 μL of E. faecalis cultivated from master broth. This was then incubated at 39°C for 5 h. Turbidity of the broth was checked to confirm bacterial growth. This product containing E. faecalis colonies was then divided based on 2 time parameters: 18 and 24 h.

Broth and organisms (BO) and broth and calcium hydroxide (BC) were decided as control groups. The PPIs were divided into 8 groups each for evaluation at 18 and 24 h, respectively.

The groups were designated as follows:

BO - Broth and organism
BC - Broth and calcium hydroxide
P6 - Calcium hydroxide and pantoprazole 6.25 μg/ml
P25 - Calcium hydroxide and pantoprazole 25 μg/ml
L6 - Calcium hydroxide and lansoprazole 6.25 μg/ml
L25 - Calcium hydroxide and lansoprazole 25 μg/ml
O6 - Calcium hydroxide and omeprazole 6.25 μg/ml
O25 - Calcium hydroxide and omeprazole 25 μg/ml
R6 - Calcium hydroxide and rabeprazole 6.25 μg/ml
R25 - Calcium hydroxide and rabeprazole 25 μg/ml

The evaluation of optical density (OD) was done at 18 and 24 h at 630 nm after addition of test solution. Three readings per sample were taken and the mean value was calculated [Table 1] and [Table 2]. Percentage inhibition was calculated based on the method of Gomes et al.

Table 1: Mean optical density at 18 h

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Table 2: Mean optical density at 24 h

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Statistical analysis

Statistical analysis was done using SPSS 16.0 software (SPSS, Chicago, IL, USA) using one-way ANOVA for comparison between groups and post hoc Tukey's test for multiple comparison. P value was set at 0.05.

Results

The results from ANOVA test indicated a significant difference in OD in the groups at 18 and 24 h (P < 0.5) [Table 3] and [Table 4].

Table 3: ANOVA Test Result at 18 h

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Table 4: ANOVA test result (at 24 hrs)

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There was a significant difference in OD between control group BC and all other groups at 18 and 24 h, except for L6 and O6. There was no difference in OD among all other groups at both time spans [Table 5] and [Table 6].

Table 5: Tukey's multiple comparison test (at 18 h)

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Table 6: Tukey's Multiple comparison test (at 24 h)

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It was observed that the PPIs had a specific concentration-dependent effect on the growth inhibition of E. faecalis [Table 7].

Table 7: Percentage inhibition of proton pump inhibitors

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At 18 h, O6 had the maximum percentage inhibition (51.4%), followed by L6 (40%) and O25 (31.4%). R25 showed the least inhibition percentage (8.5%).

At 24 h, O6 had the maximum percentage inhibition (38.7%), followed by L6 (19.2%) and P6 (15.3%). R6 showed the least inhibition percentage (3.8%)

Discussion

The primary objective of endodontic treatment is total eradication of microorganisms and necrotic tissue, as well as prevention of recontamination.^[7]

Microorganisms can harbor within the most intricate areas of root canal systems such as dentinal tubules, accessory canals, apical deltas, fins, and isthmus.^[7] They are found within biofilms (a self-developed polymeric matrix-based structured communities that are adherent to root surface).^[8]

While several microorganisms have been linked to cause reinfection of previous endodontically treated teeth, Enterococcus species are infamous for being more commonly present in such environments than any other bacteria known.^{[9],[10],[11],[12],[13]} An infected root canal environment is polymicrobial in nature. These microorganisms coexist in harmony in highly organized complex structures known as biofilms. E. faecalis is a highly persistent microbe that has time and again been associated with recurrent endodontic infections.^[11] It has the ability to form complex biofilms. These biofilms provide protection and resistance to the microorganisms residing within it against intracanal medicaments and antimicrobials used during root canal therapy.^[14] Most of the microorganism-associated endodontic infections get eliminated when in contact with high alkalinity of calcium hydroxide.^[15] However, several studies have already proven ineffectiveness of calcium hydroxide to eliminate E. faecalis from root canal systems.^{[16],[17],[18]} This is particularly attributed to the existent inherent proton pump within E. faecalis that helps buffer high alkalinity.^[19] Hence, it is critical for inhibition of this proton-pump mechanism in order for calcium hydroxide to effectively eliminate E. faecalis within the biofilm. This enhances the antimicrobial action of calcium hydroxide.

E. faecalis is a Gram-positive cocci and a facultative anaerobe.^[3] Previously carried out studies have made it evident that E. faecalis depends majorly on its survival traits than upon its virulence features.^[20] Its ability to maintain intracellular pH via proton-pump mechanism helps enhance its survival potential during its use of intracanal medicaments such as calcium hydroxide.^[19]

It has a unique ability to endure without nutrition in root canals which are obturated and reproduce as and when it comes in contact with human serum. This provides E. faecalis an edge over other root canal inhabitants.^[21],[22]

Since the introduction of calcium hydroxide into endodontics by Hermann in 1920, it has seen extensive applications. A noteworthy antimicrobial activity,^[23] tissue-dissolving ability,^[24],[25] capability to promote hard tissue formation,^[26] and inhibition of tooth resorption^[27] have bolstered its use in root canal treatment. Currently, calcium hydroxide is the material of choice as a root canal dressing. The highly alkalinity of calcium hydroxide (pH 12.5) due to hydroxyl ions creates an unfavorable environment within the root canal for the survival of most of the pathogens.^[23],[28] E. faecalis has been proved to be less susceptible to calcium hydroxide as compared to other bacteria. According to Pavelic et al., Ca(OH)₂ was seen to inhibit growth of microorganisms after 24 h. They used the agar diffusion method; however, there was a distinct difference in the sensitivity.^[29] The study demonstrated that E. faecalis was the least sensitive of all microorganisms, whereas Streptococcus mutans showed most sensitivity.^[29] Studies carried out by Podbielski et al. and Ferreira et al. revealed Ca (OH)₂ to be more efficacious against all experimental strains except that of E. faecalis.^[29],[30]

All PPIs block the gastric acid pump - H+/K+ adenosine triphosphatase (ATPase), which is the final step of acid secretion. Inhibition of this enzyme effectively suppresses gastric acid secretion.^[31] Omeprazole, the first PPI to be developed and introduced into clinical trial, was synthesized in 1989. Omeprazole (Losec; AstraZeneca, DE) was succeeded by lansoprazole (Prevacid; TAP Pharmaceuticals, IL), followed by pantoprazole (Protonix; Wyeth Pharmaceuticals, NJ) and rabeprazole (Aciphex; Eisai Company, NJ), respectively. Omeprazole and lansoprazole were the world's first two PPIs approved for clinical use in humans.

Besancon et al. in their study indicated a significant contrast in the rates of in vitro inhibition of H+/K+-ATPase among these drugs: omeprazole, lansoprazole, pantoprazole, and rabeprazole. The most rapid inhibition was achieved with rabeprazole; lansoprazole, omeprazole, and pantoprazole followed thereafter.^[32]

In our study, there was a significant difference in OD between control group BC and all other groups at 18 and 24 h, except for L6 and O6. There was no difference in OD among all other groups at both time spans.

Based on the percentage inhibition results, O6 showed the maximum percentage inhibition, followed by L6 at 18 and 24 h.

Omeprazole being highly lipophilic and a weak base can easily cross the cell membrane. It has been used to eradicate H. pylori, which is main causative agent in peptic ulcer by affecting the proton pump. Wagner et al. (2011) reported that association of omeprazole with Ca (OH)₂ favored superior repair of rat periapical lesions, as compared to conventional Ca (OH)₂ dressing.^[5] However, in this study, the concentration of the PPI was not studied. Deborah Cogo et al. also concluded that omeprazole potentiated the effect of Ca (OH)₂ against E. faecalis.^[33]

Lansoprazole which is often resorted to for treating gastric disorders belongs to the new generation of PPIs. A study by Ganesh C et al. showed maximum E. faecalis growth inhibition by 6.25% lansoprazole.^[6]

Pantoprazole, in our study, demonstrated intermediate results. It was superior to rabeprazole, but not as effective as omeprazole and lansoprazole. Similar results were obtained in studies by Ganesh^[6] and Suresh and Abraham.^[34]

Rabeprazole showed the least percentage inhibition. Kavita Dube et al.'s study also showed similar results, i.e., omeprazole and lansoprazole were superior to rabeprazole.^[35] Previous studies have concluded that there was no significant difference in superiority of one PPI over the other.

PPIs have shown promising results in E. faecalis elimination in endodontic infections. However, the efficacy of PPIs needs to be tested in clinical situations, in the presence of biofilms in root canals. Hence, further in vivo studies are needed to substantiate these results.

Conclusion

There was a definite concentration-dependent effect of the PPIs on growth inhibition of E. faecalis.

The highest growth inhibition of E. faecalis was shown by omeprazole (6.25%), followed by lansoprazole (6.25%) in combination with calcium hydroxide.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.

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