| Abstract|| |
Aim: The present in vitro experimental study was undertaken to evaluate and compare the antimicrobial activity of triple antibiotic paste (TAP), diclofenac, and proton-pump inhibitor (PPI) against the microorganism Enterococcus faecalis.
Materials and Methods: Three medicaments were selected for the study, TAP, diclofenac, and PPI. The experimental groups for the test were as follows: Part 1 – Group 1: TAP, Group 2: diclofenac, and Group 3: PPI; Part 2 – Group 1: TAP + PPI and Group 2: diclofenac + PPI. An agar well diffusion test was used to determine the efficacy of the experimental medicaments against E. faecalis (ATCC 29212). The diameter of inhibition zones was measured in millimeters using an inhibition zone measuring scale and the results were recorded.
Statistical Analysis: The statistical analysis was done using an analysis of variance and an unpaired t-test. P value was set at < 0.05.
Results: There was a significant difference in the diameter of growth inhibition zones, with the greatest diameter noted for TAP + PPI followed by diclofenac sodium (DS) + PPI, TAP, DS, and PPI.
Conclusions: The antimicrobial effectiveness of TAP + PPI was found to be superior to all other medicaments (DS + PPI, TAP, DS, and PPI).
Keywords: Diclofenac sodium; Enterococcus faecalis; proton-pump inhibitor; triple antibiotic paste
|How to cite this article:|
Tilokani A, Agrawal P, Pradhan PK, Patri G, Karmakar N, Sinha Y. Comparative evaluation of antibacterial efficacy of nonsteroidal anti-inflammatory medications and proton-pump inhibitor against Enterococcus faecalis: An in vitro study. J Conserv Dent 2023;26:79-82
|How to cite this URL:|
Tilokani A, Agrawal P, Pradhan PK, Patri G, Karmakar N, Sinha Y. Comparative evaluation of antibacterial efficacy of nonsteroidal anti-inflammatory medications and proton-pump inhibitor against Enterococcus faecalis: An in vitro study. J Conserv Dent [serial online] 2023 [cited 2023 Feb 6];26:79-82. Available from: https://www.jcd.org.in/text.asp?2023/26/1/79/362920
| Introduction|| |
Microorganisms play a key role in the development of periapical and pulpal disorders. The root canal treatment procedure eliminates bacteria from the infected root canal. Complex root canal anatomy usually makes complete disinfection of the root canal system very difficult. Hence, the use of antibacterial agents in the form of intracanal medicament is inevitable to eliminate endodontic microflora. Enterococcus faecalis, an essential component of the microbial flora of failed endodontically treated tooth, is a Gram-positive facultative anaerobe. It can survive in extreme conditions and has the ability to form biofilms.
Among the various intracanal medicaments available for use, recent studies do not consider calcium hydroxide (Ca[OH]2) as an effective intracanal medicament as it does not destroy the biofilm of E. faecalis, subsequently resulting in newer antibacterial compounds being proposed as a substitute.
Triple antibiotic paste (TAP), a combination of metronidazole, ciprofloxacin, and minocycline, is one of the most commonly used intracanal medicaments. Studies have concluded that TAP eradicates bacteria to a depth of 400 μm, whereas Ca(OH) 2 could only reach 200 μm into dentin., However, excessive use of TAP could develop bacterial resistance rendering the medicament ineffective. This led to a search for nonantibiotic compounds with effective antibacterial properties working through different mechanisms.
Certain nonantibiotic drugs such as antihistamines, antipsychotics, tranquilizers, and statins have shown antibacterial properties. Several investigations have revealed that certain nonsteroidal anti-inflammatory medications (NSAIDs) have antibacterial characteristics. Diclofenac sodium (DS), part of the NSAID family, has demonstrated a high bactericidal action on Gram-positive and Gram-negative bacteria by suppressing bacterial DNA synthesis.
Pantoprazole, a commonly used proton-pump inhibitor (PPI), has been effective in treating gastric disorders. Studies have revealed that it has a synergistic effect when combined with Ca (OH) 2 and can be used as a potential medicament to overcome the drawbacks of traditional intracanal medicaments.
To our knowledge, the antimicrobial efficacy of TAP and diclofenac in combination with PPI remains unexplored. Therefore, the present in vitro study was conducted to evaluate the antibacterial efficacy of DS (NSAID), TAP, and their combination with pantoprazole (PPI) against E. faecalis.
| Materials and Methods|| |
This protocol was approved by the Institutional Ethics Committee, Kalinga Institute of Medical Sciences, KIIT Deemed to be University, Bhubaneswar. (KIIT/KIIMS/IEC/863/2022).
In this study, the antibacterial efficacy of TAP, DS, and pantoprazole was evaluated. The drugs in pure form were obtained from Sigma-Aldrich, India.
Bacterial strains and media
The American-type culture collection 29212 of E. faecalis was obtained and nourished in brain-heart infusion broth. To adjust for turbidity, inoculum density was set at 0.5 McFarland (1.58 × 108 bacteria/ml). The agar diffusion test method was used to evaluate the antibacterial activity of materials.
Preparation of medicament
Ciprofloxacin, metronidazole, and minocycline for TAP, DS, and pantoprazole were procured in powder form (Sigma-Aldrich; India).
Part 1 1: (n = 60)
Group 1: DS (1:1 w/v) – One milliliter of distilled water was mixed with 400 mg powder to attain a concentration of 400 mg/ml.
Group 2: Pantoprazole (1:1 w/v) – Pantoprazole was mixed with distilled water at a concentration of 1 mg/ml and dilutions made at 12.5 μg/ml.
Group 3: TAP (1:1:1 w/v) – Equal amounts of metronidazole, ciprofloxacin, and minocycline were mixed with distilled water (1 mg/mL).
Part 2: (n = 80).
Group 1: TAP + PPI (1:1 w/v).
Group 2: Diclofenac + PPI (1:1 w/v).
Agar well diffusion assay
The first part of the experiment required a total of 60 Mueller–Hinton agar plates, 20 plates for each group. For part 2, 80 Mueller–Hinton agar plates were required, 40 for each group, to have a power of 80% and 95% confidence intervals. This study used Mueller–Hinton agar plates (Merck, Germany) with wells of 5 mm in diameter and 2 mm in depth. Cotton swabs were used to ensure an even distribution of bacteria cultivated on the agar plates. About 30 μl of the test material was filled into each well. Incubation of the plates was done aerobically at 37°C for 24 h. After incubation, a blinded examiner measured the zone of bacterial inhibition around each well as the shortest distance (mm) from the initial point of bacterial growth to the outer margin of the well with an inhibition zone measuring scale.
SPSS version 25 (Chicago, IL, USA) was used to tabulate and analyze data. The diameter of the growth inhibition zones in Part 1 was compared using analysis of variance; that between the combination medicaments in Part 2 was compared using an unpaired t-test. For all the analyses, a P < 0.05 was considered significant (standard deviation).
| Results|| |
On comparing the medicaments in Part 1, a notable difference in the diameter of the growth inhibition zones was observed between the three medicaments, with the greatest diameter seen for TAP, followed by DS and PPI (P < 0.001) [Table 1] and [Figure 1].
|Table 1: Mean and standard deviation of the diameter of the growth inhibition zones among the five medicaments using ANOVA|
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|Figure 1: Graphical representation of the diameter of the growth inhibition zone among plate 1 and plate 2 medicaments|
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On comparing the medicaments in Part 2, the diameter of the growth inhibition zone was observed to be significantly greater for TAP + PPI than diclofenac + PPI (P < 0.001) [Table 1] and [Figure 1].
On intergroup comparison, there was a significant difference in the diameter of the growth inhibition zones between each of the five groups, with the greatest diameter observed for TAP + PPI, followed by DS + PPI, TAP, DS, and PPI (P < 0.001) [Table 2].
| Discussion|| |
The development of antibiotic resistance has led to the evolution of alternative methods for the disinfection of root canals, involving the use of PPI and NSAIDs. In light of this, the antimicrobial efficacy of NSAIDs and PPI was compared with TAP to analyze their effectiveness as an intracanal medicament against E. faecalis in this study.
E. faecalis was used in this study and is considered the most resistant intracanal bacteria by Haapasalo and Orstavik. These bacteria can survive for at least 10 days within dentinal tubules without any nutrition. They are the causative organisms of persistent apical periodontitis with an ability to adhere, aggregate, and grow into a biofilm, giving them high chances of survival and increased resistance to antibiotics.
For years, dentists and pharmaceutical companies have evaluated the antimicrobial activity of dental materials and medications using the agar diffusion method. As a result, this method permits direct comparison of the efficacy of different medicaments against the target pathogens, allowing us to identify which medicament would be most effective in eliminating bacteria in the pulp space.
TAP has been used widely as an intracanal medicament against E. faecalis. TAP consists of metronidazole, minocycline, and ciprofloxacin, where the tetracycline group of drugs inhibits collagenases and matrix metalloproteinases and is not cytotoxic. They are known to increase the level of interleukin-10, which is an anti-inflammatory cytokine. Antibiotics such as metronidazole and ciprofloxacin have been proven to generate fibroblasts.
For the treatment of pain and inflammation, diclofenac is the most commonly used NSAID. It inhibits the action of cyclooxygenase-2 and reduces angiogenesis, thereby leading to programmed cell death. The reasons for its antibacterial property can be attributed to inhibiting bacterial DNA synthesis, impairing the membrane activity, antiplasmid activity, capability to alter gene encoding transport/binding proteins, and downregulating efflux pumps. A study done by Chockattu et al. confirmed that diclofenac has a profound antibacterial activity against E. faecalis.
Pantoprazole, a PPI, induces periapical healing due to its anti-inflammatory properties and pro-reparative effects. Pantoprazole acts directly on the proton pump present inside the cell of bacteria causing an increase in internal pH and enhanced antibacterial properties.
The comparison of the diameter of growth inhibition zones among the medicaments in Part 1 showed the highest diameter for TAP, followed by DS and PPI. These results accord with the study by Prabhakar et al., which concluded that all intracanal bacteria present inside an infected primary tooth could be completely eradicated with the use of a combination of three antibiotics after 1 day of application. Among the three antibiotics, minocycline inhibited protein synthesis on the surfaces of ribosomes, rendering an antimicrobial action. Apart from that, metronidazole and ciprofloxacin contributed to the development of a structural framework by helping the fibroblasts in the synthesis of extracellular matrix and collagen. The results of the present study were also in agreement with Valverde et al. and Zancan et al., who postulated that TAP has been very effective against E. faecalis biofilms.,
Comparing the diameter of the growth inhibition zones of medicaments in the second plate, values were significantly greater for TAP + PPI than for diclofenac + PPI. The result of this study is in accordance with Deepak et al., who concluded that TAP in combination with a PPI (TAP + PPI) showed significant and effective inhibition against bacterial strains of E. faecalis. This could be attributed to the synergistic effect of combining TAP with PPI. However, further studies have to be conducted on the evaluation of different combinations of antibiotics and nonantibiotics against E. faecalis. Therefore, the null hypothesis was rejected as there was a significant difference in the diameter of growth inhibition zones between each of the five groups.
| Conclusions|| |
Within the limitations of the present study, the antimicrobial effectiveness of TAP + PPI was found to be superior, followed by DS + PPI, TAP, DS, and PPI. Therefore, in clinical situations, the usage of combinations rather than a single medicament should be encouraged to better combat endodontic biofilm combinations. The usage of nonantibiotics as intracanal medicament eliminates the risk of any systemic complications and prevents antibiotic resistance.
Financial support and sponsorship
Conflicts of interest
There are no conflicts of interest.
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Dr. Akansha Tilokani
Department of Conservative Dentistry and Endodontics, Kalinga Institute of Dental Sciences, KIIT Deemed to be University, Bhubaneswar, Odisha
Source of Support: None, Conflict of Interest: None
[Table 1], [Table 2]