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Research Article | Volume 14 Issue 5 (Sept - Oct, 2024) | Pages 632 - 636
Bactericidal Effect of Triple Antibiotic Paste Against Enterococcus Faecalis in Dentinal Tubules-An Ex Vivo Study
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1
Graduate, Anil Neeru Konda Institute of Dental Sciences, Dr NTR University of Health Sciences, Andhra Pradesh, India
2
Reader, Department of Public Health Dentistry, Geetanjali Dental and Research Institute, Geetanjali University, Udaipur, India
3
Department of Prosthodontics, Dr. Ranjan’s Dental Care and Implant Centre, Saharanpur, India
4
Department of Public Health Dentistry, Indra Gandhi Institute of Dental Sciences, Pondicherry, India
5
Assistant Professor, Department of Conservative Dentistry and Endodontics, Government Dental College and Research Institute VIMS, Ballari, India
Under a Creative Commons license
Open Access
Received
Aug. 30, 2024
Revised
Sept. 15, 2024
Accepted
Sept. 28, 2024
Published
Oct. 21, 2024
Abstract

Objective: Utilizing a confocal laser scanning microscope (CLSM) and a culture of bacteria test, the goal of this research was to determine the bactericidal impact of different doses of triple antibiotic paste (TAP) against Enterococcus faecalis (E. faecalis) in dentinal tubules. Techniques: E. faecalis (ATCC 29212) infected 90 human teeth, which were then divided into 5 groups at random: 1 mg/ml, 5 mg/ml, 7.5 mg/ml, and 10 mg/ml TAP (n = 18). The negative control group did not receive TAP. Samples were taken both the root canal space and root dentin at 100-μm and 200-μm depths following a three-week TAP treatment. Ten bacterial culture assays were performed on the gathered samples. A CLSM analysis was performed on eight roots from each group in order to identify the live and dead cells of bacteria. Results: The negative control samples were all culturable, according to the results of the bacterial culture assay. Following TAP treatment, there were less culture-positive samples at 1, 5, 7.5, and 10 mg/ml, with 2, 2, 1, and 0 culturable samples, in that order. Nonetheless, there were no appreciable variations between the TAP treatments. Remarkably, the CLSM investigation showed that all samples included live bacteria in the dentinal tubules. There were 52.36%±3.24 viable bacteria in the negative control. The smallest percentage of viable bacterial cells (40.58%±5.40) was observed with 10 mg/ml of TAP treatment. This was followed by 7.5 mg/ml (44.14%±6.03), 5 mg/ml (46.31%±5.32), and 1 mg/ml (52.55%±8.82). Conclusion: TAP treatment significantly decreased the percentage of viable E. faecalis cells in the dentinal tubules and its bactericidal effect was dose-dependent.

Keywords
INTRODUCTION

The bacteria and their byproducts cause periradicular ulcers and pulp necrosis.1 The goal of endodontic treatment is to remove all germs from the root canal system and eliminate the pulp entirely in order to promote the healing of periapical tissues.2 The satisfactory result from endodontic treatment is influenced by technological preparation, irrigation, microbial control, and the mechanism for filling the root canals.2 In teeth that have had root canal therapy and have persistent lesions that are challenging to remove during retreatment procedures, Enterococcus faecalis is often found.3 They grow into large populations on canal walls that are challenging to remove from the root canal system. They can endure by utilising the fluid found in the periodontal ligament and by forming biofilms to protect themselves against the host's resistance and cleaning agents. It seems that they are immune to a variety of antibacterial substances. These processes are aided by E. faecalis's ability to develop resistance to antibiotics, specifically to erythromycin and azithromycin, as well as its ability to penetrate dentinal tubules and attach itself to collagen. Therefore, it is recommended to use an intracanal medication with strong antibacterial properties. 4,5

 

Several antimicrobial drugs have been studied in relation to E. faecalis. It is possible to eradicate E. faecalis from the deeper layers of the root canal dentine by using triple antibiotic paste (TAP), which consists of metronidazole, ciprofloxacin, and minocycline. Six TAP discolouration of treated teeth, a considerable reduction in dentin flexural strength, microhardness, and toxicity to dental papilla stem cells, as well as an inhibition of their attachment and proliferation, raise some concerns about its use. All three antibiotics combined in TAP are not marketed as a single medication. A potential substitute that was recently proposed to solve the drawbacks of not having a combination medication formulation and the problem of discolouration in existing treatment alternatives is the use of amoxicillin clavulanate paste (ACP). Amoxicillin is a bacterial cell wall production inhibitor that is selective, while clavulanic acid inhibits β-Lactamase. ACP is a combination medication with bactericidal and wide-spectrum effects. As a result, in the event that more conventional dental antibiotics are unable to treat the infection, this combination of antibiotics is advised as a "fall-back."1 One benefit of ACP is that it can be purchased professionally as a single medication combination. Antibiotic arrangements, especially when taken for brief periods of time, can increase the risk of bacterial resistance. Therefore, the use of a single antibiotic paste is preferred.4,6

 

ACP has been recommended for use as an intracanal medicine by Nosrat et al.7; however, there are no clinical or in vitro antimicrobial investigations in the literature that compare the antibacterial efficacy of TAP and ACP. In order to treat single-rooted permanent teeth endodontically, this in vitro study sought to assess and compare the antibacterial efficacy of TAP and ACP as an intracanal medication against E. faecalis.

METHODOLOGY

The Geetanjali Dental and Research Institute, part of Geetanjali University in Udaipur, at Department of Public Health Dentistry, where the study was conducted. Mandibular premolars with a single root that were removed for orthodontics purposes were utilised. From the cemento-enamel junction to the root apex, the root was ≥10 mm long. Teeth with crown restorations in place, root resorption, root caries, fractures, cracks, or history of endodontic therapy were not included. Every tooth had its mesio-distal and bucco-lingual pre-operative radiographs taken. In addition, teeth having radiograph-shown numerous root canals or calcified canals were not included. Before being utilised, the chosen teeth were kept in 0.1% thymol.

 

For the root canal and dental tubule contamination, E. faecalis ATCC 29212 was employed. The bacterial cells were cultured on agar plates with Brain Heart Infusion (BHI) and incubated at 37°C for 24 h (Heraeus B 5060 EK-CO2, Heraeus, Hanau, Germany). The bacterial inoculum was prepared by suspending 2–3 isolated E. faecalis colonies in BHI broth and incubating the broth at 37°C for 24 h.

 

The United States Pharmacy Convention (USP) grade powder of minocycline, ciprofloxacin, and metronidazole (Sigma Pharmaceutical, North Liberty, Iowa, USA) was used to create the freshly mixed TAP. The polyethylene glycol and macrogol were used as the drug vehicle. 5 mg of each antibiotic powder and 10 mg/ml TAP were combined in MP (M:P ratio: 1:1 by volume) to create the starting concentration. The car was added to the mixture little by little until the final volume of 1.5 ml was reached. The original concentration was diluted in MP to create other concentrations.

 

In all, 91 teeth were utilised in this investigation. Utilising a peri-odontal curette, the calculus and soft tissue remains on the root surfaces were eliminated. Three millimetres from the root apex, the teeth were sliced with a diamond disc submerged in water cooling. The teeth were extracted to create open-ended cylindrical root specimens by cutting them 8 mm coronally from the new apex. Dentsply Meillefer, USA) No. 1-3 peeso reamers were used to gradually extend the root canal. To get rid of the dentine fragments, sterile normal saline solution was irrigated during canal widening. To create a closed-end cylindrical tube, the apical end was sealed with a 2 mm resin composite (FiltekTM Z350 XT 3M ESPE, USA) using total etch bonding (Single Bond Universal, 3M ESPE, USA). The mear layer was removed as previously described (14). The canals were dried with paper points before subjected to dentinal tubule contamination.

 

To verify that all specimens were sterile, they were incubated for twenty-four hours at 37°C. E. faecalis infection was produced in the dentinal tubules as previously described (15). The process took four days to complete, including the steps for medium replacement and bacterial inoculation. The specimens were kept at 37°C and the operations were carried out in an aseptic manner. In summary, the first and third days were dedicated to bacterial inoculation, whereas the second and fourth days were devoted to medium replacement. 800 μl of bacterial suspension was added on the first day, following the removal of the BHI broth from each microcentrifuge tube containing the material. Every sample was centrifuged (Eppendorf 5417C, Eppendorf AG, Hamburg, Germany) for five minutes at 25°C, 1,400g, 2,000g, 3,600g, and 5,600g, 5 min each. At the end of each centrifugation step, 800 μl bacteri- al suspension added to the tube. Centrifugation was performed for 2 cycles.

 

The samples were incubated for twenty-four hours at 37°C. On the second day, 800 μl of new BHI broth was added to the culture medium, and the passageway was centrifuged for five minutes at 25°C and 3,600g. The samples were incubated for twenty-four hours at 37°C. On the third and fourth days, the identical process was carried out.

 

Using CLSM (Stellaris 8, Leica Microsystem, Wetzlar, Germany), one specimen was chosen at random following bacterial contamination in order to verify the development of bacterial biofilm in the dentinal tubules. Using a diamond saw (Isomet 1000, Buhler, New York, USA), the tooth was cut crosswise and the mid-root area was heavily irrigated with normal saline to create a dentine disc that was 0.3 mm thick. Rinsing the disc with 1% phosphate buffered saline under ultrasonic activation for 15 min to remove the smear layer. The disc was stained with 100 μl LIVE/ DEAD® stain for 20 min.

 

The imaging technique involved the use of twenty times magnification. Ninety contaminated root specimens were divided into five groups (n=18) at random: 1 mg/ml, 5 mg/ml, 7.5 mg/ml, and 10 mg/ml TAP as the negative control (no TAP). Utilising a laminar hood (NU-126-300E, NUAIRE, Minnesota, USA), the TAP was administered to the root canals. In order to examine the existence of bacteria within the dentinal tubules, eight samples from every group underwent the CLSM LIVE/DEAD assay. As previously mentioned, photos from four areas of interest were taken and processed. For each group (n = 8), the mean percentage of viable cells was given. The subsequent formula was used to calculate the proportion of living bacteria:

 

Percent live bacterial cells   = =       Green fluorescence/ Red fluorescence+Green fluorescence

Figure 1- CLSM images of the root sample after bacterial contamination. The red color (PI) represents dead bacterial cells, while the green color (SYTO9) represents live bacterial cells

 

RESULTS

Confirmation of Bacterial Contamination in the Dentinal Tubules

Following the contamination procedure, a single root sample was chosen at random for CLSM in order to verify the existence of bacteria in the dentinal tubules. According to Figure 2, the red signals (PI) indicated dead bacteria cells while the green signals (SYTO9) represented live bacteria (Fig. 2). The presence of live bacteria was detected in over 50% of the examined field, indicating the effectiveness of the bacterial contamination technique. Furthermore, at least 500 μm of bacterial penetration was found inside the dentinal tubules.

 

TABLE 1. The number of culturable samples in each experimental group and depth (n=10)

Experimental groups (n=10)

Number of culturable samples

Root canal

100 µm

200 µm

Neg control (No TAP)

10

10

10

1 mg/ml TAP

2

0

1

5 mg/ml TAP

 

2

0

1

7.5 mg/ml TAP

1

0

0

10  mg/ml TAP

0

0

0

 

Figure 2. Mean percentage (±SD) of live bacteria in the root canal dentine obtained from various concentrations of TAP medication. Superscript A and B indicate significant differences among the groups (p<0.05)

 

Figure 3. Representative CLSM images of live and dead bacteria in the root canal dentine obtained from various concentrations of TAP medi- cation. The red color (PI) represents dead bacterial cells, while the green color (SYTO9) represents live bacterial

DISCUSSION

The bactericidal activity of TAP has been proven by totally eliminating E. faecalis from infected root canals.Nonetheless, elevated TAP concentrations demonstrated cytotoxicity towards stem cells, potentially compromising the efficacy of regenerative endodontic treatments.10 Determining the ideal concentration to eradicate E. faecalis in the root canal was the goal of this investigation. Based on our findings, the null hypothesis is disproved.

 

Standard antimicrobial susceptibility studies, such as agar diffusion, have demonstrated the bactericidal action of the antibiotics in TAP; however, these techniques do not replicate the antibacterial impact of the antibiotics in endodontic diseases. The microbes invade the dentinal tubules and colonise the root canal wall, potentially lessening the bactericidal impact of antibiotics.

 

Additionally, the mixed bacterial species create biofilms that are resistant to drugs through a variety of drug-resistance mechanisms, such as gene transfer, delayed growth, or entering the stationary phase. For this reason, the dentinal tubule contamination method and specimens from extracted human teeth were used in the current study.11 Compared to the conventional bacterial contamination procedure, which takes 21–30 days, this method simulates dentinal tubule contamination more quickly. Furthermore, a significant proportion of living bacteria and deeper bacterial cell penetration in the dentinal tubules were attained. The effectiveness of different TAP concentrations in eliminating E. faecalis from the root canal and dentinal tubules was assessed using the bacterial culture test. There were no culturable samples found in the TAP group (10 mg/ml), indicating corresponded to other stud- ies where 10 mg/ml and higher TAP concentrations eliminated.

 

Biofilm of E. faecalis. After being treated with 1 mg/ml and 5 mg/ml TAP, two (20%) root canal samples showed inadequate E. faecalis clearance. Complete eradication of E. faecalis biofilm following direct contact with 1 mg/ml TAP was also seen in another investigation.12 Furthermore, only one (10%) root canal sample that received 7.5 mg/ml TAP treatment exhibited partial E. faecalis clearance. The bactericidal action of 1–10 mg/ml TAP has not been reported.

 

A portion of the culturable samples that were taken 200 μm into the dentine showed evidence of bacterial penetration. These could be caused by the different TAP concentrations and the quantity of bacteria in the dentine and root canal. While the quantity of bacteria that have entered the deeper dentine (200 μm) is fewer, the TAP concentration of TAP might also be lower based on the distance from the root canal. Thus, TAP could not effectively kill the bacteria 200 μm deep into root dentine.


It is also possible that the number of bacte- ria introduced in dentine by centrifugation was greater than those present in clinical infections. At 100 μm deep, there were no culturable samples, indicating the optimal concentration of TAP that effectively eradicated the bacteria present. Dentine samples more than 200 μm deep could not be collected due to the thin dentine on the mesial-distal side of the root.

CONCLUSION

The present investigation discovered that the number of culture-positive root canal samples was significantly reduced by 1, 5, 7.5, and 10 mg/ml TAP. Furthermore, TAP had a dose-dependent bactericidal impact on the dentinal tubules. Nonetheless, CLSM found that all specimens' dentinal tubules still contained living microorganisms.

CONCLUSION

The present investigation discovered that the number of culture-positive root canal samples was significantly reduced by 1, 5, 7.5, and 10 mg/ml TAP. Furthermore, TAP had a dose-dependent bactericidal impact on the dentinal tubules. Nonetheless, CLSM found that all specimens' dentinal tubules still contained living microorganisms.

REFERENCES
  1. Kaur M, Kendre S, Gupta P, Singh N, Sethi H, Gupta N, et al. Comparative evaluation of anti microbial effects of triple antibiotic paste and amox and its derivatives against E. faecalis: An in vitro study. J Clin Exp Dent. 2017;9:e799–804.
  2. Chinni SK, Veni AB, Srinivasan MR, Rajamani I. An in vitro investigation of a newer intracanal medicament nisin on Enterococcus faecalis in comparison with chlorhexidine and calcium hydroxide. J Int Clin Dent Res Organ. 2011;3:21–4. 
  3. Mehta S, Verma P, Tikku AP, Chandra A, Bains R, Banerjee G. Comparative evaluation of antimicrobial efficacy of triple antibiotic paste, calcium hydroxide, and a proton pump inhibitor against resistant root canal pathogens. Eur J Dent. 2017;11:53–7. 
  4. Alghamdi F, Shakir M. The influence of Enterococcus faecalis as a dental root canal pathogen on endodontic treatment: A systematic review. Cureus. 2020;12:e7257.. 
  5. Castilho AL, Saraceni CH, Díaz IE, Paciencia ML, Suffredini IB. New trends in dentistry: Plant extracts against Enterococcus faecalis. The efficacy compared to chlorhexidine. Braz Oral Res. 2013;27:109–15. 
  6. Madhukumar M, Geetha P, Nair KR, Unnikrishnan M. The effects of double antibiotic paste and amoxicillin-clavulanate paste used in endodontic regeneration on microhardness of radicular dentine: An in vitro study. J Pharm Bioallied Sci. 2021;13:S510–5. 
  7. Nosrat A, Li KL, Vir K, Hicks ML, Fouad AF. Is pulp regeneration necessary for root maturation? J Endod. 2013;39:1291–5. 
  8. Frough Reyhani M, Rahimi S, Fathi Z, Shakouie S, Salem Milani A, Soroush Barhaghi MH, et al. Evaluation of antimicrobial effects of different con- centrations of triple antibiotic paste on mature biofilm of Enterococcus faecalis. J Dent Res Dent Clin Dent Prospects 2015; 9(3):138–43.
  9. Sabrah AH, Yassen GH, Liu WC, Goebel WS, Gregory RL, Platt JA. The effect of diluted triple and double antibiotic pastes on dental pulp stem cells and established Enterococcus faecalis biofilm. Clin Oral Investig 2015; 19(8):2059–66.
  10. Ruparel NB, Teixeira FB, Ferraz CC, Diogenes A. Direct effect of intracanal medicaments on survival of stem cells of the apical papilla. J Endod 2012; 38(10):1372–5.
  11. Alyas SM, Fischer BI, Ehrlich Y, Spolnik K, Gregory RL, Yassen GH. Direct and indirect antibacterial effects of various concentrations of triple antibiotic pastes loaded in a methylcellulose system. J Oral Sci 2016; 58(4):575–82.
  12. Andrade FB, Arias MP, Maliza AG, Duarte MA, Graeff MS, Amoroso-Silva PA, et al. A new improved protocol for in vitro intratubular dentinal bac- terial contamination for antimicrobial endodontic tests: standardization and validation by confocal laser scanning microscopy. J Appl Oral Sci 2015; 23(6):591–8.
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