ORIGINAL RESEARCH |
https://doi.org/10.5005/jp-journals-10077-3245 |
Comparative Evaluation of Antimicrobial Efficacy of Silver Diamine Fluoride and Curodont™ Protect Remineralizing Agents on Streptococcus mutans: An In Vitro Microbiological Study
1-4Department of Pediatric and Preventive Dentistry, Sri Rajiv Gandhi College of Dental Sciences & Hospital, Bengaluru, Karnataka, India
Corresponding Author: Vandana Chandran, Department of Pediatric and Preventive Dentistry, Sri Rajiv Gandhi College of Dental Sciences & Hospital, Bengaluru, Karnataka, India, Phone: +91 9900124075, e-mail: vandanachandran.16@gmail.com
Received on: 16 September 2022; Accepted on: 10 November 2022; Published on: 26 December 2022
ABSTRACT
Background: Dental caries is regarded as a biofilm-induced oral disease, with Streptococcus mutans assaying a pivotal part in the onset and progression of the carious lesion. Hence, controlling the bacterial load of the oral cavity is one of the elementary objectives in the prevention of dental caries.
Objectives: To evaluate and compare the antimicrobial efficacy of silver diamine fluoride (SDF) and Curodont™ Protect remineralizing agents on S. mutans.
Materials and methods: An in vitro microbiological study was conducted to ascertain the antibacterial efficacy of SDF (group I) and Curodont™ Protect remineralizing agent (group II) against S. mutans. Chlorhexidine gluconate (CHX) gel 2.0% w/w (group III) and distilled water (group IV) formed the positive and negative controls, respectively. The antibacterial efficacy was determined by agar well diffusion method following 24 hours of incubation, and the diameter of zones of inhibition (ZOI) was quantified. The data were statistically analyzed using Statistical Package for Social Sciences (SPSS) version 22.0. One-way analysis of variance (ANOVA) test was used to compare the mean ZOI between groups, and Tukey’s post hoc test was used to compare the mean difference in the ZOI among groups (level of significance set at p < 0.05).
Results: The mean ZOI was significantly higher for the SDF group (38.33 ± 0.58 mm) as compared to the CHX group (26.67 ± 0.58 mm), with a mean difference of 11.66 [95% confidence interval (CI)] at p < 0.001. Curodont™ Protect and distilled water did not demonstrate any ZOI.
Conclusion: SDF tooth remineralizing agent exhibited higher antibacterial efficacy against S. mutans in comparison with Curodont™ Protect remineralizing agent.
How to cite this article: Chandran V, Smitha M, Azher U, et al. Comparative Evaluation of Antimicrobial Efficacy of Silver Diamine Fluoride and Curodont™ Protect Remineralizing Agents on Streptococcus mutans: An In Vitro Microbiological Study. J South Asian Assoc Pediatr Dent 2022;5(3):147-151.
Source of support: Nil
Conflict of interest: None
Keywords: Antibacterial efficacy, Dental caries, Remineralizing agents, Silver diamine fluoride, Streptococcus mutans, Zone of inhibition
INTRODUCTION
Dental caries is a chronic disease1 resulting from an ecological disparity in the equipoise between mineral content of the tooth and biofilms (plaque) in the oral cavity.2 It is one of the most common reasons for enamel loss and, subsequently, tooth loss in a clinical scenario.3 Prevalence of dental caries is 52% among the 3–18 years age-group with maximum prevalence seen in mixed dentition (58%).4 Among plaque microbiota, S. mutans have a very high hieratic status in terms of their acid tolerance and acidogenicity in an acidic plaque, which plays a significant role in the progression of the carious lesion.5,6 The tooth is subjected to a vicious cycle of demineralization and remineralization, which controls the progression or reversal of carious lesions throughout its lifetime.3 The bacterial action on dietary carbohydrates leads to acid production, which diffuses into the tooth and dissolves hydroxyapatite (HAP) mineral by process of demineralization, leading to initiation and progression of carious lesion.7
Various studies have proved that demineralized enamel could be remineralized if there is no mechanical deformation in the early stages.8 However, progression of the incipient lesion to cavitation is an irreversible condition. Therefore, interception of dental caries at the initial stage is of paramount importance than the development of treatment strategies for continuing dental caries.3 Tooth remineralization agents play a vital function in the interposing of dental caries. Application of remineralizing agents that offer both antimicrobial and remineralizing effects is of great clinical importance. With the understanding of the dynamics of dental caries and advancement in science and research, several materials have been developed that have the potential to remineralize the tooth structure. To guide and help clinicians and researchers in the proper choice and utilization of different technologies, newer materials require validation and comparison with accepted methods. SDF is regarded as an efficient agent in preventing and controlling carious lesions in pediatric dentistry. It is a solution containing ionic silver, fluoride, and ammonia.8 Remineralizing agent Curodont™ Protect (Credentis AG, Windisch, Switzerland) tooth gel remineralizes and protects the enamel. It comprises Curolox Technology based on P11-4, which are self-organizing molecules system with efficacious protection features due to their combination and stabilization with calcium phosphate.
Therefore, this in vitro study aimed to evaluate and compare the antimicrobial efficacy of SDF and Curodont™ Protect remineralizing agents on S. mutans using agar well diffusion method.
MATERIALS AND METHODS
An observational in vitro microbiological study was designed in accordance with the Checklist for Reporting In-vitro Studies (CRIS) guidelines and approved by the Institutional Review Board.
Study Groups
The samples of the present in vitro study were grouped as group I—Kids E SDF (Kids-e-Dental; composed of 38% Silver diamine fluoride (SDF)—test group), group II—Curodont™ Protect (Curodont, Credentis AG, Windisch, Switzerland; composed mainly of sodium monofluorophosphate, dicalcium phosphate, oligopeptide-104—test group), group III—Chlorhexidine (CHX) 2% w/w (Asep-RC; Anabond Stedman Pharma Research (P) Ltd—positive control), and group IV—distilled water—negative control (Fig. 1). A convenience sampling method was done and the study was conducted in triplicates, considering the loss of samples due to contamination and for homogeneity of the results.
Figs 1A to D: Study groups: (A) Group I—SDF; (B) Group II—Curodont™ Protect; (C) CHX; (D) Distilled water
Sample Preparation
The samples were prepared by adding dimethyl sulfoxide (DMSO) to the respective study groups. DMSO acted as a solvent for the dissolution of study materials. About 10 mg of Curodont™ Protect was dissolved into 1 mL of DMSO, and 10 µL (100 µg) was pipetted out and made up to 50 µL by adding DMSO. Also, 10 µL of SDF, CHX, and distilled water were made up to 50 µL by following the same method.
Culture Media Preparation for Bacterial Growth
About 30 mL of Luria–Bertani (LB) broth was prepared by adding tryptone 0.3 gm, sodium chloride 0.3 gm, yeast extract 0.18 gm, and distilled water 30 mL. The culture media was then autoclaved at 121°C for 15 minutes. The S. mutans strain (MTCC 497) was inoculated and incubated at 37°C for 24 hours. Later, 5 mL of broth culture was centrifuged at 6000 rpm for 10 minutes, the supernatant was discarded, the pellets were dissolved in 1% saline, and adjusted to absorbance 1.00 at 600 nm under UV spectrophotometer (Genesys 10S UV-VIS spectrophotometer).
Culture Media Preparation for Minimum Inhibitory Concentration (MIC)
About 100 mL of LB agar media was prepared by adding tryptone 2 gm, sodium chloride 2 gm, yeast extract 0.6 gm, and agar 2 gm to 100 mL of distilled water and autoclaved at 121°C for 15 minutes.
Plating for MIC against Organisms
Approximately 25 mL of LB agar was added to the sterilized Petri plates and left to solidify. 200 µL of the prepared inoculum of S. mutans was applied to agar plates and spread thoroughly using a plate spreader. Five wells measuring about 0.6 cm were made in each plate using the borer, and 50 µL of prepared sample was loaded into the corresponding wells; 50 µL of DMSO was loaded in the middle well as a check. The Petri plates were incubated at 37°C for 24 hours. Zone of inhibition (ZOI) was recorded in millimeters (mm).
Statistical Analysis
The null hypothesis stating that there is no difference in the antimicrobial efficacy between SDF and Curodont™ Protect was postulated. The data were statistically analyzed using SPSS version 22.0. One-way ANOVA test was used to compare the mean ZOI between groups, and Tukey’s post hoc test was used to compare the mean difference in the ZOI among groups. The level of significance was set at p < 0.05.
RESULTS
The mean ZOI was recorded for all the groups after 24 hours of incubation (Fig. 2 and Table 1). The test results demonstrate that group I SDF showed a mean ZOI of 38.33 ± 0.58 mm, whereas group III CHX showed 26.67 ± 0.58 mm (Table 2).
| Sample | ZOI of S. mutans in mm | ||
|---|---|---|---|
| Plate 1 | Plate 2 | Plate 3 | |
| SDF | 38 | 39 | 38 |
| Curodont™ Protect | – | – | – |
| Distilled water | – | – | – |
| CHX | 27 | 27 | 26 |
| DMSO | – | – | – |
| Groups | N | Mean (mm) | Standard deviation | Minimum (mm) | Maximum (mm) | p-value |
|---|---|---|---|---|---|---|
| SDF | 3 | 38.33 | 0.58 | 38.0 | 39.0 | <0.001* |
| Curodont™ Protect | 3 | 0.00 | 0.00 | 0.0 | 0.0 | |
| Distilled water | 3 | 0.00 | 0.00 | 0.0 | 0.0 | |
| CHX | 3 | 26.67 | 0.58 | 26.0 | 27.0 |
* Statistically significant
Figs 2A to C: ZOI between groups for S. mutans in triplicates
The test results also demonstrated that the mean ZOI was significantly higher for SDF group, followed by CHX group with a mean difference of 11.67 (95% CI). This difference across groups was statistically significant at p < 0.001. Curodont™ Protect and distilled water did not demonstrate any ZOI (Table 3 and Fig. 3).
| (I) Groups | (J) Groups | Mean difference (I–J) | 95% CI for the difference | p-value | |
|---|---|---|---|---|---|
| Lower | Upper | ||||
| SDF | Curodont™ Protect | 38.33 | 37.35 | 39.31 | <0.001* |
| Distilled water | 38.33 | 37.35 | 39.31 | <0.001* | |
| CHX | 11.67 | 10.69 | 12.65 | <0.001* | |
| Curodont™ Protect | Distilled water | 0.00 | −0.98 | 0.98 | 1.00 |
| CHX | −26.67 | −27.65 | −25.69 | <0.001* | |
| Distilled water | CHX | −26.67 | −27.65 | −25.69 | <0.001* |
* Statistically significant
Fig. 3: Mean ZOI in mm
DISCUSSION
Dental caries remains one of the most prevalent and notable public health diseases in the world today and is considered the leading etiology of dental pain and tooth loss.9S. mutans, which is regarded as the pioneer causative microbe for dental caries, colonize the tooth surface through adhesion to the enamel salivary pellicle and other plaque bacteria and cause damage to the hard tooth structure.10
The recent trend in dental caries management is preliminary detection and focus on noninvasive control of reversible lesions using various tooth remineralization materials.11 Remineralization connects the conventional disparity between preventive and invasive dentistry. Therefore, favorable management employing this technique necessitates the identification of caries lesions at the level of inception and correct quantification of the degree of mineral loss to secure appropriate precautionary measures. In addition to remineralization potential, the presence of antibacterial activity is advantageous and of paramount clinical importance. Thus, the present study is aimed to evaluate and compare the antimicrobial efficacy of SDF and Curodont™ Protect remineralizing agents on S. mutans.
The present study revealed that SDF had higher ZOI compared with Curodont™ Protect, exhibiting a higher antimicrobial action against S. mutans in addition to its remineralization potential, whereas Curodont™ Protect did not show any ZOI and, therefore, no antibacterial efficacy was observed. Therefore, the null hypothesis was invalidated due to differences observed in the antibacterial activities of the agents tested.
In the current study, the SDF group showed the highest antibacterial activity. The results of the present study were observed to be concordant with the results of previous studies. Klein et al. found that if SDF was applied to the teeth, the level of salivary S. mutans was significantly reduced.12 Chu et al. also reported that the number of bacteria reduced to zero after treatment of mature S. mutans biofilm with SDF.13 According to Chakraborty et al., SDF demonstrated a significant decline in CFU/mL of S. mutans count at 1, 3, and 6 months and consequently be used in the prevention and arrest of dental caries.14 Mei et al. observed that 38% of SDF arrested dental caries by decreasing the demineralization process, and inhibited the growth of multispecies cariogenic biofilm by way of high concentration of silver and fluoride ions.15
The antimicrobial activity of SDF can be attributed to silver ions. The SDF prevents the growth of virulent cariogenic bacteria, inhibits collagen degradation in dentin, retards demineralization, and encourages remineralization of both enamel and dentin.16
Curodont™ Protect (Curodont, Credentis AG, Windisch, Switzerland) features Curolox P11-4-based technology, which comprises self-organizing molecules in them. These molecules have essential protective properties due to their combination and stabilization with calcium phosphate. The peptides polymerize to form small fibers, which promotes the remineralization process. According to studies, on application of the gel onto the tooth surface, the peptide diffuses into the micropores of the subsurface and forms a three-dimensional scaffolding, which reinforces the crystallization of HAP. Since the P11-4-based self-assembling peptide has a substantial affinity to the calcium ions of HAP, which is the fundamental component of the tooth enamel, it acts comparable to bonding glue between HAP particles and enamel.17,18 However, on evaluation of the antimicrobial activity on S. mutans in the present study, Curodont™ Protect did not exhibit any antibacterial activity. Furthermore, due to the paucity of literature on the antimicrobial activity of P11-4, a direct comparison with other studies could not be done.
Limitations
The in vitro design of the present study did not permit the exact simulation of the complex oral environment, and therefore, the results must be analyzed with caveats before extrapolating to clinical conditions. Although paramount care was taken to standardize the experimental conditions, the determination of inhibition zone values depends on technical details that are different with different laboratories. The size of the inhibiting zones is dependent on the diffusibility of the test substance into agar, the uniformity of the density of the inoculum, appropriate culture medium, agar viscosity, plate storage conditions, sample size and number per plate, incubation time, and temperature.
CONCLUSION
Silver diamine fluoride remineralizing agent exhibited great growth inhibitory influence on S. mutans, and can be considered as efficacious in the prevention and interception of progression of incipient lesions, compared to self-assembling peptide-based remineralizing agent Curodont™ Protect.
REFERENCES
1. Selwitz RH, Ismail AI, Pitts NB. Dental caries. Lancet 2007;369(9555):51–59. DOI: 10.1016/S0140-6736(07)60031-2
2. Takahashi N, Nyvad B. Caries ecology revisited: microbial dynamics and the caries process. Caries Res 2008;42(6):409–418. DOI: 10.1159/000159604
3. Rajendran R, Kunjusankaran RN, Raghu S, et al. Comparative evaluation of remineralizing potential of a paste containing bioactive glass and a topical cream containing casein phosphopeptide-amorphous calcium phosphate: an in vitro study. Pesqui Bras Odontopediatria Clin Integr 2019;19(1):1–10. DOI: 10.4034/PBOCI.2019.191.61
4. Pandey P, Nandkeoliar T, Tikku AP, et al. Prevalence of dental caries in the Indian population: a systematic review and meta-analysis. J Int Soc Prev Community Dent 2021;11(3):256–265. DOI: 10.4103/jispcd.JISPCD_42_21
5. Harper DS, Loesche WJ. Growth and acid tolerance of human dental plaque bacteria. Arch Oral Biol 1984;29(10):843–848. DOI: 10.1016/0003-9969(84)90015-3
6. van Houte J. Role of micro-organisms in caries etiology. J Dent Res 1994;73(3):672–681. DOI: 10.1177/00220345940730031301
7. Hassanein OE, El-Brolossy T. An investigation about the remineralization potential of bio-active glass on artificially carious enamel and dentin using Raman spectroscopy. Egypt J Solids 2006;29:69–80. DOI: 10.21608/EJS.2006.149141
8. Ten Cate JM, Arends J. Remineralization of artificial enamel lesions in vitro: III. A study of the deposition mechanism. Caries Res 1980;14(6):351–358. DOI: 10.1159/000260477
9. Thean H, Wong ML, Koh H. The dental awareness of nursing home staff in Singapore—a pilot study. Gerodontology 2007;24(1):58–63. DOI: 10.1111/j.1741-2358.2007.00138.x
10. Forssten SD, Björklund M, Ouwehand AC. Streptococcus mutans, caries and simulation models. Nutrients 2010;2(3):290–298. DOI: 10.3390/nu2030290
11. Kamath P, Nayak R, Kamath SU, et al. A comparative evaluation of the remineralization potential of three commercially available remineralizing agents on white spot lesions in primary teeth: an in vitro study. J Indian Soc Pedod Prev Dent 2017;35(3):229–237. DOI: 10.4103/JISPPD.JISPPD_242_16
12. Klein U, Kanellis MJ, Drake D. Effects of four anticaries agents on lesion depth progression in an in vitro caries model. Pediatr Dent 1999;21(3):176–180. PMID: 10355008.
13. Chu CH, Mei L, Seneviratne CJ, et al. Effects of silver diamine fluoride on dentine carious lesions induced by Streptococcus mutans and Actinomyces naeslundii biofilms. Int J Paediatr Dent 2012;22(1):2–10. DOI: 10.1111/j.1365-263X.2011.01149.x
14. Chakraborty S, Gupta N, Gambhir N, et al. Efficacy of silver diamine fluoride on Streptococcus mutans count present in saliva. Int J Clin Pediatr Dent 2021;14(5):700–704. DOI: 10.5005/jp-journals-10005-2037
15. Mei ML, Li QL, Chu CH, et al. Antibacterial effects of silver diamine fluoride on multi-species cariogenic biofilm on caries. Ann Clin Microbiol Antimicrob 2013;12(1):4. DOI: 10.1186/1476-0711-12-4
16. Qanbar A, Abdulla AAM, Abutayyem H, et al. Comparative evaluation of the efficacy of silver diamine fluoride, sodium fluoride and GC tooth mousse plus and fluoride varnish as an antibacterial agent in childhood caries: as a literature review. Dent Res Manag 2021;5(1):1–7. DOI: 10.33805/2572-6978.150
17. Bröseler F, Tietmann C, Schleich R, et al. Effect of Curodont™ Repair in patients with buccal carious lesions: a mono-centre, single-blinded, randomized, controlled split-mouth study—intermediate report. Clin Oral Investig 2013;17:1055.
18. Schlee M, Rathe F, Bommer C. Effect of Curodont™ repair in patients with proximal carious lesions: uncontrolled, non-interventional study—intermediate report. Clin Oral Investig 2013;17:1046–1047.
________________________
© The Author(s). 2022 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (https://creativecommons.org/licenses/by-nc/4.0/), which permits unrestricted use, distribution, and non-commercial reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.