|Year : 2020 | Volume
| Issue : 2 | Page : 152-157
Evaluation of antifungal activity of six children's toothpaste on Candida albicans isolated from early childhood caries patients
TP Chandhru1, VR Anusha1, Faizal C Peedikayil1, MB Gufran Ahmed2, Soni Kottayi1, Dhanesh Narasimhan1
1 Department of Pediatric and Preventive Dentistry, Kannur Dental College, Kannur, Kerala, India
2 Department of Microbiology, Kannur Medical College, Kannur, Kerala, India
|Date of Submission||03-Jan-2020|
|Date of Decision||04-May-2020|
|Date of Acceptance||11-May-2020|
|Date of Web Publication||27-Jun-2020|
Dr. V R Anusha
Department of Pediatric and Preventive Dentistry, Kannur Dental College, Kannur, Kerala
Source of Support: None, Conflict of Interest: None
| Abstract|| |
Background: Early childhood caries (ECC) is an aggressive, destructive form of dental caries that affects the children younger than 6 years of age. Candida is known to increase the adherence of Streptococcus mutans to the oral biofilm and produce acids that cause tooth demineralization. Aim of the Study: To evaluate the efficacy of six commercially available children's toothpaste on clinical isolates of Candida albicans obtained from ECC patients. Materials and Methods: The study population comprised 60 children aged 3–6 years having ECC. Samples were divided into six groups comprising ten children in each group. Samples were collected using sterile cotton swabs, inoculated on Sabouraud dextrose agar, and incubated at 37°C for 24 h. Species identification was done by germ tube test and growth on corn meal agar. After the confirmed growth of C. albicans, the six commercially available children's toothpaste namely Organic Children's Coconut Oil Toothpaste®, Aloe Dent Children's Toothpaste®, Patanjali Dant Kanti Junior Toothpaste®, Colgate Kids Toothpaste®, Pediflor Kids Toothpaste®, and Crest Pro-Health Stages Kids Toothpaste® and distilled water as control group were subjected for antifungal activity of C. albicans. Agar plates were incubated at 37°C for 48 h, and the diameter of the zones of inhibition was measured and recorded. Data were tabulated and statistical analysis was done using Statistical Package for the Social Science (16.0) version. Results: Pediflor kids toothpaste® showed maximum antifungal activity and Aloe Dent children's toothpaste® showed minimum antifungal activity. Conclusion: All brands of children's toothpaste show antifungal activity against Candida albicans.
Keywords: Candida albicans, early childhood caries, toothpaste
|How to cite this article:|
Chandhru T P, Anusha V R, Peedikayil FC, Gufran Ahmed M B, Kottayi S, Narasimhan D. Evaluation of antifungal activity of six children's toothpaste on Candida albicans isolated from early childhood caries patients. J Indian Soc Pedod Prev Dent 2020;38:152-7
|How to cite this URL:|
Chandhru T P, Anusha V R, Peedikayil FC, Gufran Ahmed M B, Kottayi S, Narasimhan D. Evaluation of antifungal activity of six children's toothpaste on Candida albicans isolated from early childhood caries patients. J Indian Soc Pedod Prev Dent [serial online] 2020 [cited 2020 Jul 13];38:152-7. Available from: http://www.jisppd.com/text.asp?2020/38/2/152/288226
| Introduction|| |
Streptococci mutans have been implicated as the most important bacteria for early childhood caries (ECC) initiation and its progression. They exhibit a number of virulent characteristics that make the plaque or biofilm cariogenic. The low pH present in the carious plaques causes the increase in the numbers of acid-resistant organisms, such as Lactobacilli, Veillonella, and Candida species. The presence of Candida has been shown to enhance the adherence of S. mutans to the oral biofilm and carious tooth substance in vitro.
Recent researchers have shown Candida species to have cariogenic potential in early childhood period; due to immature immune system and not fully established micro flora in the oral cavity, children are more susceptible to opportunistic microbial colonization of Candida.,Candida albicans does not colonize teeth effectively on its own. Rather, C. albicans adheres mainly to oral mucosa, while interacting with commensal streptococci to cause mucosal infections.
Candida species is acidogenic and has the ability to ferment carbohydrates. Klinke et al. have shown that in an environment with a pH below 5.5, which is relevant for ECC formation, acidification by S. mutans decreases considerably and ceases around pH 4.2, whereas Candida can still secrete acid at pH 4.0. It also produces several organic acids including pyruvic acid and acetate. Previous studies showed strong synergism when C. albicans and S. mutans co-existed in biofilm, suggesting that this coexistence enhanced their virulence.,, One of the mediators for the synergistic relationship between C. albicans and S. mutans is the streptococcal GtfB enzyme., This is evident from the study done by Bachtiar and Bachtiar, which showed that C. albicans contributes to increasing concentration of S. mutans by inducing the expression of GtfB mRNA in the ECC-related biofilm. Therefore, it becomes necessary to prevent the formation of biofilm at the earliest.
Since the biofilms are caused by plaque-associated microorganisms, they tend to be localized. Brushing with the suitable toothpaste is the best preventive measure considered essential for plaque biofilm., Nowadays, various toothpastes are available in the market such as herbal- and nonherbal-based products. Most of the studies on antimicrobial activity of the dentifrices have focused on inhibition of bacterial growth but are less concerned about the antifungal activity. In the literature, only few studies were done on children's toothpaste on antifungal activity. Hence, the present study was carried out to evaluate the antifungal activity of commercially available children's toothpaste both herbal and nonherbal on clinical isolates of C. albicans obtained from ECC patients.
| Materials and Methods|| |
An ex vivo study was conducted in the Department of Pediatric and Preventive Dentistry, Kannur Dental College. Study population comprised sixty children who reported to the department for various therapeutic reasons. Sample size was calculated using the formula n = 2([Zα/2 + Zβ] σ/d) 2. The inclusion criteria of the participants in this study are children in the age group of 3–6 years diagnosed with Early Childhood Caries (ECC). The exclusion criteria includes children who has been on topical or systemic antimicrobial for the previous one month, and those who had undergone any dental treatment or fluoride therapy in the preceding 6 months. Children with any congenital, systemic or contagious diseases, and those with definitely negative Frankl's behavior rating, were also excluded.
Ethical approval was obtained from the Institutional Review Board Committee. The study and treatment protocol were explained in detail to the parents and the children, and their consent was obtained. A purposefully designed form was used to record demographic data, medical history, and caries status of individuals. The children were screened for the caries experience, using mouth mirror and explorer. Caries index was recorded using the decayed-extracted-filled teeth score. Samples were collected using sterile cotton swabs. Swabbing was done over the buccal, lingual, proximal, and cervical portion of the tooth, and cotton swabs were immediately transferred to the laboratory for microbiological analysis.
The obtained samples were inoculated for culture on Sabouraud dextrose agar (SDA) plates supplemented with 1% chloramphenicol with pH 6.6 to prevent bacterial overgrowth. The plates were incubated at 37°C for 24–72 h. Isolates were identified by colony morphology on the SDA plates. The growth was considered to be positive if creamy, smooth, convex pasty colonies with a moldy odor appear in 1–2 days [Figure 1]. Culture was said to be negative if there is no growth even after 72 h of incubation. The positive cultures were stocked in the SDA stock vials. Species identification was carried by germ tube test [Figure 2] and corn meal agar test [Figure 3].
After obtaining the confirmed growth of C. albicans, six various commercially available dentifrices were subjected for antifungal activity by classifying them under the following groups.
- Group I: Organic Children's Coconut Oil Toothpaste (n = 10)
- Group II: Aloe Dent Children's Toothpaste (n = 10)
- Group III: Patanjali Dant Kanti Junior Toothpaste (n = 10)
- Group IV: Colgate Kids Toothpaste (n = 10)
- Group V: Pediflor Kids Toothpaste (n = 10)
- Group VI: Crest Pro-Health Stages Kids Toothpaste (n = 10)
- Group VII: Distilled water (control group).
The selected dentifrices' solution was made by mixing 3 g of each toothpaste in 3 ml of distilled water to give 1:1 dilution; the dentifrice was further diluted with distilled water to produce 1:2, 1:4, 1:8, and 1:16 dilutions. Before this, a pilot study was conducted to know the maximum effectiveness of dilutions and 1:1 dilution showed maximum effectiveness. Suspension of C. albicans was prepared in 5 ml of sterile saline solution. The turbidity of the inoculum will be adjusted to the turbidity of a McFarland 0.5 standard, and 0.1 ml of the inoculum was dropped on the agar and spread to whole surface by a sterile glass applicator. Then, seven wells, each in 4 mm depth and 6 mm diameter, were punched on the agar surface with equal distance from each other. Six wells were filled with 0.02 ml dentifrice solution and one well with distilled water. Agar plates were incubated at 37°C for 48 h, and the diameter of the zones of inhibition was measured and recorded [Figure 4]. The average of vertically and horizontally measured diameter of obtained zone of inhibition was taken. The size of the zone of inhibition is usually related to the level of antimicrobial activity present in the sample or product, and a larger zone of inhibition usually means that the antifungal activity is more.
| Results|| |
The results obtained were tabulated and subjected for statistical analysis. The data were expressed in mean and standard deviation. SPSS Version 16 (SPSS Inc. Chicago USA) version was used for analysis. ANOVA (post hoc) followed by Dunnet's t-test was applied to find the statistical significance between the groups. P < 0.05 was considered statistically significant at 95% confidence interval.
[Table 1] shows the mean zone of inhibition of different groups. All the groups showed statistically significant result: Group I with mean value of 12.60 ± 1.40; Group II with least zone of inhibition with a mean value of 8.96 ± 0.86; Group III with mean value of 10.63 ± 1.22; Group IV with mean of 14.53 ± 2.17; Group V with highest zone of inhibition of 18.20 ± 2.13; Group VI with mean of 15.67 ± 2.38, while Group VII, distilled water as the control group, did not show zone of inhibition with value 0.00 ± 0.00.
[Table 2] shows multiple comparison of mean zone of inhibition between the groups. Group I, II, III, V, and VI between the multiple group comparison of zone of inhibition showed high significant difference with the P = 0.001. Comparison between Group IV and V showed less significant difference with the P = 0.036. Group V has shown maximum zone of inhibition and Group II has shown minimum zone of inhibition.
|Table 2: Multiple comparison of mean zone of inhibition between the groups|
Click here to view
| Discussion|| |
The present study used six commercially available children's toothpaste namely Organic Children's Coconut Oil Toothpaste®, Aloe Dent Children's Toothpaste®, Patanjali Dant Kanti Junior Toothpaste®, Colgate Kids Toothpaste®, Pediflor Kids Toothpaste®, Crest Pro-Health Stages Kids Toothpaste®. Ingredients of these toothpastes are given in [Table 3]. On evaluation of anticandidal activity of all these toothpastes, it was observed that Pediflor Kids Toothpaste® showed the maximum zone of inhibition followed by Crest Pro-Health Stages Kids Toothpaste®, Colgate Kids Toothpaste®, Organic Children's Coconut Oil Toothpaste®, Patanjali Dant Kanti Junior Toothpaste®, and the least zone of inhibition was observed in Aloe Dent Children's Toothpaste®. Distilled water was used as the negative control group.
Pediflor Kids Toothpaste® has shown maximum anticandidal activity when compared to all other commercially available toothpastes used in this study. The highest anticandidal activity of this paste may be due to its synergetic effect between the active ingredients of this toothpaste formulation. The ingredients such as sodium fluoride, neem, and meswak have got antifungal activity. Many clinical studies have demonstrated the efficiency of sodium fluoride in reducing cavities, helping diminish demineralization of tooth enamel, and even enhancing the remineralization of potential-decay spots. However, based on a variety of mechanisms, fluorides also demonstrate some antibacterial and antifungal effects, such as metabolic interference and reduction of dental plaque acidogenicity., Studies done by Varma et al. showed that toothpaste containing meswak as an active ingredient has antifungal efficacy against C. albicans. Another study done by Tadikonda et al. showed that neem which is one of the active ingredient in the toothpaste contains isoprenoids such as nimbin, nimbidin, and nimbidni, which has antibacterial effects against oral streptococci and also prevents adhesion to tooth surface. The pathogenic form of albicans binds to the tooth structure with the help of hyphae, and therefore, we can infer that isoprenoids present in the toothpaste can prevent the Candida adhesion to the host.
Crest Pro-Health Stages Kids Toothpaste® has shown significant anticandidal activity. On intergroup comparison of Crest Pro-Health Stages Kids Toothpaste® and Colgate Kids Toothpaste®, it has shown statistically less significance. The antifungal activity of Crest Pro-Health Stages Kids Toothpaste® may be due to ingredients such as sodium fluoride and triclosan. It has been suggested that triclosan blocks lipid biosynthesis by specifically inhibiting the enzyme enoylacyl carrier protein reductase. This feature of Crest Pro- Health toothpaste can be attributed to the antimicrobial efficiency. Study done by Prasanth revealed that the inhibitory effect of triclosan on C. albicans. Studies by Giuliana and Yu et al. have also shown that triclosan is an effective antifungal agent against human pathogen, i.e., C. albicans.
Colgate Kids Toothpaste® showed significant anticandidal activity. When Colgate Kids Toothpaste® and Pediflor Kids Toothpaste® were compared, it The antifungal activity of Colgate Kids Toothpaste may be due to the active ingredient sodium fluoride. This was in accordance with the study done by Adwan et al., in which high Candida activity was shown by sodium fluoride. Fluoride inhibits glycolysis and prevented the transfer of glucose into cells.
Organic Children's Coconut Oil Toothpaste® has shown significant anticandidal activity. The anticandidal activity of this paste observed in our study may be due to the presence of organic coconut oil as the main ingredient and synergetic effect between the active ingredients of this toothpaste formulation. The reason attributed for the antifungal activity of coconut oil could be due to the presence of three medium chain fatty acids, i.e., lauric acid, caprylic acid, and capric acid, all of which is evident from the study done by Ogbolu et al. They also stated that the efficacy of coconut oil was comparable with fluconazole which is an antifungal agent. Another study by Shino et al. has shown that coconut oil has shown significant antifungal activity which is comparable with ketoconazole. Currently, there are no correlative and comparative studies to compare coconut oil toothpaste with other commercially available children's toothpaste which were used in this study.
Patanjali Dant Kanti Junior Toothpaste® which was used in our study has shown significant anticandidal activity and it is an herbal-based toothpaste. The reason for significant anticandidal activity of this toothpaste could be due to its active ingredients such as neem that has got antiseptic, antibacterial, and antifungal activity. Neem is a plant with medicinal values; its antifungal activity is evident from the studies done by Mahmoud et al. Similarly, another study by Tadikonda et al. has also shown the antifungal activity of neem. Studies done by Varma et al. have shown that toothpaste containing meswak as an active ingredient has shown antifungal efficacy against C. albicans.
Aloe Dent Children's Toothpaste® was found to be effective against C. albicans but had minimal anticandidal activity showing the least zone of inhibition, when compared to other toothpastes which were used in the study. The advantage of this toothpaste is being fluoride free, anti-inflammatory, anti-bacterial, antifungal, and antioxidant due to the presence of active ingredients such as anthraquinone, aloenin, barbaloin, and isobarbaloin. The study done by Diaz et al. had showed that the toothpaste containing Aloe vera extract is effective against C. albicans. Another study conducted by Shireen et al. had also showed antifungal activity of Aloe. vera extract on C. albicans. The reason having minimal anticandidal activity in our study could be due to inappropriate dilutions, inability of the toothpaste to diffuse into the SDA agar media, and variation in the method that was adopted for conducting the study.
Distilled water has shown no zone of inhibition which means that there was no anticandidal activity. The reason for using distilled water as a negative control is due to the fact that most of the toothpaste preparations in our study were mixed with distilled water before subjecting for microbiological analysis. It was observed that distilled water was unable to initiate any zone of inhibition, thereby considering that the ingredients of toothpaste were reasons for anticandidal activity. This was in accordance to the study done by Adwan et al., in which they used distilled water as the negative control group.
The C. albicans has got synergistic effect with S. mutans on initiation and progression of ECC. Therefore, it is advisable to use toothpastes with additive antifungal activity for prevention and control of the dental caries. The research does not lead to prescribe a specific brand of toothpaste for effective inhibition of Candida colonization in ECC patients. However, it is concluded that the use of toothpastes containing antifungal ingredients like meswak, neem extracts and sodium fluoride can effectively control C. albicans. Further,in vivo studies are required to know the role of saliva, plaque, and mechanism of action of these active ingredients.
| Conclusion|| |
The following conclusions were drawn from the present study:
- All toothpastes used in this study namely Organic Children's Coconut Oil Toothpaste®, Aloe Dent Children's Toothpaste®, Patanjali Dant Kanti Junior Toothpaste®, Colgate Kids Toothpaste®, Pediflor Kids Toothpaste®, Crest Pro-Health Stages Kids Toothpaste® showed antifungal activity on C. albicans
- Pediflor Kids Toothpaste® had shown maximum antifungal activity while Aloe Dent Children's Toothpaste® had shown minimum antifungal activity
- The high anticandidal activity of Pediflor kids toothpaste® may be due to the synergistic effect of the active ingredients like sodium fluoride, neem extract and meswak extract.
Financial support and sponsorship
Conflicts of interest
There are no conflicts of interest.
| References|| |
Gibbons RJ, Armstrong WD. Bacteriology of dental caries. J Dent Res 1964;43:1021-8.
Barbieri DD, Vicente VA, Fraiz FC, Lavoranti OJ, Svidzinski TI, Pinheiro RL. Analysis of thein vitro
adherence of Streptococcus mutans
and Candida albicans
. Braz J Microbiol 2007;38:624-31.
Nikawa H, Yamashiro H, Makihira S, Nishimura M, Egusa H, Furukawa M, et al
cariogenic potential of Candida albicans
. Mycoses 2003;46:471-8.
Jose T, Thomas A. Candida
a probable etiology in early childhood caries – A literature review. J Odontol Res 2015;3:15-22.
Klinke T, Kneist S, de Soet JJ, Kuhlisch E, Mauersberger S, Forster A, et al
. Acid production by oral strains of Candida albicans
and lactobacilli. Caries Res 2009;43:83-91.
Samaranayake LP, Hughes A, Weetman DA, MacFarlane TW. Growth and acid production of Candida
species in human saliva supplemented with glucose. J Oral Pathol 1986;15:251-4.
Falsetta ML, Klein MI, Colonne PM, Scott-Anne K, Gregoire S, Pai CH, et al
. Symbiotic relationship between Streptococcus mutans
and Candida albicans
synergizes virulence of plaque biofilms in vivo
. Infect Immun 2014;82:1968-81.
Koo H, Bowen WH. Candida albicans
and Streptococcus mutans
: A potential synergistic alliance to cause virulent tooth decay in children. Future Microbiol 2014;9:1295-7.
Sztajer H, Szafranski SP, Tomasch J, Reck M, Nimtz M, Rohde M, et al
. Cross-feeding and interkingdom communication in dual-species biofilms of Streptococcus mutans
and Candida albicans
. ISME J 2014;8:2256-71.
Gregoire S, Xiao J, Silva BB, Gonzalez I, Agidi PS, Klein MI, et al
. Role of glucosyltransferase B in interactions of Candida albicans
with Streptococcus mutans
and with an experimental pellicle on hydroxyapatite surfaces. Appl Environ Microbiol 2011;77:6357-67.
Bachtiar EW, Bachtiar BM. Relationship between Candida albicans
and Streptococcus mutans
in early childhood caries, evaluated by quantitative PCR. F1000Res 2018;7:1645.
Long SR, Santos AS, Nascimento CMO. Evaluation of contamination of toothbrushes by enterobacteria. Rev Odontol Univ St Amaro 2000;5:21-5.
van Rijkom HM, Truin GJ, van't Hof MA. A meta-analysis of clinical studies on the caries-inhibiting effect of chlorhexidine treatment. J Dent Res 1996;75:790-5.
Adwan G, Salameh Y, Adwan K, Barakat A. Assessment of antifungal activity of herbal and conventional toothpastes against clinical isolates of Candida albicans
. Asian Pac J Trop Biomed 2012;2:375-9.
Ogbolu DO, Oni AA, Daini OA, Oloko AP.In vitro
antimicrobial properties of coconut oil on Candida
species in Ibadan, Nigeria. J Med Food 2007;10:384-7.
Shino B, Peedikayil FC, Jaiprakash SR, Ahmed Bijapur G, Kottayi S, Jose D. Comparison of antimicrobial activity of chlorhexidine, coconut oil, probiotics, and ketoconazole on Candida albicans
isolated in children with early childhood caries: Anin vitro
study. Scientifica (Cairo) 2016;2016:7061587. doi:10.1155/2016/7061587.
Varma SR, Sherif H, Serafi A, Fanas SA, Desai V, Abuhijleh E, et al
. The antiplaque efficacy of two herbal-based toothpastes: A clinical intervention. J Int Soc Prev Community Dent 2018;8:21-7.
Tadikonda A, Pentapati KC, Urala AS, Acharya S. Anti-plaque and anti-gingivitis effect of papain, bromelain, miswak and neem containing dentifrice: A randomized controlled trial. J Clin Exp Dent 2017;9:e649-53.
Kurian M, Geetha RV. Effect of herbal and fluoride toothpaste on Streptococcus mutans
– A comparative study. J Pharm Sci Res 2015:7:864-5.
Prasanth M. Antimicrobial efficacy of different toothpastes and mouthrinses: Anin vitro
study. Dent Res J (Isfahan) 2011;8:85-94.
Giuliana G, Pizzo G, Milici ME, Musotto GC, Giangreco R.In vitro
antifungal properties of mouthrinses containing antimicrobial agents. J Periodontol 1997;68:729-33.
Yu L, Ling G, Deng X, Jin J, Jin Q, Guo N.In vitro
interaction between fluconazole and triclosan against clinical isolates of fluconazole-resistant Candida albicans
determined by different methods. Antimicrob Agents Chemother 2011;55:3609-12.
Ellepola AN, Samaranayake LP. Adjunctive use of chlorhexidine in oral candidoses: A review. Oral Dis 2001;7:11-7.
Mahmoud DA, Hassanein NM, Youssef KA, Abou Zeid MA. Antifungal activity of different neem leaf extracts and the nimonol against some important human pathogens. Braz J Microbiol 2011;42:1007-16.
Shireen F, Manipal S, Prabu D. Anti-fungal activity of Aloe vera
study. SRM J Res Dent Sci 2015;6:92. [Full text]
[Figure 1], [Figure 2], [Figure 3], [Figure 4]
[Table 1], [Table 2], [Table 3]