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ORIGINAL ARTICLE
Year : 2019  |  Volume : 37  |  Issue : 1  |  Page : 46-54
 

Antimicrobial efficacy of nanosilver and chitosan against Streptococcus mutans, as an ingredient of toothpaste formulation: An in vitro study


1 Department of Pedodontics and Preventive Dentistry, V S Dental College and Hospital, Bengaluru, Karnataka, India
2 Department of Pedodontics and Preventive Dentistry, R V Dental College and Hospital, Bengaluru, Karnataka, India

Date of Web Publication25-Feb-2019

Correspondence Address:
Dr. Faiyaz Ahmed
Dr. Ahmed's Dental Home, B/63, Road No. A/13, Alinagar Colony, Anisabad, Patna - 800 002, Bihar
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/JISPPD.JISPPD_239_18

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   Abstract 


Context: Dental caries is the most prevalent oral infection affecting the humankind worldwide, and Streptococcus mutans is the major microrganism involved in its pathology. Mechanical plaque control in children is not performed efficiently and thus necessitates the inclusion of certain antimicrobial agents in the toothpaste. Aim: To analyze the antibacterial efficacy of nanosilver, chitosan, and fluoride as an ingredient in the dentifrices against S. mutans strains and comparing them with each other. Subjects and Methods: Pure culture of S. mutans strain (MTCC 890) was obtained from the Institute of Microbial Technology, Chandigarh, India. Nanosilver (Group 1)- and chitosan (Group 2)-containing toothpastes were obtained from the respective dealers, and fluoride (Group 3) toothpaste was obtained from the local market. The antimicrobial activity of the three toothpastes was determined by modified agar well diffusion method. Saline was kept as the control. Statistical Analysis Used: Unpaired t-test was done for intergroup comparisons. Results: Statistically significant differences between Groups 1 and 2, Groups 1 and 3, and Groups 2 and 3 were seen. Conclusions: Nanosilver-containing toothpaste has the highest antibacterial efficacy against S. mutans, followed by fluoride- and chitosan-containing toothpaste.


Keywords: Chitosan, dental caries, dentifrices, fluoride, nanosilver, Streptococcus mutans


How to cite this article:
Ahmed F, Prashanth S T, Sindhu K, Nayak A, Chaturvedi S. Antimicrobial efficacy of nanosilver and chitosan against Streptococcus mutans, as an ingredient of toothpaste formulation: An in vitro study. J Indian Soc Pedod Prev Dent 2019;37:46-54

How to cite this URL:
Ahmed F, Prashanth S T, Sindhu K, Nayak A, Chaturvedi S. Antimicrobial efficacy of nanosilver and chitosan against Streptococcus mutans, as an ingredient of toothpaste formulation: An in vitro study. J Indian Soc Pedod Prev Dent [serial online] 2019 [cited 2019 Mar 26];37:46-54. Available from: http://www.jisppd.com/text.asp?2019/37/1/46/252856





   Introduction Top


Oral health has a strong correlation with the overall health of an individual.[1] Continuous oral microbial exposure leads to the early development of caries in children.[2] To combat these potentially harmful microbes, ingredients possessing antimicrobial activity are included in several oral hygiene products. The cooperative effects of brushing and using toothpaste containing certain chemical agents aid in the removal of the hazardous biofilm on the enamel.[3],[4],[5],[6] Further, this prevention is much easier and economical than a later intervention.[7]

The inclusion of certain chemical agents in toothpaste to combat oral pathologies has been widely accepted. Chitosan and nanosilver particles are two such ingredients. Chitosan is present in insects' exoskeletons.[8] Nanosilver is pure deionized water with silver in suspension.[9] Endogenous oral bacterial species such as Streptococcus mutans play a major role in the initiation and progression of dental caries.[2] This study focuses on the efficacy of chitosan, nanosilver, and fluorides as an ingredient in the dentifrices against S. mutans.


   Subjects and Methods Top


The present study was conducted in vitro on Mitis Salivarius agar culture media by modified agar well diffusion method. Data were collected by measuring the diameters of the inhibition zones.

Instruments and materials

(1) S. mutans (MTCC 890) strain, (2) nanosilver-containing toothpaste, (3) chitosan-containing toothpaste, (4) fluoridated toothpaste, (5) Mitis Salivarius agar, (6) gloves and mask, (7) kidney tray, (8)  Petri dish More Detailses, (9) test tubes, (10) glass rods, (11) swabs, (12) dropper, (13) 8-mm sterile copper tube, (14) 5-ml plastic syringe, (15) saline, (16) distilled water, (17) vernier caliper, and (18) incubation chamber.

Procurement of the dentifrices

Fluoride toothpaste was obtained from the local market.

Nanosilver and chitosan toothpastes were obtained from the respective dealers [Figure 1].
Figure 1: (From top to bottom) Nanosilver-, chitosan-, and fluoride-containing toothpastes

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The ingredients used in the manufacturing of the dentifrices as listed on the respective packages are shown in [Table 1].
Table 1: Ingredients of the dentifrices

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Nanosilver- and chitosan-containing toothpastes did not contain any fluoride as their constituent.

Procurement of the microorganism

Pure cultures of S. mutans (MTCC 890) were obtained from the Institute of Microbial Technology, Chandigarh, India. It was cultured in brain–heart infusion broth (Hi-Media Laboratories Pvt. Ltd., Mumbai, India) at 37°C for 24 h [Figure 2]. This 24-h broth culture was later used to streak the dried agar plates with the microorganism.
Figure 2: Twenty-four hour broth culture of Streptococcus mutans

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Evaluation of dentifrices

The three different toothpastes were procured for the assessment of their in vitro antimicrobial activity against S. mutans. The selected dentifrice solutions were then made. For this, first 2.0 g of the toothpastes was measured by a sensitive scale. This calculated amount was transferred to a beaker to make the solutions. Now, 2.0 ml of sterile pyrogen-free distilled water was added to the beaker. The mixture was stirred carefully not to produce froth. This gave us a 1:1 dilution of the toothpastes for the experiment. The solutions were transferred to smaller test tubes. The samples hence produced were labeled as A, B, C, and D [Figure 3] as follows:
Figure 3: Prepared samples

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  • Sample A – Nanosilver
  • Sample B – Chitosan
  • Sample C – Fluoride
  • Sample D – Saline (control).


These samples later acted as the test Groups 1, 2, 3, and 4 for the antimicrobial assay.

Antimicrobial assay

The antimicrobial activity of the toothpastes was determined by modified agar well diffusion method. In this method, 20 Mitis Salivarius agar plates were prepared containing 10% sucrose [Figure 4]. These plates were then seeded with 0.5 ml of 24 h broth cultures of S. mutans [Figure 2]. The plates were allowed to dry for 1 h.
Figure 4: Mitis Salivarius agar plate

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A sterile 8-mm copper tube [Figure 5] was used to cut four equidistant wells in each of the plates [Figure 6]. A volume of 0.2 ml of nanosilver toothpaste (Group A) solution was introduced into one of the four wells. Similarly, 0.2 ml of chitosan (Group B) and fluoride toothpaste (Group C) solutions was introduced in two other wells. One well had 0.2 ml of saline (Group D) to act as control [Figure 7].
Figure 5: 8-mm copper tube used for making holes in agar plates

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Figure 6: Equidistant 8-mm holes made in the plates

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Figure 7: Samples introduced into the agar plates and kept for incubation

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All the wells were oriented and labeled in the same manner. The plates were then incubated at 37°C for 24 h. [Figure 8] After 24 h, the plates were observed for zones of inhibition [Figure 9]. The antimicrobial activity was evaluated by measuring the diameter of zones of inhibition (in millimeter) with a vernier caliper [Figure 10]. The minimum diameter was taken as the observed value. The data hence collected were tabulated for analysis [Table 2].
Figure 8: Incubation chamber

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Figure 9: Zones of inhibition viewed after 24 h incubation

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Figure 10: Digital vernier caliper

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Table 2: Diameter of the zone of inhibition (in millimeter)

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Statistical analysis

Statistical technique used: Unpaired t-test.

Certain assumptions were anticipated for this test as follows:

  1. The sample drawn is random
  2. There is independence between the groups
  3. The data follow a normal distribution with mean and variance
  4. The variances of the two groups are equal.


Null hypothesis: There was no significant difference in the mean score recorded in the four groups, i.e., μ1 = μ2 = μ3 = μ4.

Decision criterion: The decision criterion was to reject the null hypothesis if P < 0.05. Otherwise, we accept the null hypothesis.

Statistical software: The statistical software used for analysis of the data was SPSS 20, IBM, Armonk, NY, USA. Microsoft Word and Excel were used to generate graphs and tables.

Distribution of scores in each group: Sample size in each group was 20.


   Results Top


The highest mean score was recorded in Group 1 followed by Group 3 and Group 2, whereas Group 4 showed no zone of inhibition [Table 3] and [Figure 11].
Table 3: Mean values of the diameters of zones of inhibition with standard deviations

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Figure 11: Comparison of mean values of diameters of zones of inhibition of the four groups

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Unpaired t-test was used for intergroup comparisons.

Group 1 (nanosilver) and Group 2 (chitosan)

Mean score difference calculated was 9.3 with P < 0.0001. This result suggests that there was a statistically significant difference between Groups 1 and 2 [Table 4] and [Figure 12].
Table 4: Differences between Group 1 and Group 2 with respect to mean score difference

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Figure 12: Comparison of Groups 1 and 2

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Group 1 (nanosilver) and Group 3 (fluoride)

Mean score difference calculated was 4.13 with P < 0.0001. This result suggests that there was a statistically significant difference between Groups 1 and 3 [Table 5] and [Figure 13].
Table 5: Differences between Group 1 and Group 3 with respect to mean score difference

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Figure 13: Comparison of Groups 1 and 3

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Group 2 (chitosan) and Group 3 (fluoride)

Mean score difference calculated was 5.17 with P < 0.0001. This result suggests that there was a statistically significant difference between Groups 2 and 3 [Table 6] and [Figure 14].
Table 6: Differences between Group 2 and Group 3 with respect to mean score difference

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Figure 14: Comparison of Groups 2 and 3

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   Discussion Top


Maintenance of good oral hygiene is the key to the prevention of dental diseases. The primary etiological factor for dental diseases is dental plaque.[2],[10] The formation of plaque on the tooth surface is characterized by the progression from a limited number of pioneer microbial species to the complex flora of mature dental plaque. This progression involves initial adherence of bacteria to the salivary pellicle and subsequent accumulation by growth and interbacterial adherence. Ultimately, the tooth surface gets coated with a dense, complex microcommunity that ends up in the destruction of hard enamel tissue.[11]

S. mutans was selected as the test organism in this study because of there being promising evidence about it being the primary human odontopathogen in the etiology of dental caries.[12] As such, dental caries is a diagnosable and treatable infection. Aciduricity appears to be the most consistent attribute of S. mutans that can be associated with both its selection in stagnant areas and cariogenicity. Other aciduric species such as S. sobrinus appears to be important primarily in smooth surface decay and as such, may be a cariogenic determinant when rampant decay occurs. Colonization by S. mutans occurs after tooth eruption, and if the fissures become colonized in their depths, then decay may be inevitable. However, if this colonization is delayed until the fissure depths are occupied by other bacteria, there is possibility that decay will not occur or its occurrence will be greatly reduced. This understanding of the ecology of S. mutans suggests that treatment strategies which interfere with the colonization of S. mutans may have a profound effect on the incidence of dental decay in human populations.[13]

The modified agar well diffusion method is the method of choice for antimicrobial sensitivity test, in which the bacterial growth is done by streaking pure culture on agar plates. The same method was utilized by Prasanth (2011) to check the antimicrobial efficacy of five toothpastes and five mouthwashes.[14] To culture facultative anaerobic S. mutans under microaerophilic/aerobic conditions in the absence of an anaerobic jar, the agar double-layer technique is a standard method. Cury et al. and Gomes et al. used the pour plate technique to culture S. mutan s for antibacterial activity test.[15],[16] In the present investigation, as in previous reports, the strain of S. mutans was cultured by the agar double-layer technique, and Mitis Salivarius agar, which contains 10% of sucrose, was used as the medium in order to simulate the oral conditions. Dental caries is caused by the combined effects of plaque accumulation and complex microbial communities, especially S. mutans, which is the first important colonizer of tooth surface in people who consume large amounts of sugary foods.[12],[17] Under this culture method, S. mutans strain could grow as a uniform lawn, although incubation was done under aerobic conditions. Hence, any antimicrobial agent is considered effective if the size of inhibition zone produced by it measures 2 mm or more.[18]

The activities of oral microflora being responsible for mouth odor and most oral diseases are not in doubt. The need to keep these oral organisms to a level consistent with oral health by antimicrobial agent inclusion in dentifrice has been stressed.[19] When these substances are added to oral products, they kill microorganisms by disrupting their cell walls and inhibiting their enzymatic activity. They also prevent bacterial aggregation, slow multiplication, and release endotoxins.[51] Several clinical studies including that by Ciancio S (2003) have demonstrated the inhibitory effects of antimicrobial dentifrice on oral bacteria and gingiva.[19]

Data from the present study are in support of this assertion as all the three investigated dental care products exhibited wide variations in their effectiveness against the test microorganism, S. mutans, a feature that may have been largely due to their antimicrobial active ingredients [Table 1]. All the tested dentifrices (Groups 1, 2, and 3) showed diameter of zones of inhibition more than 2 mm (which is considered positive) in contrast to the control (Group 4) which showed no zone of inhibition. The selected dentifrices had most of the ingredients similar in their manufacturing. Such a selection was done to avoid any deviation from the intended active ingredient's antimicrobial action. Other than the antimicrobial agent to be tested, all the other ingredients were regularly present in common dentifrices. Nanosilver- and chitosan-containing toothpastes did not contain any fluoride in their composition. Hence, the evaluation of the antimicrobial action can be regarded as more accurate in relation to the agent tested.

Among all the investigated toothpastes, Group 1 emerged as the most effective, based on the mean diameter of the zone of microbial inhibition produced by the toothpastes in agar well diffusion method, against the tested microorganism, i.e., S. mutans. The mean diameter of zone of inhibition was 20.14 ± 0.96 mm. This exceptional ability of Group 1 to retain its in vitro antimicrobial activity against S. mutans is notable. This might be due to the presence of nanosilver in its formulation.[20],[21],[22],[23],[24],[25],[26],[27],[28],[29],[30],[31],[32],[33] This becomes more plausible as the utility and effectiveness of silver (Ag) nanoparticles in health-care industries has been reviewed by Prabhu and Poulose.[34]

The mechanism of the inhibitory effects of Ag ions on microorganisms is partially known. Some studies have reported that the positive charge on the Ag ion is crucial for its antimicrobial activity through the electrostatic attraction between negatively charged cell membrane of microorganism and positively charged nanoparticles.[35],[36],[37] In contrast, Sondi and Salopek-Sondi reported that the antimicrobial activity of Ag nanoparticles on Gram-negative bacteria was dependent on the concentration of Ag nanoparticle and was closely associated with the formation of “pits” in the cell wall of bacteria. Then, Ag nanoparticles accumulated in the bacterial membrane caused the permeability, resulting in cell death.[38] However, because those studies included both positively charged Ag ions and negatively charged Ag nanoparticles, it is insufficient to explain the antimicrobial mechanism of positively charged Ag nanoparticles. Therefore, we expect that there is another possible mechanism. Amro et al. suggested that metal depletion may cause the formation of irregularly shaped pits in the outer membrane and change membrane permeability, which is caused by progressive release of lipopolysaccharide molecules and membrane proteins.[39]

When evaluating any toothpaste, its possible adverse effects have to be taken into consideration. Gaillet and Rouanetindicated that Ag nanoparticles in toothpaste and other products may be associated with the development of inflammations of the gastrointestinal tract.[40] However, because toothpaste is indicated for topical rather than systemic use and that too in small quantities, adverse reactions are unlikely.

Next to nanosilver, Group 3 showed the maximum antibacterial effect against S. mutans. The mean diameter of inhibition was evaluated to be 16.01 ± 2.68 mm, which was significant. This may be due to the ingredients present in their formulations. This dentifrice contained sodium fluoride (1000 ppm) as an active ingredient. Fluorides are abundantly used in many oral health products including toothpastes and mouthrinses as they help in caries prevention. If formulated correctly and used as directed, fluoride toothpaste helps to safely and effectively prevent tooth decay. The ability of fluoride to inhibit or even reverse the initiation and progression of dental caries has been well documented.[41] However, if the bacterial challenge is too high, it is not possible for fluoride to overcome the challenge completely.[42] In a previous study, Jenkins stated that fluoride products such as toothpaste formulations have shown to reduce caries between 30% and 70% compared with no fluoride therapy.[43] A combination of nanosilver and fluoride was created as nanosilver fluoride (NSF) and tested by Santos et al. and the authors demonstrated that NSF is effective to arrest active dentinal caries and does not cause staining.[44]

Group 2 was the least effective as compared to the other two toothpastes, but still showed significant effect when compared to the control group (Group 4). The mean diameter of zone of inhibition in this group was 10.84 ± 0.27 mm. This may be due to the presence of chitosan as the active ingredient. The general properties and applications of chitosan have been reviewed by Dutta et al. (2002) where they have suggested greater antierosive and antiplaque activities of chitosan in contrast to its antimicrobial activity.[45] A possible explanation of the lesser antimicrobial effect of chitosan can be the chemical interaction between chitosan and an anionic-forming detergent as chitosan is itself a multicationic compound.[46]

Chitosan is a natural polysaccharide derived from chitin, mainly from the shells of shrimps and other crustaceous. It is a biopolymer which is positively charged and strongly binds with its amino groups to surfaces with negative zeta potential, such as dental hard tissues.[45] The reactive molecule chitosan reacts with the enamel surface and leads to a coverage of, at least, a thin, ubiquitous chitosan layer. Such chitosan-based layers have a thickness of few nanometers and are, therefore, not visible under scanning electron microscope. Various studies have evaluated the antierosive property of chitosan.[47],[48],[49] Schlueter et al. (2013) suggested that a positive control of chitosan without abrasive challenges was able to reduce tissue loss by 78%.[50]

It is known that a balance exists in a person's oral microbial population. If this balance is lost, opportunistic microorganisms can proliferate, enabling the initiation of disease processes. Therefore, the formulation identified as having the largest microbial inhibition zone, and thus probably having the strongest antimicrobial properties, may not be necessarily superior to that with smaller diameter inhibition zones. Because the formulation used in vivo is likely to be diluted by saliva, the level to which antimicrobial properties are buffered or lost in dilution in vitro is of interest.[51]

This testing method also functioned as a screening method, which may not have been able to detect the effects of a chemical agent that does not diffuse through the agar matrix. More importantly, the test was conducted in vitro, so it cannot be assumed that the results of antimicrobial efficacy could be proportional or transferable to the oral cavity and translated into clinical effectiveness. Studies have demonstrated that the bacteria in biofilm forms such as plaque have decreased sensitivity to antibacterial agents. Moreover, formulations for topical antimicrobial oral use, such as dentifrices, must be able to penetrate the biofilm matrix and deliver the active agents quickly because exposure times are limited under actual conditions. Nevertheless, the in vitro method is a well-established technique that is used commonly in screening the antimicrobial efficacy of chemicals before in vivo testing.


   Conclusions Top


This in vitro comparative study assessed the antimicrobial efficacy of nanosilver-, chitosan-, and fluoride-containing toothpastes against S. mutans through agar well diffusion method. It was hence concluded that:

  • All the tested experimental groups were found to have an antibacterial efficacy against S. mutans
  • Nanosilver had the highest antibacterial efficacy against S. mutans followed by fluoride toothpaste and chitosan
  • The control group, that is, saline showed no antibacterial effect.


Hence, it can be concluded that the toothpaste formulations containing nanosilver particles can be an effective method of plaque control.

Furthermore, chitosan, having an acceptable antibacterial effect as well as having an already established antierosive property, can be a promising plaque control method.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.



 
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    Figures

  [Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5], [Figure 6], [Figure 7], [Figure 8], [Figure 9], [Figure 10], [Figure 11], [Figure 12], [Figure 13], [Figure 14]
 
 
    Tables

  [Table 1], [Table 2], [Table 3], [Table 4], [Table 5], [Table 6]



 

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    Abstract
   Introduction
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   Results
   Discussion
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  2005 - Journal of Indian Society of Pedodontics and Preventive Dentistry | Published by Wolters Kluwer - Medknow 
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