Antimicrobial Effect of Newly Formulated Toothpastes and a Mouthrinse on Specific Microorganisms: An In Vitro Study.

OBJECTIVE: The aim of this in vitro study was to assess the antimicrobial properties of newly formulated toothpastes (four toothpastes for adults and two toothpastes for kids/babies) and a mouthrinse.
MATERIALS AND METHODS: Newly formulated six different toothpastes and one mouthrinse of a single brand and commercially available five toothpastes and three mouthrinse were investigated for their antimicrobial activity against two oral pathogens, Streptococcus mutans and Candida albicans, by agar well diffusion assay. After incubation, the inhibition zone diameters were measured in millimeters and statistical analyses were performed.
RESULTS: All experimental adult toothpastes exhibited good antimicrobial activity against S. mutans and C. albicans except the experimental toothpaste D. Experimental toothpaste B exhibited the highest antibacterial activity against C. albicans and S. mutans. Experimental toothpaste for kids showed the best antimicrobial activity against S. mutans when kids' toothpastes were compared. None of the tested toothpastes for kids/babies showed antibacterial effects for C. albicans. Among the mouthrinse tested, Sensodyne mouthrinse showed the best results. Experimental mouthrinse showed significantly lower antibacterial activity against S. mutans then Sensodyne, Eludril, and chlorhexidine mouthrinse.
CONCLUSION: Although experimental toothpaste and mouthrinse formulations revealed good results in terms of antimicrobial activity to some specific microorganisms, further studies involving more bacterial species or analyzing the quality and efficacy of these products by other in vitro or in vivo tests are needed. Dental Investigation Society.

Introduction

Dental caries is a localized, progressive, and transmissible bacterial disease caused by the acidic by-products of bacterial metabolism, which results in dissolution of the dental hard tissues. 1 Dental plaque that is an oral microbial biofilm formed on dental surfaces is composed of a large variety of different oral microbial strains and species. Microbiological shifts and biochemical activities of complex microcommunities within the dental plaque result in dental caries. 2 Streptococcus strains are the main group of microorganisms associated with the caries process. Streptococcus mutans is one of the major cariogenic pathogens, which metabolizes fermentable carbohydrates and synthesizes an extracellular polysaccharide matrix that allows tight adherence of the organisms to the tooth surface and leads to the decalcification of the tooth structure. 3 4 In addition to S. mutans , other microorganisms have been shown to be involved in the formation of cariogenic biofilms. Candida albicans is the most frequently yeast group isolated from the oral cavity and primarily associated with the mucosal infections (oral candidiasis) and denture-related stomatitis. 5 Several studies report that it has been detected together with S. mutans in the dental plaque of children with carious teeth. 6 7

Poor oral hygiene that leads to the accumulation of dental plaque on tooth surfaces is the main reason of carious lesions. The most effective measure for the prevention of plaque formation is the mechanical removal of dental plaque by regular tooth brushing, but its efficacy is highly dependent on the ability and cooperation of the individual. 8 9 Therefore, chemical plaque control agents such as toothpastes and mouthrinse should be used due to their potential role as a delivery system for antimicrobials. 9 10 Various chemical agents such as triclosan, chlorhexidine (CHX), metal ions, and essential oils have been added to toothpastes and mouthrinse to provide antimicrobial activity. The antibacterial effect of fluoride is well established and depends on the influx of hydrogen fluoride into bacterial cells and the dissociation to the H + and F – ions in the cytoplasm. Fluoride also has been known as an inhibitor of bacterial enzymes, such as adenosine triphosphatase and enolase. 11 Despite the proven antibacterial efficiency of single chemical agents, this antibacterial activity may diminish or increase due to their interaction with other constituents in the toothpastes or mouthrinses. 12 For instance, fluoride can be inactivated when calcium containing abrasives were used. Similarly, fluoride may react with silica to form fluorosilicates if a sufficient amount of detergent is not present. 11 As a result, it is essential to examine every new toothpaste or mouthrinse formulations in their complete form. Therefore, the aim of this in vitro study is to assess the antimicrobial properties of newly formulated toothpastes (four toothpastes for adults and two toothpastes for kids) and a mouthrinse containing natural compounds.

Materials and Methods

Newly formulated oral hygiene products (six different toothpastes and one mouthrinse) developed by a single company and commercially available products of varying companies (five toothpastes and three mouthrinse) were investigated for their antimicrobial activity against two oral pathogens, S. mutans (RSKK 07038) and C. albicans (ATCC 10231) by agar well diffusion assay. The manufacturer's name and the ingredients of the toothpastes and mouthrinse evaluated in the present study are shown in Table 1 .

Table 1

The tested products and their ingredients

Product name	Manufacturer	Ingredients as listed on packages
Experimental toothpaste A (for sensitive teeth)	Biota Laboratory/Istanbul	Glycerin, sorbitol, xanthan gum, stevia, abrasive silica, thickener silica, titanium dioxide, sodium lauryl sulfate, potassium nitrate, clove oil, miswak powder
Experimental toothpaste B (whitening and stain removal)	Biota Laboratory/Istanbul	Glycerin, sorbitol, xanthan gum, stevia, xylitol, abrasive silica, thickener silica, titanium dioxide, sodium lauryl sulfate, mint flavor, miswak powder
Experimental toothpaste C (anticavity)	Biota Laboratory/Istanbul	Glycerin, sorbitol, xanthan gum, stevia, xylitol, abrasive silica, thickener silica, titanium dioxide, sodium lauryl sulfate, mint flavor, miswak powder, propolis extract
Experimental toothpaste D (anti-adenomatous lesions)	Biota Laboratory/Istanbul	Glycerin, sorbitol, xanthan gum, xylitol, abrasive silica, thickener silica, titanium dioxide, cocamidopropyl betaine, sodium lauroyl sarcosinate, mint flavor, miswak powder, liquorice, blackberry extract
Experimental toothpaste for kids	Biota Laboratory/Istanbul	Glycerin, sorbitol, xanthan gum, stevia, xylitol, abrasive silica, thickener silica, titanium dioxide, cocamidopropyl betaine, sodium lauroyl sarcosinate, miswak powder, caseine, preprobiotic
Experimental toothpaste for babies	Biota Laboratory/Istanbul	Glycerin, sorbitol, gellan gum, xylitol, silica, aroma
Colgate anticavity toothpaste with miswak	Colgate-Palmolive Co.	Sodium monofluorophosphate 1.1%, calcium carbonate, aqua, sorbitol, sodium lauryl sulfate, hydrated silica, aroma, cellulose gum, magnesium aluminum silicate, sodium carbonate, sodium saccharin, benzyl alcohol, sodium bicarbonate, Commiphora myrrha oil, chamomilla recutita flower extract, melaleuca alternifolia oil, salvia officinalis oil, Salvadora persica , Mentha piperita oil, eucalyptus globulus oil, citrus medica limonum oil, limonene, Cl 77492, Cl 12085
Sensodyne full protection and whitening toothpaste	GlaxoSmithKline	Water, hydrated silica, sorbitol, glycerin, pentasodium triphosphate, PEG-8, flavor, titanium dioxide, cocamidopropyl betaine, sodium methyl cocoyl taurate, xanthan gum, sodium hydroxide, sodium saccharin, sucralose, potassium nitrate (5%), sodium fluoride (0.15% w/v fluoride ion)
Sensodyne repair and protect toothpaste	GlaxoSmithKline	Glycerin, PEG-8, Calcium sodium phosphosilicate (NOVAMIN), sodium lauryl sulfate, sodium monofluorophosphate, aroma, titanium dioxide, carbomer, potassium acesulfame, limoneneContains sodium monofluorophosphate 1.08% w/w (1450 ppm fluoride)
Ipana Pro-Expert All in One	Procter and Gamble Co.	Aqua, sorbitol, hydrated silica, sodium lauryl sulfate, cellulose gum, aroma, sodium gluconate, stannous chloride, carrageenan, CI 77891, zinc citrate sodium fluoride 0.31%
R.O.C.S kids toothpaste	WSD Laboratory.	Aqua, silica, glycerin, xylitol (10%), olaflur, hydroxyethyl cellulose, polysorbate-20, aroma, cocamidopropyl betaine, titanium dioxide, sodium saccharin, methylparaben, propylparaben, potassium hydroxide, benzyl alcohol, fluoride (500 ppm)
R.O.C.S baby toothpaste	WSD Laboratory.	Aqua, glycerin, xylitol (10%), silica, chamomilla recutita ( Matricaria ) flower extract, potassium (sodium) alginate, sodium benzoate, xanthan gum
Experimental mouthrinse	Biota Laboratory/Istanbul	Glycerin, sorbitol, polysorbate 20, stevia, poloxamer 407, CPC, mint flavor, menthol, propolis extract, eucalyptol, thymol, chlorhexidine digluconate
Listerine zero mouthrinse	Johnson and Johnson Cons. Co	Water, sodium fluoride 0.02%, sorbitol, propylene glycol, poloxamer 407, sodium lauryl sulfate, flavor, sodium benzoate, phosphoric acid, eucalyptol, methyl salicylate, thymol, sodium saccharin, menthol, disodium phosphate, sucralose, red 40, blue 1
Sensodyne mouthrinse	GlaxoSmithKline Co.	Aqua, glycerin, sorbitol, poloxamer 338, PEG-60 hydrogenated castor oil, potassium chloride, aroma, cetylpyridinium chloride, sodium fluoride (250 ppm), methylparaben, propylparaben, sodium benzoate, sodium saccharin, disodium phosphate, sodium phosphate
Eludril antibacterial mouthrinse	Pierre Fabre Oral Care	Glycerin, alcohol, aqua, aroma, benzyl alcohol, chlorhexidine digluconate, chlorobutanol, Cl 16255, citral, citronellol, diethylhexyl sodium sulfosuccinate, eugenol, limonene, linalool, menthol

Experimental toothpastes were cultivated in Tryptic soy agar (TSA) (Merck), MacConkey agar (Merck), and yeast extract glucose chloramphenicol (YGC) agar (Merck) media to assess the microbial contamination before the antimicrobial activity test. TSA and MacConkey agar were incubated at 35°C for 24 hours, while YGC agar was at 25°C for 5 days. After the incubation period, no bacteria, yeast, or molds growth was observed in the samples. All mouthrinse and toothpastes were used at 1:1 dilution. Sterile deionized water was used as a negative control group in toothpaste and mouthrinse comparisons. A 0.2% solution of CHX digluconate was used as a positive control group when comparing mouthrinse.

S. mutans was cultivated in Columbia medium supplemented with 5% sheep blood (COS medium, bioMérieux) and C. albicans was cultivated in Sabouraud 4% dextrose agar. Both were incubated at 32.5°C ± 2.5°C for 44 to 52 hours. Fresh microorganism cultures were adjusted to McFarland 0.5 (10 8 ) turbidity standards in 0.9% NaCl 2 solution. Nutrient agar plates were seeded with 100 µL broth cultures of each isolate. Three wells per plate of 8 mm in diameter were prepared in each seeded agar plate and each well was filled with 50 μL of the diluted solutions. The Petri dishes were incubated under the same growth conditions mentioned above. After the incubation period, the inhibition zones formed were measured in millimeters.

Statistical Analysis

The data were analyzed using SPSS 15.0 (IBM, Armonk, New York, United States) program. Kruskal–Wallis test was used for multiple comparisons and Mann–Whitney U-test was used in analyzing the differences between two groups.

Results

The results indicated that all toothpastes for adults were effective in inhibiting the growth of S. mutans and C. albicans , except Colgate anticavity with miswak toothpaste and experimental toothpaste D that did not produce inhibition zone for C. albicans ( Table 2 ). Experimental toothpaste D showed the lowest antibacterial effect against S. mutans among all groups. Experimental toothpaste B exhibited the highest antibacterial activity against C. albicans and S. mutans .

Table 2

Zones of inhibition produced by adult toothpastes against Streptococcus mutans and Candida albicans

	Mean ± SD (median)
	Streptococcus mutans	Candida albicans
Abbreviation: SD, standard deviation.Kruskal–Wallis test. ** p < 0.01. Note: Different superscript letters indicate statistical significance.
Colgate anti-cavity toothpaste with miswak a	21.5 ± 0.5 (21.5) b,c,d,f,h,i	0 ± 0 (0) b,c,d,e,f,g
Sensodyne full protection and whitening toothpaste b	19.7 ± 0.6 (20) a,d,f,g,h,i	15 ± 1 (15) a,c,d,g,h,i
Sensodyne repair and protect toothpaste c	20 ± 0 (20) a,d,f,g,h,i	12.7 ± 0.6 (13) a,b,d,f,h,i
Ipana Pro-Expert All in One d	18.2 ± 0.3 (18) a,b,c,e,f,g,h,i	10 ± 0 (10) a,b,c,f,h,i
Experimental toothpaste A (for sensitive teeth) e	21.3 ± 1.2 (22) d,f,h,i	11.7 ± 2.9 (10) a,h,i
Experimental toothpaste B (whitening and stain removal) f	23.7 ± 0.6 (24) a,b,c,d,e,h,i	15 ± 0 (15) a,c,d,g,h,i
Experimental toothpaste C (anticavity) g	22.7 ± 0.6 (23) b,c,d,h,i	11.3 ± 1.2 (12) a,b,f,h,i
Experimental toothpaste D (antiadenomatous lesions) h	12.3 ± 0.6 (12) a,b,c,e,f,g,i	0 ± 0 (0) b,c,d,e,f,g
Sterile deionized water i	0 ± 0 (0) a,b,c,d,e,f,g,h	0 ± 0 (0) b,c,d,e,f,g
p- Value	0.003**	0.005**

When the toothpastes for kids/infants were compared, experimental toothpaste for kids exhibited largest inhibition zone for S. mutans ( p < 0.01) ( Table 3 ). None of the tested toothpastes for kids/babies showed antibacterial effect for C. albicans .

Table 3

Zones of inhibition produced by children toothpastes against Streptococcus mutans

	Streptococcus mutans , mean ± SD (median)
Abbreviation: SD, standard deviation.Kruskal–Wallis test. ** p < 0.01. Note: Different superscript letters indicate statistical significance.
R.O.C.S kids tooth paste a	9.3 ± 0.6 (9) b,c,d,e
R.O.C.S baby tooth paste b	0 ± 0 (0) a,c
Experimental toothpaste for kids c	16.8 ± 1.9 (16) a,b,d,e
Experimental toothpaste for babies d	0 ± 0 (0) a,c
Sterile deionized water e	0 ± 0 (0) a,c
p- Value	0.008**

All tested mouthrinse demonstrated a significant antimicrobial activity against S. mutans and C. albicans ( p < 0.01), and the negative control showed no activity ( Table 4 ). Sensodyne mouthrinse showed the highest effect on C. albicans , while Listerine Zero mouthrinse showed the lowest effect on S. mutans ( p < 0.05). Experimental mouthrinse showed significantly lower antibacterial activity against S. mutans than Sensodyne, Eludril, and CHX mouthrinse ( p < 0.05). There was no significant difference between the levels of S. mutans and C. albicans in other mouthrinse ( p > 0.05).

Table 4

The mean values and standard deviations of the microbial inhibition zones induced by mouthrinse against Streptococcus mutans and Candida albicans

	Mean ± SD (median)
	Streptococcus mutans	Candida albicans
Abbreviation: SD, standard deviation.Kruskal–Wallis test. ** p- Value<0.01. Note: Different superscript letters indicate statistical significance.
Listerine zero mouthrinse a	8 ± 0 (8) b,c,d,e,f	0 ± 0 (0) b,e
Sensodyne mouthrinse b	11 ± 0 (11) a,d,f	13 ± 0 (13) a,c,d,e,f
Eludril antibacterial mouthrinse c	10.7 ± 0.3 (10.5) a,d,f	0 ± 0 (0) b,e
Experimental mouthrinse d	10 ± 0 (10) b,c,e	0 ± 0 (0) b,e
Chlorhexidine digluconate e	11 ± 0 (11) a,d,f	14 ± 0 (14) a,b,c,d,f
Sterile deionized water f	0 ± 0 (0) a,b,c,d,e	0 ± 0 (0) b,e
p- Value	0.006**	0.004**

Discussion

It has been known that a sensitive bacterial balance exists in oral microflora and the loss of this balance results in emergence of potentially pathogenic bacteria and the initiation of disease process. 13 Since S. mutans is the major pathogen involved in dental plaque and caries formation, it has been chosen as the main test organism in the present study. C. albicans , the most common fungal pathogen that is involved in candidiasis, systemic infections, and even dental caries, was chosen as another pathogen for testing. If a toothpaste or mouthrinse has good inhibition properties against C. albicans, then it can be recommended for the patients who is susceptible to oral fungal infections. 14

The use of toothpaste as an adjunct to tooth brushing or the use of mouthrinse may assist to improve the effectiveness of oral hygiene practices in several ways. These include inhibiting the bacterial adhesion to tooth surface, reducing the overall rate of accumulation of plaque, and decreasing bacterial counts of saliva. 15 Antimicrobial activity was assessed by the agar diffusion method that is based on the measurements of the microbial inhibition zones against the tested microorganisms. Diffusion phenomenon depends on physical and chemical properties of tested substance, such as the diffusion coefficient and the medium where the diffusion occurs. Moreover, bacteria colonized in the dental plaque have reduced sensitivity to antimicrobial agents compared with the planktonic bacteria. 14 16 Therefore, this method can be used as a preliminary test for detecting antimicrobial activity in substances or products. The clinical relevance of these inhibition zones needs to be evaluated using in vivo models.

The largest inhibition zone for S. mutans was observed in experimental whitening and stain removal toothpaste. Three experimental toothpastes (whitening and stain removal, for sensitive teeth, anticavity types) and Colgate miswak showed higher antibacterial activity. These results can partly be attributed to the miswak content of the toothpastes. 17 A review evaluating the therapeutic effects of miswak ( Salvadora persica ) has concluded that several studies investigating the antimicrobial activity of miswak found significant antibacterial effects against bacteria implicated in periodontitis and caries progression. 18 On the contrary, Almas et al investigated the antimicrobial activity of 50% miswak extract against seven microorganisms and concluded that miswak extract had mild antimicrobial activity against S. mutans . 19 In the present study, another miswak containing toothpaste (experimental toothpaste D) showed the worst antibacterial activity against S. mutans . This controversial result may be explained by the fact that experimental toothpaste D contains cocamidopropyl betaine and sodium lauroyl sarcosinate instead of sodium lauryl sulfate that has a stronger antibacterial activity. Experimental toothpaste B and Sensodyne full protection and whitening toothpaste showed the largest inhibition zone for C. albicans compared with others. The ingredients such as titanium dioxide, xanthan gum, 20 and miswak extract possess antifungal properties.

Inhibiting the growth of S. mutans in young children during the early phases of colonization may prevent its long-term establishment in the oral cavity. Therefore, tooth brushing with toothpastes containing compounds with antibacterial activity can contribute to the prevention of caries in children. 21 In the present study, when children toothpastes were compared, only two of them (experimental toothpaste for kids and R.O.C.S kids toothpaste) were found to have antimicrobial activities. The antimicrobial activity of these products is due to the presence of ingredients such as fluoride, cocamidopropyl betaine, and sodium lauroyl sarcosinate. Toothpastes for babies that are free of these compounds did not show any antibacterial activity.

With respect to mouthrinse, Sensodyne mouthrinse showed the highest reduction in S. mutans counts. This may be due to the presence of cetylpyridinium chloride (CPC) as a major ingredient in its formulation. In a study by Latimer et al, CPC-containing mouthrinse, with and without fluoride, exhibited significant antibacterial efficacy against S. mutans . 22 Watanabe et al investigated the antibacterial efficacy of a CPC-containing mouthrinse and found that all clinical isolates of mutans streptococci were inhibited by this product. 23 Eludril antibacterial mouthrinse that contains CHX digluconate showed similarly high antibacterial activity against S. mutans. CHX has been demonstrated in in vivo and in vitro studies to reduce oral bacteria as well as inhibiting plaque formation and reducing gingivitis. 24 25 Although the experimental mouthrinse has been expected to show high antibacterial activity, since it contains CHX digluconate and CPC in its formulation, significantly lesser antimicrobial activity than Sensodyne, Eludril, and the positive control group (CHX digluconate) was observed. This difference might be attributed to the differences in the CHX concentration and the modifications in the whole formulation that may have led to discrepancies among ingredients. 25 26 Only Sensodyne mouthrinse and positive control group showed inhibitory effect against C. albicans. Although other mouthrinse contained ingredients such as CHX gluconate and CPC that have a proven efficacy against C. albicans , the lack of antifungal effect might be due to the fact that their effect may have been vitiated by another ingredient.

While the present study provides insight into the antimicrobial activity of the tested toothpastes and mouthrinse, certain limitations exist such as a limited number of tested microorganisms selected for this study. Furthermore, the test was conducted in vitro and the clinical implications were not investigated. It should be noted that in vitro results of antimicrobial efficacy may not be fully transferable to the oral cavity. Therefore, further studies should be conducted to find out the clinical efficacy of the tested toothpastes and mouthrinse that are not available commercially.

Conclusion

Based on the results of the present study, it can be concluded that all experimental adult toothpastes exhibited good antimicrobial activity against S. mutans and C. albicans except the experimental toothpaste developed for using in patients experiencing adenomatous lesions. With regard to kids/babies toothpastes, experimental toothpaste formulation prepared for kids showed the best antimicrobial activity against S. mutans . Among the mouthrinse tested, a conventionally available mouthrinse involving sodium fluoride and castor oil showed the best results. Although experimental toothpaste and mouthrinse formulations revealed good results in terms of antimicrobial activity to some specific microorganisms, further studies involving more bacteria species or analyzing the quality and efficacy of these products by other in vitro or in vivo tests are needed.