Australian Dental Journal

Australian Dental Journal The official journal of the Australian Dental Association Australian Dental Journal 2015; 60: 368 374 doi: 10.1111/adj.12247 Antibacterial activity of fluoride compounds and herbal toothpastes on Streptococcus mutans: an in vitro study JP Randall,* WK Seow,* LJ Walsh* *School of Dentistry, The University of Queensland, Brisbane. ABSTRACT Background: Streptococcus mutans is an important bacterial species implicated in dental caries. This laboratory study compared the antimicrobial activity of a number of fluoride containing and herbal dentifrices and their components against S. mutans. Methods: An agar diffusion method was used with Mueller Hinton agar. Wells were filled with either 10 commercial fluoride or 6 herbal dentifrices, or with solutions of various fluoride compounds, sodium lauryl sulphate, sodium benzoate, chlorhexidine digluconate or triclosan. Diameters of zones of bacterial growth inhibition surrounding the wells were measured using a micrometer. Results: Significant differences were found for growth inhibition between the 10 fluoridated dentifrices (p < ), with Colgate Total having the greatest effect. There was not a direct correlation with fluoride type or fluoride concentration. The antibacterial activities of the 6 herbal toothpastes varied, with Herbal Fresh being the strongest. Sodium lauryl sulphate showed strong antimicrobial activity against S. mutans at the levels used in dentifrices. Conclusions: Antimicrobial activity of commercial dentifrices against S. mutans may be exerted by components other than fluoride. Ingredients such as triclosan and sodium lauryl sulphate have larger antimicrobial effects than fluorides in this model. Keywords: Antibacterial, fluoride, herbal, Streptococcus mutans, toothpaste. Abbreviations and acronyms: SLS = sodium lauryl sulphate. (Accepted for publication 22 October 2014.) INTRODUCTION Dental caries is a common condition which reflects the emergence of cariogenic bacteria and an imbalance between mineral loss and mineral gain. 1,2 Of the species implicated in dental caries, a large body of epidemiologic evidence links Streptococcus mutans to the initiation of dental caries. 3 Most individuals harbour several strains of this bacteria, 4 and the organism is often found at high levels in patients with rampant caries, 5 particularly in association with lactobacilli. 4,6 The effectiveness of fluoride containing dentifrices in preventing dental caries is well documented. At fluoride levels of 1000 ppm, effects on the balance of demineralization and remineralization are more important than any influence on bacterial metabolism. 7,8 Past studies have found that different fluoride containing dentifrices have widely differing antimicrobial effects, 9 which may explain why some confer greater protection than others. The antimicrobial actions of different forms of fluoride also vary considerably. 10 12 With regard to cariogenic bacteria, the extent of the antimicrobial activity of dentifrices is unclear. Accordingly, the aims of the present study were to measure the anti-s. mutans activity of 10 over-the-counter fluoridated dentifrices and 6 herbal dentifrices, and compare this to the antimicrobial activity of key ingredients. These toothpastes were selected because they are popular among consumers and contain different ingredients. MATERIALS AND METHODS An agar diffusion method was used to assess anti- S. mutans activity. Mueller Hinton agar plates (Thermo Scientific, Thebarton, Australia) that were 90 mm in diameter were used. Up to five wells were created in the agar of each plate by removing plugs cut with a sterile 5 mm diameter stainless steel biopsy punch. The wells were spaced approxi- 368 2015 Australian Dental Association

Antibacterial activity of toothpaste on Streptococcus mutans mately 30 mm apart, and were 20 mm from the outer edge of the plate. Each well held 0.1 ml of the undiluted test sample of dentifrice. Each plate contained a negative control well with 150 mmol/l phosphate buffered saline (PBS) solution (ph 7.20), and a positive control of 0.25% chlorhexidine diacetate in PBS. The first part of the study examined the anti-s. mutans activity of 15 common over-the-counter dentifrice brands used in Australia, and a stannous fluoride gel. Details of the products are listed in Tables 1 and 2. The sample included the available herbal dentifrices used in Australia as well as prominent products in the market, so that there could be a sufficient range of fluoride concentrations. The second part of the study assessed the anti-s. mutans activity of specific ingredients found in the above products, namely sodium lauryl sulphate (SLS) (Amresco, Solon, Ohio, USA), sodium fluoride (BDH Laboratory Supplies, Poole, England), stannous fluoride, sodium monofluorophosphate, triclosan, chlorhexidine diacetate and sodium benzoate (all obtained from Sigma Aldrich, St Louis, USA). The final concentrations used for testing the individual components ranged from 0.25 to 15% w/v in sterile PBS. As with the dentifrices, a negative control (sterile PBS) and positive control (0.25% chlorhexidine) was included in each agar plate. To prevent dislodgement during inoculation, all wells with liquid components (rather than dentifrices) contained inserts of sterile filter paper. After loading the wells, the plates were inoculated with S. mutans ATCC 25175, which was obtained from the American Type Culture Collection (ATCC; Manassas, USA). Stocks were maintained in 15% glycerol at 80 C. The bacteria were revived from freeze-dried vials and cultured on trypticase soy agar (Becton Dickinson, Franklin Lakes, USA) supplemented with 5% defibrinated sheep blood (Equicell, Melbourne, Australia) for 7 days at 37 C, and then subcultured in brain heart infusion broth (BHI; Becton Dickinson, Franklin Lakes, USA) and incubated for 2 3 days at 37 C. Viability was checked by subculture, and the purity of cultures was monitored by Gram staining and colonial morphology. Bacterial identification was carried out using the API rapid ID 32 Strep and API 50 CHL identification kits (BioMerieux SA, Marcy l Etoile, France). Suspensions of S. mutans were prepared to a McFarland optical density standard no. 1 (Biomerieux, Marcy l Etoile, France), and 3 ml added to 3 ml of Mueller Hinton agar melted at 45 C. The S. mutans suspensions in agar were mixed thoroughly, and then poured evenly over the surface of each agar plate. The plates were inverted and incubated at 37 C in5%co 2 for 24 hours. At the end of this incubation time, bacterial growth was confluent on the agar surface except at areas of growth inhibition where there was a clear area around the test well. The diameters of each area of growth inhibition were measured to the nearest 0.5 mm by viewing the bottom of the agar plate. A total of 15 replicates were performed for each agent. STATISTICAL ANALYSIS Means and standard deviations for zones of bacterial growth inhibition were calculated. As datasets showed normal distributions, one-way analysis of variance (ANOVA) and Tukey Kramer post hoc tests assessed differences between groups. The threshold for significance was p < 0.05. RESULTS Fluoride dentifrices Data for zones of inhibition are shown in Table 1. All 10 fluoride dentifrices demonstrated antimicrobial activity. Colgate Total showed the largest zone of growth inhibition (38.3 2.2 mm) and Colgate Gel Kam the least (7.2 0.2 mm). There was wide variation between these extremes for the products tested. The relative effectiveness of products is shown in Fig. 1. There was no correlation of the ranking with fluoride concentrations. Differences between products are shown in Table 2. Herbal toothpastes Data for zones of inhibition are shown in Table 1. Only two herbal dentifrices demonstrated antimicrobial activity (Herbal Fresh and Macro), neither of which contained fluoride. The only herbal dentifrice with fluoride (Herbal Brite with 1000 ppm) did not give clear zones of growth inhibition. Pure fluoride compounds The zones of growth inhibition for the three fluoride compounds tested are shown in Table 2. Sodium fluoride and sodium monofluorophosphate did not show any growth inhibition against S. mutans at the concentrations found in dentifrices. In contrast, stannous fluoride gave growth inhibition of 24.3 mm for a 15% solution and a 9.5 mm for a 2% solution. Other components The zones of growth inhibition for chlorhexidine and triclosan are shown in Table 3. Each showed a clear dose response curve. Chlorhexidine was superior to triclosan at each concentration tested. At 0.25%, 2015 Australian Dental Association 369

JP Randall et al. Table 1. Dentifrices and their inhibition zones Product and manufacturer Ingredients Fluoride type and ppm Sensodyne Repair (England) calcium sodium phosphosilicate 5%, novamint flavour 1.03%, titanium dioxide 1%, limonene, potassium acesulfame, carbomer 1.08% di-sodium fluorophosphate = 1450 ppm Sensodyne Total Care sorbitol, glycerol, silica, titanium dioxide, saccharin sodium, mint flavour, cocamidopropyl betaine, xanthan gum, macrogol 0.221% sodium fluoride = 1000 ppm Pronamel (USA) potassium nitrate 5%, titanium dioxide 0.1%, sorbitol, glycerol, silica, macrogol, cocamidopropyl betaine, flavour, xanthan gum, saccharin sodium, titanium dioxide 0.32% sodium fluoride = 1450 ppm Colgate Pro Relief (Brazil) calcium carbonate 30-60%, sorbitol 10-30%, L-arginine <10%, flavour, sodium silicate, carmellose sodium, sodium bicarbonate, titanium dioxide, acesulfame potassium, xanthan gum, sucralose, limonene. 1.1% sodium MFP = 1450 ppm Colgate Sensitive Multi Colgate Total sorbitol 10-30%, synthetic amorphous silica 10-30%, glycerin <10%, potassium citrate <10%, lauryl alchol <0.1%, sodium chloride <0.1%, sodium chloride <0.1%, pentapotassium triphosphate <0.1%, di-potassium hydrogen orthophosphate, sodium fluoride <0.1% silicon dioxide, glycerol, sorbitol, PVM/MA co-polymer, flavour, carrageenan, titanium dioxide, saccharin sodium 0.76% sodium MFP = 1000 ppm 0.22% sodium fluoride = 1000 ppm Colgate Neutrafluor 5000 plus (USA) Colgate Sparkling Gel (Spiderman) amorphous silica 10-20%, polyethylene glycol 1-5%, di-potassium hydrogen orthophosphate 0-0.1% sorbitol 30-60%, synthetic amorphous silica 10-30%, tetrasodium pyrophosphate <1%, spearmint flavour <1% 1.1% sodium fluoride = 5000 ppm 0.22% sodium fluoride = 1000 ppm Antibacterial agents identified Inhibition zone Comparison P value SLS 1.1% 30.6 2.9 SensTot Pronam ColProRel ColSensMult SLS 9.8 0.7 Pronam ColProRel ColSensMult - 10.9 1.4 ColProRel ColSensMult SLS <5% 27.2 3.5 ColSensMult SLS <5% 28.7 1.7 MacAdvEb 0.1711 (NS) 0.0093 0.0156 <0.0054 SLS triclosan 0.3% 38.3 2.29 SLS 1-2% 22.8 1.1 SLS <2% 25.8 1.7 0.0008 0.3030 (NS) (continued) 370 2015 Australian Dental Association

Antibacterial activity of toothpaste on Streptococcus mutans Table 1 continued Product and manufacturer Ingredients Fluoride type and ppm Antibacterial agents identified Inhibition zone Comparison P value Macleans Advanced Enamel Lock (England) Gel Kam (USA) Herbal Brite GS America (USA) Herbal Fresh Red Seal Natural Health (New Zealand) Grants Lateral Food Corporation (Australia) Alfree Alfree Laboratories (Australia) Woolworths (Australia) Select Woolworths (China) hydrated silica, sorbitol, glycerin, PEG-6, flavours, xanthan gum, limonene, titanium dioxide, carbomer, saccharin sodium, carrageenan, sodium hydroxide, thioindigoid colour, phthalocyanine blue glycerin, hydroxyethylcellulose, flavouring, stannous fluoride 0.4% olive leaf, glycerin, silicon dioxide, xylitol, canola oil, grape seed oil, peppermint leaf, olive oil, aluminium oxide, xanthan gum, papain, d&c green no. 5 dye eucalyptus, peppermint, lemon, orange, spearmint, nutmeg, aniseed, basil, thyme, cinnamon, clove, pimento, menthol, calcium carbonate, sorbitol, glycerin, sodium lauryl sulphate, silica, light mineral oil, carboxymethyl cellulose, magnesium aluminium silicate, sodium saccharin, hydroxyl benzoates, flavours, colour. calcium carbonate, glycerin, aloe barbadensis leaf juice, xylitol, silica, sodium lauroyl sarcosinate, cellulose gum, dicalcium phosphate dihydrate, flavours, stevioside, magnesium hydroxide, potassium chloride calcium carbonate, sorbitol, glycerine, silica, sodium cocyl isethionate, cellulose gum, sodium magnesium silicate, methylparaben sorbitol, hydrated silica, glycerin, peg-8, cellulose gum, sodium lauroyl sarcosinate, sodium ascorbyl phosphate, carum petroselinum(parsley), seed oil, titanium dioxide, sodium saccharin, methylparaben, disodium EDTA sorbitol, hydrated silica, sodium sarcosinate, glycerin, PEG-14, cellulose gum, flavour, titanium dioxide, sodium saccharin, trisodium phosphate, sodium benzoate, honeysuckle extract. 0.3152% sodium fluoride = 1450 ppm 0.4% stannous fluoride = 1000 ppm F 0.22 NaF = 1000 ppm fluoride 0 SLS 24.7 1.2 7.2 0.2 SLS 21.7 0.5 0 methylparaben 0 sodium lauroyl sarcosinate methylparaben 20.6 0.9 0 The country of manufacture is shown for each product. MFP = monofluorophosphate. SLS = sodium lauryl sulphate (also known as sodium dodecyl sulphate). Data for the diameter of inhibition zones are shown as the mean (in mm) and standard deviation from 15 replicates. NS = not significant. 2015 Australian Dental Association 371

JP Randall et al. Table 2. Inhibition zones for pure fluoride components (mm) Agent 2% (2000 ppm) 4% (4000 ppm) 8% (8000 ppm) 10% (10 000 ppm) 15% (15 000 ppm) Stannous fluoride 9.5 0.4 13.2 0.8 18.8 0.8 22.2 0.7 24.3 1.1 Sodium fluoride ND ND ND ND ND Sodium MFP ND ND ND ND ND ND = none detected. MFP = monofluorophosphate. triclosan did not give growth inhibition. Growth inhibition for SLS is detailed in Table 4, which shows a dose response relationship. Sodium benzoate did not give bacterial growth inhibition. DISCUSSION The results for in vitro antimicrobial activity of commercially available dentifrices against S. mutans indicate that the overall activity exerted by any one product is not simply dependent on the concentration of fluoride present. Rather, the majority of antimicrobial activity against S. mutans is dependent on the presence of other agents, such as triclosan and SLS. By using standardized bacterial inocula, consistent storage conditions, the size and number of wells per plate, some insight into the relative performance of pure compounds and mixtures has been gained. The present study focused on recognized active agents and did not assess the flavouring components, which could also contribute to the antibacterial activity seen for each dentifrice. Colgate Total, which contains both agents as well as fluoride, gave the largest zone of growth inhibition. This dentifrice contains more antimicrobial agents than the other toothpastes tested. The result for Colgate Total is in agreement with previous assessments of triclosan/copolymer dentifrices which showed significantly stronger antimicrobial effects compared to the dentifrices that did not contain triclosan. 9 Likewise, Davies et al. 16 found that this particular triclosan/copolymer/fluoride dentifrice provided more effective plaque control when compared with nontriclosan containing dentifrices. There was no association between fluoride levels and the effects on S. mutans in the assay system used in this study. The two herbal toothpastes that showed growth inhibition both lacked fluoride, but contained SLS, which explains the antibacterial activity seen. 13 Stronger results for inhibition of growth by fluoride containing dentifrices would be expected if the study was repeated using low ph growth conditions, under which S. mutans becomes more sensitive to the effects of fluoride ions. Mueller Hinton agar has a ph of 7.3 Fig. 1 Zones of inhibition listed in order of decreasing effectiveness. Bars show standard deviations. 372 2015 Australian Dental Association

Antibacterial activity of toothpaste on Streptococcus mutans Table 3. Inhibition zones for dentifrice components (mm) Agent 0.25% 0.5% 1.25% 2.5% Chlorhexidine 16.3 0.5 17.3 0.5 21.6 1.3 24.1 2.5 Triclosan 0.0 0 6.9 3.9 9.1 1.4 18.4 2.3 Table 4. Inhibition zones of sodium lauryl sulphate (mm) Concentration Zone of inhibition (mm) 2% 28.3 1.2 1% 23.9 1.0 0.5% 14.6 2.0 0.25% 10.5 1.1 which is close to physiological ph, but does not reflect the acidic ph conditions which could be encountered in the oral cavity, particularly at sites such as the interdental regions. The ph used in any laboratory study of bacterial growth may influence the bacterial generation time and the lag time for bacterial division, which in the agar diffusion assay may impact the bacterial population that forms at the edge of the cleared zone in agar. 15 The agar diffusion method used in the study is useful for screening and comparing products for their potential antimicrobial activity. Both complex mixtures and solutions of pure compounds differ in their solubility and other physical properties, and this can affect the diffusion of the agent through the agar, and thus the growth inhibition seen. The effects seen are influenced by the diffusion coefficient of each antimicrobial agent through the water-based agar matrix. Antimicrobial agents vary in their molecular size and water solubility, and these larger molecules such as chlorhexidine would diffuse less than smaller fluoride or stannous ions. Bacteria present in biofilms tend to be much less susceptible to antimicrobial agents compared with planktonic bacteria, hence the need to use clinical trials to assess effects on dental plaque under realistic conditions in which bacteria are living in a structured biofilm. 9 Previous studies have reported large variations in the antimicrobial effects of herbal dentifrices, 17 which is consistent with the current results. Whilst Macro and Herbal Fresh demonstrated clear zones of bacterial inhibition, Select, Grants and Alfree demonstrated a reduced density of growth around the holes containing the dentifrice, but not clear inhibition. This activity could be attributed to the low solubility of their contents such as essential oils (as listed in Table 1) failing to produce clear zones of growth inhibition. Adding to this, it is known that the antimicrobial of plant-derived essential oils against bacteria varies considerably. 18 In terms of implications for clinical practice, taking into account the caveats and limitations described above, the findings of this in vitro study suggest that Colgate Total has greater antimicrobial activity against S. mutans than the other commercial dentifrices tested. While the present laboratory findings cannot be translated directly into recommendations for clinical practice, it is noteworthy that the status of dentifrices which contain triclosan is currently undergoing considerable change, with some manufacturers discontinuing such products. Recently, the European Union has banned a number of consumer products that contain triclosan because of concerns regarding possible long-term adverse effects on human health or on the environment. In contrast, recent reviews 19,20 found little evidence exists that triclosan exposure through personal care product use presents a risk of endocrine disruptive adverse health effects in humans. CONCLUSIONS Overall, Colgate Total was found to have the greatest anti-s. mutans activity among the commercial dentifrices tested. Sodium lauryl sulphate appears to exert greater antimicrobial activity than both triclosan and chlorhexidine under the conditions used in the agar diffusion assay. Finally, the results of the present study demonstrate that a significant amount of the antimicrobial activity of fluoridated dentifrices against S. mutans may reflect the combined action of nonfluoride components. Further analysis of interactions between fluoride and non-fluoride components may lead to improvements in the formulation of dentifrices to optimize their anti-caries activity. ACKNOWLEDGEMENTS We thank Dr Shaneen Leishman for assistance with laboratory work, and Janet Palmer for statistical advice. REFERENCES 1. Loesche W. Role of Streptococcus mutans in human dental decay. Microbial Rev 1986;50:353 380. 2. Marsh P. The role of microbiology in models of dental caries. Adv Dent Res 1995;9:244 254. 3. Van Houte J. Role of micro-organisms in caries etiology. J Dent Res 1994;73:672 681. 4. Corby Y, Lyons-Weiler J, Bretz WA, et al. Microbial risk indicators of early childhood caries. J Clin Microbiol 2005;43: 5753 5759. 5. Aas JA, Griffen AL, Dardis SR, et al. Bacteria of dental caries in primary and permanent teeth in children and young adults. J Clin Microbiol 2008;46:1407 1417. 2015 Australian Dental Association 373

JP Randall et al. 6. Chhour LK, Nadkarni MA, Byun R, et al. Molecular analysis of microbial diversity in advanced caries. J Clin Microbiol 2005;43:843 849. 7. Ten Cate J. Current concepts on the theories of the mechanism of action of fluoride. Acta Odontol Scand 1999;57:325 329. 8. Hamilton IR. Effects of fluoride on enzymatic regulation of bacterial carbohydrate metabolism. Caries Res 1977;11:262 291. 9. Haraszthy VI, Zambon JJ, Sreenisavan PK. Evaluation of the antimicrobial activity of dentifrices on human oral bacteria. J Clin Dent 2010;21:96 100. 10. Arnold WA, Dorow A, Langenhorst S, Ginter Z, Banoczy J. Effect of fluoride toothpastes on enamel demineralization. BMC Oral Hlth 2006;6:1 6. 11. Duckworth RM, Knoop DT, Stephen KW. Effect of mouthrinsing after toothbrushing with a fluoride dentifrice on human salivary fluoride levels. Caries Res 1991;25:287 291. 12. Den Besten P, Ko HS. Fluoride levels in whole saliva of preschool children after brushing with 0.25 g (pea sized) as compared to 1.0 g (full brush) of a fluoride dentifrice. Pediatr Dent 1996;18:277 280. 13. Moran J, Addy M, Newcombe R. The antibacterial effect of toothpastes on the salivary flora. J Clin Periodontol 1988;15:193 199. 14. Athanassiadis B, Abbott P, George N, Walsh LJ. An in vitro study of the antimicrobial activity of some endodontic medicaments and their bases using an agar well diffusion assay. Aust Dent J 2009;54:141 146. 15. Nathan P, Law E, Murphy DF, Macmillan BG. A laboratory method for selection of topical antimicrobial agents to treat infected burn wounds. Burns 1978;4:177 187. 16. Davies RM, Ellwood R, Davies GM. The effectiveness of a toothpaste containing triclosan and polyvinyl-methyl ether maleic acid copolymer in improving plaque control and gingival health: a systematic review. J Clin Periodontol 2004;31:1029 1033. 17. Lee SS, Zhang W, Li Y. The antimicrobial potential of 14 natural herbal dentifrices. J Am Dent Assoc 2004;135:1133 1141. 18. Inouye S, Takizawa T, Yamaguchi H. Antibacterial activity of essential oils and their major constituents against respiratory tract pathogens by gaseous contact. J Antimicrob Chemothy 2001;47:565 573. 19. Witorsch RJ, Thomas JA. Personal care products and endocrine disruption: a critical review of the literature. Crit Rev Toxicol 2010;40(Suppl 3):1 30. 20. Witorsch RJ. Critical analysis of endocrine disruptive activity of triclosan and its relevance to human exposure through the use of personal care products. Crit Rev Toxicol 2014;44:535 555. Address for correspondence: Dr Jasyn Randall School of Dentistry The University of Queensland 200 Turbot Street Brisbane QLD 4000 Email: jasyn.randall@uqconnect.edu.au 374 2015 Australian Dental Association