|Year : 2016 | Volume
| Issue : 4 | Page : 463-468
Antimicrobial efficacy of commercially available mouthrinses: An in vitro study
Roopavathi Kallahalli Mruthyuenjaya1, Sanjay Venugopal1, Chikkanayakanahalli Parashuramayya Sateesh1, Darshana Bennadi2, BV Renushree3
1 Department of Periodontics, Sree Siddhartha Dental College and Hospital, Tumkur, Karnataka, India
2 Department of Public Health Dentistry, Sree Siddhartha Dental College and Hospital, Tumkur, Karnataka, India
3 Department of Microbiology, Sree Siddhartha Dental College and Hospital, Tumkur, Karnataka, India
|Date of Web Publication||15-Dec-2016|
Roopavathi Kallahalli Mruthyuenjaya
Department of Periodontics, Sree Siddhartha Dental College and Hospital, Agalkote, Tumkur - 572 107, Karnataka
Source of Support: None, Conflict of Interest: None
Introduction: Oral cavity ecosystem represents a dynamic pattern. An effective plaque control measure should target plaque formation before the mature plaque is formed. Various types of chemotherapeutic agents are coming up with different antimicrobial agents in them. Hence, this study has been undertaken to know whether these antimicrobial agents are effective on common microorganisms of oral cavity which directly and indirectly contributes to plaque formation Aim: The aim of this study was to determine antimicrobial efficacy of different mouthrinses against the oral pathogens in vitro. Materials and Methods: A total of seven mouthrinses were tested for their antimicrobial activity against three oral pathogens, namely, Streptococcus mutans (MTCC 890), Escherichia coli (ATCC 25922), and Candida albicans (ATCC 10231) by well agar diffusion assay. Statistical analysis was performed using Kruskal–Wallis test. The level of significance used was P< 0.05. Results: Mouthrinse with chlorhexidine (CHX) gluconate, triclosan as main ingredients showed maximum zone of inhibition (P = 0.003) against streptococcal mutans and E. coli at 1:16 dilution and mouthrinse with CHX gluconate and zinc chloride showed maximum zone of inhibition at 1:16 dilution against Candida among seven mouthrinses used in the present study. It was also observed that zone of inhibition of all the mouthrinses decreased with the increase in dilution. Conclusion: Among mouthrinses formulations, CHX combined with other active ingredients was found to be more effective.
Keywords: Antimicrobial activity, antimicrobial agents, chlorhexidine gluconate, mouthrinse
|How to cite this article:|
Mruthyuenjaya RK, Venugopal S, Sateesh CP, Bennadi D, Renushree B V. Antimicrobial efficacy of commercially available mouthrinses: An in vitro study. J Indian Assoc Public Health Dent 2016;14:463-8
| Introduction|| |
Oral cavity ecosystem represents a dynamic pattern and it is not advisable to eliminate all bacterial microflora. The ideal condition is removing the most cariogenic and periodontopathic agents from dental plaque., Dental plaque formation begins with accumulation of Gram-positive streptococci, developed by Gram-negative anaerobic bacterial aggregation. Plaque is one of the major causes for tooth loss. Plaque leads to gingival and periodontal diseases, which affects patient's oral health as well as contribute to number of systemic diseases. Hence, to prevent plaque accumulation, many mechanical and chemical plaque control measures are available. Studies ,,,, have shown that tooth brushing alone will remove 50% of dental plaque, so additional mechanical and chemical measures are required to further reduce the bacterial load. Mouthwashes are nonsterile aqueous solutions. They are used for reducing oral bacteria, cleaning food remnants, and decreasing oral malodor.,,,,
An effective plaque control measure should target plaque formation before the mature plaque is formed. Now-a-days, we are coming across various chemical measures with different antimicrobial agents in them. Hence, this study has been undertaken to know whether these antimicrobial agents are effective on common microorganisms of oral cavity which directly and indirectly contributes to plaque formation and tooth loss. Hence, the aim of the study was to compare the antimicrobial efficacy of commercially available seven mouthrinses against the oral pathogens.
| Materials and Methods|| |
An in vitro study conducted to assess the antimicrobial efficacy of seven commercially available mouthrinses on three organisms, namely, Streptococcus mutans, E. coli, Candida albicans. The study period was about 6 months from July 2013 to December 2013.
The seven mouthrinses selected for the study were Listerine, Colgate Plax, Freshclor, Guard – OR, Amflor, Rexidin Plus, Hexidine [Table 1]. The following procedures were adapted to carry out the study.
Selection of the mouthrinses
Before the start of the study, various brands of commercially available mouthrinses were obtained. Owing to the constraints pertaining to the available time and expenditure, only seven mouthrinses were selected for the present study. Only those test products with a date of manufacturing within the last 6 months were selected for the present study for the purpose of standardization.
Preparation of subcultures of microorganisms
Pure cultures of C. albicans (ATCC 10231) and Escherichia coli (ATCC 25922) were obtained from the Department of Microbiology, Sri Siddhartha Medical College, Tumkur. Pure cultures of S. mutans (MTCC 890) were obtained from the Institute of Microbial Technology, Chandigarh, India. These test microorganisms were subcultured on specific media, recommended for different microorganisms such as brain-heart infusion for S. mutans and nutrient agar for C. albicans and E. coli.
Preparation of nutrient agar plates
A specified amount of nutrient agar powder was mixed with 200 ml of water. Then, the solution was transferred to a glass bottle, stirred, and closed with a lid and allowed to heat for approximately 30 min. This allowed sufficient time for the solution to become a homogenous and uniform liquid. A total of 12 ml of agar solution was transferred into individual glass plates and allowed to solidify. Then, the nutrient agar plates were placed in incubator for 24 h to ensure sterility. Thus, the nutrient plates were prepared for the growth of E. coli and Candida. The preparation of the agar plates for the streptococci followed the same procedures as described for E. coli and Candida. However, the medium used was brain-heart infusion agar.
Preparation of serial dilutions of mouthrinses
Mouthrinse (2 ml) mixed with 2 ml of sterile distilled water and serial dilutions of 1:1, 1:2, 1:4, 1:8, and 1:16 were made.
Determination of antimicrobial activity
The antimicrobial activity of different concentrations of the mouthrinses was determined by modified agar well-diffusion method where nutrient agar plates were seeded with 0.5 ml of 24 h broth cultures of each isolate. Brain-heart infusion agar was used for S. mutans strain and allowed to dry for 1 h. A sterile 8-mm cork borer was used to cut one central and five wells at equidistance in each of the plates. 0.2 ml of the mouthrinse dilutions was introduced into each of the five wells while the same amount of distilled water was introduced into the central well. The plates were incubated at 37°C for 24 h (48 h for yeast species). The antimicrobial activity was evaluated by measuring the diameter of zones of inhibition (in mm). All the plates were in triplicates and the experiments were repeated thrice.
The collected data were subjected for statistical analysis. The descriptive statistics and Kruskal–Wallis test were done. The level of significance used is P < 0.05.
| Results|| |
This was an in vitro study conducted to assess the antimicrobial efficacy of seven commercially available mouthrinses on three organisms, namely, S. mutans, E. coli, and C. albicans.
Comparison of Streptococcus mutans among the different mouthrinse in concentration of 1:1, 1:2, 1:4, 1:8, and 1:16
Mouthrinse F with chlorhexidine (CHX) gluconate and triclosan as a main ingredient showed maximum zone of inhibition (21.40 ± 0.40) at the dilution of 1:16 and statistically significant (P = 0.003). It was also observed that zone of inhibition of all the mouthrinses decreased with the increase in dilution [Table 2].
|Table 2: Antimicrobial efficacy of seven mouthrinses against the Streptococcus mutans at different dilutions of 1:1, 1:2, 1:4, 1:8, and 1:16|
Click here to view
Comparison of Escherichia coli among the different mouthrinses in concentration of 1:1, 1:2, 1:4, 1:8, and 1:16
Mouthrinse F with CHX gluconate and triclosan as a main ingredient shows maximum zone of inhibition (18.17 ± 0.15) and statistically significant (P = 0.003). It is also observed that zone of inhibition decreased with the increase in dilution for all mouthrinses [Table 3].
|Table 3: Antimicrobial efficacy of seven mouthrinses against the Escherichia coli at different dilutions of 1:1, 1:2, 1:4, 1:8, and 1:16|
Click here to view
Comparison of Candida among the different mouthrinses in concentration of 1:1, 1:2, 1:4, 1:8, and 1:16
Mouthrinse D with the CHX gluconate and zinc chloride as a main ingredient showed maximum zone of inhibition against Candida (25.10 ± 0.00) at the dilution of 1:16 and statistically significant (P = 0.003). Mouthrinse G with CHX gluconate as a main ingredient showed minimum zone of inhibition against Candida. It was also observed that zone of inhibition decreased with the increase in dilution [Table 4].
|Table 4: Antimicrobial efficacy of seven mouthrinses against the Candida at different dilutions of 1:1, 1:2, 1:4, 1:8, and 1:16|
Click here to view
| Discussion|| |
Clinical studies that have shown significant antiplaque activity after using antimicrobial mouthrinses frequently have used gingivitis as the clinically relevant endpoint and very few studies used the caries causing microorganisms as endpoint.
It has been established that S. mutans is the leading cause of dental caries and is considered to be the most cariogenic of all of the oral streptococci. S. mutans plays a major role in the tooth decay by metabolizing sucrose to lactic acid. S. mutans has ability to metabolize dietary sucrose and synthesize glucan by cell surface and extracellular glucosyltransferase which in turn helps the establishment of S. mutans in the dental plaque.C. albicans a yeast-like fungus is the chief causative factor of candidiasis and is the most opportunistic infection in the world. This organism is a relatively common inhabitant of the oral cavity, gastrointestinal tract, and vagina of clinically normal individuals. Thus, owing to high frequency of occurrence of oral diseases caused by Candida, it would be beneficial if oral formulations having established antimicrobial properties against Candida organism are utilized. E. coli is a Gram-negative, facultative anaerobic, rod-shaped bacterium of the genus Escherichia, that is, commonly found in the lower intestine. Studies have also shown the growth of E. coli in subgingival flora of aggressive and chronic periodontitis. Hence, there is association of E. coli to periodontal disease.
The mouthrinses, formulation F (Rexidin Plus) with CHX gluconate and triclosan, have shown maximum zone of inhibition against S. mutans and E. coli. This may be due to the presence of CHX gluconate and triclosan as major ingredients in their formulations. This observation is similar to the earlier studies carried out by the Spets-Happonen et al.,
CHX gluconate is a cationic biguanide with broad-spectrum antimicrobial action, whose effectiveness in decreasing the formation of dental biofilm (plaque) and gingivitis have been demonstrated in several clinical studies. It is considered as the positive control (gold standard), to which all other anti-plaque agents should be compared. Its advantages are based on its substantivity property. Its antibacterial action is due to an increase in cellular membrane permeability followed by coagulation of the cytoplasmic macromolecules.
Studies have shown that triclosan itself does not produce optimal plaque inhibitory effects without the addition of other chemicals which increase its antibacterial effect. Most commonly used are copolymer polyvinyl methyl ether/maleic acid (PVM/MA) copolymer and zinc citrate. They enhance surface retention of triclosan. Next to CHX, triclosan showed maximum zone of inhibition against streptococcal mutans and E. coli organism. For both triclosan toothpastes and mouthrinses, formulations usually contain one of two chemicals which increase substantivity or antibacterial activity, namely, zinc citrate or copolymer PVM/MA. Triclosan mouthrinses containing zinc citrate have been proven to reduce plaque and gingivitis in both short- and long-term studies.,,,
Similarly in short-term clinical studies carried out with triclosan/PVM/MA mouthrinses, reductions in plaque of 45% compared to a placebo rinse have been recorded using a 4-day plaque regrowth model. In longer term studies over periods as long as 6 months, reductions in the region of 20% ± 30% in plaque and gingivitis have been noted. Combination of CHX with triclosan or zinc chloride or fluoride showed more effectiveness compared to CHX gluconate alone. It was also observed that zone of inhibition of all the mouthrinses decreased with the increase in dilution. Similar observations were seen in other studies as well.,
Next to CHX formulations, essential oil-containing mouthrinses such as Listerine showed maximum zone of inhibition against Candida. Listerine contains thymol, menthol, eucalyptol, methyl salicylate as main ingredients. Its mechanism of action is through alteration of the bacterial cell wall. It has low substantivity and it is uncharged, so it favors compliance because of no dentifrice interactions. Early short-term controlled clinical studies demonstrated that the essential oil mouthrinse could also produce significant plaque and gingivitis reductions when tested in an experimental gingivitis model. A series of later longer term studies, designed in accordance with guidelines to assess the antiplaque and antigingivitis effectiveness of chemotherapeutic products (Council on Dental Therapeutics 1986), confirmed the effectiveness of the essential oil mouthrinse when used as an adjunct to usual oral hygiene procedures over a 6-month period. These studies also showed that the essential oil mouthrinse did not promote either calculus formation or extrinsic tooth stain.
It has been shown that CHX and essential oil-containing mouthrinses to be significantly more effective than the 5% hydroalcohol as negative control in reducing supragingival plaque and gingivitis. Although both mouthrinses had comparable antigingivitis effectiveness, the CHX mouthrinse was significantly more effective than the essential oil mouthrinse in reducing plaque. In addition, the CHX mouthrinse group had significantly more extrinsic tooth stain and calculus than either the essential oil or the control group.
One of the systematic reviews has reported that in long-term use, the standardized formulation of essential oil mouthrinse appeared to be a reliable alternative to CHX mouthwash with respect to parameters of gingival inflammation.
In other study, it was concluded that triclosan-containing mouthrinse is less effective in reducing the mutans streptococci count when compared to CHX.
It is followed by formulations containing amine fluoride against E. coli. It exhibited least efficacy as the ingredient lack antimicrobial activity. The amine fluoride/stannous fluoride (ASF) mouthrinse has also shown promise, and a significant amount of evidence is available that supports the use of this rinse as an adjunct to oral hygiene. A number of short-term studies demonstrated that ASF rinses reduced plaque accumulation in the absence of normal oral hygiene measures; however, these studies compared the products against a negative control or placebo. Two short-term studies compared the antiplaque activity of one product to another, but the results on the relative plaque control benifits provided by the two rinses have been equivocal. Moreover, some studies reported confilcting result on the antiplaque effects of different dosages of ASF rinse.
The plaque-inhibiting properties of ASF were demonstrated in short-term plaque regrowth studies in which the rinse was used in the absence of mechanical oral hygiene procedures. Moreover, there is scant research on the antiplaque effects of different dosages of ASF rinse. The manufacturer of ASF rinse recommends a dosage of 10 ml, whereas it was reported that when ASF is used at a dosage of 15 ml, its antiplaque activity is quite similar to that elicited by CHX.
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 the strongest antimicrobial properties may not be necessarily superior to those 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 of interest.
Some in vitro studies have shown that CHX gluoconate has the best antimicrobial activity against streptoccoccal mutans, lactobacilli, E. coli, and C. albicans.,
This testing method also functioned as a screening method and 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 mouthrinses and 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 commonly is used in screening the antimicrobial efficacy of chemicals before in vivo testing. Further studies, in vivo studies are recommended with persons' acceptance values as well.
| Conclusion|| |
In the present study, among mouthrinses formulations, CHX was found to be more effective against streptococcal mutuns, E. coli, and Candida.
Financial support and sponsorship
Conflicts of interest
There are no conflicts of interest.
| References|| |
Ghapanchi J, Lavaee F, Moattari A, Shakib M. The antibacterial effect of four mouthwashes against Streptococcus mutans
and Escherichia coli
. J Pak Med Assoc 2015;65:350-3.
Slee AM, O'Connor JR.In vitro
antiplaque activity of octenidine dihydrochloride (WIN 41464-2) against preformed plaques of selected oral plaque-forming microorganisms. Antimicrob Agents Chemother 1983;23:379-84.
Lewis K. Riddle of biofilm resistance. Antimicrob Agents Chemother 2001;45:999-1007.
Barnett ML. The role of therapeutic antimicrobial mouthrinses in clinical practice: Control of supragingival plaque and gingivitis. J Am Dent Assoc 2003;134:699-704.
Peck MT, Africa CW, Stephen LX, Johan M, Abdul M. An in-vitro
analysis of the antimicrobial efficacy of herbal toothpastes on selected primary plaque colonizers. Int J Clin Dent Sci 2011;2:28-32.
Maza JL, Elguezabal N, Prado C, Ellacuría J, Soler I, Pontón J. Candida albicans
adherence to resin-composite restorative dental material: Influence of whole human saliva. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 2002;94:589-92.
Rajendran R, Sivapathasundaram B. Shafers Textbook of Oral Pathology. 5th
ed. India: Elsevier; 2005. p. 530.
Jonarta LA. Systemic IL-1β and TNF-α production of E. coli
lipopolysaccharide-induced periodontitis model on rats. Indones J Dent Res 2010;1:49-54.
Amel Y, Bouziane D, Leila M, Ahmed B. Microbiolical study of periodontitis in the West of Algeria. World J Med Sci 2010;1:7-12.
Spets-Happonen S, Markkanen H, Pöllänen L, Kauppinen T, Luoma H. Salivary Streptococcus mutans
count and gingivitis in children after rinsing with a chlorhexidine-fluoride solution with and without strontium. Scand J Dent Res 1985;93:329-35.
Hefti AF, Huber B. The effect on early plaque formation, gingivitis and salivary bacterial counts of mouthwashes containing hexetidine/zinc, aminefluoride/tin or chlorhexidine. J Clin Periodontol 1987;14:515-8.
Herrera D, Roldán S, Santacruz I, Santos S, Masdevall M, Sanz M. Differences in antimicrobial activity of four commercial 0.12% chlorhexidine mouthrinse formulations: An in vitro
contact test and salivary bacterial counts study. J Clin Periodontol 2003;30:307-14.
Haraszthy VI, Zambon JJ, Sreenivasan PK. Evaluation of the antimicrobial activity of dentifrices on human oral bacteria. J Clin Dent 2010;21:96-100.
Arancibia R, Cáceres M, Martínez J, Smith PC. Triclosan inhibits tumor necrosis factor-alpha-stimulated urokinase production in human gingival fibroblasts. J Periodontal Res 2009;44:726-35.
Parkar SM, Thakkar P, Shah K. Antimicrobial activity of four commercially available mouthwashes against Streptococcus mutans
: An in vitro
study. Univ Res J Dent 2013;3:108-12.
Prasanth M. Antimicrobial efficacy of different toothpastes and mouthrinses: An in vitro
study. Dent Res J (Isfahan) 2011;8:85-94.
Charles CH, Mostler KM, Bartels LL, Mankodi SM. Comparative antiplaque and antigingivitis effectiveness of a chlorhexidine and an essential oil mouthrinse: 6-month clinical trial. J Clin Periodontol 2004;31:878-84.
Van Leeuwen MP, Slot DE, Van der Weijden GA. Essential oils compared to chlorhexidine with respect to plaque and parameters of gingival inflammation: A systematic review. J Periodontol 2011;82:174-94.
Moran J, Addy M, Newcombe RG, Marlow I. A study to assess the plaque inhibitory activity of a new triclosan mouthrinse formulation. J Clin Periodontol 2000;27:806-9.
Kulkarni VV, Damle SG. Comparative evaluation of efficacy of sodium fluoride, chlorhexidine and triclosan mouth rinses in reducing the mutans streptococci count in saliva: An in vivo
study. J Indian Soc Pedod Prev Dent 2003;21:98-104.
Schaeffer LM, Szewczyk G, Nesta J, Vandeven M, Du-Thumm L, Williams MI, et al. In vitro
antibacterial efficacy of cetylpyridinium chloride-containing mouthwashes. J Clin Dent 2011;22:183-6.
Gordon JM, Lamster IB, Seiger MC. Efficacy of Listerine antiseptic in inhibiting the development of plaque and gingivitis. J Clin Periodontol 1985;12:697-704.
Pizzo G, La Cara M, Licata ME, Pizzo I, D'Angelo M. The effects of an essential oil and an amine fluoride/stannous fluoride mouthrinse on supragingival plaque regrowth. J Periodontol 2008;79:1177-83.
Malhotra N, Rao SP, Acharya S, Vasudev B. Comparative in vitro
evaluation of efficacy of mouthrinses against Streptococcus mutans
, lactobacilli and Candida albicans
. Oral Health Prev Dent 2011;9:261-8.
Moeintaghavi A, Arab H, Khajekaramodini M, Hosseini R, Danesteh H, Niknami H.In vitro
antimicrobial comparison of chlorhexidine, persica mouthwash and miswak extract. J Contemp Dent Pract 2012;13:147-52.
[Table 1], [Table 2], [Table 3], [Table 4]