Author: Aneja, Kamal Rai
Date published: January 1, 2010
Despite great improvements in the global oral health status, dental caries still remains one of the most prevalent diseases [I]. The early stage of dental caries is characterized by a destruction of superficial dental structures caused by acids which are byproducts of carbohydrate metabolism by Streptococcus mutans, a cariogenic bacterium . Colonization of teeth by cariogenic bacteria is one of the most important risk factors in the development of dental diseases . S. mutans and Candida albicans are the two microbes often implicated in oral diseases, C albicans is the most common yeast isolated from the oral cavity and a common cause of oral thrush, endocarditis, septicemia, vaginitis and infection of skin, nails and lungs [3-5]. It is by far the fungal species most commonly isolated from infected root canals, showing resistance to intercanal medication [6,7]. Staphylococcus aureus is a major human pathogen, responsible for a number of hospital-acquired infections, initially colonizes several locations in the human body, but the mouth and hands are the main reservoirs for propagation of this pathogen in the hospital environment .
Individuals heavily colonized by cariogenic bacteria are considered to be at high risk for dental caries. Hence eradication of these microorganisms is important for dental treatment [H]. Prevention of oral diseases is easier than a cure. The widespread use of mouthwashes as an aid to oral hygiene is a relatively recent phenomenon in the developing countries of the world. Development work on the mouthwashes has been done mostly by the manufacturers, and the little work that has been done relates to the individual ingrethents they contain rather than to their complete formulations [12-14].
While their primary appeal is as an aid to breath freshness and cleansing the mouth, the majority of the newer mouthwashes also claim to have antiseptic properties [5,13,1517]. While many manufacturers claim that their mouthwashes have antimicrobial properties, the aim of this study was to determine the antimicrobial properties of ten commonly available mouthwashes against four oral pathogens related to caries and to oral fungal infections since these organisms have now gained more importance due to the increased incidence of AIDS/HIV }, to verify the claims made by the manufacturers to provide information to dental professionals about the efficacy of their products in vitro and to use these mouthwashes as a base for the evaluation of antimicrobial plant products.
Materials and methods
Collection of mouthwashes
Ten often used mouthwash products (Table 1) were purchased from the drug stores of Kurukshetra and Gurgaon, Haryana, India.
The test microorganisms S. mutans (MTCC *497), S aureus (MTCC 740), C albicans (MTCC 227) and Saccharomyces cerevisiae (MTCC 170) were procured from MTCC, IMTECH, Chandigarh. These were subcultured on specific media, procured from HiMedia Laboratory Pvt. Ltd., Bombay, India, recommended for different microorganisms such as Brain Heart Infusion Agar, BHI (S. mutans), Nutrient Agar (S. aureus) and Malt Yeast Agar (C. albicans and S. cerevisiae) and incubated aerobically at 370C. The identification of all the microbes was confirmed by standard biochemical and staining methods [19-21].
Screening for antimicrobial activity
Antimicrobial effectiveness of various mouthwashes was assessed by using two techniques:
Turbidity measurement by spectrophotometer: One percent of nutrient broth (HiMedia Ltd.) was prepared containing a 10% concentration of the mouthwashes. After autoclaving, the broth and mouthwashes were inoculated with ???µ? of the microbial inoculums adjusted equal to 106cfu/ml (with turbidity equating to a McFarland standard of 0.5) and were incubated aerobically at 37°C for 24h. The inoculated broths were suspended and their optical density was measured by spectrophotometer at a wavelength of 490nm as a guide to microbial growth. The experiments were performed in triplicates for each mouthwash and the mean for each test microorganism was calculated. Broth without mouthwash was used as control .
Agar well diffusion method: The mouthwashes were tested at four different concentrations: 1 :4(25%), 1 : 1 (50%), 3:4(75%) and füll strength (100%), taking sterile distilled water as the diluent, using agar well diffusion method or cup plate method [22-23]. In this method, pure isolate of each microbe was subcultured on the recommended specific media for each microorganism at 37°C for 24h. From each inoculated agar plate, a minimum of four colonies were touched with a sterile loop and transferred into a tube containing normal saline (0.85%) and density of each microbial suspension was adjusted equal to that of 106 cfu/ml (standardized by 0.5McFarland standard) and was used as the inoculum [23-27].
A 100µl volume of each mouthwash concentration (full strength, 3:4, 1:1, 1:4) and the control was propelled directly into the wells (in triplicates) of the inoculated specific media agar plates for each test organism. The plates were allowed to stand for ten minutes for diffusion of the mouthwash to take place and incubated at 37°C for 24h, 48h and 72h [28-29]. The antimicrobial activity, indicated by an inhibition zone surrounding the well containing the mouthwash, was recorded if the zone of inhibition was greater than 8mm . The experiments were performed in triplicates and the mean values of the diameter of inhibition zones with ± standard deviation were calculated. Nutrient broth without mouthwash in turbidity measurement method and sterile distilled water in agar well diffusion method were used as negative control.
The mouthwashes were measured at 10% concentration for turbidity by spectrophotometer, Hexidine showed no turbidity at all thus having excellent activity, while Chlohex and Triguard showed very little turbidity thus having equally good activity against all the microorganisms. Zytee and Chlohex plus possessed comparatively lesser potential while Hexnor, Chlorhexidine and Listerine showed average ability to inhibit the microbial growth. Toss-K and Senquel-AD showed even more turbidity than the control, thus showing total inability to control the dental caries pathogens. Figure 1 shows the antimicrobial activity of ten mouthwashes against S. mutans in BHI broth by measuring the optical density.
Although few negligible changes in the inhibition zones were observed in some mouthwashes after 48h and 72h, most of the antimicrobial activity was observed, in all the ten mouthwashes tested, during the initial 24h of incubation when tested by agar well diffusion method. Six test mouthwashes, namely Chlorhexidine (Blue Cross Laboratories Ltd., Nasik, India), Hexnor (Dynor Pharmaceuticals Pvt. Ltd., Mumbai, India), Hexidine (ICPA Health Products Ltd., Ankleshwar, India), Chlohex and Chlohex plus (Dr. Reddy's Laboratories Ltd., Hyderabad, India) and Triguard (FDC Ltd., Aurangabad, India) produced consistent antimicrobial activity against all the four test organisms i.e. S. mutans, S. aureus, S. cerevisiae and C. albicans at all the four test concentrationsfull strength, 3:4, 1:1 and 1:4 (Fig. 2). At 3:4, 1:1 and 1:4 dilutions, the differences among the ten test mouthwashes shown to inhibit the growth of microorganisms at full strength became less evident (Table 2).
Out of these, Hexidine showed the highest antimicrobial activity against all the four microorganisms, the maximum inhibition zone produced against S. aureus (28.3mm to 33.9mm) followed by S. mutans (23.6mm to 26mm), S. cerevisiae (20.6mm to 26.3mm) and minimum against C albicans (11.9mm to 22.9mm), at different concentrations with the maximum inhibition zone being produced at full strength (Fig. 3(a, b, c, d)). One of the tested mouthwashes, Zytee (Raptakos, Brett and Co. Ltd., Mumbai, India) showed inhibition of S. aureus at all the four concentrations ranging between 14mm and 3 1 .3mm at 24h which gradually reduced to 25.9mm at 48h and further 20.6mm at 72h, thus showing the bacteriostatic nature of this mouthwash. Zytee showed inhibitory activity against S. mutans at 1:1, 3:4 and full strength ranging between 10.3mm and 13.9mm but had no inhibitory effect on the two fungi C albicans and S. cerevisiae. Another tested mouthwash Listerine cool mint (Pfizer Ltd., Kolhapur, India) showed inhibitory zones against the two bacteria S. mutans and S. aureus ranging betweenl2.6mm and 18.6mm at all the four concentrations. It produced zones of inhibition ranging between 10.3mm and 12.6mm against C albicans and S. cerevisiae at 1:1, 3:4 and full strength but no zone of inhibition against the two yeasts at 1:4 concentrations (Table 2).
Two of the ten tested mouthwashes, Toss-K (Ind-Swift Ltd., Chandigarh, India) and Senquel-AD (Dr. Reddy's Laboratories Ltd., Hyderabad, India) did not show any inhibitory effect against any of the four microorganisms at any of the four tested concentrations. The inhibitory activity of Hexnor against S. aureus was found to increase from 27.3mm to 30.3mm at 3:4 concentration and from 29.9mm to 33.0mm at full strength when measured after 72h of incubation. Rest all mouthwashes (except Zytee) showed almost the same zone diameter after 24h, 48h or 72h, while the zone diameter increased slightly in all the mouthwashes when moving from 1:4 concentration to full strength showing that full strength is the most effective concentration against all the tested microorganisms.
Following the completion of the two different techniques to assess the antimicrobial potential of the mouthwashes, statistical ranking procedures were used to place the ten different mouthwashes in order of antimicrobial effectiveness. The results reveal wide variations in their effectiveness against the four tested microorganisms. Of the ten mouthwashes tested, Hexidine mouthwash emerged as the most effective antimicrobial mouthwash, based on the optical density in liquid nutrient media and the mean diameter of the zones of microbial inhibition produced by the mouthwashes in agar well diffusion method, against all the four tested microorganisms followed by Chlohex and Triguard, all of which showed excellent level of activity. Following Triguard were Zytee, Chlohexplus, Hexnor and Chlorhexidine that showed good antimicrobial activity and finally, displaying very little antimicrobial activity was Listerine while Toss-K and SenquelAD totally lacked antimicrobial activity.
Interestingly, all the three mouthwashes that showed excellent antimicrobial activities had Chlorhexidine gluconate as the active ingrethent. Chlorhexidine gluconate is a cationic biguanide with broad-spectrum antimicrobial action, whose effectiveness in decreasing the formation of dental biofilm (plaque) and gingivitis has been demonstrated in several clinical studies [31-34]. Its mechanism of action is that the cationic molecule binds to the negatively-charged cell walls of the microbes, destabilising their osmotic balance [16,35].
Its substantivity, the ability of an agent to be retained in particular surroundings, is due to its ability to bind to the carboxyl groups of the mucin that covers the oral mucus and be steadily released from these areas in an active form, displaced by the calcium ions segregated by the salivary glands . Chlorhexidine formulations are considered to be the "gold standard" antiplaque mouthrinses due to their prolonged broad spectrum antimicrobial activity and plaque inhibitory potential [16, 17].
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 mouthwash identified as having the largest microbial inhibition zone-and thus probably the strongest antibacterial and antifungal properties-may not be necessarily superior to those found to have smaller diameter inhibition zones. Because a mouthwash used in vivo likely is diluted by saliva, the level to which antimicrobial properties are buffered or lost in dilution in vitro is of interest . In addition, dentists should keep in mind that the mean average inhibition zone of one mouthwash may not be directly comparable with that of another mouthwash because different mouthwashes are constituted of different active ingrethents and may diffuse at different rates.
This testing method also functioned as a screening method, and it may not have been able to detect the effects of a chemical 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 the effectiveness of rinsing with an antimicrobial mouthrinse in significantly reducing both salivary [38-40] and mucosal [41-42] levels of bacteria. Thus, from the overall results obtained, it is evident that various mouthwashes listing Chlorhexidine gluconate as the active ingrethent presented different antimicrobial activities.
This is probably due to the different formulations in different mouthwashes in association with other ingrethents. The possible explanation may be the active product concentration and its interaction with other constituents, in addition to differences in the formulations, might be responsible for different effects. The result justifies the antimicrobial claims of the mouthwashes, made by earlier workers [13, 43-44].
Hexidine mouthwash (ICPA Health Products Ltd., Ankleshwar, India) showed excellent antimicrobial activity against the four dental caries causing microorganisms in vitro. The six mouthwashes found to be effective against all the four tested microorganisms at all the four concentrations, comprising of Chlorhexidine gluconate as the basic constituent, presented different antimicrobial activities. The possible explanation may be the active product concentration and its interaction with other constituents, in addition to differences in the formulations, would be responsible for different effects.
We would like to thank Dr. Tapan Chakrabarti, Institute of Microbial Technology, Chandigarh, for providing the microbial cultures and the Chairperson of the Department of Microbiology for providing laboratory facilities.
1) van Gemert-Schricks MCM, van Amerongen WE, ten Cate JM, Aartman IHA. The effect of different treatment strategies on the oral health of children: a longitudinal randomized controlled trial. Clin Oral Invest. 2008; 12: 361-8.
2) Loesche WJ. Role of Streptococcus mutans in human dental decay. Microbiol Rev. 1986; 50: 353-80.
3) Agbelusi GA, Odukoya OA, Otegbeye AF. In vitro screening of chewing stick extracts and sap on oral pathogens: immune compromised infections. Biotechnology. 2007; 6(1): 97-100.
4) Bagg J. Essentials of microbiology for dental students. New York, Oxford University Press, 1999; 1-326.
5) Lee SS, Zhang W, Li Y. The antimicrobial potential of 14 natural herbal dentifrices: Results of an in vitro diffusion method study. J Am Dent Assoc. 2004; 135: 1133-41.
6) Odds FC. Candida and candidosis: a review and bibliography. 2nd ed. London, Bailiere Tindall, 1988; 252-78.
7) Oztan MD, Kiyan M, Gerceker D. Antimicrobial effect, in vitro, of guttapercha points containing root canal medications against yeasts and Enterococcus faecalis. Oral Surg Oral Med Oral Pthol Oral Radio Endod. 2006; 102: 410-6.
8) Knighton HT. Study of bacteriophage types and antibiotic resistance of staphylococci isolated from dental students and faculty members. J Dent Res. 1960; 39: 906-11.
9) Lowy FD. Staphylococcus aureus infections. N Engl J Med. 1998; 339(8): 520-32.
10) Piochi BJ, Zelante F. Contribution to the study of Staphylococcus isolated in the mouth. III. Staphylococcus isolated from dental plaque. Rev Fac Odontol Sao Paulo. 1975; 13(1): 91-7.
11) Rodis OM, Shimono T, Matsumura S, Hatomoto K, Matsuo K, Kariya N. Cariogenic bacteria and caries risk in elderly Japanese aged 80 and older with at least 20 teeth. JAm Geriatr Soc. 2006; 54: 1573-7.
12) Granby TH, Saldanha MG. The antimicrobial activity of modern mouthwashes. Br Dent J. 1984; 157: 239-42.
13) Mat Ludin CM, Md Radzi J. The antimicrobial activity of different mouthwashes in Malaysia. Malay J Med Sci. 2001; 8: 14-8.
14) Tanzer JM, Slee AM, Kamary B, Schaer ER. In vitro evaluation of seven cationic detergents as anti-plaque agents. Antimicrob Agnts Chemother. 1979; 15: 408-14.
15) Akande 00, Alada ARA, Aderinokun GA, Ige AO. Efficacy of different brands of mouthrinses on oral bacterial load count in healthy adults. Afr J Biomed Res. 2004; 7: 125-8.
16) Amornchat C, Kraivaphan P, Dhanabhumi C, Tandhachoon K, Trirattana T, Choonhareongdej S. Effect of Cha-em Thai mouthwash on salivary levels of mutans streptococci and total IgA. Southeast Asian J Trop Med Pub Hlth. 2006; 37: 528-31.
17) Sheen S, Owens J, Addy M. The effect of toothpaste on the propensity of Chlorhexidine and cetyl pyridinium chloride to produce staining in vitro: a possible predictor of inactivation. J Clin Periodontol. 2001; 28: 46-51.
18) Samaranayake LP. Introduction and historical aspects. In: Samaranayake LP, MacFarlane TW. (eds), Oral candidosis. London, Butterworth & Co. Ltd, 1990; 21-46.
19) Aneja KR. Experiments in microbiology plant pathology and biotechnology. 4nd ed, New Delhi, New Age International Publishers, 2003; 608.
20) Benson HJ. Microbiological applications: laboratory manual in general microbiology. USA, McGraw Hill Publication, 2004; 478.
21) Cappuccino JG, Sherman N. Microbiology lab manual. USA, Benjamin-Cummings Publishing Company, 1995; 477.
22) Okeke MI, Iroegbu CU, Eze EN, Okaoli AS, Esimone CO. Evaluation of extracts of the root of Landolphia owerrience for antibacterial activity. J Ethnopharmacol. 2001;78:119-27.
23) Mbata TI, Debiao L, Saikia A. Antibacterial activity of the crude extract of Chinese green tea (Camellia sinensis) on Listeria monocytogenes. Internet J Microbiol. 2006; 2.
24) Aneja KR, Joshi R. Antimicrobial activity of Amomum subulatum and Elettaria cardamomum against dental caries causing microorganisms. Ethnobot Lealf 2009; 13: 840-9.
25) Aneja KR, Joshi R, Sharma C. Antimicrobial activity of Dalchini (Cinnamomum zeylanicum bark) extracts on some dental caries pathogens. J Pharm Res. 2009; 2(9): 1387-90.
26) Aneja KR, Joshi R. Evaluation of antimicrobial properties of the fruit extracts of Terminalia chebula against dental caries pathogens. Jundishapur J Microbiol. 2009; 2(3): 105-11.
27) Ncube NS, Afolayan AJ, Okoh AI. Assessment techniques of antimicrobial properties of natural compounds of plant origin: current methods and future trends. Afr J Biotechnol. 2008; 7: 1797-806.
28) Khokra SL, Prakash O, Jain S, Aneja KR, Dhingra Y. Essential oil composition and antimicrobial studies of Vitex negundo Linn, extracts. Ind J Pharma Sci. 2008; 70: 522-6.
29) Rios JL, Reico, MC, Villar, A. Screening methods for natural products with antimicrobial activity: a review of the literature. J Ethnopharmacol. 1988; 23: 127-49.
30) Hammer KA, Caon, CF, Riley TV. Antimicrobial activity of essential oils and other plant extracts. J Appi Microbiol. 1999; 86: 985-90.
31) Adams D, Addy M. Mouthrinses. Adv Dent Res. 1994;8:291-301.
32) Bascones A, Morante S, Mateos L, Mata M, Poblet L. Influence of additional active ingrethents on the effectiveness of nonalcoholic Chlorhexidine mouthwashes: a randomized clinical trial. J Periodontol. 2005; 76: 1469-75.
33) 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.
34) Lorenz K, Brunn G, Heumann C, Netuschil L, Brecx M, Hoffmann T. Effect of two new Chlorhexidine mouthrinses on the development of dental plaque, gingivitis and discoloration. A randomized, investigator-blind, placebo-controlled, 3week experimental gingivitis study. J Clin Periodontol. 2006; 33: 561-7.
35) Hugoson A, Koch G, Johansson S. Consensus Klorhexidin inom tandvârden. Lie Forlag, Solna, 1990; 123.
36) Silla MP, Company JMM, Silla JMA. Use of Chlorhexidine varnishes in preventing and treating periodontal disease. A review of the literature. Med Oral Patol Oral Cir Bucal. 2008; 13(4): E257-60.
37) Barry AL, Thornsberry C. Susceptibility tests: diffusion test procedures. In: Balows A, (ed). Manual of clinical microbiology. 5nd ed, Washington, American Society for Microbiology, 1991; 1117-25.
38) Dahlen G. Effect of antimicrobial mouthrinses on salivary microflora in healthy subjects. Scand J Dent Res. 1984; 92(1): 38-42.
39) Jenkins S, Addy M, Wade W, Newcombe RG. The magnitude and duration of the effects of some mouthrinse products on the salivary bacterial counts. J Clin Periodontol. 1994; 21(6): 397-401.
40) DePaola LG, Minah GE, Overholser CD. Effect of an antiseptic mouthrinse on salivary microbiota. Am J Dent. 1996; 9(3): 93-5.
41) Pitts G, Pianotti R, Feary TW, McGuiness J, Masura T. The in vivo effects of an antiseptic mouthwash on odor producing microorganisms. J Dent Res. 1981; 60(11): 1891-6.
42) Fine DH, Furgang D, Sinatra K, Charles C, McGuire A, Kumar LD. In vivo antimicrobial effectiveness of an essential oil containing mouth rinse 12h after a single use and 14days use. J Clin Periodontol. 2005; 32(4): 335-40.
43) Barnett ML. The rationale for the daily use of an antimicrobial mouthrinse. J Am Dent Assoc. 2006; 137: 16S-21S.
44) Pourabbas R, Delazar A, Chitsaz MT. The effect of German chamomile mouthwash on dental plaque and gingival inflammation. Iranian J Pharma Res. 2005; 2: 105-9.
Kamal Rai Aneja, Radhika Joshi, Chetan Sharma
Department of Microbiology, Kurukshetra University, Kurukshetra-1 361 1 9, Haryana, India
How to cite this article:
Aneja KR, Joshi R, Sharma C. The antimicrobial potential often often used mouthwashes against four dental caries pathogens. Jundishapur J Microbiol. 2010; 3(1): 15-27.
Received: November 2009 Accepted: January 2010
Address for correspondence:
Radhika Joshi, Department of Microbiology, Kurukshetra University, Kurukshetra-136119, Haryana, India
Tel: +9355566163; Fax: +9111 23557580
Email: joshi_radhika3 firstname.lastname@example.org