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Evaluation of antimicrobial effects of commercial mouthwashes utilized in South Korea
Evaluation of antimicrobial effects of commercial mouthwashes utilized in South Korea
BMB Reports. 2015. Jan, 48(1): 42-47
Copyright © 2015, Korean Society for Biochemistry and Molecular Biology
This is an open-access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/3.0) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
  • Received : April 29, 2014
  • Accepted : May 08, 2014
  • Published : January 31, 2015
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About the Authors
Su-Jeong Yang
Department of Microbiology, College of Medicine, The Catholic University of Korea, Seoul 137-701
Sang-Ha Han
Department of Microbiology, College of Medicine, The Catholic University of Korea, Seoul 137-701
Ah-Ra Lee
Department of Microbiology, College of Medicine, The Catholic University of Korea, Seoul 137-701
Joon-Ho Jun
Pharmaceutical Product Research Aboratories, Dong-A ST Research Institute, Yonin 449-905
Mi-Won Son
Pharmaceutical Product Research Aboratories, Dong-A ST Research Institute, Yonin 449-905
Se-Hwan Oh
Apgugeong St. Mary’s Eye Center, Seoul 135-894
Jaehong Kim
Department of Biochemistry, School of Medicine, Gachon University, Incheon 406-799, Korea
paik@catholic.ac.kr
Soon-Young Paik
Department of Microbiology, College of Medicine, The Catholic University of Korea, Seoul 137-701
paik@catholic.ac.kr

Abstract
Streptococcus mutans is frequently associated with dental caries. Bacterial fermentation of food debris generates an acidic environment on the tooth surface, ultimately resulting in tooth deterioration. Therefore, various mouthwashes have been used to reduce and prevent Streptococcus mutans . The aim of this study was to evaluate the antimicrobial activities of 4 commercial mouthwashes and those of 10% and 20% ethanol solutions (formula A, B, C, D, E and F) against Streptococcus mutans using biofilm and planktonic methods. The range of reduction in the viable cell count of Streptococcus mutans as estimated by the biofilm and planktonic methods was 0.05-5.51 log (P ≤ 0.01) and 1.23-7.51 log (P ≤ 0.001) compared with the negative control, respectively, indicating that the planktonic method had a stronger antibacterial effect against S. mutans . Among the tested formulations, formula A(Garglin regular mouthwash) was the most effective against Streptococcus mutans (P ≤ 0.001). [BMB Reports 2015; 48(1): 42-47]
Keywords
INTRODUCTION
The oral environment provides favorable conditions for the proliferation of bacteria, where pathogen infection may cause dental caries by creating an acidic environment and biofilm around teeth and gums (1) .
Numerous types of bacteria are associated with tooth decay, and the most prominent one among these is Streptococcus mutans . Research into the relationship of S. mutans growth and dental caries has progressed steadily (2) since its initial description in 1924 (3) . S. mutans is a gram-positive, coccoidal, and aciduric bacterium (3) that achieves critical pH with unusual rapidity (4) . S. mutans produces three genetically distinct glycosyltransferases: GtfB, GtfC, and GtfD (5) . These Gtfs play an important role in the formation of dental plaque, such as in the absorption of various oral microorganisms to the dental surface and cohesion of the enamel and plaque (5 , 6) . Owing to these characteristics, S. mutans is most often found on dental surfaces prior to the formation of a cavity (7) . Consequently, S. mutans has been used as an indicator for cariogenic biofilm in the diagnosis of dental caries (1 , 8) .
The development of dental caries is highly dependent upon an individual’s lifestyle and oral hygiene (9) , therefore dental caries is likely to remain a very common infection throughout an affected individual’s lifetime (10) .
The prevalence of pathogens that cause dental caries can be successfully reduced through effective strategies such as tooth-brushing, inter-dental cleaning, and the use of commercial antimicrobial mouthwashes (11) . Daily use of mouthwashes is recommended for proper oral hygiene (12) . Mouthwashes contain various antibacterial agents such as cetylpyridinium chloride (CPC) and essential oils (EO) (13) . Mouth-rinsing was first described in Chinese medicine in 2700 B.C as a folk remedy (14) . Since the 1960s, the scientific record of antimicrobial mouthwashes has been well documented (15 , 16) . The effectiveness of antibacterial agents has been discussed elsewhere (13 , 17 - 22) .
The aim of this study was to examine the antibacterial activity of four commercial mouthwashes available in South Korea against S. mutans by evaluating colony reduction using both biofilm and planktonic methods.
RESULTS AND DISCUSSION
This study was performed to compare the antimicrobial activity and efficacy of commercially available mouthwashes in South Korea as well as ethanol solutions (10% and 20%) against S. mutans colony reduction. S. mutans cells exhibit various shapes depending on their microenvironments such as with biofilm-attaching cells at the tooth surface and planktonic cells in an oral environment (23 , 24) . Mouthwashes must possess antibacterial activity against both biofilms and planktonic cells. Therefore, we used biofilm and planktonic methods to investigate the reduction of viable cells elicited by the action of the mouthwashes.
The biofilm method showed that the reduction of biofilm cells exposed to the four commercial mouthwashes, as well as the ethanol solutions (10% and 20%), for 30 s ranged from 0.05-5.54 log when compared with the negative control ( Table 1 ). All data demonstrated a statistically significant reduction in bacterial colonies (A-D, F: P ≤ 0.001; E: P ≤ 0.01). Among the tested mouthwashes, bacterial colony reductions in formulas B, C, and D did not exceed 1-log reduction (range, 0.08-0.54 log [P ≤ 0.001]). However, formula A displayed the highest antimicrobial activity, beyond 5-log reduction (P ≤ 0.001), indicating complete removal of S. mutans ( Fig. 1 ). Formulas E and F displayed an approximate 0.05-log reduction (P ≤ 0.01). Fig. 1 shows the log CFU/ml of S. mutans after treatment with each formulation for 30 s using biofilm methods.
Effect of antimicrobial mouthwashes on biofilm and planktonic cells ofS. mutansss
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*Mean (SD) Log-transformed counts to test solution. Test formulas compared to PBS (P ≤ 0.001). 10% ethanol compared to PBS (P ≤ 0.01).
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The log CFU/ml of S. mutans using the biofilm method. (A) negative control and formula A (P ≤ 0.001), (B) negative control and formula B (P ≤ 0.001), (C) negative control and formula C (P ≤ 0.001), (D) negative control and formula D (P ≤ 0.001), (E) negative control and formula E (P ≤ 0.01), (F) negative control and formula F (P ≤ 0.001).
Mouthwashes consisting primarily of chemotherapeutic agents have antibacterial effects on oral microorganisms (25) . These components include chlorohexidine, CPC, sodium fluoride (NaF), and ethanol (25) . In the present study, formula A (0.02% NaF and 0.05% CPC) demonstrated significantly greater antibacterial effects (5-log reduction; P ≤ 0.001) than the other formulas. Between the two agents, CPC was determined to be the more effective antimicrobial agent, since CPC was present exclusively in formula A, whereas NaF was also present in formulas B and D (0.02%).
CPC is freely soluble in water and organic solvents such as chloroform, alcohol, and acetone (19) . With a positive charge and a single aromatic ring, it is an amphiphilic molecule and has weak surface tension (19) . Owing to these properties, the compound may associate with negatively charged macromolecules on the bacterial surface (26) . Moreover, CPC may permeate into thin or thick oral biofilms (20) , a feature that accounts for its high germicidal and bacteriostatic potencies (20 , 26) . Therefore, the use of mouthwashes containing CPC, along with tooth brushing, may be highly effective for the prevention of dental plaque and inflammation of gums (21 , 27 - 30) . Sreenivasan et al . investigated the antibacterial capabilities of mouthwashes containing 0.05% CPC (18) , reporting greater than 90% colony reduction (18) . In the present study, formula A, containing 0.05% CPC, demonstrated greater than 99% antibacterial activity (P ≤ 0.001). Similarly, the antimicrobial ability of CPC has been described in other studies (19 - 22 , 25 - 41) . Therefore, the use of 0.05% CPC as an antibacterial agent in mouthwashes may be appropriate for preventing dental caries.
The planktonic method was also used to evaluate the antimicrobial activity of planktonic cells. Reductions in S. mutans colonies using this method ranged from 1.02-7.56 (P ≤ 0.001). Formulas A, B and C displayed over a 6-log reduction in antibacterial activity (P ≤ 0.001). In contrast, formulas D, E and F displayed almost 1-log reduction (P ≤ 0.001). All data displayed statistically significant differences (P ≤ 0.001) compared with the negative control. Viable S. mutans that were treated with the test solutions using the planktonic method are displayed in Fig. 2 .
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The log CFU/ml of S. mutans using the planktonic method. (A) negative control and formula A (P ≤ 0.001), (B) negative control and formula B (P ≤ 0.001), (C) negative control and formula C (P ≤ 0.001), (D) negative control and formula D (P ≤ 0.001), (E) negative control and formula E (P ≤ 0.001), (F) negative control and formula F (P ≤ 0.001).
Overall, the antimicrobial activity was found to be significantly more effective than when using the biofilm method (P ≤ 0.001). Among the tested solutions, formulas B and C showed particularly lower antibacterial activity when evaluated by the biofilm method (range, 0.08-0.54 log in the biofilm method and 6.33-6.50 log [P ≤ 0.001] than when evaluated by the planktonic method). The precise reason for this enhanced antibacterial activity in the planktonic versus the biofilm methods is unknown, although we surmise that the penetrative activity of both formulas was weak in the biofilm method.
Although the sole component of formula B is 0.02% NaF, formula B displayed greater antibacterial activity (6-log reduction; P ≤ 0.001) in the planktonic evaluation. Fluoride ions, possessing antibacterial activity (42 - 45) , have been widely used in oral hygiene studies for the past 50 years, to reduce the formation of dental caries and to prevent periodontitis (44) . Formula D also contained NaF (0.02% NaF and 0.02% IPMP); however, it had the weakest antimicrobial activity in the present study (P ≤ 0.001). While few reports concerning the antibacterial effects of isopropylene methyl phenol (IPMP) and NaF have been published, both IPMP and NaF are widely used antimicrobial agents for reducing dental caries (42 - 48) . Wakamatsu et al . (47) reported that IPMP-containing mouthwashes displayed a 2-log bacterial reduction in S. mutans biofilm. In the present study, formula D showed reductions of 0.12 log (P ≤ 0.001) in the biofilm method and 1.29 log (P ≤ 0.001) in the planktonic method. The antimicrobial effect of formula D was lower than that of formula B (range: 1.29 log and 6.33 log, respectively; P ≤ 0.001). The underlying reason for this difference in microbial activities between formula B and D is unknown. Further research will be required to unravel the interaction between NaF and IPMP.
Formula C contained EO and 27% ethanol. EO has been used as an antiseptic agent for the prevention of dental caries and gingivitis (49) , as discussed in previous studies (17 , 22 , 49 - 52) . Albert-Kiszely et al . reported that oral health was improved with the use of mouthwashes containing EO and CPC (twice a day for 6 months) after 1 min of tooth-brushing (22) . Thus, EO is an effective component of mouthwashes and plays a role in pathogen removal.
Formula A completely eliminated S. mutans in both biofilm and planktonic evaluations ( Fig. 1 and Fig. 2 ), displaying the highest antimicrobial activity in both biofilm and planktonic method (ranges: 5.54 log and 7.56 log, respectively; Table 1 ).
On comparison of the two techniques, the planktonic method showed higher antimicrobial activity than that exhibited by the biofilm method (ranges: 0.05-5.54 log and 1.02-7.56 log, respectively). In previous study, the differential result existed against antibacterial activity of Aggregatibacter actinomycetemcomitans between biofilm and planktonic methods (53) . Biofilm methods were effective in evaluating the antimicrobial competence of mouthwashes, as mouthwashes should affect the survival capacity and penetrate the biofilm matrix of bacteria in the oral environment (53) . Therefore, various modified biofilm methods have been widely used in many studies using planktonic methods (20 , 39 , 40 , 45 , 46 , 54) .
MATERIALS AND METHODS
- Bacterial strain and cultivation
The Water borne Virus Bank (Seoul, South Korea) purchased S. mutans (ATCC 25175) from the American Type Culture Collection (ATCC, Manassas, VA, USA) and supplied the bacteria for this study. The strain was cultured at 37℃ for 24 h in brain heart infusion broth (BHI; BD Biosciences, San Jose, CA, USA).
- Preparation of formulations
Six formulations were used in this study (A-F). Formula A was Garglin Regular (Dong-A Pharmaceutical Co. Seoul, South Korea). Formula B was Garglin Mint (Dong-A Pharmaceutical Co.). Formula A contained CPC and NaF, whereas formula B contained NaF alone. Formulas C and D were purchased from a market in Seoul, South Korea. Formula C consisted of EO and ethanol, and formula D consisted of IPMP and NaF. Formulas E and F contained 10% and 20% ethanol, respectively— a common antimicrobial agent present in mouthwashes (55) . Phosphate-buffered saline (PBS; HyClone Laboratories, Inc, South Logan, UT, USA) was used as a negative control. The components of the six solutions tested are presented in Table 2 .
Components of the tested solutions
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Components of the tested solutions
- Biofilm method
The biofilm evaluations were performed according to the method described by Fine et al . (53) with slight modifications. Biofilms of S. mutans were formed in BHI broth in a 75-cm 3 cell-culture flask (T75 cell culture flask; SPL Life Sciences Co., Gyeonggi-do, South Korea). S. mutans was inoculated in BHI broth for 24 h; the broth was then replaced every 24 h and incubated for 5 days to obtain a sufficient population of bacteria (approximately 1 × 10 5 CFU/ml). Biofilms were washed three times in sterile PBS to remove the cell suspension. Then, the biofilms were treated with 5 ml each of the six formulations and PBS (negative control) for 30 s, followed by removal of the test solutions. After removal of the residual mouthwash, the biofilm in each tube was washed three times with sterile PBS. Subsequently, the biofilms of S. mutans were scraped using a cell scraper (SPL Life Sciences Co.), and the volume of biofilm was adjusted to 1000 μl by addition of PBS. S. mutans was transferred into 1.5-ml tubes and dispersed for 15 s using a vortex mixer (VELP Scientifica, Usmate Velate MB, Italy). The cell suspensions were serially diluted, inoculated on a BHI agar plate, and incubated at 37℃ for 3 days.
- Planktonic method
S. mutans was inoculated in BHI broth and cultivated at 37℃ for 24 h as described above. The planktonic evaluation was performed according to the method described by Fine et al . (53) . Viable cells were measured by the plate count agar method, whereby a cell density of 1 × 10 7 CFU/ml was obtained. The absorbance of S. mutans was measured using a Beckman DU 530 spectrophotometer (Beckman Coulter, Inc., Pasadena, CA, USA) at an excitation wavelength of 600 nm, and the measured OD value was approximately 0.7. For the planktonic method, S. mutans was isolated by centrifugation at 13,500 ×g for 1 min followed by removal of the supernatant. The remaining pellets of S. mutans were added to 500 μl each of the six formulations and PBS for 30 s. Next, the S. mutans was centrifuged at 13,500 ×g for 30 s, and the supernatant was removed. The remaining pellets of S. mutans were washed three times with sterile PBS to remove the residual mouthwash. Next, 1,000 μl of PBS was added to resuspend the pellets. S. mutans was serially diluted, and a plate count was performed to determine the viable cell count. S. mutans was then incubated at 37℃ aerobic atmosphere for 3 days for enumeration of viable bacteria.
- Statistical analysis
All experiments were repeated three times, and the mean and standard deviation (SD) values were calculated. The colony density was log-transformed, and the log reductions of the tested mouthwashes were compared to the PBS. All data were analyzed using IBM SPSS statistics 21 program (56) . The P-values were calculated, and significant differences with a confidence level of 95% were measured.
Acknowledgements
The research leading to these results was supported by funding from the Mid-career Researcher Program (2012R1A2A2A010 45078).
References
Loesche WJ (1986) Role of Streptococcus mutans in human dental decay. Microbiol Rev 50 353 - 380
Zhou Y , Yang J , Zhi Q , Tao Y , Qiu R , Lin H (2013) Factors associated with colonization of Streptococcus mutans in 8- to 32-month-old children: a cohort study. Aust Dent J 58 507 - 513    DOI : 10.1111/adj.12113
Clarke JK (1924) On the Bacterial Factor in the Ætiology of Dental Caries. Br J Exp Pathol 5 141 - 147
Hamilton IR , Buckley ND (1991) Adaptation by Streptococcus mutans to acid tolerance. Oral Microbiol Immunol 6 65 - 71    DOI : 10.1111/j.1399-302X.1991.tb00453.x
Bowen WH , Koo H (2011) Biology of Streptococcus mutans-derived glucosyltransferases: role in extracellular matrix formation of cariogenic biofilms. Caries Res 45 69 - 86
Monchois V , Willemot RM , Monsan P (1999) Glucansucrases: mechanism of action and structure-function relationships. FEMS Microbiol Rev 23 131 - 151
Tanzer JM , Livingston J , Thompson AM (2001) The microbiology of primary dental caries in humans. J Dent Educ 65 1028 - 1037
Loesche WJ , Rowan J , Straffon LH , Loos PJ (1975) Association of Streptococcus mutans with Human Dental Decay. Infect Immun 11 1252 - 1260
Selwitz RH , Ismail AI , Pitts NB (2007) Dental caries. Lancet 369 51 - 59    DOI : 10.1016/S0140-6736(07)60031-2
Featherstone JD (2000) The science and practice of caries prevention. J Am Dent Assoc 131 887 - 899    DOI : 10.14219/jada.archive.2000.0307
Gurenlian JAR (2007) The Role of Dental Plaque Biofilm in Oral Health. J Dent Hyg 81 4 - 12
Barnett ML (2003) The role of therapeutic antimicrobial mouthrinses in clinical practice: control of supragingival plaque and gingivitis. J Am Dent Assoc 134 699 - 704    DOI : 10.14219/jada.archive.2003.0255
DePaola LG , Spolarich AE (2007) Safety and Efficacy of Antimicrobial Mouthrinses in Clinical Practice. J Dent Hyg 81 13 - 25
Weinberger BW (1948) Introduction to the history of dentistry. Mosby, St. Louis
Loe H , Theilade E , Jensen SB (1965) Experimental Gingivitis in Man. J Periodontol 36 177 - 187    DOI : 10.1902/jop.1965.36.3.177
Barnett ML (2006) The rationale for the daily use of an antimicrobial mouthrinse. J Am Dent Assoc 137 16s - 21s    DOI : 10.14219/jada.archive.2006.0408
Pan P , Barnett ML , Coelho J , Brogdon C , Finnegan MB (2000) Determination of the in situ bactericidal activity of an essential oil mouthrinse using a vital stain method. J Clin Periodontol 27 256 - 261    DOI : 10.1034/j.1600-051x.2000.027004256.x
Sreenivasan PK , Haraszthy VI , Zambon JJ (2013) Antimicrobial efficacy of 0.05% cetylpyridinium chloride mouthrinses. Lett Appl Microbiol 56 14 - 20    DOI : 10.1111/lam.12008
Hucky CL (1945) The Effect of Cetylpyridinium Chloride on the Bacterial Growth in the Oral Cavity. J Pharm Sci 34 5 - 11    DOI : 10.1002/jps.3030340103
Sandt C , Barbeau J , Gagnon MA , Lafleur M (2007) Role of the ammonium group in the diffusion of quaternary ammonium compounds in Streptococcus mutans biofilms. J Antimicrob Chemother 60 1281 - 1287    DOI : 10.1093/jac/dkm382
Stookey GK , Beiswanger B , Mau M , Isaacs RL , Witt JJ , Gibb R (2005) A 6-month clinical study assessing the safety and efficacy of two cetylpyridinium chloride mouthrinses. Am J Dent 18 24a - 28a
Albert-Kiszely A , Pjetursson BE , Salvi GE (2007) Comparison of the effects of cetylpyridinium chloride with an essential oil mouth rinse on dental plaque and gingivitis - a six-month randomized controlled clinical trial. J Clin Periodontol 34 658 - 667    DOI : 10.1111/j.1600-051X.2007.01103.x
Watnick P , Kolter R (2000) Biofilm, city of microbes. J Bacteriol 182 2675 - 2679    DOI : 10.1128/JB.182.10.2675-2679.2000
Yoshida A , Kuramitsu HK (2002) Streptococcus mutans biofilm formation: utilization of a gtfB promoter-green fluorescent protein (PgtfB::gfp) construct to monitor development. Microbiology 148 3385 - 3394
Sreenivasan P , Gaffar A (2002) Antiplaque biocides and bacterial resistance: a review. J Clin Periodontol 29 965 - 974    DOI : 10.1034/j.1600-051X.2002.291101.x
Hucky CL (1944) Cetyl pyridinium chloride. J Pharm Sci 116 50 - 59
Zimmer S , Kolbe C , Kaiser G , Krage T , Ommerborn M , Barthel C (2006) Clinical efficacy of flossing versus use of antimicrobial rinses. J Periodontol 77 1380 - 1385    DOI : 10.1902/jop.2006.050362
Mankodi S , Bauroth K , Witt JJ (2005) A 6-month clinical trial to study the effects of a cetylpyridinium chloride mouthrinse on gingivitis and plaque. Am J Dent 18 9a - 14a
Allen DR , Davies R , Bradshaw B (1998) Efficacy of a mouthrinse containing 0.05% cetylpyridinium chloride for the control of plaque and gingivitis: a 6-month clinical study in adults. Compendium of continuing education in dentistry Compend. Contin Educ Dent 19 20 - 26
Moran J , Addy M (1991) The effects of a cetylpyridinium chloride prebrushing rinse as an adjunct to oral hygiene and gingival health. J Periodontol 62 562 - 564    DOI : 10.1902/jop.1991.62.9.562
Ayad F , Prado R , Mateo LR (2011) A comparative investigation to evaluate the clinical efficacy of an alcohol-free CPC-containing mouthwash as compared to a control mouthwash in controlling dental plaque and gingivitis: a six-month clinical study on adults in San Jose, Costa Rica. J Clin Dent 22 204 - 212
Barnes VM , Arvanitidou E , Szewczyk G (2011) Evaluation of the antiplaque efficacy of two cetylpyridinium chloride-containing mouthwashes. J Clin Dent 22 200 - 203
Gunsolley JC (2010) Clinical efficacy of antimicrobial mouthrinses. J Dent 38 S6 - 10    DOI : 10.1016/S0300-5712(10)70004-X
Haps S , Slot DE , Berchier CE , Van der Weijden GA (2008) The effect of cetylpyridinium chloride-containing mouth rinses as adjuncts to toothbrushing on plaque and parameters of gingival inflammation: a systematic review. Int J Dent Hyg 6 290 - 303    DOI : 10.1111/j.1601-5037.2008.00344.x
Herrera D (2009) Cetylpyridinium chloride-containing mouth rinses and plaque control. Evid Based Dent 10 44 -    DOI : 10.1038/sj.ebd.6400647
Lim K , Mustapha A (2007) Inhibition of Escherichia coli O157:H7, Listeria monocytogenes and Staphylococcus aureus on sliced roast beef by cetylpyridinium chloride and acidified sodium chlorite. Food Microbiol 24 89 - 94    DOI : 10.1016/j.fm.2006.04.005
Nelson RF , Rodasti PC , Tichnor A , Lio YL (1991) Comparative study of four over-the-counter mouthrinses claiming antiplaque and/or antigingivitis benefits. Clin Prev Dent 13 30 - 33
Quirynen M , Soers C , Desnyder M , Dekeyser C , Pauwels M , van Steenberghe D (2005) A 0.05% cetyl pyridinium chloride/0.05% chlorhexidine mouth rinse during maintenance phase after initial periodontal therapy. J Clin Periodontol 32 390 - 400    DOI : 10.1111/j.1600-051X.2005.00685.x
Rao D , Arvanitidou E , Du-Thumm L , Rickard AH (2011) Efficacy of an alcohol-free CPC-containing mouthwash against oral multispecies biofilms. J Clin Dent 22 187 - 194
Schaeffer LM , Szewczyk G , Nesta J (2011) In vitro antibacterial efficacy of cetylpyridinium chloride-containing mouthwashes. J Clin Dent 22 183 - 186
Williams MI (2011) The antibacterial and antiplaque effectiveness of mouthwashes containing cetylpyridinium chloride with and without alcohol in improving gingival health. J Clin Dent 22 179 - 182
Liu J , Ling JQ , Zhang K , Huo LJ , Ning Y (2012) Effect of sodium fluoride, ampicillin, and chlorhexidine on Streptococcus mutans biofilm detachment. Antimicrob Agents Chemother 56 4532 - 4535    DOI : 10.1128/AAC.00885-12
Mayhew RR , Brown LR (1981) Comparative effect of SnF2, NaF, and SnCl2 on the growth of Streptococcus mutans. J Dent Res 60 1809 - 1814    DOI : 10.1177/00220345810600101301
Robinson C , Connell S , Kirkham J , Brookes SJ , Shore RC , Smith AM (2004) The effect of fluoride on the developing tooth. Caries Res 38 268 - 276    DOI : 10.1159/000077766
Yost KG , VanDemark PJ (1978) Growth inhibition of Streptococcus mutans and Leuconostoc mesenteroides by sodium fluoride and ionic tin. Appl Environ Microbiol 35 920 - 924
Khan R , Adil M , Danishuddin M , Verma PK , Khan AU (2012) In vitro and in vivo inhibition of Streptococcus mutans biofilm by Trachyspermum ammi seeds: an approach of alternative medicine. Phytomedicine 19 747 - 755    DOI : 10.1016/j.phymed.2012.04.004
Wakamatsu R , Takenaka S , Ohsumi T , Terao Y , Ohshima H , Okiji T (2014) Penetration kinetics of four mouthrinses into Streptococcus mutans biofilms analyzed by direct time-lapse visualization. Clin Oral Investig 18 625 - 634    DOI : 10.1007/s00784-013-1002-7
Bibby BG , Van Kesteren M (1940) The effect of fluorine on mouth bacteria. J Dent Res 19 391 - 402    DOI : 10.1177/00220345400190040601
Fine DH , Furgang D , Barnett ML (2000) Effect of an essential oil-containing antiseptic mouthrinse on plaque and salivary Streptococcus mutans levels. J Clin Periodontol 27 157 - 161    DOI : 10.1034/j.1600-051x.2000.027003157.x
Chen Y , Wong RW , Seneviratne CJ , Hagg U , McGrath C , Samaranayake LP (2011) Comparison of the antimicrobial activity of Listerine and Corsodyl on orthodontic brackets in vitro. Am J Orthod Dentofacial Orthop 140 537 - 542    DOI : 10.1016/j.ajodo.2011.01.022
Pan PH , Finnegan MB , Sturdivant L , Barnett ML (1999) Comparative antimicrobial activity of an essential oil and an amine fluoride/stannous fluoride mouthrinse in vitro. J Clin Periodontol 26 474 - 476    DOI : 10.1034/j.1600-051X.1999.260710.x
Tufekci E , Casagrande ZA , Lindauer SJ , Fowler CE , Williams KT (2008) Effectiveness of an essential oil mouthrinse in improving oral health in orthodontic patients. Angle Orthod 78 294 - 298    DOI : 10.2319/040607-174.1
Fine DH , Furgang D , Barnett ML (2001) Comparative antimicrobial activities of antiseptic mouthrinses against isogenic planktonic and biofilm forms of Actinobacillus actinomycetemcomitans. J Clin Periodontol 28 697 - 700    DOI : 10.1034/j.1600-051x.2001.028007697.x
Malic S , Emanuel C , Lewis MAO , Williams DW (2013) Antimicrobial activity of novel mouthrinses against planktonic cells and biofilms of pathogenic microorganisms. Microbiol Discov 1 11 -    DOI : 10.7243/2052-6180-1-11
Lachenmeier DW , Keck-Wilhelm A , Sauermann A , Mildau G (2008) Safety Assessment of Alcohol-Containing Mouthwashes and Oral Rinses. SOFW 134 70 - 78
Lee HS , Lim JH (2013) SPSS 20.0 Manual. Jiphyunjae Republis of Korea 143 - 150