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 Table of Contents  
Year : 2017  |  Volume : 3  |  Issue : 1  |  Page : 40-44

Evaluation and comparison of the antimicrobial effect of two different mouthwashes on selected periodontal pathogens: An in vitro study

Department of Periodontology, Maratha Mandals Nathajirao G Halgekar Institute of Dental Sciences and Research Center, Belgaum, Karnataka, India

Date of Submission10-Nov-2016
Date of Acceptance18-Mar-2017
Date of Web Publication12-Jul-2017

Correspondence Address:
Gunjan Richa
403, Santosha Complex, Bandar Bagicha, Patna - 800 001, Bihar
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Source of Support: None, Conflict of Interest: None

DOI: 10.4103/jcrsm.jcrsm_40_16

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Introduction: Antimicrobial mouth rinses as an adjunct to nonsurgical periodontal therapy can play an important role in maintaining oral health.
Aim: The aim of this study is to evaluate the antimicrobial effect of Listerine and HiOra® mouthrinses and compare their efficacy on four specific standard bacterial strains, namely, Aggregatibacter actinomycetemcomitans (Aa), Porphyromonas gingivalis (Pg), Prevotella intermedia (Pi), and Fusobacterium nucleatum (Fn).
Settings and Design: Ethical clearance for the study was granted by the Institutional Ethics Committee.
Materials and Methods: Aa, Pg, Pi, and Fn were maintained on enriched tryptic soy agar. Listerine and HiOra were tested against these bacterial strains using agar diffusion and broth dilution assay method where minimal inhibitory concentrations (MICs) were defined as the lowest concentration of test agent, either showing no or few bacterial growth colonies and by lack of turbidity, respectively. Distilled water was used as the control group. The tests were run three times for each mouthrinse against each organism. The results obtained were compared by their median values.
Results: All the strains showed sensitivity towards both the test solutions. Listerine showed a lower MIC value than HiOra against all the strains, except Fn where the MIC value by broth dilution was 3.12 mcg/ml and by agar method was 90% concentration for both the test solutions. Both the test solutions had antibacterial effect at various concentrations.
Conclusion: Listerine, the essential oil-based mouthrinse, was observed to be more potent than the herbal mouthrinse HiOra where both had antimicrobial effect.

Keywords: Antimicrobial agent, dental plaque, Gram-negative bacteria, Listerine

How to cite this article:
Richa G, Pudakalkatti PS, Joshi V. Evaluation and comparison of the antimicrobial effect of two different mouthwashes on selected periodontal pathogens: An in vitro study. J Curr Res Sci Med 2017;3:40-4

How to cite this URL:
Richa G, Pudakalkatti PS, Joshi V. Evaluation and comparison of the antimicrobial effect of two different mouthwashes on selected periodontal pathogens: An in vitro study. J Curr Res Sci Med [serial online] 2017 [cited 2023 May 30];3:40-4. Available from: https://www.jcrsmed.org/text.asp?2017/3/1/40/210344

  Introduction Top

Dental plaque has been recognized as the primary etiological factor for dental caries, gingivitis, and periodontal disease.[1] Elimination of plaque by means of mechanical home-care methods includes tooth brushing by manual or electric means combined with interdental cleaning,[2] which requires time, motivation, and manual dexterity,[3] and is of limited use in hard to reach areas, malpositioned teeth, geriatric and physically disabled individuals.[4],[5] Thus, to overcome the limitations of the mechanical plaque control, the combination of mechanical and chemotherapeutic approaches has been used effectively to control plaque and prevent periodontal diseases by acting against both Gram-positive and Gram-negative organisms.[1] Among the chemotherapeutic agents, antimicrobial mouthrinses play an important role.

Listerine ® is an essential oil containing mouthrinse that has antiplaque and antigingivitis effects as chlorhexidine without staining of teeth and taste sensation alteration.[7] One adverse effect reported during the use of Listerine ® is burning sensation.[8] Its antimicrobial property involves bacterial cell wall destruction, bacterial enzymatic inhibition, and extraction of bacterial lipopolysaccharides.[9]

Herbs have been scientifically proven to be safe and effective medicine against various oral health problems without any side effect till date.[10] One such herbal product is HiOra ®, a mouthwash known for its antiseptic, antimicrobial, antiplaque, and analgesic property.[11]

The objective of this study was to evaluate the antimicrobial effect of Listerine, a known mouthrinse, and HiOra, a herbal mouthwash, and to compare the efficacy of the two on standard bacterial strains of common Gram-negative periodontal pathogens - Aggregatibacter actinomycetemcomitans (Aa), Porphyromonas gingivalis (Pg), Prevotella intermedia (Pi), and Fusobacterium nucleatum (Fn).

  Materials and Methods Top

This study included standard strains of

  • Aa (ATCC 43718)
  • Pg (ATCC 33277)
  • Pi (ATCC 25611)
  • Fn (ATCC 25586).

They were obtained from the in-house bacterial bank Central Research Laboratory at Maratha Mandals Nathajirao G Halgekar Institute of Dental Sciences and Research Centre, Belgaum.

The mouthrinses used for the study were the following:

  • HiOra ® from Himalaya Drug Company, Bangalore, India, consisting of Nagavalli (Piper betle), Bibhitaki (Terminalia bellerica), Pilu (Salvadora persica) commonly known as Meswak, Gandharpura Tailum, Yavani, Ela, Peppermint satva
  • Listerine ® from Johnson & Johnson consisting of eucalyptol 0.092%, menthol 0.042%, methyl salicylate 0.060%, and thymol 0.064%[8]
  • Control solution – distilled water.


The bacterial strains along with respective mouthrinse were subjected for culture by means of broth dilution method and agar method to identify the minimal inhibitory concentrations (MICs) and antibacterial properties of the mouthwashes towards each strain. The following culture tests were done three times for each mouthrinse in relation to each bacterial strain.

The MIC was defined as the lowest concentration of test agent that inhibited bacterial growth either no bacterial colonies or only a few small colonies.[12]

Preparation of stock culture strains

Bacterial strains were maintained on enriched tryptic soy agar, supplemented with 5% defibrinated sheep blood; 5 μg/ml hemin and 0.5 μg/ml Vitamin k1.[12]

Minimal inhibitory concentration by broth dilution method [13]

Test strains were inoculated in 10 tubes containing thioglycollate. Nine dilutions of each mouthrinse were done with thioglycollate broth for MIC. In the initial tube, 20 μl of drug was added to the 380 μl of thioglycollate broth. For dilutions, 200 μl of thioglycollate broth was added into the next nine tubes separately. Then, from the initial tube, 200 μl was transferred to the first tube containing 200 μl of thioglycollate broth. This was considered as 10−1 dilution. From 10−1 diluted tube, 200 μl was transferred to second tube to make 10−2 dilution. The serial dilution was repeated up to 10−9 dilution for each mouthwash. From the 10th tube which was the last tube, 200 μl final solution was discarded. The concentrations of the aqueous extract achieved by this serial dilution method were as follows – 100, 50, 25, 12.5, 6.25, 3.1, 1.6, 0.8, 0.4, 0.2. From the maintained stock cultures of required organisms, 5 μl was taken and added into 2 ml of thioglycollate broth. In each serially diluted tube, 200 μl of above culture suspension was added. The tubes were incubated for 48–72 h in anaerobic jar at 37°C and observed for turbidity. The MIC value was obtained by visualizing each series of tubes, and the last tube with clear supernatant was taken as the MIC value. The clear supernatant was considered to be without any growth. Turbidity in the MIC tube indicated growth of the bacteria implying that the bacteria were resistant to that concentration of mouthwash [Figure 1].
Figure 1: Turbidity implying the growth of bacteria

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Antibacterial susceptibility test by agar diffusion method [14]

A specified amount of blood agar was added to tubes containing sterilized distilled water in appropriate concentrations and was heated until homogeneity was obtained in each tube. The solutions were autoclaved at 121°C for 15 min. Serial dilutions of 10%, 20%, 40%, 80%, and 100% of the two test solutions were then added 1% by volume to agar solution in tubes in a sterile environment and then were transferred to individual  Petri dish More Detailses making a total of five Petri dishes each for four microorganisms. Each bacterial strain was added to in accordance with McFarland standards of inoculation by a sterile loop. Petri dishes were incubated at 37°C for 48 h and the colony counts were measured [Figure 2].
Figure 2: Agar Petri dish showing bacterial growth after inoculation

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  Results Top

[Table 1] and [Graph 1] shows the MICs obtained by means of broth dilution method for the two test mouthrinses used against all the four strains when the tests were run three times. All the strains showed sensitivity towards both the test solutions. Listerine shows a lower MIC value than HiOra in all the strains, except Fn. Distilled water shows no antibacterial property.
Table 1: Antibacterial susceptibility by broth dilution method

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[Graph 2] shows that Listerine has a higher of percentage antibacterial susceptibility toward all the strains when compared to HiOra, except in case of Fn.

[Table 2] and [Graph 3] show the median CFU counts at various dilutions of the test solutions. The MICs at which no growth or little growth were seen to be lower for Listerine, except Fn, where growth inhibition was seen at 100% concentration for both test solutions.
Table 2: Minimum inhibitory concentration and median colony forming unit counts agar diffusion method

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  Discussion Top

The present study is one of its kinds where the antimicrobial efficacy of mouthrinses has been evaluated against Gram-negative putative periodontal pathogens Aa, Pg, Pi, Fn by MIC of the respective mouthrinses using broth dilution and agar diffusion technique.

Listerine is known to have antimicrobial property due to the presence of thymol and eucalyptol being one of its constituents. 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.[8] In the present study, all the test strains showed antimicrobial susceptibility against Listerine having least shown by Fn. Charles et al. in 2000[15] found 43.8% reduction in recoverable plaque bacteria following rinsing with the Listerine mouthrinse and attributed it to the rapid kill and plaque permeabilizing properties of the formulation of Listerine. Pan et al. in 2000[16] observed 78.7% bactericidal effect by Listerine against Aa, Fn, Pi, and other strains in their study which supports the current study, wherein subsequent sensitivity and growth reductions were observed with the use of Listerine ® against Aa, Pg, Fn, Pi which could be attributed to the presence of thymol and eucalyptol, the bactericidal agents in Listerine ®. Fine et al. in 2000[17] and Chen et al. in 2011[18] similarly suggested strong bactericidal effect of Listerine against Streptococcus mutans.

HiOra is an herbal mouthrinse whose antimicrobial potency is yet to be proved. In the present study, the particular mouthrinse showed antimicrobial activity against all the test strains, yet the susceptibility was seen to be least toward Aa and Fn. Such variation in the microbial composition in both the mouthrinses could be either attributed to the virulence factors of the organisms or the efficiency of the formulations used in MIC tests. Haffajee et al. in 2008[14] observed the antimicrobial effectiveness of the herbal mouthrinse exhibiting significantly lower MICs for Actinomyces species, periodontal pathogens Eubacterium nodatum, Tannerella forsythia, and Prevotella species when compared to essential oil mouthrinse. Bhat et al. in 2013[19] when compared the antimicrobial properties of HiOra with chlorhexidine mouthwash in vitro found equal effectiveness for the zones of inhibition in periodontal patients. Shetty et al. in 2013[20] observed more reduction of Aa in the chlorhexidine group and of S. mutans in the HiOra group comparatively, suggesting the synergistic action of potent herbal components to exert their antimicrobial activities. The antimicrobial and antiplaque property of HiOra in the present could be attributed to Nagavalli leaf and Salvadora ingredients. Oil extracted from S. persica L. leaves consists of benzyl nitrile, eugenol, thymol, isothymol, eucalyptol, isoterpinolene, and beta-caryophyllene, which have considerable antibacterial effect on several different oral aerobic bacteria with comparable results to known antibiotics.[21]

MIC methods depend on the insolubility of the agent(s) in the test medium; unforeseen interaction of a medium constituent with one or more of the test agents (for example, precipitation); or volatility of an important ingredient of the test mixture, such as alcohol and the range of expected MICs, leading to difficulty in dissolving the test product.[14] Substantivity as well cannot be described in the in vitro study. Thus, above mentioned could be considered as the limiting factors in the present study.

  Conclusion Top

The present study was conducted in interest of finding a potent alternative to the currently used mouthrinses which may have least or no side effects. It could be hence concluded within the limits of the study that the herbal mouthrinse had antimicrobial properties with lesser or equal potency against the test strains when compared to essential oil-based mouthrinse. Further microbial and clinical researches are required to identify the exact mechanism of action towards different organisms as well as their substantivity in clinical scenario for long-term benefits.

Financial support and sponsorship


Conflicts of interest

There are no conflicts of interest.

  References Top

Witt JJ, Walters P, Bsoul S, Gibb R, Dunavent J, Putt M. Comparative clinical trial of two antigingivitis mouthrinses. Am J Dent 2005;8:15A-7A.  Back to cited text no. 1
Al Habashneh R, Qubain TG, Alsalman W, Khader Y. The effect of listerine mouthwash on dental plaque, gingival inflammation and C-reactive protein (CRP). Dentistry 2014;4.  Back to cited text no. 2
DePaola LG, Overholser CD, Meiller TF, Minah GE, Niehaus C. Chemotherapeutic inhibition of supragingival dental plaque and gingivitis development. J Clin Periodontol 1989;16:311-5.  Back to cited text no. 3
Baker K. Mouthrinses in the prevention and treatment of periodontal disease. Curr Opin Periodontol 1993:89-96.  Back to cited text no. 4
Ciancio SG. Use of mouthrinses for professional indications. J Clin Periodontol 1988;15:520-3.  Back to cited text no. 5
Briner WW, Kayrouz GA, Chanak MX. Comparative antimicrobial effectiveness of a substantive (0.12% chlorhexidine) and a nonsubstantive (phenolic) mouthrinse in vivo and in vitr o. Compendium 1994;15:1158, 1160, 1162.  Back to cited text no. 6
Moran JM. Home-use oral hygiene products: Mouthrinses. Periodontol 2000 2008;48:42-53.  Back to cited text no. 7
Jangid K, Doraiswamy JN, Saji VS, Malaiappan S. Efficacy of herbal mouthwashes compared to chlorhexidine in gingivitis – A systematic review. Int J Ethnobiol Ethnomed 2014;1:1-12.  Back to cited text no. 8
Mandel ID. Antimicrobial mouthrinses: Overview and update. J Am Dent Assoc 1994;125 Suppl 2:2S-10S.  Back to cited text no. 9
Ciancio SG. Mouth rinses and their impact on oral hygiene. Lead Story. 2008.  Back to cited text no. 10
Malhotra R, Grover V, Kapoor A, Saxena D. Comparison of the effectiveness of a commercially available herbal mouthrinse with chlorhexidine gluconate at the clinical and patient level. J Indian Soc Periodontol 2011;15:349-52.  Back to cited text no. 11
[PUBMED]  [Full text]  
Haraszthy VI, Reynolds HS, Sreenivasan PK, Subramanyam R, Cummins D, Zambon JJ. Media-and method-dependent variations in minimal inhibitory concentrations of antiplaque agents on oral bacteria. Lett Appl Microbiol 2006;43:256-61.  Back to cited text no. 12
Schwalve, Moore, Goodwin. Antimicrobial Susceptibility Testing Protocols. CRC Press; 2007.  Back to cited text no. 13
Haffajee AD, Yaskell T, Socransky SS. Antimicrobial effectiveness of an herbal mouthrinse compared with an essential oil and a chlorhexidine mouthrinse. J Am Dent Assoc 2008;139:606-11.  Back to cited text no. 14
Charles CH, Pan PC, Sturdivant L, Vincent JW.In vivo antimicrobial activity of an essential oil-containing mouthrinse on interproximal plaque bacteria. J Clin Dent 2000;11:94-7.  Back to cited text no. 15
Pan P, Barnett ML, Coelho J, Brogdon C, Finnegan MB. Determination of the in situ bactericidal activity of an essential oil mouthrinse using a vital stain method. J Clin Periodontol 2000;27:256-61.  Back to cited text no. 16
Fine DH, Furgang D, Barnett ML, Drew C, Steinberg L, Charles CH, et al. Effect of an essential oil-containing antiseptic mouthrinse on plaque and salivary Streptococcus mutans levels. J Clin Periodontol 2000;27:157-61.  Back to cited text no. 17
Chen Y, Wong RW, Seneviratne CJ, Hägg U, McGrath C, Samaranayake LP. Comparison of the antimicrobial activity of Listerine and Corsodyl on orthodontic brackets in vitro. Am J Orthod Dentofacial Orthop 2011;140:537-42.  Back to cited text no. 18
Bhat N, Mitra R, Reddy JR, Oza S, Km V. Evaluation of efficacy of chlorhexidine and a herbal mouthwash on dental plaque: An in vitro comparative study. Int J Pharm Bio Sci 2013;4:(B) 625-32.  Back to cited text no. 19
Shetty S, Pillai S, Sridharan S, Satyanarayana A, Rahul A. Comparative efficacy of chlorhexidine and a herbal mouth rinse in patients with gingival inflammation – A clinical & microbiologic study. Asian J Pharm Technol Innov 2013;1.  Back to cited text no. 20
Alali F, Al-Lafi T. GC-MS analysis and bioactivity testing of the volatile oil from the leaves of the toothbrush tree Salvadora persica L. Nat Prod Res 2003;17:189-94.  Back to cited text no. 21


  [Figure 1], [Figure 2]

  [Table 1], [Table 2]

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