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Evaluation and comparison of the antimicrobial effect of two different

mouthwashes on selected periodontal pathogens: An in vitro study

Article  in  Journal of Current Research in Scientific Medicine · July 2017

DOI: 10.4103/jcrsm.jcrsm_40_16

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Original Article

Evaluation and comparison of the antimicrobial effect of two

different mouthwashes on selected periodontal pathogens:
An in vitro study
Gunjan Richa, Pushpa S. Pudakalkatti, Vinayak Joshi
Department of Periodontology, Maratha Mandals Nathajirao G Halgekar Institute of Dental Sciences and Research Center, Belgaum,
Karnataka, India

Abstract 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

Address for correspondence: Dr. Gunjan Richa, 403, Santosha Complex, Bandar Bagicha, Patna ‑ 800 001, Bihar, India.
E‑mail: gunric@gmail.com
Received: 10.11.2016, Accepted: 18.03.2017

INTRODUCTION Elimination of plaque by means of mechanical home‑care

methods includes tooth brushing by manual or electric
Dental plaque has been recognized as the primary etiological
factor for dental caries, gingivitis, and periodontal disease.[1] This is an open access article distributed under the terms of the Creative Commons
Attribution‑NonCommercial‑ShareAlike 3.0 License, which allows others to remix, tweak,
Access this article online and build upon the work non‑commercially, as long as the author is credited and the
new creations are licensed under the identical terms.
Quick Response Code:
Website:
http://www.jcrsmed.org For reprints contact: reprints@medknow.com

How to cite this article: Richa G, Pudakalkatti PS, Joshi V. Evaluation

DOI: and comparison of the antimicrobial effect of two different mouthwashes
10.4103/jcrsm.jcrsm_40_16 on selected periodontal pathogens: An in vitro study. J Curr Res Sci Med
2017;3:40-4.

40 © 2017 Journal of Current Research in Scientific Medicine | Published by Wolters Kluwer - Medknow
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Richa, et al.: Antimicrobial effect of two mouthwashess against periodontal pathogens
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
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
Journal of Current Research in Scientific Medicine | Volume 3 | Issue 1 | January-June 2017 41
• 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.
Methodology
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].
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Richa, et al.: Antimicrobial effect of two mouthwashess against periodontal pathogens

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 dishes 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].
RESULTS
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.
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.
DISCUSSION
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

Figure 1: Turbidity implying the growth of bacteria Figure 2: Agar Petri dish showing bacterial growth after inoculation

Table 1: Antibacterial susceptibility by broth dilution method

mcg/ml Aa Pg Pi Fn
Listerine® HiOra® Listerine® HiOra® Listerine® HiOra® Listerine® HiOra®
100 S S S S S S S S
50 S S S S S S S S
25 S S S S S S S S
12.5 S S S S S S S S
6.25 S S S S S S S S
3.12 S S S S S S S S
1.6 S S S S S S R R
0.8 S R S R S R R R
0.4 S R S R S R R R
0.2 S R S R R R R R
S: Sensitive, R: Resistant, Aa: Aggregatibacter actinomycetemcomitans, Pg: Porphyromonas gingivalis, Pi: Prevotella intermedia, Fn: Fusobacterium
nucleatum

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Richa, et al.: Antimicrobial effect of two mouthwashess against periodontal pathogens

dentifrice interactions.[8] In the present study, all the test
strains showed antimicrobial susceptibility against Listerine
Graph 1: Minimal inhibitory concentration by broth dilution method
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

Graph 2: Comparison of antimicrobial susceptibility of the test solutions
Graph 3: Minimal inhibitory concentration by agar diffusion
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]

Table 2: Minimum inhibitory concentration and median colony forming unit counts agar diffusion method
Concentration (%) Aa Pg Pi Fn
Listerine® HiOra® Listerine® HiOra® Listerine® HiOra® Listerine® HiOra®
10 6 300 300 250 200 300 450 600
20 1 300 169 214 78 300 400 500
40 0 268 90 72 0 200 250 400
80 0 200 0 12 0 14 90 100
100 0 10 0 0 0 0 0 0
Aa: Aggregatibacter actinomycetemcomitans, Pg: Porphyromonas gingivalis, Pi: Prevotella intermedia, Fn: Fusobacterium nucleatum

Journal of Current Research in Scientific Medicine | Volume 3 | Issue 1 | January-June 2017 43

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Richa, et al.: Antimicrobial effect of two mouthwashess against periodontal pathogens
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
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
Nil.
Conflicts of interest
There are no conflicts of interest.
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