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Comparative in vitro evaluation of efficacy of

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 pyrig
ORIGINALoARTICLE
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Comparative In Vitro Evaluation of Efficacy of ublicat

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ion
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Mouthrinses against Streptococcus mutans, sse nc e
ot

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fo r

Lactobacilli and Candida albicans

Neeraj Malhotraa/Sugandhi P. Raob/Shashirashmi Acharyac/Ballal Vasudevd

Purpose: The present study was undertaken to compare the relative antimicrobial efficacy of two commercially available
mouthrinses, Hexidine (0.12% chlorhexidine mouthrinse) and S-Flo (0.2% sodium fluoride), and a laboratory-manufac-
tured propolis mouthrinse (10%) tincture with a dilution of 1:5 with water and their combinations against Streptococcus
mutans, lactobacilli and Candida albicans.

Materials and Methods: Unstimulated saliva samples were obtained from the patients using the spitting method and
the isolates of Streptococcus mutans, Lactobacillus and Candida albicans were obtained. The agar diffusion method was
used to evaluate the antimicrobial activity of these test solutions and their combinations.

Results: Hexidine (0.12% chlorhexidine gluconate mouthrinse) showed the best antimicrobial efficacy against all the
tested microorganisms. The laboratory manufactured propolis mouthrinse showed an effective antimicrobial action only
against Streptococcus mutans. The antimicrobial efficacy of propolis (P) against Streptococcus mutans was similar to that
of chlorhexidine (CHX) and the combination of propolis with chlorhexidine (CHX+P). S-Flo mouthrinse (0.2% sodium fluor-
ide) showed the least efficacy against Streptococcus mutans among all tested solutions, but had better efficacy than
propolis against lactobacilli and Candida albicans. The antimicrobial efficacy of the combination of chlorhexidine and
fluoride mouthrinse and the combination of chlorhexidine and propolis mouthrinse was less than chlorhexidine mouth-
wash alone. Among all the tested combinations, the combination of fluoride and propolis showed the least efficacy
against all the tested microorganisms.

Conclusion: The results of the study indicate that 0.12% chlorhexidine gluconate mouthrinse (Hexidine) has the best
anti-microbial efficacy against all the tested microorganisms, with laboratory-manufactured propolis mouthrinse showing
an equivalent efficacy against Streptococcus mutans only. No added advantage of using the tested mouthrinse combin-
ations was observed.

Keywords: Candida albicans, chlorhexidine mouthrinse, lactobacilli, propolis, Streptococcus mutans

Oral Health Prev Dent 2011; 9: 261-268 Submitted for publication: 07.09.09; accepted for publication: 28.08.10

a Assistant Professor, Department of Conservative Dentistry and En-

dodontics, Manipal College of Dental Sciences, Manipal University,
Mangalore, Manipal, India.
D evelopment of dental caries involves a triad of
indispensable factors, including bacteria (den-
tal plaque), carbohydrates (the diet), and suscepti-
b Professor, Department of Microbiology, Kasturba Medical College, ble teeth (the host) (al-Sharbati et al, 2000). The
Manipal University, Manipal, India. first organisms to be implicated as caries-etiologic
c Professor, Department of Conservative Dentistry and Endodontics, agents were the lactobacilli, which possess the
Manipal College of Dental Sciences, Manipal University, Manipal,
India. property of acid tolerance and acidogenicity in an
d Associate Professor, Department of Conservative Dentistry and acid milieu (Brambilla et al, 1999). The data so far
Endodontics, Manipal College of Dental Sciences, Manipal Univer- indicate that lactobacilli are more important for the
sity, Manipal, India. progression of caries rather than its initiation (Bra-
Correspondence: Dr Neeraj Malhotra, Department of Conservative billa et al, 1999; Smith et al, 2001). The associa-
Dentistry and Endodontics, Manipal College of Dental Sciences, Man-
galore 576104, Karnataka, India 575001. Tel: +91-0824-98445- tion of members of the mutans streptococci family
79329, Fax: +91-0824-2422653. Email: [email protected] with caries is well established. Mutans streptococ-

Vol 9, No 3, 2011 261

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Malhotra et al
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mouthrinse, a fluoride mouthrinse and a rlaboratory-

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ci (MS) are considered the main caries etiological Pu
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agents (de Soet and de Graaff, 1998), and their manufactured propolis mouthrinse against Strepto- ca
counts exhibit a strong positive correlation with car- coccus mutans, lactobacilli, and Candida albicans.tion
te ot

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ies development (Brambilla et al, 1999). They have Also, the relative efficacy of the combinations
ss e n c eof
fo r
the properties of acidogenicity, acid tolerance and these three mouthrinses was evaluated against the
the ability to synthesise extracellular glucans from three test microorganisms.
dietary sucrose, which is an important virulence
trait of these microorganisms. Streptococcus
bacteria are mainly responsible for the initial phase MATERIALS AND METHODS
of the caries lesion especially in the enamel (initia-
tion), whereas Lactobacillus is more involved with The efficacy of three mouthrinses (Table 1) and
the progression of caries (Brambilla et al, 1999). their combinations were evaluated against Strepto-
Although yeasts (Candida albicans) have no signifi- coccus mutans, lactobacilli and Candida albicans.
cant role in either the initiation or progression of The mouthrinses were divided into 6 groups of test
caries, their salivary counts correlate significantly solutions as follows (Fig 1):
with caries activity in an individual and high caries
prevalence (Pienihakkinen, 1988). This can be at- r Group 1: 0.12% chlorhexidine gluconate (Hex-
tributed to their opportunistic behaviour. idine mouthrinse, ICPA Health Products; Mum-
A variety of agents and therapeutic protocols have bai, India) - CHX
been advocated for caries prevention. A daily r Group 2: 0.2% sodium fluoride (S-Flo mouthrinse,
mouthrinse with 0.12% CHX solution has proven to Dr. Reddy’s Lab; Hyderabad, India ) - F
reduce the number of Streptococcus mutans and r Group 3: Laboratory-manufactured propolis
Lactobacillus in the saliva of elderly persons at risk mouthrinse (10% tincture diluted 1:5 with water)
of caries (Persson et al, 1991). Fluoride mouthrins- -P
es (neutral sodium fluoride) are widely used as car- r Group 4: Combination of Groups 1 + 2 - CHX+F
ies preventive agents (Petersson, 1993), as they in- r Group 5: Combination of Groups 1 + 3 - CHX+P
hibit demineralisation and enhance remineralisation r Group 6: Combination of Groups 2 + 3 - F+P
of tooth structure (enamel). Over the last few dec-
ades, there has been a worldwide increase in the Normal saline served as the negative control group.
use of natural products for pharmacological purpos-
es to minimise the side-effects caused by synthetic
drugs. One such product is propolis, which is a natu- Preparation of the propolis mouthrinse
ral resinous bee-hive product collected by Apis mel-
lifera bees from tree buds (Ghisalberti, 1979). Many Propolis was obtained in the form of finely ground
biological activities, such as antimicrobial, cytostat- powder (Drapers SuperBee Apiaries; Millerton, PA,
ic, and anti-inflammatory properties have been at- USA). A propolis mouthrinse preparation as de-
tributed to the ethanolic extract of propolis (Persson scribed by Schmidt et al (1980) was used. A 10%
et al, 1991). Recent studies have shown the anti- tincture of propolis mouthwash with a dilution of
caries potential of propolis obtained from the south- 1:5 with water was prepared. To prepare this, 10 g
ern (type-3) and southeastern (type 12) regions of of propolis powder was dissolved in 70% alcohol
Brazil (Koo et al, 1999; Park et al, 1998). (100 ml) and kept for a day to obtain the extract.
Another novel approach advocated for caries pre- The extract was then filtered with 41 Whatman filter
vention is to use combination preparations of differ- paper to obtain the tincture of active propolis. The
ent therapeutic agents. The use of a mouthrinse tincture was further diluted with distilled water at a
containing fluoride and chlorhexidine has also ratio of 1:5.
shown beneficial effects in reducing the develop-
ment of new caries lesions (Joyston-Bechai et al,
1992; Zickert et al, 1985). So far, the effects of the Sample collection
combination of propolis with other therapeutic
agents such as fluoride and chlorhexidine have not Unstimulated salivary samples were obtained from
been tested. the patients using the spitting method. In this meth-
Thus, the present study was undertaken to deter- od, the subject is seated with the head inclined for-
mine the relative efficacy of a chlorhexidine ward so that the saliva is collected anteriorly in the

262 Oral Health & Preventive Dentistry

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lica
Groups tio
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ss e n c e
fo r

Chlorhexidine Fluoride Propolis

CHX F P
Group 1 Group 2 Group 3

CHX+F CHX+P F+P

Group 4 Group 5 Group 6

Fig 1  Flowchart depicting distribution of different mouthrinses (and combinations) in various groups.

Table 1 Mouthrinses, their composition and manufacturer

Mouthrinse Commercial name Composition Manufacturer

Chlorhexidine gluconate Hexidine Chlorhexidine gluconate, propylene ICPA Health Products;

0.12% glycol, menthol, color Mumbai, India

Sodium fluoride 0.2% S-Flo Sodium fluoride, Ponccau 4R, Dr Reddy’s Lab;
erythrosine Hyderabad, India

Propolis None Propolis powder, water Laboratory manufactured

10% tincture diluted 1:5
with water

floor of the mouth. He/she then spits the collected using Mitis Salivarius Agar (MSA), Lactobacillus
saliva into a filter funnel, once every minute for 5 MRS Agar (LBA), and Sabouraud Dextrose Agar
min. A bench clock is placed in front of the subject (SDA), respectively. Further subculturing was done
so that the spitting procedure can be self-regulated. for proper isolation of colonies (Fig 2). The agar dif-
Individual salivary samples from the patients were fusion method was used to evaluate the antimicro-
kept separately for further processing. bial activity of the test solutions (Bruschi et al,
2006; Koo et al, 2000; Silici et al, 2005). Fresh
isolates of Streptococcus mutans, Lactobacillus,
Antimicrobial testing and Candida albicans were inoculated onto MSA,
LBA, and SDA, respectively, and swabbed to pro-
The collected saliva samples were processed within duce a lawn culture. Using a borer which had been
30 min of collection to obtain the isolates of Strep- flamed red hot and subsequently cooled, wells hav-
tococcus mutans, lactobacilli, and Candida albicans ing a diameter of 6 mm and depth of 4 mm were

Vol 9, No 3, 2011 263

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Malhotra et al
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among the three tested microorganisms,rwhich

by N
Pu was
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equivalent to the combination of chlorhexidine ca
fluoride (CHX+F). Among the combination prepara-tion
te ot

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tions, chlorhexidine with fluoride (CHX+F)ss eshowed
nc e
fo r
a b c the significantly best efficacy against Streptococcus
mutans as compared to other combinations (P =
Fig 2  Colonies obtained following subculturing: a) Strepto- 0.001). Fluoride mouthrinse (F) and the combina-
coccus mutans; b) Lactobacillus; c) Candida albicans. tion of propolis with fluoride (F+P) showed the
smallest zones of inhibition. No statistically signifi-
cant difference was observed between the combi-
nation of chlorhexidine with propolis (CHX+P) and
the combination of propolis with fluoride (F+P).

Lactobacilli

made in the agar plates. Five wells were punched in Chlorhexidine (CHX) showed the best efficacy
each Petri plate. Each experimental and control against lactobacilli followed by the combination of
agent was placed over five wells (n = 5) in each of chlorhexidine with fluoride (CHX+F) and the combi-
the three prepared agar bases. 100 μl of each nation of propolis with chlorhexidine (CHX+P). Sta-
agent/combination was pipetted into the wells. For tistically significant differences were observed in
the combination groups, 50 μl of each agent was efficacy against lactobacilli between chlorhexidine
mixed (total = 100 μl) and then pipetted into the (CHX) and all other tested solutions (P < 0.001),
wells. The plates were kept for 1 h at room tem- except for the combination of chlorhexidine with
perature for the diffusion of agents through the fluoride (CHX+F). Fluoride mouthrinse (F) showed
agar. Afterwards, the plates were incubated in an better efficacy than propolis (P), which had the
appropriate gaseous condition at 37°C for 48 h. smallest zone of inhibition against lactobacilli. No
Following this incubation, inhibition zone diameters statistically significant difference was observed be-
(in mm) were measured along the most uniform tween the fluoride mouthrinse (F) and the combina-
diameter using a transparent plastic scale. Mean tion of fluoride with propolis (F+P).
inhibition zone diameters were recorded and results
were subjected to two-way ANOVA (analysis of vari-
ance) using statistical software (SPSS for Windows, Candida albicans
Version 11.0.0, SPSS; Chicago, IL, USA). Mean val-
ues were compared using the nonparametric Although all the test groups showed the smallest
Kruskal-Wallis test and Tukey’s HSD test (P < 0.05). zone of inhibition against Candida albicans as com-
pared to Streptococcus mutans and lactobacilli,
chlorhexidine (CHX) produced the largest zone of
RESULTS inhibition and propolis (P) the smallest. Statistically
significant differences were observed in efficacy
Mean values and standard deviations of zones of against Candida albicans between chlorhexidine
inhibition for the microorganisms by the tested so- (CHX) and all other tested solutions (P < 0.001),
lutions are given in Table 2. ANOVA test results are except for the combination of chlorhexidine with
shown in Table 3. propolis (CHX+P). Among the combinations tested,
the combination of fluoride and propolis (F+P)
showed the smallest zone of inhibition, with no
Streptococcus mutans statistically significant difference between the com-
binations of chlorhexidine with fluoride (CHX+F) and
Chlorhexidine (CHX) showed the statistically signifi- the combination of propolis with chlorhexidine
cantly largest inhibition zone against Streptococcus (CHX+P).
mutans as compared with the other tested solu- In the control group (saline), no zone of inhibition
tions (P = 0.001). Propolis (P) had the best was observed for any of the tested microorgan-
antimicrobial efficacy against Streptococcus mutans isms.

264 Oral Health & Preventive Dentistry

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Table 2 Mean diameter (in mm) and standard deviation of the zones of microbial growth inhibition by the test solutions
ub

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(mouthrinses and their combinations) lica
tio
Test solutions Microorganism t n ot

n
(n = 5) Mean (standard deviation) e ss e n c e fo r
Streptococcus mutans Lactobacilli Candida albicans

Group 1 27.6 (0.55) a,b,c,d 30 (1.73) a,d 19 (1.73) a,c,d

CHX

Group 2 18.2 (1.64) d,f 17.6 (0.89) a,b 14.4 (0.55) c

F

Group 3 22 (1.22) a,b,d 11.2 (0.45) a,b,c 10.4 (0.55) a,b,c

P

Group 4 23.2 (0.84) c,e,f 27.8 (2.05) b,c 16.2 (0.45) a

CHX+F

Group 5 19.8 (0.45) b,c 25.4 (0.55) a,b,c 17.6 (0.89) b,e
CHX+P

Group 6 18 (0.71) a,e 17.8 (0.45) c,d 11.4 (0.55) d,e

F+P
Same superscript letters in the same column (individual microorganism) indicate statistical significance (P < 0.05).

Table 3 Two-way ANOVA for the microorganisms and tested solutions (mouthrinses and their combinations)

Variable df Sum of squares Mean of squares F Significance

Microorganism (A) 2 994.47 497.23 8.53 0.007

Test Solution (B) 5 1341.20 268.24 4.60 0.019

A*B 10 582.73 58.27 29.55 0.001

Intercept 1 33177.60 33177.60 123.69 0.001

P < 0.05 denotes statistically significant difference.

DISCUSSION death (Seymour, 1992). Additionally, it is actively

adsorbed to the negative groups on the bacterial
In the present study, chlorhexidine mouthrinse was cell surface, thereby preventing them from adhering
found to be statistically significantly more effica- to the tooth pellicle. The fungicidal activity of chlor-
cious against Streptococcus mutans and Lactobacil- hexidine is also due to its penetration in the region
lus (P = 0.001) than all other tested mouthrinses. of the glycerol moieties with subsequent break-
The efficacy of the chlorhexidine mouthrinse against down of the permeability barrier and leaking of cyto-
Candida albicans was significantly less than against plasmic content (Elferink and Booij, 1974). Thus,
Streptococcus mutans and Lactobacillus. Similar re- the lower efficacy against Candida albicans can be
sults have been reported in studies conducted by attributed to the basic differences in outer cell
Nakamoto et al (1995) and Bruschi et al (2006). structure between bacteria and fungi, with the lat-
Chlorhexidine is bacteriostatic at low concentra- ter possessing a rigid outer chitin wall.
tions and bactericidal at high concentrations. At The efficacy of the fluoride mouthrinse was less
low concentrations, it increases the permeability of than chlorhexidine against all the tested micro-
the bacterial cell membrane with leakage of intra- organisms (P = 0.001) and less than propolis (P)
cellular components. At high concentrations, it only against Streptococcus mutans. As mentioned
causes precipitation of bacterial cytoplasm and cell earlier, the main mechanism of the action of fluo-

Vol 9, No 3, 2011 265

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ride is to maintain equilibrium between the demin- ments, the flavonoids are considered to be ub

Q ui
eralisation and remineralisation of dental hard tis- lica
cipal components for its biological activities (Bank-
sues rather than antimicrobial action. Most of the ova, 2000) and anticariogenic effects (Koo et al,tion
te ot

n
in vivo studies have shown a cariostatic effect of 2000). Studies have shown that a wide svariability
se nc e
fo r
fluoride gels or mouthrinses at 1% or 2% fluoride exist in the quantity and quality of flavonoids pre-
(Meurman, 1988). Thus, the observed lower effica- sent among different types of propolis. Propolis
cy of fluoride mouthrinse (S-Flo) could also be at- type-3 is a rich source of flavonoids such as
tributed to the lower percentage of fluoride (0.2% of pinobanksin, kaempferol, apigenin, pinocembrin,
NaF) used in this study. Meurman (1988) studied chrysin and galangin. In contrast, propolis type-12
the ultrastructure, growth, and adherence of Strep- contains kaempferide, kaempferol, isosakuranetin
tococcus mutans ATCC 27351 to hydroxyapatite, and pinobanksin (Park et al, 1997). A high variabil-
after treating bacterial suspensions for 1 h with ity in flavonoids concentrations have also been ob-
0.1% chlorhexidine gluconate (CHX), 0.1% sodium served in ten different commercially available etha-
fluoride (F), and a combination of the two. The fluo- nolic solutions of propolis (Kosalec et al, 2005). As
ride-treated specimens appeared the same as the the cariostatic effect of propolis is dependent on
controls, where the ultrastructure was mostly nor- its composition (flavonoid content), the variability in
mal. Treatment with fluoride alone did not cause the efficacy of propolis observed against different
alterations in the ultrastructure or reduction in ad- microorganisms can be attributed to its variable
sorption of S. mutans. composition, dependent on the region from which it
The best efficacy of the laboratory-manufactured is obtained (Koo et al, 2000; Koru et al, 2007;
propolis mouthrinse was against Streptococcus Libério et al, 2009).
mutans, as compared to Lactobacillus and Candida The combination of chlorhexidine and fluoride
albicans. Also, the efficacy of the laboratory-manu- mouthrinse was more effective against Streptococ-
factured propolis was less than that of the chlor- cus mutans and lactobacilli as compared to the two
hexidine mouthrinse for all the three tested micro- other combinations. However, the antimicrobial ef-
organisms (P = 0.001). The efficacy of propolis was ficacy of the combination of chlorhexidine and fluor-
statistically significantly less than fluoride ide mouthrinse was significantly less effective than
mouthrinse against Lactobacillus and Candida the chlorhexidine mouthwash alone against Strep-
albicans (P = 0.001). Similar results were observed tococcus mutans (P = 0.001) and Candida albicans
by Bruschi et al (2006) and Ugur and Arslan (2004). (P = 0.005). Similar results have been reported in
Bruschi et al (2006) evaluated the in vitro anti- other studies, in which the efficacy of a chlorhex-
microbial activity of gelatin microparticles contain- idine mouthrinse is better when compared to the
ing a propolis ethanolic extract solution (PES), pre- combination preparation containing chlorhexidine
pared by the spray-drying technique, against the and fluoride (Herrera, 2003). It has been suggested
following microorganisms of oral importance: Ente- that the presence of anionic fluorides can interfere
rococcus faecalis, Streptococcus salivarius, Strepto- with the action of chlorhexidine (Roldan et al,
coccus sanguinis, Streptococcus mitis, Streptococ- 2004).
cus mutans, Streptococcus sorbinus, Candida In the present study, it was observed that the ad-
albicans, and Lactobacillus casei. Results showed dition of propolis to the chlorhexidine mouthrinse
that the efficacy of propolis and PES was less than led to a decrease in efficacy of the chlorhexidine
that of chlorhexidine for all the tested microorgan- mouthrinse for all the tested microorganisms. On
isms. Streptococcus sanguis and mutans groups the other hand, no statistically significant differ-
were significantly inhibited by propolis, but there ence was noted between the fluoride mouthrinse
was only a small inhibition zone for Candida (F) and the combination mouthrinse with fluoride
albicans. Recently, Duailibe et al (2007) observed and propolis (F+P) against all the tested micro-
that propolis extract possesses in vivo antimicro- organisms. It is known that propolis is a resinous
bial activity against S. mutans. This can be attribu- product obtained from beehives (Ghisalberti, 1979)
ted to the following properties: antibacterial activity, with a resin content of 45% to 55% and 23% to 35%
inhibition of cell adherence and inhibition of water- waxes (Krell, 1996). Thus, it can be hypothesised
insoluble glucan formation. More than 300 constit- that the resins and waxes present in propolis could
uents (flavonoids, aromatic acids, diterpenic acids interfere with the action of chlorhexidine and de-
and phenolic compounds etc.) have been identified crease its efficacy. It might also be attributed to the
in different propolis samples. Among these ele- highly variable composition of propolis, which is

266 Oral Health & Preventive Dentistry

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ACKNOWLEDGEMENTS rP

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highly dependent on the region from which it is ob- ub

Q ui
tained (Koo et al, 1999; Park et al, 2002). lic
The most clinically relevant in vitro method to Heartfelt thanks to Mr S C Deshpande, the former pharmacist inatio
te the pro- n
ot

n
charge at Kasturba Medical College, Manipal, for preparing
test the efficacy of mouthrinses is a dental biofilm polis solution as required for this research project.
fo rss e n c e
model, since the aforementioned anticariogenic
properties – e.g., inhibition of cell adherence and
formation of water-insoluble glucan – are more re-
lated to dental biofilms. However, in the present REFERENCES
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