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Beena Antony et al , J Biosci Tech, Vol 1 (2),2010, 59-63.
SEMIQUANTITATION AND CHARACTERIZATION OF
STREPTOCOCCUS MUTANS FROM PATIENTS
UNDER GOING ORTHODONTIC TREATMENT
Beena Antony *, Rekha.B, Anup Kumar Shetty,
Thomas Kuruvilla, Ramanathan.K
Dept. of Microbiology, Fr. Muller Medical College, Kankanady,
Mangalore - Karnataka -575 002
*email: beenafmmc @ gmail. com
Abstract
Streptococcus mutans (S.mutans) is a primary aetiological agent in dental
caries and is reported to colonize orthodontic patients in significant numbers.
The aim of the study was to characterize and semiquantitate S.mutans from
subjects undergoing active orthodontic treatment in comparison with an
age matched healthy control group. Salivary samples from 75 orthodontic
patients and controls were diluted ,dispersed, inoculated in appropriate media
and incubated in CO2. An attempt was made to evaluate the efficacy of a
Chlorhexidine mouth wash in reducing the count of S.mutans, by testing
samples on two more visits with a gap of 3 weeks each. Significant
growth of S.mutans was noticed in 56 out of 75 subjects in the test
group, but none in the control group. Though reduction in the count was
noticed in the test group, S.mutans could not be completely eliminated
after using Chlorhexidine mouth wash. Due attention should be given to
control this organism reinforcing oral hygiene, especially during orthodontic
treatment.
Key Words:
Chlorhexidine,
S.mutans,
S.viridans
1. INTRODUCTION:
S. mutans is a significant pathogen of
oral cavity and initiates dental caries. This
organism was isolated for the first time
from the dental plaque by Clarke in 1924.
The name ‘mutans’ was chosen because
of its tendency to exhibit both coccal
and
rod
shaped (mutant)
cell
[1]
morphology . S.mutans belongs to the S
viridans group, which is a poorly defined,
heterologous group of the Genus
Streptococcus. Members of this group are
indigenous to the oral cavity. Though the
classification of this particular group is
oversimplified, it includes a variety of
strains with different physiological and
serological characteristics comprising
S. mutans group, S. sanguis group, S. mitis
group, S. salivarius group and S. milleri
group [2].
rapid generation time, fermentation of
wide range of carbohydrates, ability to
withstand low pH, presence of enzyme
glucosyl transferase that converts sucrose
to glucose to enhance adhesion.[1] S.
mutans is reported to cause systemic
infections
like
endocarditis
and
Increased
intravascular
infections..
proportion of S. mutans is also reported
in patients undergoing active orthodontic
treatment [3] Altered oral ecological
changes during orthodontic therapy leads
to the elevated level of this organism [3].
According to the present trend, all the
Streptococci which exhibit α haemolysis
(other than pneumococci) are assigned to
the S. viridans group. Hence,
it is
advisable to make Microbiologists
familiarize with the identity of this oral
pathogen.
Many factors contribute to the
cariogenicity of this organism such as
The
present
study
involves
the
semiquantitative isolation and detailed
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Beena Antony et al , J Biosci Tech, Vol 1 (2),2010, 59-63.
characterization of S. mutans from patients
undergoing active orthodontic treatment in
comparison with an age matched control
group. An attempt is also made to study
the efficacy of a Mouthwash Clohex plus
(chlorhexidine gluconate -0.2%) in
controlling S. mutans.
2. MATERIALS AND METHODS:
This cross sectional study was done for a
period of 1 year at the dept. of
Microbiology of a tertiary care hospital in
Mangalore. Test group constituted 75
healthy subjects between the age group of
13-25 years, being 19 males and 56
females undergoing active orthodontic
treatment
at
the
department
of
Orthodontics in a Dental College in
Mangalore. For comparative evaluation,
an age matched control group with good
oral hygiene is also included. The criteria
for patient selection included:
• 20 permanent teeth that were either
banded or bonded.
• Subjects were free of oral and
systemic disease.
• Had periodontal packets not
greater than 5 mm.
• Not on antibiotic therapy for at
least 3 months before the
commencement of the study.
2.1. Collection of sample and
processing:
Salivary samples were obtained from these
subjects (after chewing sterile paraffin to
stimulate salivary flow) into wide mouthed
vials and transported to the Microbiology
laboratory without delay. 0.5 ml of saliva
was transferred to a sterile screw capped
tube that contained 4.5 ml of Reduced
Transport Fluid (RTF) [4]. With aseptic
precautions, vortex mixed for 1minute, to
disperse the bacteria.
A loopful of
dispersed salivary samples was inoculated
on various media using a standard
calibrated loop (26 swg.4mm. diameter
that can hold 0.01ml of the sample).
2.2. Media:
1. Brain Heart Infusion Blood Agar (Non
selective media)
2. Gold’s Medium [5] – (Mitis Salivarius
Sucrose Bacitracin Agar - selective media
for S. mutans) [Figure-1]
3. Sucrose Agar & Sucrose broth (To
detect extracellular glucan production)
[Figure-2]
4. Carbohydrate Fermentation Media
[Figure-3]
The plates were streaked in 4 quadrants
without heating to facilitate a rough
quantitation as 1+ to 4+ according to the
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Beena Antony et al , J Biosci Tech, Vol 1 (2),2010, 59-63.
Wadsworth Laboratory Manual [6]. All
the plates were incubated at CO2 rich
environment for 24-48 hrs. Colonies of
S.mutans appeared as dark blue, 1-2 mm in
diameter, with smooth surface and an
entire edge, dry and adherent in the Gold’s
medium, dry and adherent refractile
colonies on Sucrose agar & formation of a
complete or partial gel in Sucrose broth as
reported by Emilson & Bratthal [7].
Colonies morphologically resembling
S.mutans in the Gold’s medium were
quantitated and characterized according
to the identification criteria described
by Facklam [8] & Coykendall [9].
Effect of Chlorhexidine on S. mutans is
given in the Table 2. In the second visit,
17 had 4+ growth and 35 had 3+ growth
(significant in 52) but in the third visit
only 4 had 4+ growth and 12 had 3+
growth (i.e. significant growth in 16) and
no significant growth in rest of the 59.
S mutans could not be completely
eliminated even after using mouthwash for
6 weeks.
2.3. Evaluation of Chlorhexidine in
controlling S. mutans:
In order to evaluate the efficacy of
Chlorhexidine mouthwash in controlling
the count of
S. mutans, the test group
was advised to use 10 ml quantity of the
above said mouth wash for rinsing the
mouth twice daily. A second sample of
the saliva was collected after 3 weeks and
a third sample after 3 more weeks and
processed like the first sample.
3. RESULTS:
Significant growth of S. mutans (3+ or
4+) was noticed in 56 out of 75 subjects in
the test group, but none in the control
group as shown in the Table 1.
Biochemical characterization employed
for S.mutans is given in Table 3
4. DISCUSSION:
S. mutans is the major component of oral
streptococci which are indigenous to oral
cavity. It can produce large amounts of
extracellular glucan from sucrose by the
enzyme glucosyl transferase. The
production of glucan and large amounts
of lactic acid by fermentation of
carbohydrates constitutes major virulence
factors in the causation of dental caries
which is proved beyond doubts. [1]
The usual trend of assigning all αhaemolytic Streptococci (other than
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Beena Antony et al , J Biosci Tech, Vol 1 (2),2010, 59-63.
pneumococci) as S. viridans group has
limited its detailed characterization. Oral
Streptococci can easily be differentiated
by characteristic growth on selective
media like Mitis salivarius agar or Gold’s
medium and by simple biochemical
analysis.[8,9].
A detailed account of
differential growth characteristics on
various media was described by Emilson
& Bratthal [7].
Rosenbloom et.al [3], reported high
incidence of S. mutans in subjects
undergoing active orthodontic treatment.
Altered oral ecological changes like low
pH, increased retentive sites and increased
retention of food particles are contributing
factors. The present study is also in
correlation with these findings. In the
present study, significant growth of S.
mutans (3+or 4+) was noticed in 74.7%
(56/75) of subjects, compared to the age
matched control group.
Streptococcus viridans accounts for more
than 50% of all cases of infectious
endocarditis [10], is a matter of concern.
The predilection to the damaged heart
tissue plays a major role in causing
endocarditis [10]. S.viridans group is also
reported to cause other systemic infections
like
meningitis,
lung
abscess,
osteomyelitis [11-15]. An Indian study
reported 25.93% of viridans Streptococci
from cases of bacteraemia following
dental
procedures
[16].
Transient
bacteraemia after dental manipulation may
have long term sequelae, which warrants
regular, follow up and prophylactic
antibiotic therapy.
In the present study, an attempt was made
to evaluate a recently introduced
antiplaque mouth wash Clohex plus
(which contains 0.2% chlorhexidine
gluconate, 0.05% sodium fluoride and
0.09% Zn chloride).
Even though
effective suppression was obtained, S.
mutans could not be completely eliminated
even after using this mouth wash for 6
weeks. A recent study from Madras
reported
that
0.4%
chlorhexidine
gluconate was found to be effective in
inhibiting S.mutans by broth dilution
technique, in vitro. [17]
Orthodontic treatment lasting for 1-2 years
has become the part and parcel of the
beauty conscious teenagers and young
adults. An increased association of S.
mutans has been noticed in the present
study as well as in the existing literature.
As it is a primary cariogenic organism and
an opportunistic pathogen in periodontal
and systemic infections, due attention
should be given to control this organism
reinforcing
oral
hygiene
during
orthodontic treatment.
Chlorhexidine
gluconate is helpful in suppressing S.
mutans to a great extent and maintaining
oral hygiene.
5. CONCLUSIONS
S.mutans , a primary aetiological agent
in dental caries is reported to colonize
orthodontic patients in siginificant
numbers. Present study investigate the
semiquantitation and characterization of
S.mutans
from
healthy
subjects
undergoing active orthodontic treatment
in comparison with an age matched
healthy control group. The efficacy of
a
mouthwash, Chlorhexidine
was
evaluated in reducing the count of
S.mutans. Even after using this mouth
wash for 6 weeks, complete elimination of
S.mutans was not possible. As it is a
primary cariogenic organism that can
disseminate to systemic infections, control
measures should be adopted reinforcing
oral hygiene.
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Beena Antony et al , J Biosci Tech, Vol 1 (2),2010, 59-63.
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