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292 Volume 6 Number 2 Spring 2023
Antibacterial and Anti-biofilm Effects of Chitosan Nanoparticles
on Streptococcus Mutans Isolates
Azam Valian1, Hossein Goudarzi2, Mohammad Javad Nasiri3, Amin Roshanaei4 and Farzaneh
Sadeghi Mahounak1*
1. Department of Restorative, School of Dentistry, Shahid Beheshti University of Medical Science, Tehran, Iran
2. Department of Microbiology, School of Medicine, Infectous Disease and Tropical Medicine Research Center, Shahid
Beheshti University of Medical Science, Tehran, Iran
3. Department of Microbiology, School of Medicine, Shahid Beheshti University of Medical Science, Tehran, Iran
4. Private Practice, Tehran, Iran
Journal of Iranian Medical Council, Volume 6, Issue no. 2
http://dx.doi.org/10.18502/jimc.v6i2.12238
Original Article
Abstract
Background: Dental caries is an infectious disease caused by bacterial
colonization and biofilm formation. Streptococcus mutans (S. mutans)
is mainly responsible for dental caries development. Considering the
side effects of synthetic antibacterial agents, attempts are ongoing to
find antimicrobial agents with minimal or no side effects for preventing
dental caries. Based on the reported antibacterial activity of chitosan,
this in vitro study aimed to assess the antibacterial and anti-biofilm
effects of chitosan nanoparticles on S. mutans clinical isolates.
Methods: S. mutans isolates were isolated from supragingival plaque
and carious lesions of patients by standard biochemical tests and
Polymerase Chain Reaction (PCR) of the gtfB gene. The antibacterial
activity and Minimum Inhibitory Concentration (MIC), Minimum
Bactericidal Concentration (MBC) of chitosan nanoparticles against S.
mutans was evaluated by the agar well-plate and broth micro-dilution
test, respectively. Also, the effect of chitosan nanoparticles on biofilm
formation was evaluated using micro-titer plate method. Data were
analyzed using ANOVA.
Results: Fifteen S. mutans isolates were collected from patients.
The chitosan nanoparticles synthesized had a diameter of 20–30 nm.
The chitosan nanoparticles showed antibacterial activity against S.
mutans isolates. MICs and MBCs ranged from 0.625-2.5 µg/ml and
1.25-5 µg/ml, respectively. All isolates evaluated in this study were
biofilm-forming and 5 of these produced a strong biofilm. The chitosan
nanoparticles inhibited biofilm formation at 0.75 µg/ml concentration.
Conclusion: Chitosan nanoparticles had antibacterial and anti-
biofilm activity on S. mutans clinical isolates. This study suggests
the potential of chitosan nanoparticles as antimicrobial agents against
cariogenic Streptococci.
Keywords: Biofilm, Chitosan, Dental caries, Nanoparticle, Strep-
tococcus mutans
* Corresponding author
Farzaneh Sadeghi Mahounak, DDS
Department of Restorative, Shahid
Beheshti University of Medical Science,
Tehran, Iran
Tel: +98 9132931893
Email: farzaneh.sadeghi.m@gmail.
com
Received: 24 Jul 2022
Accepted: 15 Oct 2022
Citation to this article:
Valian A, Goudarzi H, Nasiri MJ,
Roshanaei A, Sadeghi Mahounak
F. Antibacterial and Anti-biofilm Ef-
fects of Chitosan Nanoparticles on
Streptococcus Mutans Isolates.J Iran
Med Counc. 2023;6(2):292-8.
293293293
Volume 6 Number 2 Spring 2023
Introduction
Based on the World Health Organization reports,
dental caries still remains a main health problem,
especially among poor social groups (1). Dental caries
is a multifactorial, sugar- and biofilm-dependent
disease that initiates decalcification of tooth
structure and degradation of the organic matrix (2,3).
Cariogenic bacteria such as Streptococcus mutans (S.
mutans) play an important role in the pathogenesis of
dental caries. This bacterial species is a gram-positive
cocci that is a normal inhabitant of the oral cavity and
is a key participant to the formation of Extracellular
Polysaccharides (EPS) matrix in dental biofilms (3,4).
Dental biofilm production is a biological process
mediated by the adhesion of oral planktonic bacteria
to dental surfaces and proliferation (5).
Due to its multifactorial etiology, treatment of oral
dental biofilm-related disease is complicated. In
addition, biofilms are composed of more than 90%
EPS that make biofilms more resistant to antimicrobial
substances due to their limited diffusion to access
microorganism cells (6).
Today, several antimicrobial substances including
metronidazole, chlorhexidine, and quaternary am-
monium compounds are used for the deletion of
cariogenic microorganisms and the prevention of dental
caries, but they have side effects such as increasing
calculus formation, staining, and causing diarrhea
by changing the gastrointestinal normal microbial
flora (1,5,7). Thus, new strategies for prevention of
dental biofilm-related disease are required. One of
the strategies that have been investigated widely is by
using nanoparticles. Nanoparticles are proven to have
superior penetration ability, effective antimicrobial
activity, and cost effective, compared to treatment
with naturally derived anti-biofilm agents (6).
Chitosan is a linear cationic polysaccharide with
optimal biocompatibility and biodegradability. It
is non-toxic and has no immunological effects. It is
abundant in nature as a biopolymer, and has been
used for treatment of neural diseases, rheumatism,
and cerebrovascular accident (8,9). Antibacterial
properties of chitosan nanoparticles have been
previously documented (1,5-7). The exact mechanism
of the antimicrobial activity of chitosan and its
derivatives has yet to be fully understood. However,
some theories have been proposed in this respect.
According to one suggested theory, positively charged
chitosan molecules interact with the negatively
charged bacterial cell membrane, and lead to leakage
of proteins and other intracellular components of the
bacteria. Also, chitosan acts as a chelating agent,
binds to metals, and inhibits the microbial growth as
such (10).
Advances in nanotechnology have enabled the
production of dental materials with unique properties.
Considering the reported antibacterial activity of
chitosan nanoparticles, this study aimed to assess
the antibacterial and anti-biofilm effects of chitosan
nanoparticles on S. mutans clinical isolates.
Materials and Methods
Bacterial strains
The study was approved by the ethics committee of
Shahid Beheshti University of Medical Sciences (IR.
SBMU.RIDS.REC.1396.594).
This in vitro, experimental study was conducted on
standard strain S. mutans (ATCC25175) purchased
from the Iranian Industrial Bacterial Collection and
15 clinical isolates of S. mutans collected from the
supragingival plaque and carious lesions of patients
presenting to the dental clinic of Shahid Beheshti
Dental School. Collected microbial samples were
transferred to the microbiology laboratory of the
university in thioglycolate broth medium. Then,
the samples were cultured on Mitis Salivarius Agar
medium, and incubated at 37°C in presence of
5% CO2, 10% H2 and 80% N2 for 48 hr (11). The
obtained colonies were evaluated by conventional
biochemistry tests including Gram-staining, catalase
test, oxidase test, mannose fermentation test, and
mannitol salt agar test.
Polymerase chain reaction
The obtained isolates were also confirmed by
Polymerase Chain Reaction (PCR) of the gtfB gene
amplification using forward: 5′-ACTACACTTT
CGGGTGGCTTGG- 3′ and reverse: 5′ CAGTATAA
GCGCCAGTTTCATC- 3′ primers. DNA extraction
was done by the boiling method as described
previously. The PCR reaction was prepared in a
final volume of 20 μl, containing 10 μl Mastermix
(Ampliqon, Denmark), 0.5 μl of each primer (10 pM),
5 μl (50 ng) DNA template and 4 μl distilled water.
Antibacterial and Anti-biofilm Effects of Chitosan
294294 Volume 6 Number 2 Spring 2023
Then, the PCR assay was carried out as follows: an
initial denaturation at 94°C for 5 min, followed by 30
cycles at 94°C for 30 s, 55°C for 30 s, 72°C for 30 s,
and a final extension at 72°C for 5 min (12). The PCR
distilled water and S. mutans strain (ATCC25175)
were used as the negative and positive control,
respectively.
Preparation of chitosan nanoparticles
Chitosan with 95% purity was purchased from Sigma
Aldrich (Sigma Aldrich, USA). Low molecular
weight chitosan (500,000 D) was used in this study.
The 10 mg/ml stock solution of chitosan was prepared
as follows: 2 g of chitosan was added to 100 ml of
distilled water; 2 ml acetic acid was also added, and
the mixture was stirred by a magnetic stirrer for 24
hr. Next, the volume of the solution was increased
to 200 ml with distilled water and the pH was
adjusted at 5 by adding NaOH. This stock solution
was utilized to prepare 320 μg/ml concentration of
chitosan nanoparticles. Also, Transmission Electron
Microscopy (TEM) was used to determine the
chitosan nanoparticles’ size and shape.
Antibacterial activity determination
Bacterial suspensions (1×108 CFU) were cultured in
Muller-Hinton agar medium with 5% sheep blood.
Then, using sterile Pasteur pipettes, wells were
created over the culture plates. Next, 100 ml of
chitosan nanoparticles was added to into the wells.
The plates were then incubated 37°C for 24 hr. To
ensure the accuracy of testing, it was repeated 3
times for each bacterial isolates (1). The diameter of
the growth inhibition zones for the 15 isolates was
measured and means value was reported. Acetic acid
without chitosan nanoparticles served as the negative
control. The S. mutans strain (ATCC25175) was
positive control.
MIC and MBC determination
Minimum Inhibitory Concentration (MIC) and
Minimum Bactericidal Concentration (MBC) of
chitosan against S. mutans isolates were determined
by broth microdilution assay according to the
CLSI guidelines. The stock solution of chitosan
nanoparticles was prepared with a concentration of 320
μg/ml. Next, 100 µl Mueller Hinton broth was added
to each well of a 96-well plate. After that 100 μl of the
chitosan were added to first well, and two-fold serial
dilutions concentrations ranging from 160 to 1.25
mg/ml were made. A suspension with a turbidity of
0.5 McFarland standard (∼1.5 x 108 Colony-Forming
Units [CFU]/ml) was prepared in Phosphate-Buffered
Saline (PBS) and was subsequently diluted 1:20.
Then, 10 μl was added to each well. Then, the plates
were incubated for 24 hr at 37°C under anaerobic
conditions. Acetic acid without chitosan served as
the negative control. Quality control was done under
similar conditions to those of the experiment using
S. mutans (ATCC25175). For MIC determination,
the plates were read for Optical Density (OD) under
PLATE reader at 600 nm.
The last well that did not have turbidity indicated
the MIC, to determination of the MBC, 20 µl of
MIC, 2MIC and 4MIC well were cultured on MHA
medium and incubated at 37°C for 18 hr. and the least
concentration with the colony growth not over 0.1%
compared to the initial concentration was considered
as the MBC value.
Effect of chitosan nanoparticles on biofilm formation
Effect of chitosan nanoparticles on biofilm formation
was evaluated using micro-titer plate method. Tryptic
Soy Broth (TSB) supplemented with 1% (w/v)
sucrose was used for biofilm formation assay in this
study. All 15 clinical isolates and the control strain
were cultured in TSB and incubated overnight at
37°C. Two to three colonies of the fresh culture of
bacteria were cultured in sterile tubes containing 10
ml of TSB and incubated at 37°C in a shaker incubator
operating at 200 rpm for 15-18 hr. The OD of each
liquid culture was adjusted using fresh medium with
an OD of 0.1 at 600 nm wavelength (6).
Next, 100 µL of the microbial suspension was added
to 100 µL and served as the control. Then, 100 µL
of the microbial suspension along with 100 µL of
chitosan nanoparticles was cultured on a 96-well
plate to assess its effect on biofilm formation and
incubated overnight at 37°C. The overlaying medium
was removed from the wells, and the microorganisms
were rinsed with phosphate buffer three times.
Biofilm-forming bacteria adhering to the walls and
bottom of the plate were fixed with methanol for 15
min. Next, the plate was exposed to air upside down
to dry. The fixed biofilm layer at the bottom and on
Valian A, et al
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Volume 6 Number 2 Spring 2023
the walls of the plate was stained with 200 µL of 1%
crystal violet aqueous solution for 15 min. The dye
was then discarded, and the biofilm was rinsed with
phosphate buffer three times. The plate was dried at
room temperature and the dye absorbed by the biofilm
was rinsed off with 200 µL of 33% acetic acid, and
the OD of each well was read with an ELISA Reader
(BiotTek, UK) at 570 nm wavelength. The medium
without bacteria served as the negative control. Each
experiment was performed in triplicate (6,13).
The OD values were categorized as follows:
- ODcut = ODavg of negative control+3×standard
deviation (SD) of ODs of negative control.
OD ≤ODcut = Non-biofilm
ODcut <OD ≤2×ODcut = Weak biofilm
2×ODcut <OD ≤4×ODcut = Moderate biofilm
OD >4×ODcut = Strong biofilm.
Statistical analysis
Data were analyzed using SPSS software version
20. The difference between OD values and Mean
were compared using one-way analysis of variance
(ANOVA), and the significance level in the tests was
considered 0.05.
Results
PCR results indicated that all 15 obtained isolates
carried the gtfB biofilm formation gene (517 bp) and
confirmed as S. mutans (Figure 1). Figure 2 showed
an image of chitosan nanoparticles by TEM. The
diameter of the synthesized chitosan nanoparticle
ranged between 20 and 30 nm. Also, the chitosan
nanoparticle shape was nearly spherical with a smooth
surface. The well-plate technique indicated that the
growth inhibition zone for the clinical isolates ranged
from 18 to 21 mm (mean value of 19 mm).
MICs and MBCs of the chitosan nanoparticles against
S. mutans isolates are presented in table 1. MICs and
MBCs ranged from 0.625-2.5 µg/ml and 1.25-5 µg/
ml, respectively. Twelve (80%) of the isolates had
MIC 1.25 µg/ml and MBC 2.5 µg/ml.
Six isolates (40%) showed moderate biofilm form-
ation, 4 isolates (26.7%) indicated weak biofilm
formation while 5 isolates (33.3%) formed a
strong biofilm. Among these 5 isolates, chitosan
nanoparticles in 0.75 µg/ml concentration inhibited
the biofilm formation.
Figure 1. PCR product of the gtfB biofilm formation gene:
(1) ladder, (2) positive control, (3 and 4) specimens, (5)
negative control.
Figure 2. The figure shows that chitosan nanoparticles
have a particle size below 30 nm in TEM image (scale bar
30 nm).
Discussion
Considering the side effects of synthetic antibacterial
agents, attempts are ongoing to find antimicrobial
agents with minimal or no side effects for preventing
dental caries. According to the reported antibacterial
activity of chitosan, this study aimed to assess the
antibacterial and anti-biofilm effects of chitosan
nanoparticles on S. mutans isolates. Our results
demonstrate that chitosan nanoparticles have
antibacterial effect, and that it can reduce biofilm
formation in vitro. The results showed a MIC of 0.625-
2.25 µg/ml and MBC of 1.25-5 µg/ml for chitosan
nanoparticles against S. mutans isolates. Also, 80%
of isolates had MIC of 1.25 mg/ml. This finding is
consistent with the result of Aliasghari et al’s study
(1) that reported an MIC of chitosan nanoparticles for
Antibacterial and Anti-biofilm Effects of Chitosan
296296 Volume 6 Number 2 Spring 2023
Table 1. The MIC, MBC and biofilm formation of chitosan
nanoparticles among S. mutans isolates
Sample MIC
(µg/ml)
MBC
(µg/ml)Biofilm
1 1.25 2.5 Weak
2 1.25 2.5 Strong
3 1.25 2.5 Weak
4 2.5 5 Moderate
5 1.25 2.5 Strong
6 1.25 2.5 Moderate
7 1.25 2.5 Strong
8 1.25 2.5 Moderate
9 0.62 1.25 Weak
10 1.25 2.5 Moderate
11 0.62 1.25 Moderate
12 1.25 2.5 Moderate
13 1.25 2.5 Weak
14 1.25 2.5 Strong
15 1.25 2.5 Strong
S. mutans of 1.25 µg/ml. But Khoshmaram et al (14)
found an MIC of 0.114 mg/ml that was higher than
our results
On the other hand, results indicated that 5 isolates
formed strong biofilm. Chitosan nanoparticles at
0.75 µg/ml concentration inhibited biofilm formation
by these isolates. These results supported the results
of Costa et al (15). Divya et al (16) evaluated the
antimicrobial activity of chitosan nanoparticles
against Escherichia coli, Klebsiella pneumoniae,
Staphylococcus aureus, and Pseudomonas aeruginosa
by calculation of MIC. They reported that chitosan
nanoparticles had antimicrobial activity against all
the tested microorganisms. They also assessed the
anti-biofilm activity of chitosan nanoparticles using
ELISA and Congo red agar test. They confirmed
the anti-biofilm effects of chitosan nanoparticles.
Fujiwara et al (17) evaluated the effect of pH and
polymerization rate of chitosan on inhibition of S.
mutans. They evaluated chitosan polymer, oligomer,
and monomer at three pH levels and found that water-
soluble chitosan directly inhibited the proliferation
of standard strain S. mutans even at a pH of 6.5
without causing tooth surface degradation. In the
present study, the pH was 5, and the results indicated
the antibacterial effects of chitosan even in more
acidic conditions than that in the study by Fujiwara
et al (18). Sarasam et al (19) demonstrated that
chitosan scaffolds had antibacterial activity against
S. mutans and Actinomyces actinomycetemcomitans.
They showed that chitosan inhibited bacterial
adhesion, and prevented biofilm formation, which
is in harmony with the present findings. Mirhashemi
et al (20) reported that biofilm formation and
proliferation of S. mutans significantly decreased
due to the effect of chitosan nanoparticles, findings
that agree with the present results. Also, Rajabnia
et al (21) demonstrated that chitosan-containing
sealants had antibacterial effects on S. mutans that
were intensified by increasing the concentration of
chitosan. They concluded that addition of 500 µg/
ml of chitosan to a mouthwash eliminated 99% of
S. mutans bacteria after 5 s. Their findings are in
agreement with the present results and highlight the
high potential of chitosan for use in the composition
of mouthwashes (22). Kim and Shin (23) pointed to
the inhibitory effect of chitosan incorporated in resin
composites on S. mutans. They concluded that all
chitosan-containing resin composites had inhibitory
activity; however, addition of chitosan caused some
unfavorable changes in the mechanical properties of
some resin composites. Their results regarding the
antimicrobial activity of chitosan were in line with the
present results. Chávez de Paz et al (24) evaluated the
effect of molecular weight of chitosan nanoparticles
on S. mutans. They showed that chitosan nanoparticle
complexes with high molecular weight had lower
antimicrobial activity than complexes with lower
molecular weights. Considering the low molecular
weight of particles used in the present study (50000
D), our findings supported the results of Chávez de
Paz et al (24).
Future studies are required to assess the effect
of chitosan on human cell lines to assess its
biocompatibility in greater detail.
Conclusion
Chitosan nanoparticles have antibacterial and anti-
biofilm activity on S. mutans clinical isolates. This
Valian A, et al Antibacterial and Anti-biofilm Effects of Chitosan
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Volume 6 Number 2 Spring 2023
study suggests the potential of chitosan nanoparticles
as antimicrobial agents against cariogenic
Streptococci.
Acknowledgements
The study was approved by the ethics committee of
Shahid Beheshti University of Medical Sciences (IR.
SBMU.RIDS.REC.1396.594).
Conflict of Interest
Not applicable.
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