Antibacterial Effectiveness of 2% Chitosan and 2% Chlorhexidine Against PDF
Antibacterial Effectiveness of 2% Chitosan and 2% Chlorhexidine Against PDF
Antibacterial Effectiveness of 2% Chitosan and 2% Chlorhexidine Against PDF
ISSN - 0975-7058
Research Article
ANTIBACTERIAL EFFECTIVENESS OF 2% CHITOSAN AND 2% CHLORHEXIDINE AGAINST
ENTEROCOCCUS FAECALIS IN BIOFILM (LABORATORY EXPERIMENT)
ABSTRACT
Objective: Enterococcus faecalis can form biofilms and has a major role in the etiology of persistent lesions after root canal. We analyzed the efficacy
of chitosan and chlorhexidine against E. faecalis in biofilms.
Methods: Polymerase chain reaction was used to analyze E. faecalis DNA that survived and lived after immersing the biofilm in an antibacterial
solution.
Results: A statistically significant difference was noted in living E. faecalis between chitosan and control and between 2% chlorhexidine and control
groups (p≤0.05). No significant difference was noted between chitosan and chlorhexidine groups (p>0.05).
Conclusions: Antibacterial effectivity of chitosan is equal to that of chlorhexidine against E. faecalis in biofilm.
© 2019 The Authors. Published by Innovare Academic Sciences Pvt Ltd. This is an open access article under the CC BY license (http://creativecommons.
org/licenses/by/4. 0/) DOI: http://dx.doi.org/10.22159/ijap.2019.v11s1.163
The 3rd International Conference on Global Health (ICGH), Universitas Indonesia, Bali, Indonesia
Simanjuntak et al.
Int J App Pharm, Vol 11, Special Issue 1, 2019
METHODS a b c
This laboratory experimental study was conducted at Bogor
Agricultural Institute Laboratory (IPB) from June 2014 to October
2014. The objective was to analyze the antibacterial efficacy of chitosan
2% solution and chlorhexidine 2% solution on E. faecalis bacteria in
biofilm by observing the amount of bacteria alive after exposure to the
test material.
d e
A chitosan 2% solution was obtained by mixing 2 g low molecular
Fig. 1: (a) Enterococcus faecalis American Type Culture Collection
weight chitosan (85% deacetylation; Sigma-Aldrich Corp., St. Louis, MO,
(ATCC) 29212TM bacterial preparations. (b) Breeding of E. faecalis
USA) into 100 mL acetic acid 1% (vol/vol) up to 2%. The chlorhexidine
ATCC 29212TM bacteria in Brain Heart Infusion Agar. (c) Colonies
2% solution used in our study was obtained commercially (Consepsis;
of bacteria formed after incubation for 24 h at 37°C. (d) Collecting
Ultradent Products, Inc., South Jordan, UT, USA). E. faecalis bacteria
bacterial colonies using ose needles to be inserted in a reaction
American Type Culture Collection (ATCC) 29212 was obtained from
tube containing NaCl. (e) Density adjustment in accordance with
KWIK-STIKTM (Microbiologics, St. Cloud, MN, USA).
McFarland standard 0.5
E. faecalis ATCC 29212 was applied evenly to the top of Brain Heart
Infusion Agar (BHIA) and incubated for 24 h at 37°C. The cultured
E. faecalis then was obtained using an ose needle and inserted into
a reaction tube containing 10 mL sterile saline. The density of the
suspension was standardized with McFarland standard of 0.5 to
obtain 108 colony-forming units (CFU)/mL. The cellulose nitrate filter
membrane located in the BHIA then was covered with 25 μL bacterial
suspension and incubated at 37°C for 72 h in aerobic conditions (Fig. 1).
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Table 1: Mean amount of E. faecalis bacteria in biofilm that lived (CFU/mL) after exposure to chitosan 2% and chlorhexidine 2%
Table 2: Mean E. faecalis amount between treatment groups from biofilms appear to be distributed evenly on the 3rd day, whereas
in the first 24 h, no or only a few EPS are found on biofilms. Stoodley
Test material Chitosan 2% Chlorhexidine 2% Control et al. [24] also investigated the growth of biofilms microscopically and
Chitosan 2% ‑ 0.827 0.05* found that E. faecalis biofilms began to mature and stabilize.
Chlorhexidine 2% 0.82700 ‑ 0.05*
Control 0.05* 0.05* ‑ The biofilms in our study formed on a cellulose nitrate membrane as
Post hoc Mann–Whitney U‑test, p≤0.05. E. faecalis: Enterococcus faecalis was used by some investigators to examine antibacterial efficacy of
a substance against E. faecalis biofilm. Chai et al. [25] stated that this
method allows for the growth of biofilms on standardized surfaces, thus
providing a more accurate assessment of the efficacy of an antibacterial
material.
We determined the number of E. faecalis bacteria that lived after biofilm Chlorhexidine is widely used to kill E. faecalis in the endodontic field. It
exposure to chitosan antibacterial 2% and chlorhexidine 2% for 10 min, is a broad-spectrum antimicrobial active against Gram-positive, Gram-
in absolute quantification form. The obtained data then were analyzed negative, and fungal bacteria. Studies on the antibacterial efficacy of
using SPSS 20.0 program. various chlorhexidine concentrations against E. faecalis that were
inoculated into root canals reported that chlorhexidine 2% provided
Data were analyzed statistically. The number of surviving E. faecalis the best antibacterial power [27,28]. On the basis of these results, we
bacteria from the control and material groups was first analyzed for used chlorhexidine 2% as a positive control in our study.
normality and homogeneity. If the distribution data were normal and
With increasing treatment using natural ingredients, chitosan became
homogeneous, then the test continued using one-way analysis of variance.
a preferred ingredient that is widely used in the medical world. On
If the difference was significant, a multiple comparison test with post hoc
the basis of several studies, chitosan is believed to have antimicrobial
least significant difference was performed. If the data distribution was
power and is not toxic to tissue [18,29].
not normal or homogeneous, a non-parametric test, such as the Kruskal–
Wallis and post hoc Mann–Whitney U-tests, was performed.
Two main factors that affect the antibacterial power of chitosan are
the molecular weight and degrees of chitosan deacetylation (DD). We
RESULT
used low molecular weight chitosan (87.875 Da) with an 84% DD. The
The distribution of data on the number of bacteria was not homogenous, smaller the weight of the chitosan molecule, the greater its ability to
so the non-parametric Kruskal–Wallis test was used to assess the inhibit growth and multiplication of microorganisms [30]. This was in
significance of the bacterial yield number. accordance with the results of a study by Liu et al., who showed that
low molecular weight chitosan had the highest antibacterial power
The lowest and highest E. faecalis amounts were found in the chitosan against Escherichia coli bacteria [31]. Thus, the use of low molecular
(average, 2.953×103 CFU/mL) and control (average, 1.66×108 weight chitosan allows for easier mobility and ion interaction, thereby
CFU/mL) groups, respectively (Table 1). enhancing effective bonding with bacterial membrane surfaces [16].
There was a significant difference in bacterial amount between the The DD, the percentage of units (glucosamine monomer) that is
control and chitosan 2% groups (p≤0.05 and p=0.05) and also between deacetylated in the chitosan chain affects the chemical, physical, and
the control and chlorhexidine 2% groups (p≤0.05 and p=0.05; Table 2). biological properties of chitosan, such as the strain strength of a film,
However, there was no significant difference between the chitosan 2% ability to clamp metal ions, and immunoadjuvant activity. DD also affects
and chlorhexidine 2% groups (p>0.05 and p=0.827). the intrinsic pKa of chitosan, thus affecting the solubility of chitosan in
acid. For chitosan to dissolve in acid, its DD must be ≥40% [29]. DD
DISCUSSION affects the amount of positive charge of chitosan. The higher the DD, the
higher the positive charge and the better the antibacterial power [31].
This study analyzed the antibacterial efficacy of chitosan on E. faecalis The high DD (84%) in our study aimed to obtain acid-soluble chitosan
biofilm. E.faecalis is commonly found in endodontic treatment failure with high antibacterial power.
and it is highly resistant to various antibacterial agents because it can
form biofilm in root canal. In our study, antibacterial power was analyzed using real-time PCR with
PMA staining. Real-time PCR has higher sensitivity and higher accuracy
A study of biofilm resistance relationship by the age of the biofilm than the culture method and also can provide more detailed and
suggests that mature biofilms are more difficult to destroy than younger accurate quantitative results [32]. Venieri et al. [33] compared the value
biofilms. Santos et al. [23] stated that extracellular polysaccharides (EPS) of post-exposure bacterial number using real-time PCR and culture
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Int J App Pharm, Vol 11, Special Issue 1, 2019
methods and found that real-time PCR provided more detailed and Our results are different from those of Ballal et al. [36], who stated that
accurate results. This is because PCR can detect viable but non-cultivable chlorhexidine 2% is more effective than chitosan 2%. This can be due to
(VBNC) bacteria, as shown by E. faecalis, whereas conventional culture the different research methods. Ballal et al. [36] used an agar diffusion
methods are only capable of culturing live bacterial cells that can form method, whereas we assessed antibacterial efficacy using the direct
colonies on nutrient media, without detecting dead cells, VBNC cells, or contact method. With the agar diffusion technique, the diffusion ability
bacteria requiring special conditions to grow [33,34]. of these two materials can affect their antibacterial efficacy. In addition,
Ballal et al. [36] used planktonic bacteria, whereas we used biofilms.
One disadvantage of real-time PCR is that it detects all DNA (living and
dead). This can be solved by a widely used intercalation material in CONCLUSIONS
real-time PCR to discriminate and count the number of living and dead Chitosan 2% had antibacterial power against E. faecalis in biofilm.
cells in a microbiological sample. This method uses PMA, a derivative of The antibacterial efficacy of chitosan 2% was proportional to the
PI that is widely used in microscopic cytometry to dye dead cells. PMA antibacterial efficacy of chlorhexidine 2% in killing E. faecalis bacteria
penetrates into the damaged cell membrane and binds the DNA of the in biofilms.
cell, so it cannot be amplified in the real-time PCR process [34]. With
the use of PMA in our study, the number of living bacterial DNA in the CONFLICTS OF INTEREST
test material and control groups could be detected.
The author report no conflicts of interest.
Chlorhexidine 2% is widely used in the endodontic field due to its
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