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This study investigated the effect of hypochlorous acid (HOCl) rinses and chlorhexidine (CHX)
on the bacterial viability of S. mutans, A. israelii, P. gingivalis, A. actinomycetemcomitans,
E. corrodens, C. rectus, K. oxytoca, K. pneumoniae and E. cloacae. The percentage of live
bacteria was tested by fluorescence method using Live/Dead kit® and BacLight (Molecular
Probes®) and compared between groups by the Kruskal-Wallis and U Mann-Whitney tests
with Bonferroni correction (p value<0.012). The effect of HOCl and CHX on total proteins
of P. gingivalis and S. mutans was determined by SDS-PAGE. CHX showed a higher efficacy
than HOCl against S. mutans, A. israelii, E. corrodens and E. cloacae (p<0.001) while HOCl
was more effective than CHX against P. gingivalis, A. actinomycetemcomitans, C. rectus
and K. oxytoca (p=0.001). CHX and HOCl had similar efficacy against K. pneumoniae.
Proteins of P. gingivalis and S. mutans were affected similarly by HOCl and CHX. HOCl
reduced the bacterial viability especially in periodontopathic bacteria, which may support
its use in the control of subgingival biofilm in periodontal patients.
Viability and Effects on Bacterial
Proteins by Oral Rinses with
Hypochlorous Acid as Active Ingredient
Diana Marcela Castillo1, Yormaris Castillo1, Nathaly Andrea Delgadillo1,
Yineth Neuta1, Johana Jola2, Justo Leonardo Calderón3, Gloria Inés Lafaurie1
1Basic Oral Research Unit-UIBO,
Dental School, Universidad El
Bosque, Bogotá, Colombia
2Dental School, Universidad El
Bosque, Bogotá, Colombia
3Aquilabs S.A., Bogotá, Colombia
Correspondence: Prof. Dr. Gloria
Inés Lafaurie, Carrera 7 B Bis
No. 132 - 11, Bogotá, 110121,
Colombia. Tel: +57-648-9000.
e-mail: institutouibo@gmail.com
Key Words: hypochlorous acid,
chlorhexidine, bacterial viability,
proteins, Porphyromonas
gingivalis, Aggregatibacter
actinomycetemcomitans,
Streptococcus mutans.
Introduction
Dental plaque is the most studied biofilm and it is the
most common form of bacterial growth in the oral cavity
(1). Many substances with antimicrobial effects have
been developed and evaluated for the control of dental
biofilm and gingivitis (2). Chlorhexidine (CHX) has been
extensively studied in the inhibition of dental plaque,
gingivitis reduction and is widely used in full-mouth
disinfection protocols (3). However, CHX does not seem
to be more effective than an essential-oil mouthwash to
reduce gingivitis in long-term studies and its main action
is the reduction of dental plaque (4,5).
There is a great interest in the development of
new molecules with antimicrobial effect against oral
microorganisms with a significant control of dental caries
and periodontal disease. Hypochlorous acid (HOCl) is a
non-antibiotic antimicrobial solution developed during the
First World War by the dilution and acidification of sodium
hypochlorite (Dakin solution) for the treatment of infected
wounds (6). HOCl is part of a group of small molecules
known as reactive oxygen species (ROS) synthesized by
immune system cells during phagocytosis of antigens (7).
The molecule has demonstrated a broad antimicrobial
spectrum for the inhibition of multiple Gram positive
and Gram negative microorganisms, without causing
side effects such as irritation of the mucosa or extrinsic
pigmentation on the tooth surface or restorations. HOCl
also has an important anti-inflammatory and proliferative
activity (8-10).
The purpose of this study was to evaluate the
antimicrobial activity of hypochlorous acid rinses on oral
bacteria (Gram positive and Gram negative) and study its
effects on proteins of two of the major opportunistic and
pathogenic oral microorganisms: Porphyromonas gingivalis
and Streptococcus mutans.
Material and Methods
HOCl Preparation
A special formulation of HOCl for oral health was
developed according to formulations and technology
patented by Aquilabs S.A. (US patent: US2009/0258083A1)
(11). The solutions were obtained with a concentration of
available chlorine as HOCl of 250 ppm (0.025%) and 500
ppm (0.05%) according to NTC 1847 with an ORP (oxide
reduction potential) of 950-1100 MV and a conductivity
of 25.3 ds/m, at a density of 1.01 g/mL at pH 5.8±0.2. HOCl
solution was subjected to an accelerated stability test to
ensure a stable solution for more than 12 months before
losing 10% of its active ingredient.
Antimicrobial Mechanisms
The viability for Streptococcus mutans ATCC 25175,
Actinomyces israelii ATCC 12012, Porphyromonas gingivalis
ATCC 33277, Aggregatibacter actinomycetemcomitans ATCC
29523, Eikenella corrodens ATCC 23834, Campylobacter
rectus ATCC 33238, Klebsiella oxytoca ATCC 43086,
Klebsiella pneumoniae ATCC 700603, Enterobacter cloacae
ATCC 13047 was tested by fluorescence method using Live/
ISSN 0103-6440
Brazilian Dental Journal (2015) 26(5): 519-524
http://dx.doi.org/10.1590/0103-6440201300388
Braz Dent J 26(5) 2015
520
D.M. Castillo et al.
Dead kit® and BacLight (Molecular Probes®).
Bacterial Culture and Inoculum Standardization
ATTC strains of P. gingivalis, A. israelii, E. corrodens and
C. rectus were grown in supplemented brucella agar (0.3%
Bacto agar, 0.2% yeast extract, 5% defibrinated sheep
blood, 0.2% hemolyzed blood, 0.0005%, hemin, 0.00005%
and menadione) and incubated at 37 °C for 4 days in
anaerobic conditions (Anaerogen, Oxoid, Hampshire, UK).
A. actinomycetemcomitans was seeded in agar Dentaid 1
and incubated at 37 °C for 72 h with 5-10% CO2. S. mutans
was grown in blood agar (Blood Agar Base with 5% sheep
blood) and incubated at 37 °C for 24 h with 5-10% CO2.
K. oxytoca, K. pneumoniae and E. cloacae were plated on
BHI agar (Brain Heart Infusion) and incubated for 24-48
h at 37 °C in aerobic atmosphere. Subsequently, inocula
were performed in BHI broth (Brain Heart Infusion) and
were quantified by spectrophotometry for specific optical
densities (OD) of 1 x 108 cells/mL or higher according to
Time Kill Assay protocol (12). After obtaining the expected
OD base 10, serial dilutions were plated on appropriate agar
for each type of microorganism and incubated under the
conditions referred above, to confirm count colony forming
units (CFU). Assays were made in triplicate and the results
are shown as mean and standard deviation (Table 1).
Bacterial Viability Test
To evaluate the number of live and dead bacteria
after exposure to different concentrations of HOCl and
CHX 0.2%, the inocula of all previously described bacteria
were adjusted to a 1 x 108 bacteria/mL concentration and
cultured in BHI broth. 230 μL of each bacteria in culture
were exposed by for 30 s to 700 μL of HOCl at 250 ppm
and 500 ppm concentrations at pH=5.8. Reactions were
neutralized with 70 μL of sodium thiosulfate 0.10 N and the
mixture (HOCl + bacteria + neutralizer),
and centrifuged at 14,000 rpm during 5
min. The pellet was re-suspended in 50
μL of the supernatant and the excess
was discarded. 0.15 μL of the viability
mixture (Live/Dead® BacLight Bacterial
Viability) was added to the 50 μL pellet.
The mixture contains two dyes: SYTO 9
3.34 mM and propidium iodide 20 mM.
Samples were incubated in low-light
conditions for 15 min. The dyes were used
to differentiate the bacteria with intact
membrane (green fluorescence) from
the bacteria with abnormal membrane
(red fluorescence). Positive control
with CHX 0.2% and negative control
without treatment were used in the same
experimental conditions. The laminas were preincubated
with 2 μL of formaldehyde at 2%, in order to immobilize
the bacteria. Images were observed in a fluorescence
microscope (Axio-Imager A2; Zeiss, Jena, Germany) in
greater magnification and digitized using the AxioVision
LE 4.8 software (Zeiss Microscopy). Quantification was
determined by the number of pixels for live and dead
bacteria using the ImageJ software (National Institutes
of Health, Bethesda, MD, USA) and data were expressed
as percent of viability.
Evaluation of the Protein Integrity
P. gingivalis ATCC 33277 and S. mutans ATCC 25175
were cultured as described above. A suspension of each
bacterium was made in BHI broth and standardized to
a concentration of 1 x108 bacteria/mL and adjusted
spectrophotometrically as above described. Bacterial
suspensions were exposed to HOCl at 250 ppm and 500
ppm at pH=5.8. CHX 0.2% was used as positive control and
untreated inoculum as negative control. The cell pellet was
washed in PBS (Phosphate Buffer Saline) and re-suspended
in 2 mL of lysis buffer (50 mM Tris pH=7.5, 50 mM NaCl, 5%
glycerol). For P. gingivalis, bacterial inhibitor and proteinase
K (10 mg/mL) was added and incubated for 15 min at 65
°C. For S. mutans, buffer plus bacterial protease inhibitor
and 20 mg/mL of lysozyme was added and incubated at 37
°C overnight. The samples ere sonicated on ice in a Sonics
Vibra-Cell VCX 130 ultrasonic processor (Sonics & Materials,
Inc. Newtown, CT, USA) with 40% amplitude, totalizing 7
cycles (30 s on, 15 s off). The extracts were stored at -20 °C.
The protein concentration was determined using the
bicinchoninic acid reagent (BCA) to quantify the outer
membrane protein by a colorimetric method (13) and
determining the concentration of the protein according to
Table 1. Standardized bacterial inoculum
Bacteria Wavelength OD ± 0.02 Bacteria/mL (CFU)
mean ± SD x 108
Streptococcus mutans ATCC 25175 580 nm 0.570 1.5±0.7
Actinomyces israelii ATCC 12012 600 nm 1.000 1±0
Klebsiella oxytoca ATCC 43086 580 nm 0.700 1.66±0.5
Klebsiella pneumoniae ATCC 700603 580 nm 1.000 1.66±1.5
Enterobacter cloacae ATCC 13047 580 nm 0.980 1±0.5
A. actinomycetemcomitans ATCC 29523 480 nm 1.700 2.33±1.5
Porphyromonas gingivalis ATCC 33277 620 nm 0.900 1.66±1.1
Eikenella corrodens ATCC 23834 620 nm 1.500 1.5±0.7
Campylobacter rectus ATCC 33238 620 nm 1.000 1.33±0.5
Braz Dent J 26(5) 2015
521
Oral bacterial viability with hypochlorous acid
the manufacturer’s instructions (Thermo Specific).
The integrity of the proteins was evaluated using
electrophoresis in polyacrylamide gel in 10% SDS (sodium
dodecyl sulfate) and a standard pattern of known molecular
weight was used to determine the molecular weights of
the different bacterial proteins. Separating gels (12%
acrylamide/bisacrylamide) were used and the samples
diluted in Laemmli buffer in each well with the same protein
concentration (20 µg/µL) to evaluate the differences in
the electrophoretic profile of the bacteria treated with
the different HOCl concentrations. A run buffer with 3%
Tris base, 14% glycine, 1% SDS, pH=8.3 was used and the
gels were run for 80 min at 100 V. Past the
runtime each gel was stained using Silver
Stain Kit Pierce® (Thermo Fisher Scientific,
Inc., Waltham, MA, USA.), following the
manufacturer’s recommendations.
Statistical Analysis
The average of live bacteria for each
experiment was calculated as percentage.
The viability reduction was calculated as
the difference of living bacteria between
the untreated controls per experiment.
Descriptive analysis of the means and
standard deviations were made. Viability
percentages between different rinses were
compared through the Kruskal Wallis and
U Mann-Whitney test with Bonferroni
correction. A p-value of <0.012 was
established for differences between groups.
Results
Inoculum Standardization
Table 1 shows the average and standard
deviation of CFU counts for reference strains
used in the standardization phase of the
bacterial inoculum, required to perform the
tests at a concentration of 1x108 bacteria/mL
or higher according to the recommendations
of the ASTM E2315-03 for the Time Kill
Method for antimicrobial agents (12).
Bacterial Viability Assessment
Viability percentages for Gram positive
observed in Fig.1A. The viability of S. mutans
and A. israelii was significantly affected by
CHX to 0.2% and HOCl at 500 ppm when
compared with the untreated control and
with HOCl at 250 ppm (p=0.001). However,
0.2% CHX showed a higher efficacy against
these microorganisms when compared with
all groups (p<0.001). HOCl 250 ppm showed
greater effect than the control for A. israelii
(p=0.0032), but did not show differences
against S. mutans (p= 0.64) (Fig. 1). 500
ppm HOCl showed to be the most effective
substance reducing the viability of Gram
Figure 1. Percentage of bacterial viability after 30 seconds treatment with CHX, HOCl
de 250 ppm and 500 ppm. A: Gram positive bacteria: S. mutans ATCC 25175 and A.
israelii ATCC 12012. B. Gram negative bacteria associated to periodontal disease: P.
gingivalis ATCC 33277, A. actinomycetemcomitans ATCC 29523, C. rectus ATCC 33238
and E. corrodens ATCC 23834. C. Enteric rods: E. cloacae ATCC 13047, K. oxytoca ATCC
43086 and K. pneumoniae ATCC 700603. p-values come from t-test or Kruskal Wallis
y U Mann-Whitney (p≤0,012). a: p-value for differences with untreated; b: p-value for
differences with chlorhexidine; c: p-value for differences with HOCl 250 ppm; d: p-value
for differences with HOCl 500 ppm.
Braz Dent J 26(5) 2015
522
D.M. Castillo et al.
negative organisms associated with periodontal disease.
P. gingivalis, A. actinomycetemcomitans and C. rectus
showed significant differences for HOCl at 500 ppm in all
evaluated groups including CHX 0.2% (p=0.001). CHX 0.2%
showed better effectiveness in E. corrodens compared to
all groups (p<0.001). However, HOCl to 250 ppm and 500
ppm were more effective than the control group for this
microorganism (p<0.001) Figure 1B. Viability of P. gingivalis
after treatments is observed in Figure 2.
The enteric rods commonly found as contaminants in
samples of saliva and subgingival plaque were affected
by the solutions in study. K. pneumoniae showed a similar
reduction to 0.2% CHX and the two concentrations of HOCl
(p>0.05). K. oxytoca showed reduced viability for HOCl 500
ppm for all evaluated groups (p<0.001). CHX 0.2% showed
a better efficacy against K. oxytoca than HOCl at 250
ppm and the control group (p<0.001). E. cloacae showed
a significant decrease in bacterial viability for 0.2% CHX
compared to all evaluated groups (Fig. 1C).
Evaluation of the Protein Integrity
After the treatment with HOCl at 500 ppm, 250 ppm
and CHX there was an important change in the banding
pattern in bacterial proteins for the reference strains of P.
gingivalis and S. mutans. A disappearance of a large part of
the bands is observable compared to the untreated control.
For P. gingivalis some of the low molecular weight
proteins are conserved, on the other side the high molecular
weight proteins lose their sharpness as if the proteins
concentrations decreased after the treatment with HOCl.
After treatment with CHX thin proteic profile is observed.
However the protein concentration seems to be affected,
suggesting some kind of protein alterations, but less than
with HOCl. In S mutans is more evident the disappearance
of the bands with high molecular weight and a band of
approximately 28 kD is conserved for all treatments. A band
of 70 kD for the HOCl at 250 ppm is conserved but in minor
concentration. All experiments were made by triplicate and
presented the same behavior (Fig. 3).
Discussion
There is an agreement on the effect of various antiplaque
substances to reduce gingivitis in long term studies (4).
However, the effective reduction of plaque and gingivitis
in the short term is still under study. Chlorhexidine remains
as the gold standard as antiplaque agent in short and long
term effect (5). Despite their wide use, some adverse effects
have discouraged its use, as tooth discoloration as it is easily
Figure 2. Bacterial viability of P. gingivalis ATCC 33277. Green fluorescent bacteria are alive and red fluorescing bacteria are dead. A: untreated; B:
0.2% chlorhexidine; C: HOCl 250 ppm, pH 5.8 and D: HOCl 500 ppm, pH 5.8.
Braz Dent J 26(5) 2015
523
Oral bacterial viability with hypochlorous acid
mixed with the dietary chromogens, weak microbicidal
activity at low concentrations and at high concentrations
may produce dermatitis and desquamation of oral mucosa
as well as delay in healing (14,15).
Hypochlorous acid rinses are proposed for plaque
control and as a wound healing agent for its use in oral
health, due to its low toxicity, high antimicrobial efficacy,
anti-inflammatory effect, induction to cell proliferation
and its background as a topical solution in the antisepsis of
wounds in clinical medicine (16). HOCl has also the ability
to oxidize the amino acid taurine and induce the formation
of chlorine-taurine (TauCl) which has broad spectrum
antimicrobial activity. The TauCl has a significant protective
effect on tissues because it can inhibit the production of
inflammatory mediators and thereby contribute to the
processes of tissue protection (10).
In this study, HOCl showed a significant effect on
Gram positive bacteria but did not exceed the effect of
chlorhexidine. Chlorhexidine has shown better effect on
Gram positive microorganisms but less on Gram negative
microorganisms (17,18). The antimicrobial action of HOCl
appears to be greater in Gram negative than in Gram positive
possibly because Gram negative bacteria has sulfur and
hem groups (rich in iron) in its membrane which causes an
irreversible reaction HOCl/membrane proteins, producing
structural damage, and altering cell permeability, affecting
bacterial viability in Gram negative bacteria (19,20). The
HOCl oxidizes and/or chlorinates endotoxins and exotoxins
such as lipopolysaccharides and gingipains as Rgp and Kgp
neutralizing their action. In Gram positive bacteria HOCl
oxidizes glycine residues present in the peptidoglycan,
on the other hand chlorination reactions in this group of
microorganisms differs in the action on the target (21).
Different authors have controversial uses of
microbiological culture methods to evaluate the efficacy
of antimicrobials. The bacterial viability assessment with
specific methods such as fluorochromes or epifluorescence
has been proposed for evaluation of antimicrobials (22).
In the present study a method of epifluorescence was
used for the evaluation of the bacterial viability similar
to the reported by other studies that have evaluated the
substantivity of antiplaque substances (23,24).
Proteins of P. gingivalis and S. mutans were affected
after 30 s of treatment with the test solutions. For P.
gingivalis, after HOCl and CHX treatments it is observed
a similar reduction of the protein concentrations in many
bands when is compared with the untreated control. In S.
mutans there is an elimination of almost all the proteins for
the different treatments when compared with the untreated
control. Cheung et al., in 2011 have shown changes in the
proteic profile in Bacillus subtilis and E. coli after treatment
with CHX, suggesting that the mechanism of action of CHX
is related with an alteration of lipidic stability of the cell
membrane (25). It is not clear which is the action of HOCl
on bacteria, however oxidation and chlorination of amino
groups in some functional and structural proteins in Gram
positive and Gram negative bacteria is suggested (7). In
further it is important to identify the proteins affected
by HOCl action and thus explain how this antimicrobial
molecule affects the bacterial viability and why in some
Gram negative bacteria as E. cloacae does not have an
important reduction in the viability.
The formulation of HOCl has been stabilized in Colombia
and patented as an substance with antimicrobial effects
Figure 3. Total protein electrophoretic profile of P. gingivalis ATCC 33277 (A) and S. mutans ATCC 25175 (B). Lanes: 1 molecular weight marker; 2.
Treatment with HOCl 250ppm; 3. Treatment with HOCl 500ppm; 4. Treatment with CHX; 5. Without treatment.
Braz Dent J 26(5) 2015
524
D.M. Castillo et al.
for medical applications such as the treatment of chronic
and non-healing wounds (11). Findings of this study could
support future research of HOCl as antimicrobial and
antiplaque agent in dentistry.
HOCl showed better effects on bacterial viability
than CHX in Gram negative microorganism specially in P.
gingivalis, A. actinomycetemcomitans and C. rectus. HOCl
could have a significant effect on periodontophatic bacteria
that could colonize and aggregates as dental biofilm.
Resumo
Este estudo investigou o efeito de enxaguantes à base de ácido hipocloroso
(HOCl) e clorexidina (CHX) sobre a viabilidade bacteriana de S. mutans,
A. israelii, P. gingivalis, A. actinomycetemcomitans, E. corrodens, C.
rectus, K. oxytoca, K. pneumoniae e E. cloacae. O percentual de bactérias
sobreviventes foi testado pelo método de fluorescência utilizando Live/
Dead kit® e BacLight (Molecular Probes®), fazendo comparação entre os
grupos com os testes de Kruskal-Wallis e U Mann-Whitney e correção de
Bonferroni (p<0,012). O efeito de HOCl e CHX sobre P. gingivalis e S. mutans
foi determinado por SDS-PAGE. O CHX mostrou eficácia superior ao HOCl
contra S. mutans, A. israelii, E. corrodens e E. cloacae (p<0,001), ao passo
que P. gingivalis, A. actinomycetemcomitans, C. rectus e K. oxytoca foram
melhores que o CHX para o HOCl (p=0,001). O K. pneumoniae teve efeito
similar para o CHX e para o HOCl. As proteínas de P. gingivalis e S. mutans
foram afetadas de modo semelhante por CHX e HOCl. O HOCl reduziu
a viabilidade bacterial, especialmente nas bactérias periodontopáticas,
o que pode recomendar o uso no controle do biofilme subgingival em
pacientes periodentais.
Acknowledgements
The authors thanks to the Colombian Department for Science, Technology
and Innovation (COLCIENCIAS) as the sponsor of this project through the
Grant No 130850227678 To Dr. Marcela Buitrago for it assistance in the
development of the SDS-Page.
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Received December 13, 2014
Accepted June 23, 2015
