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Accepted Article
Revised Date : 10-Aug-2014
Department of Preventive and Institute of Oral Bioscience, BK21 Plus Program, School of
Corresponding author:
Aim To determine the relationships between the antibacterial activity of NaOCl and
treatment time and biofilm age in early Enterococcus faecalis biofilms using a linear-fitting
procedure.
relationship between the antibacterial activity of NaOCl and biofilm age, 22-, 46-, 70-, and
94-h-old biofilms were exposed to NaOCl (0 3%) for 5 min. To investigate the relationship
between the antibacterial activity of NaOCl and treatment time, 70-h-old biofilms were
exposed to NaOCl (0 3%) for 1, 3, 5, and 7 min. After treatment, colony forming units
(CFUs) were counted. To determine the relationships between these variables, linear-fitting
was performed.
Results The change in the minimum biofilm eradication concentration (MBEC) of NaOCl
dependence (R = 0.948, R2 = 0.898). Below the MBEC, the fitting lines for bacterial CFU
count versus NaOCl concentration (R 0.973, R2 0.948) in the 22-, 46-, 70-, and 94-h-old
biofilms implied that the antibacterial activity of NaOCl decreased as the biofilm age
increased. The fitting lines for bacterial CFU count versus NaOCl concentration (R 0.970,
R2 0.942) in the 1, 3, 5, and 7 min treatments implied that the antibacterial activity of
Conclusions These results suggest that the antibacterial activity of NaOCl against early E.
faecalis biofilms in root canals may follow a linear pattern depending on biofilm age or
treatment time.
infections in the body; by one estimate, nearly 80% of all infections involve biofilms (Biel
disease as a biofilm-mediated infection (Bhuva et al. 2010), suggesting that the elimination
or the significant reduction of biofilms may be an essential factor for the successful treatment
that at least 20 taxa of bacteria are related to endodontic infection (Munson et al. 2002),
matrix (Barnes & Patel 2011). Of the endodontic pathogens, Enterococcus faecalis, a
facultative anaerobic Gram-positive coccus, has been extensively studied since this species
al. 2003, Ras et al. 2004, Stuart et al. 2006). This bacterium possesses various virulence
lipoteichoic acid, which contribute to its survival in the harsh environment of the root canal
(Ras et al. 2004). Furthermore, the ability of this bacterium to form biofilms provides it with
Biofilms develop after the initial attachment of bacteria to a surface, followed by the
surface and enmeshed in a complex extracellular matrix (Mohammadi et al. 2013). It has
been reported that bacterial cells in biofilms show increased resistance to antibacterial
antimicrobial agents are the impenetrable character of biofilms, the slow growth rate of
bacteria, and the induction of resistance mechanisms (Donlan & Costerton 2002); however,
since the composition, structure, and physiology of biofilms vary with the nature of its
resident bacteria and the local environment (Koo et al. 2010), the resistance of biofilms to
The number of bacteria in infected root canals can be reduced by the antibacterial
sodium hypochlorite (NaOCl) has been in popular use for many years and has become the
gold standard for irrigation in root canal treatment because of its ability to dissolve necrotic
tissue and its potent antibacterial action (Zehnder et al. 2002, Mohammadi 2008). Recently,
many studies have revealed the strong antibacterial activity of NaOCl against E. faecalis in
the planktonic state (Gomes et al. 2001) as well as on their biofilms (Bhuva et al. 2010,
Meire et al. 2012, Chen et al. 2013). Furthermore, previous studies have shown that NaOCl
is more effective in young biofilms than old biofilms and is more effective with 3 min
treatment than with 1 min treatment (Wang et al. 2012, Stojicic et al. 2013), suggesting that
biofilm age and treatment time are important factors related to the antibacterial activity of
NaOCl. However, little has been reported on the statistical relationships between the anti-
biofilm activity of NaOCl and treatment time and biofilm age, especially in early E. faecalis
biofilms.
The aim of the present study was to determine the statistical relationships between the
antibacterial activity of NaOCl and treatment time and biofilm age using an in vitro early E.
faecalis biofilm model. The null hypothesis is that there is no relationship between the
Test solution
NaOCl solutions at concentrations of 0.001, 0.003, 0.01, 0.03, 0.1, 0.3, 1, and 3% were
tested. The test solution was diluted from 12% NaOCl solution (Yakuri Pure Chemicals Co.,
LTD., Kyoto, Japan) with sterilized distilled water and kept at room temperature. The test
Before biofilm formation, three colonies of E. faecalis (KCCM 11814) were inoculated into a
brain heart infusion (BHI) broth and incubated overnight (37C, 5% CO2). A part of the
bacterial suspension (100 L) was sub-inoculated into 4 mL of BHI and incubated for 2 h to
arrive at an optical density (OD) of 0.2 at 600 nm. The bacteria at OD 0.2 were in the
exponential phase, and the number of colony forming units (CFUs) was 1 2 108 mL-1
(data not shown). The 40-fold diluted E. faecalis solution (2.5 5 106 CFU mL-1) with BHI
broth was used for biofilm formation. E. faecalis biofilms were formed on hydroxyapatite (HA)
discs (0.50 in. x 0.04 0.06 in.; Clarkson Chromatography Products Inc., South
Williamsport, PA, USA) placed in a vertical position in 24-well plates (Fig. 1). For biofilm
formation, the HA discs were kept in sterile distilled water for 30 min and then transferred to
a 24-well plate containing BHI broth (2.8 mL well-1) with E. faecalis (2.5 5 106 CFU mL-1).
The biofilms grew undisturbed for 22 h to allow initial biofilm growth. From this time (22 h
old), the culture medium (BHI broth) was changed daily (9 AM) until it was 94 h old. The
culture medium was changed a total of three times (at 22, 46, and 70 h).
Fig. 1 shows early E. faecalis biofilms on HA discs and the experimental scheme for this
study. To investigate the relationship between the antibacterial activity of NaOCl and biofilm
age in early E. faecalis biofilms, the 22-, 46-, 70-, and 94-h-old biofilms were exposed to
each concentration of NaOCl for 5 min, dip-rinsed three times in sterile water (to remove
excess NaOCl), and transferred to glass tubes containing 2 mL of 0.89% NaCl solution. To
investigate the relationship between the antibacterial activity of NaOCl and its treatment
time, the 70-h-old biofilms were exposed to each concentration of NaOCl for 1, 3, 5, and 7
using an ultrasonic water bath (Powersonic 410, Hwashin Technology Co., Seoul, Korea),
and then the biofilms were removed by a sterile spatula. An aliquot (100 L) of the dispersed
solution was serially diluted and plated onto the BHI agar plates to count the CFUs. Each
The data are presented as mean standard deviation. The intergroup differences were
estimated by one-way ANOVA, followed by a post hoc multiple comparison (Tukey test).
Values were considered statistically significant when the P value was < 0.05.
To determine the relationships between the antibacterial activity of NaOCl and treatment
time and biofilm age in early E. faecalis biofilms, linear-fitting (linear regression analysis) was
performed using a linear regression analysis program (Origin 7.0; Microcal Inc.,
Northampton, MA, USA). For the relationship between the antibacterial activity of NaOCl and
biofilm age, linear-fittings were performed for the minimum biofilm eradication concentration
(MBEC) of NaOCl versus biofilm age, bacterial CFU count versus NaOCl concentration in
the 22-, 46-, 70-, and 94-h-old biofilms, and bacterial CFU count versus biofilm age. In this
study, the MBEC was defined as the lowest concentration of NaOCl capable of killing all of
the biofilm bacteria. For the relationship between the antibacterial activity of NaOCl and
treatment time, linear-fittings were performed for MBEC of NaOCl versus treatment time,
bacterial CFU count versus NaOCl concentration in the 1, 3, 5, and 7 min treatments, and
bacterial CFU count versus treatment time. The correlation coefficient (R) and determination
coefficient (R2) of each fitting line were calculated, which can show the strength of the
relationship between the two variables and the proportion of the total variation of one
variable that is explained by its linear relationship with the other variable, respectively (Quinn
The antibacterial activity of NaOCl against early E. faecalis biofilms was closely related to
biofilm age. As shown in Fig. 2a, bacterial CFU counts in the 22- and 46-h-old biofilms
started to diminish at 0.001% NaOCl (P < 0.05), while those in the 70- and 94-h-old biofilms
started to decrease at 0.003% NaOCl (P < 0.05). The MBECs of NaOCl in the 22- and 46-h-
old biofilms were 0.1 and 0.3%, respectively. In the 70- and 94-h-old biofilms, the MBEC was
1%. Generally, this result suggests that the antibacterial activity of NaOCl against early E.
faecalis biofilms could be related to the biofilm age at all tested concentrations.
However, since Fig. 2a did not show a definite relationship between the antibacterial
activity of NaOCl and biofilm age, linear-fitting was performed to determine the statistical
relationship using the data shown in Fig. 2a. Fig. 2b d shows the results of the linear-fitting
procedure. As shown in Fig. 2b, the change in MBEC followed a linear pattern of biofilm age
dependence (P < 0.05). The R and R2 of the fitting line for MBEC versus biofilm age were
0.941 and 0.886, respectively. Furthermore, the fitting lines for bacterial CFU count versus
NaOCl concentration in the 22-, 46-, 70-, and 94-h-old biofilms at concentrations lower than
MBEC, which showed high R (0.997 0.973) and R2 (0.948 0.996) values, were right-
shifted with increasing biofilm age (Fig. 2c). In addition, the CFU count of the E. faecalis
biofilms treated with the control increased with increasing biofilm age (R = 0.997, R2 = 0.996)
(Fig. 2d).
The antibacterial activity of NaOCl against early E. faecalis biofilms was also closely related
to treatment time. As shown in Fig. 3a, the MBECs in the 1, 3, 5, and 7 min treatments were
3, 1, 1, and 0.3%, respectively, suggesting that the antibacterial activity of NaOCl against
early E. faecalis biofilms might be related to treatment time. However, since NaOCl reduced
biofilm cell viability from 0.003% regardless of the treatment time (Fig. 3a), and since the
using the data shown in Fig. 3a. As shown in Fig. 3b, the change in MBEC followed a linear
pattern of treatment time dependence (P < 0.05). The R and R2 of the fitting line for MBEC
versus treatment time were 0.948 and 0.898, respectively. Furthermore, the fitting lines for
bacterial CFU count versus NaOCl concentration in the 1, 3, 5, and 7 min treatments at
concentrations lower than MBEC (P < 0.05, respectively), which showed high R (0.984
0.970) and R2 (0.942 0.970) values, were left-shifted as the treatment time increased (Fig.
3c). In this study, there was no significant difference among the CFU counts of 94-h-old E.
faecalis biofilms treated with the control for 1, 3, 5, and 7 min (P > 0.05) (Fig. 3d).
Discussion
Many studies have shown that the antibacterial activity of NaOCl depends on its
concentration, contact time, pH, and temperature (Vianna et al. 2004, DeQueiroz & Day
2008, Mercade et al. 2009); however, few studies have reported on the precise relationship
between the antibacterial activity of NaOCl and the factors influencing the antibacterial
between early and mature E. faecalis biofilms can show different patterns of antimicrobial
resistance (Shen et al. 2011), little has been reported on the precise relationship in early or
mature E. faecalis biofilms. In this study, the focus was on early E. faecalis biofilms and
linear-fitting (linear regression analysis) was performed to reveal the statistical relationship
between the antibacterial activity of NaOCl against early E. faecalis biofilms and treatment
time and biofilm age. It is well accepted that linear regression analysis is useful to describe
the linear relationship between two variables and to determine how much of the variation in
one variable can be explained by the linear relationship with the other variable (Quinn &
Keough 2002).
of the fitting line for MBEC versus the biofilm age was 0.941, suggesting that MBEC is
strongly and positively correlated with biofilm age; however, since R only shows the strength
of the relationship between two variables, R2 was also calculated. In the fitting line, the R2
was 0.886 (Fig. 2b), indicating that the line can appropriately describe the change in MBEC
according to the biofilm age, and at least 88.6% of the variation in MBEC can be explained
by the biofilm age. In addition to the relationship with MBEC, the relationship at
concentrations lower than MBEC was also investigated. As shown in Fig. 2c, the fitting lines
for bacterial CFU count versus NaOCl concentration in the 22-, 46-, 70-, and 94-h-old
biofilms were right-shifted as the biofilm age increased, indicating that the antibacterial
activity of NaOCl at concentrations lower than MBEC decreased as the biofilm age
increased. Overall, the data clearly showed that the antibacterial activity of NaOCl against
early E. faecalis biofilms decreased as the biofilm age increased at all tested concentrations.
In this study, the difference in the antibacterial activity of NaOCl against the 22-, 46-, 70-,
and 94-h-old biofilms may be related to the number of biofilm cells. As shown in Fig. 2d, the
number of biofilm cells was strongly and positively correlated with the biofilm age (R = 0.997,
R2 = 0.996), suggesting that the number of biofilm cells increases thus affording better
The relationship between the antibacterial activity of NaOCl against early E. faecalis
biofilms and treatment time was also determined. As shown in Fig. 3b, the R and R2 of the
fitting line for MBEC of NaOCl versus the treatment time (1, 3, 5, and 7 min) were 0.948
and 0.898, respectively, indicating that MBEC is strongly and negatively correlated with the
treatment time, and at least 89.8% of the variation in MBEC can be explained by the
treatment time. Furthermore, the fitting lines for bacterial CFU count versus NaOCl
Overall, the data showed that the antibacterial activity of NaOCl against early E. faecalis
biofilms is strongly and positively correlated with the treatment time at all tested
concentrations, confirming a previous finding that the antibacterial effect of NaOCl increases
In addition, the data showed the relationship between the antibacterial activity of NaOCl
against early E. faecalis biofilms and its concentration. As shown in Fig. 2c and 3c, the R
and R2 of the respective biofilm age and treatment time in the fitting lines for the biofilm cell
viability versus NaOCl concentration ranged from 0.997 0.970 and 0.942 0.996,
respectively, suggesting that the antibacterial activity of NaOCl is closely correlated with the
In this study, the data also demonstrated the relationship between the antibacterial activity
of NaOCl against early E. faecalis biofilms and NaOCl concentration. As shown in Fig. 2c
and 3c, the change in bacterial CFU count followed a linear pattern of NaOCl concentration
dependence at all of the biofilm ages and treatment times tested (R 0.970, R2 0.942).
These results suggest that the antibacterial activity of NaOCl against early E. faecalis
In this study, the data clearly showed that the antibacterial effect of NaOCl against early E.
faecalis biofilms is closely correlated with i) biofilm age, ii) treatment time, and iii) NaOCl
concentration. This result suggests that the suitable NaOCl concentration for the eradication
of early E. faecalis biofilm cells in root canals can vary since at least three factors can jointly
influence the antibacterial effect of NaOCl. As reported in previous studies (Gomes et al.
2001, Radcliffe et al. 2004, Vianna et al. 2004, Rematozo et al. 2010), the treatment time
and concentration of NaOCl required to completely eradicate E. faecalis biofilm cells were
various, confirming the difficulty of choosing the suitable concentration of NaOCl. Thus, a
in endodontic therapy.
Conclusions
The antibacterial activity of NaOCl against early E. faecalis biofilms followed a linear pattern
0.886) at MBEC. Furthermore, at concentrations lower than MBEC, the antibacterial activity
also increased as biofilm age decreased or treatment time increased. These results suggest
that the antibacterial activity of NaOCl against early E. faecalis biofilms in root canals may
follow a linear pattern of biofilm age or treatment time dependence. However, additional in
Acknowledgement
This work was supported under the framework of international cooperation program
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Figure legends
Figure 1 Early E. faecalis biofilm formation on hydroxyapatite (HA) discs and the
experimental plan.
Figure 2 Relationship between the antibacterial activity of NaOCl and biofilm age in early E.
faecalis biofilms. (a) Antibacterial activity of NaOCl (5 min treatment) against the 22-, 46-,
70-, and 94-h-old biofilms. (b) Linear-fitting for the minimum biofilm eradication concentration
(MBEC) of NaOCl versus biofilm age. (c) Right shift of the linear-fitting line with an increase
in biofilm age at concentrations lower than MBEC. (d) Linear-fitting for CFU of the biofilms
treated with the control (sterile water) versus biofilm age. The data in b, c, and d were from a.
followed by the same superscript text are not significantly different from each other (P >
0.05). N/S: not significantly different from the control (P > 0.05).
Figure 3 Relationship between the antibacterial activity of NaOCl and treatment time in 70-
h-old E. faecalis biofilms. (a) Effect of 1, 3, 5, and 7 min treatments with NaOCl on the 70-h-
old biofilms. (b) Linear-fitting for the minimum biofilm eradication concentration (MBEC) of
NaOCl versus treatment time. (c) Left shift of the linear-fitting line with an increase in
treatment time at concentrations lower than MBEC. (d) The CFU of the biofilms treated with
followed by the same superscript text are not significantly different from each other (P >
0.05). N/S: not significantly different from the control (P > 0.05).
Preparation of HA discs
NaOCl treatment NaOCl treatment NaOCl treatment NaOCl treatment
(5 min) (5 min) (5 min) (5 min)
Fig. 1
Fig. 2
Fig. 3