Received Date : 04-Apr-2014

Accepted Article
Revised Date : 10-Aug-2014

Accepted Date : 20-Aug-2014

Article type : Original Scientific Article

Relationships between the antibacterial activity of sodium hypochlorite and treatment

time and biofilm age in early Enterococcus faecalis biofilms

N P T Chau, N-H. Chung, J.G. Jeon

Department of Preventive and Institute of Oral Bioscience, BK21 Plus Program, School of

Dentistry, Chonbuk National University, Jeonju, Republic of Korea

Running head: Anti-biofilm activity of sodium hypochlorite

Keywords: Antibacterial activity, biofilm age, Enterococcus faecalis biofilms, sodium

hypochlorite, treatment time.

Corresponding author:

Dr. Jae-Gyu Jeon,

Department of Preventive Dentistry, School of Dentistry, Institute of Oral Bioscience and BK

21 plus program, Chonbuk National University, Jeonju 561-756, Republic of Korea

Tel.: + 82 63 270 4036, Fax: + 82 63 270 4035

This article has been accepted for publication and undergone full peer review but has not
been through the copyediting, typesetting, pagination and proofreading process which may
lead to differences between this version and the Version of Record. Please cite this article as
an 'Accepted Article', doi: 10.1111/iej.12376

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 E-mail address: dentjjk@jbnu.ac.kr (J.-G. Jeon)
Accepted Article
Abstract

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.

Methodology E. faecalis biofilms were formed on hydroxyapatite discs. To investigate the

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

followed a linear pattern of biofilm age (R = 0.941, R2 = 0.886) or treatment time

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

NaOCl increased with treatment time.

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.

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 Introduction
Accepted Article
It has been well known that biofilms are closely related to a wide variety of microbial

infections in the body; by one estimate, nearly 80% of all infections involve biofilms (Biel

2010). The current perspective in endodontic microbiology also conceptualizes endodontic

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

of endodontic infections (Mohammadi 2012). Recently, molecular techniques have revealed

that at least 20 taxa of bacteria are related to endodontic infection (Munson et al. 2002),

indicating that endodontic biofilms are complex communities of microbes embedded in a

matrix (Barnes & Patel 2011). Of the endodontic pathogens, Enterococcus faecalis, a

facultative anaerobic Gram-positive coccus, has been extensively studied since this species

is frequently found in endodontic infections, especially in post-treatment disease (Pinheiro et

al. 2003, Ras et al. 2004, Stuart et al. 2006). This bacterium possesses various virulence

factors such as lytic enzymes, cytolysin, aggregation substances, pheromones, and

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

an ecologic advantage of increased resistance to the antibacterial activity of endodontic

medicaments (Donlan & Costerton 2002, Sandoe et al. 2006).

Biofilms develop after the initial attachment of bacteria to a surface, followed by the

formation of a bacterial community characterized by cells that are firmly attached to a

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

agents. Mechanisms that may be related to the increased resistance of biofilms to

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

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 antibacterial agents can depend on biofilm age and environmental changes (Shen et al.
Accepted Article
2011, Stojicic et al. 2013).

The number of bacteria in infected root canals can be reduced by the antibacterial

activity of endodontic medicaments (Siqueira et al. 2007). Of the endodontic medicaments,

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

antibacterial activity of NaOCl, treatment time and biofilm age

Materials and Methods

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

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 solution was prepared before the experiments and was used within one week. Sterile
Accepted Article
distilled water was used as a control.

Early E. faecalis biofilm formation

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).

Experimental scheme and NaOCl treatment

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

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 min, dip-rinsed three times in sterile water, and transferred to glass tubes containing 2 mL of
Accepted Article
0.89% NaCl solution. The glass tubes containing the biofilms were sonicated for 10 min

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

assay was performed in duplicate in at least four different experiments (n = 8).

Statistical analysis and linear-fitting procedure

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

& Keough 2002).

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 Results
Accepted Article
Relationship between the antibacterial activity of NaOCl and biofilm age

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).

Relationship between the antibacterial activity of NaOCl and treatment time

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

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 change in MBEC according to the treatment time only showed a tendency for a relationship
Accepted Article
between the antibacterial activity of NaOCl and treatment time, linear-fitting was performed

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

activity of NaOCl in E. faecalis biofilms. Furthermore, although the physiological differences

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).

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 In this study, the relationship between the antibacterial activity of NaOCl and biofilm age in
Accepted Article
the 22-, 46-, 70-, and 94-h-old biofilms was investigated initially. As shown in Fig. 2b, the R

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

resistance to antibacterial agents with increasing biofilm age.

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

concentration in 1, 3, 5, and 7 min treatments at concentrations lower than MBEC shifted to

This article is protected by copyright. All rights reserved.

 the left as the treatment time increased (Fig. 3c), indicating that the antibacterial activity of
Accepted Article
NaOCl increased as the treatment time increased at concentrations lower than MBEC.

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

with increasing exposure time (Rematozo et al. 2010).

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

concentration of NaOCl regardless of biofilm age or treatment time.

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

biofilms is positively correlated with the concentration of NaOCl.

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

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 more sophisticated study including factors related to the antibacterial activity of NaOCl
Accepted Article
against early E. faecalis biofilms will be needed to determine the appropriate concentration

in endodontic therapy.

Conclusions

The antibacterial activity of NaOCl against early E. faecalis biofilms followed a linear pattern

of treatment time (R = 0.948, R2 = 0.898) and biofilm age dependence (R = 0.941, R2 =

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

vivo studies are required to confirm the relationships in endodontic therapy.

Acknowledgement

This work was supported under the framework of international cooperation program

managed by the National Research Foundation of Korea (2012K2A1A2029691).

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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.

In a, we approximated 0 with an X coordinate of 1E5 and a Y coordinate of 0.1. In d, values

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

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 the control (sterile water) for 1, 3, 5, and 7 min. The data in b, c, and d are from a. In a, we
Accepted Article
approximated 0 with an X coordinate of 1E5 and a Y coordinate of 0.1. In d, values

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).

Biofilm start (E. faecalis inoculation, 2.5 5 x 106 CFU mL-1)

Culture medium (BHI) replacement

0 h (11 AM) 22 h (9 AM) 46 h (9 AM) 70 h (9 AM) 94 h (9 AM)

Preparation of HA discs
NaOCl treatment NaOCl treatment NaOCl treatment NaOCl treatment
(5 min) (5 min) (5 min) (5 min)

Relationship between antibacterial activity of NaOCl and biofilm age

E. faecalis biofilms on HA discs placed NaOCl treatment (1, 3, 5, and 7 min)

in a vertical position in a 24-well plate
Relationship between antibacterial activity of NaOCl and treatment time

Fig. 1

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Accepted Article

Fig. 2

Fig. 3

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