Accepted Manuscript Title: The effects of sodium hypochlorite and chlorhexidine irrigants on the antibacterial activities of alkaline media against Enterococcus faecalis Author: Jinglei Ma Zhongchun Tong Junqi Ling Hongyan Liu Xi Wei PII: DOI: Reference:
S0003-9969(15)00097-7 http://dx.doi.org/doi:10.1016/j.archoralbio.2015.04.008 AOB 3380
To appear in:
Archives of Oral Biology
Received date: Revised date: Accepted date:
17-1-2015 16-4-2015 19-4-2015
Please cite this article as: Ma J, Tong Z, Ling J, Liu H, Wei X, The effects of sodium hypochlorite and chlorhexidine irrigants on the antibacterial activities of alkaline media against Enterococcus faecalis, Archives of Oral Biology (2015), http://dx.doi.org/10.1016/j.archoralbio.2015.04.008 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Highlights: 1. E. faecalis survival decreased in CHX or 5.25% NaOCl and next alkaline challenge. 2. E. faecalis biofilm survival were reduced in CHX and next alkaline challenge.
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4. CHX might be more effective in improving the alkaline than NaOCl.
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3. E. faecalis biofilm survival increased in NaOCl and next alkaline challenge.
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The effects of sodium hypochlorite and chlorhexidine irrigants on the antibacterial activities of alkaline media against Enterococcus faecalis Jinglei Ma a b 1, Zhongchun Tong a b 1, Junqi Ling a b*, Hongyan Liu a, Xi Wei a Department of Operative Dentistry and Endodontics, Guanghua School of Stomatology, Sun
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a
Yat-sen University, Guangzhou, Guangdong, China.
Guangdong Provincial Key Laboratory of Stomatology, Sun Yat-sen University, Guangzhou,
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b
1
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Guangdong, China. Jinglei Ma and Zhongchun Tong contributed equally to this work.
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* Corresponding author: Junqi Ling
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Postal address: Prof Junqi Ling, Department of Operative Dentistry and Endodontics, Guanghua School of Stomatology, Sun Yat-sen University, Guangzhou 510055, Guangdong, China. Fax: +86 20 83822807
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E-mail: [email protected]
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Telephone: +86 20 83862621
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Abstract Objective: Sodium hypochlorite (NaOCl), chlorhexidine (CHX) and calcium hydroxide are common intracanal medicaments. The present study aimed to evaluate the effects of NaOCl and CHX on the antibacterial activities of alkaline media against Enterococcus faecalis.
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Designs: The survival rates of planktonic and biofilm E. faecalis were evaluated by plate counts after 1 min of pretreatment with NaOCl and CHX, and time-kill assays were then used to assess subsequent pH alkaline challenges. Dead and living cells in the E. faecalis biofilm were assessed
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with SYTO 9 and PI staining in combination with confocal laser scanning microscopy following
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exposure to NaOCl or CHX and subsequent alkaline challenges by common root canal irrigation and dressing procedures.
Results: One minute of pretreatment with 2% CHX, 0.2% CHX, or 5.25% NaOCl in combination
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with a subsequent alkaline challenge significantly decreased planktonic E. faecalis survival rates, but pretreatment with 1% NaOCl did not. The E. faecalis biofilm survival rates were reduced in the
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subsequent alkaline challenge following CHX pretreatment but gradually increased following NaOCl pretreatment. Similarly, CLSM analysis revealed that the greatest proportions of dead E.
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faecalis cells in the biofilms were presented in the CHX and alkaline treatment group.
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Conclusion: CHX might be more effective in improving the antibacterial activities of alkaline root
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canal medicaments against E. faecalis than NaOCl during routine root canal therapy procedures.
Keywords: sodium hypochlorite, chlorhexidine, alkaline challenge, Enterococcus faecalis.
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Introduction The objectives of root canal therapies are to eradicate pathogenic bacteria within the root canal and to prevent recurrent infections. Although biomechanical instrumentation can eliminate most bacteria in root canals, antibacterial irrigants and intracanal dressings are indispensable for
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improving the success of root canal therapy. Sodium hypochlorite (NaOCl) and chlorhexidine (CHX) are two common intracanal irrigants that have demonstrated good antibacterial activity.1
After root canal irrigation, alkaline calcium hydroxide is generally utilized as an intracanal dressing
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to kill any residual bacteria.2 Currently, studies concerning irrigants have primarily focused on the
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effects of intracanal medicaments on bacteria in their normal physiological states in the root canals and on the removal of smear layers.3–7 However, due to the challenge of intracanal medicaments with high antibacterial activities, for example, irrigation with NaOCl and CHX, the residual viable
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bacteria and the biofilm bacteria in root canals could possibly be in stressed states. Stressed bacteria exhibit altered physiologies and might be resistant to intracanal dressings, and this issue deserves
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further study.
The harbouring of microorganisms in the root canal system is the major cause of intraradicular
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infection, which is divided into three categories: primary, secondary, and persistent infections.8,9
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Enterococcus faecalis is generally considered to be closely related to secondary and persistent root canal infections despite representing only low proportions of the diverse microflora that cause
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primary root canal infections.10,11 E. faecalis can survive disadvantageous environments, including those with extreme alkaline pH values, high salt concentrations, bile salts, and starvation, and are resistant to many antimicrobials. Thus, these bacteria are often considered test bacteria for evaluations of the effectiveness of intracanal medicaments.12–15 The disadvantageous environments place E. faecalis in a stressed state, and at present, a few studies have studied E. faecalis that has been stressed by starvation and alkaline pH.15–18 However, little is known about the stress response of E. faecalis to exposure to NaOCl and CHX or its ability to resist an alkaline pH. In the present study, we attempted to investigate the sensitivity of planktonic and biofilm E. faecalis to alkaline pH after short-duration challenges with NaOCl and CHX and to estimate the survival ability of E. faecalis in alkaline intracanal dressings.
Materials and methods Bacterial culture Page 4 of 18
E. faecalis ATCC 29212 was used in this study. The strain was routinely streaked on brain heart infusion (BHI, BD Difco, Sparks, MD, USA) agar and cultured aerobically at 37°C for 24 h. A single bacterial colony was inoculated into 5 mL of BHI medium and was grown overnight. The preparation of planktonic bacteria for the alkaline challenge assay was accomplished by adding 50
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mL of BHI broth to 500 µL of overnight E. faecalis culture, followed by an additional 12 h of culturing. The concentration of E. faecalis was adjusted to an OD600 value of 0.8, which
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corresponded to approximately 109 colony-forming units per millilitre (CFU/mL). Preparation of the antibacterial agents
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Sodium hypochlorite (NaOCl, Sigma, St. Louis, MO, USA) and chlorhexidine acetate (CHX, Sigma, St. Louis, MO, USA) solutions were prepared for this study. The 2% and 0.2% solutions of CHX were prepared by dissolving CHX powder in sterile distilled water, and 5.25% and 1%
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solutions of NaOCl were also prepared by dilution with sterile distilled water. For the alkaline challenges, fresh sterile BHI broth and tryptic soy broth (TSB, BD Difco, Sparks, MD, USA) were
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adjusted to pH values of 8, 9, 10 and 11 with sodium hydroxide.
Alkaline challenge of the planktonic and biofilm E. faecalis following NaOCl and CHX
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pretreatment
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E. faecalis was grown to a concentration of approximately 109 CFU/mL according to growth curves. After the five groups of E. faecalis cultures (48 mL culture in each group) were centrifuged
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at 6,000 rpm for 5 min at 4 °C, the pellets were challenged by resuspension in five common intracanal irrigants (A: 5.25% NaOCl; B: 1% NaOCl; C: 2% CHX; D: 0.2% CHX; and E: 0.9% NaCl [control]) for 1 min. The antibacterial activity of CHX was stopped with 0.5% tween 80 and 0.07% lecithin, and the NaOCl was inactivated with 0.6% sodium thiosulfate.19,20 To prove complete inactivation, the inactivated CHX and NaOCl were added to E. faecalis cultures, and the lack of significant changes in the E. faecalis survival rates indicated complete inactivation of the drugs. Following 1 min of pretreatment with the common intracanal irrigants, each group of E. faecalis was washed twice using PBS by centrifugation (6,000 rpm for 5 min at 4 °C) and then was divided into four subgroups. After additional centrifugation, the supernatant was discarded and the bacterial pellet was challenged by resuspension in 12 mL of BHI broth with different alkalinities (pH 8, pH 9, pH 10 and pH 11). Afterward, the E. faecalis in each subgroup was divided equally among 12 freezing tubes (2-mL volume, Corning Costar, Cambridge, MA, USA). At the 0, 2 h, 4 h, 6 h, 8 h, 10 h, 12 h, 1 d, 2 d, 4 d, 7 d, and 14 d time points, 100 µL of bacterial solution in a random Page 5 of 18
freezing tube was 10-fold serially diluted and spread on a BHI agar plate. After 48 h of incubation, the survival counts of the E. faecalis were determined. To maintain a constant alkaline pH, the E. faecalis that were challenged in alkaline media were enclosed in freezing tubes to reduce exposure to CO2 in the air. Freezing tubes without bacteria were used as negative controls to ensure the
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absence of contamination. Each test was repeated three times on different days. For the biofilm assays, E. faecalis culture (approximately 109 CFU/mL) and TSB containing 1% glucose at a ratio of 1:100 were added to 48-well plates (1 mL per well). After 24 h of
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incubation, the supernatant and non-adhesive cells were discarded and the biofilm on the bottom of
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the well was gently washed with sterile PBS. Subsequently, 1-mL aliquots of the test intracanal irrigants (A: 5.25% NaOCl; B: 1% NaOCl; C: 2% CHX; D: 0.2% CHX; and E: 0.9% NaCl [control]) were added to the 48-well plates. The challenges lasted for 1 min and were then stopped
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by neutralizers as described above. The solutions in the wells were removed, and the biofilms on the bottom were gently washed once with PBS. The biofilms were immediately exposed to TSB at
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different pH values (pH 8, pH 9, pH 10, and pH 11). At the 0, 2 h, 4 h, 6 h, 8 h, 10 h, 12 h, 1 d, 2 d, 4 d, 7 d, and 14 d time points, the alkaline TSB solutions were discarded. The biofilms on the
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bottom were repeatedly scraped using a pipette tip and resuspended with 1 mL of sterile PBS.21 The
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resuspended biofilm cells were adequately agitated in a vortex mixer for 5 min, and the bacterial counts were assessed as the plate counts of serial 10-fold dilutions. During the course of the alkaline
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challenges, to maintain the constant alkalinity of the broth, bacterial challenges from the same time points were enclosed in the same 48-well plates using a sealing strip. The pH of the broth was assayed to ensure a constant pH before the plate counts at each time point. All procedures were independently performed three times on different days, and the means are reported. Confocal laser scanning microscope A confocal laser scanning microscope (CLSM) was used to evaluate the E. faecalis biofilms that had been subjected to an alkaline challenge after NaOCl or CHX pretreatment. For the E. faecalis biofilms, E. faecalis cultures (approximately 109 CFU/mL) and TSB containing 1% glucose at a ratio of 1:100 were added to plastic petri dishes with glass bottoms (D: 35 mm, Hangzhou Shengyou Biotechnology, China). After incubation for 24 h, the biofilms on the bottom were treated with the test intracanal irrigants (A: 5.25% NaOCl; B: 1% NaOCl; C: 2% CHX; D: 0.2% CHX; and E: 0.9% NaCl [control]) for 1 min and were then exposed to TSB at a pH of 9 or 11 for 24 h. E. faecalis biofilms were stained with a mixture of 6 µM SYTO 9 stain and 30 µM PI at room Page 6 of 18
temperature in the dark for 15 min according to the specifications of the Live/Dead BacLight Bacterial Viability Kit (L13152; Molecular Probes, Invitrogen, Inc., Eugene, OR, USA). Images were then captured using a Carl Zeiss confocal laser scanning microscope (CLSM) and ZEN software (Zen 2012 light edition, Carl Zeiss MicroImaging, Inc., Thornwood, NY). SYTO 9 and PI
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were excited at 488 nm and 543 nm, respectively. The viable cells were stained green, and the dead cells were stained red. Five random (four angular vertexes and a central point in the square) areas were captured from each treatment group. The viable and dead cells were analysed based on
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fluorescence intensity with ZEN software, and the average percentages of the viable cells in the
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biofilms were obtained. Statistical analyses
The statistical analyses were performed using SPSS 18.0 software. In the analyses of the
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fluorescence intensities, Kruskal–Wallis H and Nemenyi nonparametric tests were used to analyse the live bacterial percentages following challenges with 2% CHX, 0.2% CHX, 5.25% NaOCl, 1%
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Results
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level of significance was set at p < 0.05.
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NaOCl and 0.9% NaCl for 1 min and the 24-h alkaline challenges in each of the five groups. The
The survival of planktonic E. faecalis
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In the intracanal irrigant challenge tests, the survival rates of the planktonic E. faecalis significantly decreased after 1-min exposures to pretreatment with 5.25% NaOCl, 1% NaOCl, 2% CHX and 0.2% CHX, and 5.25% NaOCl and 2% CHX were more effective than 1% NaOCl and 0.2% CHX. After the 5.25% NaOCl and 2% CHX pretreatments, the E. faecalis survival rates decreased to zero after 2 h of alkaline challenge at pH levels of 8, 9, 10 and 11. In the challenges at pH levels of 8, 9 and 10, the E. faecalis survival rates in the 0.2% CHX pretreatment group exhibited similar reductions and dropped to zero at 7 d, and the E. faecalis survival rates in the 1% NaOCl pretreatment group gradually increased until 7 d and then decreased between 7 and 14 d. In the challenges at pH levels of 11, the survival rate of the 0.2% CHX pretreatment was more significantly reduced, and survival in the 1% NaOCl pretreatment group also decreased (Fig. 1a-d). The survival of the E. faecalis biofilm E. faecalis survival rates in the biofilms were determined following treatments with the test intracanal irrigants and the alkaline challenges (Fig. 2a-d). The E. faecalis in the biofilms were not Page 7 of 18
completely killed after 1-min exposures to 5.25% NaOCl, 1% NaOCl, 2% CHX or 0.2% CHX and the subsequent alkaline challenges. In the 5.25% NaOCl and 1% NaOCl pretreatment groups, the E. faecalis survival rates exhibited gradual increases and reached a maximum at 2 d and 4 d before finally dropping following the pH 8, pH 9, pH 10 and pH 11 alkaline challenges. However, in the
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2% CHX and 0.2% CHX pretreatment groups, the E. faecalis survival rates gradually decreased throughout the entire experimental period following the different alkaline pH challenges. In the later stage of the alkaline challenges, the E. faecalis survival rates in the CHX pretreatment group were
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far less than those of the NaOCl pretreatment group.
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Fluorescence intensity analyses of the living and dead cells in the biofilms
The live and dead cells in the biofilms that had been treated with intracanal irrigants and alkaline pHs were evaluated by CLSM (Fig. 3 and 4). Following the 1-min treatment with NaOCl
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or CHX alone, the ratios of live cells in the biofilms were significantly different between the 5.25% NaOCl, 1% NaOCl, 2% CHX, and 0.2% CHX groups and the control group (p < 0.05). In the
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5.25% NaOCl group, the ratio of live cells was less than those in the 1% NaOCl, 2% CHX and 0.2% CHX groups, and dead cells dominated in the biofilm. After 24 h of exposure to alkalinities of
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pH 9 or 11, significant differences were found in the ratios of live cells between the NaOCl and
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CHX pretreatment groups (p < 0.05). In the 5.25% NaOCl and 1% NaOCl pretreatment groups, the ratios of live cells significantly increased and the live cells dominated the biofilms in a manner
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similar to that of the control. However, in the 2% CHX and 0.2% CHX pretreatment groups, the ratios of live cells were significantly decreased and the dead cells dominated the biofilms.
Discussion
NaOCl, CHX and calcium hydroxide have been used in the field of endodontics for decades because of their wide antimicrobial spectra and other biological properties.2,22,23 In routine root canal therapy procedures, the bacteria in the root canal are initially subjected to short-duration treatments with intracanal irrigants, such as NaOCl and CHX, and are then subjected to long-term challenges with alkaline calcium hydroxide dressings. Our studies showed that small portions of planktonic and biofilm E. faecalis survived the 1-min challenges with NaOCl and CHX, and this duration is consistent with the active time of intracanal irrigants in root canals. The surviving cells exhibited different growth patterns in the subsequent alkaline challenge. The concentrations of 5.25% NaOCl and 2% CHX effectively killed planktonic E. faecalis. However, high concentrations Page 8 of 18
of irrigants cannot be ubiquitously delivered to all sites in the root canal due to dilution and the complexity of the root canal system. Therefore, low concentrations of 1% NaOCl and 0.2% CHX were tested in our study. We found that following challenge with 1% NaOCl, planktonic E. faecalis was able to grow in the subsequent alkaline challenge, and even following treatment with 5.25%
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NaOCl, the E. faecalis biofilm was still able to grow during the alkaline challenge. However, the 2% and 0.2% CHX treatments significantly decreased the planktonic and biofilm E. faecalis
survival rates in the alkaline conditions. These results suggest that in terms of antibacterial activities,
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CHX might be more effective than NaOCl in improving the subsequent application of alkaline
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calcium hydroxide dressings.
CHX is a positively charged hydrophobic and lipophilic molecule.24, 25 A few studies have indicated that its positively charged ions aid the adsorption of CHX into the dentine and generate
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residual effects on pathogens in the root canal.25–28 Furthermore, the positive charge of CHX also helps the CHX molecule bind to negatively charged phosphate groups on microbial cell walls and
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alter the cells’ osmotic equilibrium to further destroy bacteria.24 Therefore, after 1 min of pretreatment with CHX, although the nonadherent CHX molecules were rinsed away, the CHX
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molecules bound to the E. faecalis cells could continue to exert their activities. This residual effect
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might explain why E. faecalis did not grow in the subsequent pH 8 alkaline challenge, even though E. faecalis can generally grow in such low alkaline conditions. However, NaOCl did not exert such
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activity. The high concentration of 5.25% NaOCl effectively killed planktonic E. faecalis cells in 1 min, but the concentration of 1% did not. Similar to calcium hydroxide, NaOCl exerts its antibacterial effects via an amino acid chloramination reaction and high pH levels.23 Because alkalinity and a low concentration of NaOCl might create an adaptable environment for E. faecalis, the residual viable E. faecalis might have survived better during the subsequent alkaline challenge. Furthermore, OCl- is a negative ion and is not adsorbed into the bacteria. NaOCl has one advantage: the tissue dissolution capacity of NaOCl has been confirmed in a few studies.29,30 NaOCl irrigation might be inferior to CHX irrigation in improving the effects of subsequent alkaline challenge, but NaOCl is capable of dissolving organic matter. In our studies, we found that E. faecalis deposits were quickly dissolved during the 1-min treatment with NaOCl, which did not occur during the treatments with CHX. In the in vitro biofilm assay, NaOCl was superior to CHX in dissolving E. faecalis biofilm. Consequently, a high concentration of NaOCl is recommended for intracanal irrigation because it guarantees both good bactericidal activity and high Page 9 of 18
dissolution capacity and because a low concentration of NaOCl with a short duration of contact is unfavourable for both the subsequent alkaline challenge and necrotic tissue dissolution. The alkaline challenge of this study was intended to simulate the pH levels achieved by calcium hydroxide inside the root canal, and thus, different alkaline pH levels (pH 8, 9, 10, and 11)
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were used. A few studies have employed buffer solutions to maintain pH values.17,31 However, in the actual intracanal dressing, alkaline calcium hydroxide itself might be influenced by the
buffering effects of dentine and bacterial metabolites, and thus, the alkaline pH in a root canal might
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not be steadily maintained.32,33 Therefore, in our studies, buffer solutions were not used in the
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alkaline challenges because the effects of E. faecalis metabolites on the alkaline pH might more closely resemble the actual intracanal dressing, and the pH tests at each time point also indicated that the effect was negligible.
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The root canal system is very complex, and pathogens in the root canal might be confronted with various types of stress, for example, starvation.8,34 However, we did not perform alkaline
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challenges in starvation conditions. This study was conducted in a nutrient-rich condition. In the preliminary experiment, after 1 min of treatment with NaOCl and CHX, the E. faecalis survival
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rates were very low, and at such low cell concentrations, the subsequent alkaline challenges
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differentially altered the E. faecalis survival rates because E. faecalis was not grown under starvation conditions. Therefore, nutrition-rich conditions were used to establish the E. faecalis
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growth and inhibition curves to evaluate the possible effects of the use of these intracanal medicaments during root canal therapy procedures on planktonic and biofilm E. faecalis. Furthermore, E. faecalis usually exists in root canals as biofilms of a typical mushroom-shape, and endodontic bacteria often have a correlation between the capacity to form a biofilm and resistance to some antibiotics. 35,36 In our study, following pretreatment of NaOCl, the E. faecalis biofilm still regenerated a new biofilm in the subsequent alkaline challenge, but the E. faecalis biofilm pretreated with CHX did not. This indicated that the NaOCl-treated E. faecalis might have a high capacity for biofilm formation. In summary, CHX was effective in improving the subsequent antibacterial activities of alkaline intracanal dressings, and NaOCl did not exert such effects. This study implies that CHX might be more conducive to the desired effects of subsequent alkaline dressings in root canal therapy than NaOCl.
Funding Page 10 of 18
This research was supported by grants from the Guangdong Natural Science Foundation (No. S2013040014932) and the National Natural Science Foundation of China (No. 81200778). Competing interests The authors deny any conflicts of interest related to this study.
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Ethical approval Not required.
1. Zehnder M. Root canal irrigants. J Endod 2006;32(5):389-98.
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Legend of figure Fig. 1 – The inhibitory effects of NaOCl, CHX and the subsequent alkaline challenges in planktonic E. faecalis. After 1-min pretreatments with 5.25% NaOCl, 1% NaOCl, 2% CHX, 0.2% CHX and the control solution, planktonic E. faecalis were challenged with different alkaline pH levels of 8 (a),
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9 (b), 10 (c) and 11 (d). Each test was performed three times on different days, and the means and the SDs are presented. The X-axes represent the exposure times, and the Y-axes represent the
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logarithmic E. faecalis counts.
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Fig. 2 – The inhibitory effects of NaOCl, CHX and the subsequent alkaline challenges on the E. faecalis biofilms. The E. faecalis biofilms were grown on the bottoms of 48-well plates for 1 d, and the viable cells in the E. faecalis biofilms were counted in alkaline pH levels of 8 (a), 9 (b), 10 (c)
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and 11 (d) following 1-min pretreatments with 5.25% NaOCl, 1% NaOCl, 2% CHX, 0.2% CHX, or the control. Each test was performed three times on different days, and the means and SDs are
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presented. The X-axes represent the exposure times, and the Y-axes represent the logarithmic E.
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faecalis counts.
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Fig. 3 – CLSM images of the E. faecalis biofilms following 1 min of treatment with NaOCl or CHX and the subsequent alkaline challenges. (a-e) The E. faecalis biofilm after 1-min treatments
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with 5.25% NaOCl, 1% NaOCl, 2% CHX, 0.2% CHX or the control, respectively. (f-j) The E. faecalis biofilms exposed to 24 h of the alkaline pH of 9 after the 1-min treatments with 5.25% NaOCl, 1% NaOCl, 2% CHX, 0.2% CHX and the control, respectively. (k-o) The E. faecalis biofilms exposed to 24 h of the alkaline pH of 11 following 1-min treatments with 5.25% NaOCl, 1% NaOCl, 2% CHX, 0.2% CHX or the control, respectively. The viable cells are stained fluorescent green, whereas the dead cells are stained fluorescent red. All images were magnified at 400 × using an Olympus CLSM.
Fig. 4 – The percentages of live cells in the E. faecalis biofilms following 1-min treatments with 5.25% NaOCl, 1% NaOCl, 2% CHX, 0.2% CHX or the control and the subsequent 24-h alkaline challenges. Five random areas (four angular vertexes and a central point in square) were captured from each group, and the percentages of live bacteria in the biofilm were analysed based on fluorescence intensity. “＊” represents significant differences between the percentages of live bacteria following the treatments with antimicrobial agents and the controls (p < 0.05). Page 14 of 18
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S0003-9969(15)00097-7 http://dx.doi.org/doi:10.1016/j.archoralbio.2015.04.008 AOB 3380
To appear in:
Archives of Oral Biology
Received date: Revised date: Accepted date:
17-1-2015 16-4-2015 19-4-2015
Please cite this article as: Ma J, Tong Z, Ling J, Liu H, Wei X, The effects of sodium hypochlorite and chlorhexidine irrigants on the antibacterial activities of alkaline media against Enterococcus faecalis, Archives of Oral Biology (2015), http://dx.doi.org/10.1016/j.archoralbio.2015.04.008 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Highlights: 1. E. faecalis survival decreased in CHX or 5.25% NaOCl and next alkaline challenge. 2. E. faecalis biofilm survival were reduced in CHX and next alkaline challenge.
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4. CHX might be more effective in improving the alkaline than NaOCl.
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3. E. faecalis biofilm survival increased in NaOCl and next alkaline challenge.
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The effects of sodium hypochlorite and chlorhexidine irrigants on the antibacterial activities of alkaline media against Enterococcus faecalis Jinglei Ma a b 1, Zhongchun Tong a b 1, Junqi Ling a b*, Hongyan Liu a, Xi Wei a Department of Operative Dentistry and Endodontics, Guanghua School of Stomatology, Sun
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a
Yat-sen University, Guangzhou, Guangdong, China.
Guangdong Provincial Key Laboratory of Stomatology, Sun Yat-sen University, Guangzhou,
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Guangdong, China. Jinglei Ma and Zhongchun Tong contributed equally to this work.
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* Corresponding author: Junqi Ling
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Postal address: Prof Junqi Ling, Department of Operative Dentistry and Endodontics, Guanghua School of Stomatology, Sun Yat-sen University, Guangzhou 510055, Guangdong, China. Fax: +86 20 83822807
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E-mail: [email protected]
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Telephone: +86 20 83862621
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Abstract Objective: Sodium hypochlorite (NaOCl), chlorhexidine (CHX) and calcium hydroxide are common intracanal medicaments. The present study aimed to evaluate the effects of NaOCl and CHX on the antibacterial activities of alkaline media against Enterococcus faecalis.
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Designs: The survival rates of planktonic and biofilm E. faecalis were evaluated by plate counts after 1 min of pretreatment with NaOCl and CHX, and time-kill assays were then used to assess subsequent pH alkaline challenges. Dead and living cells in the E. faecalis biofilm were assessed
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with SYTO 9 and PI staining in combination with confocal laser scanning microscopy following
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exposure to NaOCl or CHX and subsequent alkaline challenges by common root canal irrigation and dressing procedures.
Results: One minute of pretreatment with 2% CHX, 0.2% CHX, or 5.25% NaOCl in combination
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with a subsequent alkaline challenge significantly decreased planktonic E. faecalis survival rates, but pretreatment with 1% NaOCl did not. The E. faecalis biofilm survival rates were reduced in the
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subsequent alkaline challenge following CHX pretreatment but gradually increased following NaOCl pretreatment. Similarly, CLSM analysis revealed that the greatest proportions of dead E.
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faecalis cells in the biofilms were presented in the CHX and alkaline treatment group.
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Conclusion: CHX might be more effective in improving the antibacterial activities of alkaline root
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canal medicaments against E. faecalis than NaOCl during routine root canal therapy procedures.
Keywords: sodium hypochlorite, chlorhexidine, alkaline challenge, Enterococcus faecalis.
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Introduction The objectives of root canal therapies are to eradicate pathogenic bacteria within the root canal and to prevent recurrent infections. Although biomechanical instrumentation can eliminate most bacteria in root canals, antibacterial irrigants and intracanal dressings are indispensable for
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improving the success of root canal therapy. Sodium hypochlorite (NaOCl) and chlorhexidine (CHX) are two common intracanal irrigants that have demonstrated good antibacterial activity.1
After root canal irrigation, alkaline calcium hydroxide is generally utilized as an intracanal dressing
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to kill any residual bacteria.2 Currently, studies concerning irrigants have primarily focused on the
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effects of intracanal medicaments on bacteria in their normal physiological states in the root canals and on the removal of smear layers.3–7 However, due to the challenge of intracanal medicaments with high antibacterial activities, for example, irrigation with NaOCl and CHX, the residual viable
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bacteria and the biofilm bacteria in root canals could possibly be in stressed states. Stressed bacteria exhibit altered physiologies and might be resistant to intracanal dressings, and this issue deserves
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further study.
The harbouring of microorganisms in the root canal system is the major cause of intraradicular
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infection, which is divided into three categories: primary, secondary, and persistent infections.8,9
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Enterococcus faecalis is generally considered to be closely related to secondary and persistent root canal infections despite representing only low proportions of the diverse microflora that cause
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primary root canal infections.10,11 E. faecalis can survive disadvantageous environments, including those with extreme alkaline pH values, high salt concentrations, bile salts, and starvation, and are resistant to many antimicrobials. Thus, these bacteria are often considered test bacteria for evaluations of the effectiveness of intracanal medicaments.12–15 The disadvantageous environments place E. faecalis in a stressed state, and at present, a few studies have studied E. faecalis that has been stressed by starvation and alkaline pH.15–18 However, little is known about the stress response of E. faecalis to exposure to NaOCl and CHX or its ability to resist an alkaline pH. In the present study, we attempted to investigate the sensitivity of planktonic and biofilm E. faecalis to alkaline pH after short-duration challenges with NaOCl and CHX and to estimate the survival ability of E. faecalis in alkaline intracanal dressings.
Materials and methods Bacterial culture Page 4 of 18
E. faecalis ATCC 29212 was used in this study. The strain was routinely streaked on brain heart infusion (BHI, BD Difco, Sparks, MD, USA) agar and cultured aerobically at 37°C for 24 h. A single bacterial colony was inoculated into 5 mL of BHI medium and was grown overnight. The preparation of planktonic bacteria for the alkaline challenge assay was accomplished by adding 50
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mL of BHI broth to 500 µL of overnight E. faecalis culture, followed by an additional 12 h of culturing. The concentration of E. faecalis was adjusted to an OD600 value of 0.8, which
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corresponded to approximately 109 colony-forming units per millilitre (CFU/mL). Preparation of the antibacterial agents
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Sodium hypochlorite (NaOCl, Sigma, St. Louis, MO, USA) and chlorhexidine acetate (CHX, Sigma, St. Louis, MO, USA) solutions were prepared for this study. The 2% and 0.2% solutions of CHX were prepared by dissolving CHX powder in sterile distilled water, and 5.25% and 1%
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solutions of NaOCl were also prepared by dilution with sterile distilled water. For the alkaline challenges, fresh sterile BHI broth and tryptic soy broth (TSB, BD Difco, Sparks, MD, USA) were
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adjusted to pH values of 8, 9, 10 and 11 with sodium hydroxide.
Alkaline challenge of the planktonic and biofilm E. faecalis following NaOCl and CHX
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pretreatment
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E. faecalis was grown to a concentration of approximately 109 CFU/mL according to growth curves. After the five groups of E. faecalis cultures (48 mL culture in each group) were centrifuged
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at 6,000 rpm for 5 min at 4 °C, the pellets were challenged by resuspension in five common intracanal irrigants (A: 5.25% NaOCl; B: 1% NaOCl; C: 2% CHX; D: 0.2% CHX; and E: 0.9% NaCl [control]) for 1 min. The antibacterial activity of CHX was stopped with 0.5% tween 80 and 0.07% lecithin, and the NaOCl was inactivated with 0.6% sodium thiosulfate.19,20 To prove complete inactivation, the inactivated CHX and NaOCl were added to E. faecalis cultures, and the lack of significant changes in the E. faecalis survival rates indicated complete inactivation of the drugs. Following 1 min of pretreatment with the common intracanal irrigants, each group of E. faecalis was washed twice using PBS by centrifugation (6,000 rpm for 5 min at 4 °C) and then was divided into four subgroups. After additional centrifugation, the supernatant was discarded and the bacterial pellet was challenged by resuspension in 12 mL of BHI broth with different alkalinities (pH 8, pH 9, pH 10 and pH 11). Afterward, the E. faecalis in each subgroup was divided equally among 12 freezing tubes (2-mL volume, Corning Costar, Cambridge, MA, USA). At the 0, 2 h, 4 h, 6 h, 8 h, 10 h, 12 h, 1 d, 2 d, 4 d, 7 d, and 14 d time points, 100 µL of bacterial solution in a random Page 5 of 18
freezing tube was 10-fold serially diluted and spread on a BHI agar plate. After 48 h of incubation, the survival counts of the E. faecalis were determined. To maintain a constant alkaline pH, the E. faecalis that were challenged in alkaline media were enclosed in freezing tubes to reduce exposure to CO2 in the air. Freezing tubes without bacteria were used as negative controls to ensure the
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absence of contamination. Each test was repeated three times on different days. For the biofilm assays, E. faecalis culture (approximately 109 CFU/mL) and TSB containing 1% glucose at a ratio of 1:100 were added to 48-well plates (1 mL per well). After 24 h of
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incubation, the supernatant and non-adhesive cells were discarded and the biofilm on the bottom of
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the well was gently washed with sterile PBS. Subsequently, 1-mL aliquots of the test intracanal irrigants (A: 5.25% NaOCl; B: 1% NaOCl; C: 2% CHX; D: 0.2% CHX; and E: 0.9% NaCl [control]) were added to the 48-well plates. The challenges lasted for 1 min and were then stopped
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by neutralizers as described above. The solutions in the wells were removed, and the biofilms on the bottom were gently washed once with PBS. The biofilms were immediately exposed to TSB at
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different pH values (pH 8, pH 9, pH 10, and pH 11). At the 0, 2 h, 4 h, 6 h, 8 h, 10 h, 12 h, 1 d, 2 d, 4 d, 7 d, and 14 d time points, the alkaline TSB solutions were discarded. The biofilms on the
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bottom were repeatedly scraped using a pipette tip and resuspended with 1 mL of sterile PBS.21 The
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resuspended biofilm cells were adequately agitated in a vortex mixer for 5 min, and the bacterial counts were assessed as the plate counts of serial 10-fold dilutions. During the course of the alkaline
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challenges, to maintain the constant alkalinity of the broth, bacterial challenges from the same time points were enclosed in the same 48-well plates using a sealing strip. The pH of the broth was assayed to ensure a constant pH before the plate counts at each time point. All procedures were independently performed three times on different days, and the means are reported. Confocal laser scanning microscope A confocal laser scanning microscope (CLSM) was used to evaluate the E. faecalis biofilms that had been subjected to an alkaline challenge after NaOCl or CHX pretreatment. For the E. faecalis biofilms, E. faecalis cultures (approximately 109 CFU/mL) and TSB containing 1% glucose at a ratio of 1:100 were added to plastic petri dishes with glass bottoms (D: 35 mm, Hangzhou Shengyou Biotechnology, China). After incubation for 24 h, the biofilms on the bottom were treated with the test intracanal irrigants (A: 5.25% NaOCl; B: 1% NaOCl; C: 2% CHX; D: 0.2% CHX; and E: 0.9% NaCl [control]) for 1 min and were then exposed to TSB at a pH of 9 or 11 for 24 h. E. faecalis biofilms were stained with a mixture of 6 µM SYTO 9 stain and 30 µM PI at room Page 6 of 18
temperature in the dark for 15 min according to the specifications of the Live/Dead BacLight Bacterial Viability Kit (L13152; Molecular Probes, Invitrogen, Inc., Eugene, OR, USA). Images were then captured using a Carl Zeiss confocal laser scanning microscope (CLSM) and ZEN software (Zen 2012 light edition, Carl Zeiss MicroImaging, Inc., Thornwood, NY). SYTO 9 and PI
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were excited at 488 nm and 543 nm, respectively. The viable cells were stained green, and the dead cells were stained red. Five random (four angular vertexes and a central point in the square) areas were captured from each treatment group. The viable and dead cells were analysed based on
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fluorescence intensity with ZEN software, and the average percentages of the viable cells in the
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biofilms were obtained. Statistical analyses
The statistical analyses were performed using SPSS 18.0 software. In the analyses of the
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fluorescence intensities, Kruskal–Wallis H and Nemenyi nonparametric tests were used to analyse the live bacterial percentages following challenges with 2% CHX, 0.2% CHX, 5.25% NaOCl, 1%
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Results
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level of significance was set at p < 0.05.
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NaOCl and 0.9% NaCl for 1 min and the 24-h alkaline challenges in each of the five groups. The
The survival of planktonic E. faecalis
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In the intracanal irrigant challenge tests, the survival rates of the planktonic E. faecalis significantly decreased after 1-min exposures to pretreatment with 5.25% NaOCl, 1% NaOCl, 2% CHX and 0.2% CHX, and 5.25% NaOCl and 2% CHX were more effective than 1% NaOCl and 0.2% CHX. After the 5.25% NaOCl and 2% CHX pretreatments, the E. faecalis survival rates decreased to zero after 2 h of alkaline challenge at pH levels of 8, 9, 10 and 11. In the challenges at pH levels of 8, 9 and 10, the E. faecalis survival rates in the 0.2% CHX pretreatment group exhibited similar reductions and dropped to zero at 7 d, and the E. faecalis survival rates in the 1% NaOCl pretreatment group gradually increased until 7 d and then decreased between 7 and 14 d. In the challenges at pH levels of 11, the survival rate of the 0.2% CHX pretreatment was more significantly reduced, and survival in the 1% NaOCl pretreatment group also decreased (Fig. 1a-d). The survival of the E. faecalis biofilm E. faecalis survival rates in the biofilms were determined following treatments with the test intracanal irrigants and the alkaline challenges (Fig. 2a-d). The E. faecalis in the biofilms were not Page 7 of 18
completely killed after 1-min exposures to 5.25% NaOCl, 1% NaOCl, 2% CHX or 0.2% CHX and the subsequent alkaline challenges. In the 5.25% NaOCl and 1% NaOCl pretreatment groups, the E. faecalis survival rates exhibited gradual increases and reached a maximum at 2 d and 4 d before finally dropping following the pH 8, pH 9, pH 10 and pH 11 alkaline challenges. However, in the
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2% CHX and 0.2% CHX pretreatment groups, the E. faecalis survival rates gradually decreased throughout the entire experimental period following the different alkaline pH challenges. In the later stage of the alkaline challenges, the E. faecalis survival rates in the CHX pretreatment group were
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far less than those of the NaOCl pretreatment group.
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Fluorescence intensity analyses of the living and dead cells in the biofilms
The live and dead cells in the biofilms that had been treated with intracanal irrigants and alkaline pHs were evaluated by CLSM (Fig. 3 and 4). Following the 1-min treatment with NaOCl
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or CHX alone, the ratios of live cells in the biofilms were significantly different between the 5.25% NaOCl, 1% NaOCl, 2% CHX, and 0.2% CHX groups and the control group (p < 0.05). In the
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5.25% NaOCl group, the ratio of live cells was less than those in the 1% NaOCl, 2% CHX and 0.2% CHX groups, and dead cells dominated in the biofilm. After 24 h of exposure to alkalinities of
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pH 9 or 11, significant differences were found in the ratios of live cells between the NaOCl and
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CHX pretreatment groups (p < 0.05). In the 5.25% NaOCl and 1% NaOCl pretreatment groups, the ratios of live cells significantly increased and the live cells dominated the biofilms in a manner
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similar to that of the control. However, in the 2% CHX and 0.2% CHX pretreatment groups, the ratios of live cells were significantly decreased and the dead cells dominated the biofilms.
Discussion
NaOCl, CHX and calcium hydroxide have been used in the field of endodontics for decades because of their wide antimicrobial spectra and other biological properties.2,22,23 In routine root canal therapy procedures, the bacteria in the root canal are initially subjected to short-duration treatments with intracanal irrigants, such as NaOCl and CHX, and are then subjected to long-term challenges with alkaline calcium hydroxide dressings. Our studies showed that small portions of planktonic and biofilm E. faecalis survived the 1-min challenges with NaOCl and CHX, and this duration is consistent with the active time of intracanal irrigants in root canals. The surviving cells exhibited different growth patterns in the subsequent alkaline challenge. The concentrations of 5.25% NaOCl and 2% CHX effectively killed planktonic E. faecalis. However, high concentrations Page 8 of 18
of irrigants cannot be ubiquitously delivered to all sites in the root canal due to dilution and the complexity of the root canal system. Therefore, low concentrations of 1% NaOCl and 0.2% CHX were tested in our study. We found that following challenge with 1% NaOCl, planktonic E. faecalis was able to grow in the subsequent alkaline challenge, and even following treatment with 5.25%
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NaOCl, the E. faecalis biofilm was still able to grow during the alkaline challenge. However, the 2% and 0.2% CHX treatments significantly decreased the planktonic and biofilm E. faecalis
survival rates in the alkaline conditions. These results suggest that in terms of antibacterial activities,
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CHX might be more effective than NaOCl in improving the subsequent application of alkaline
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calcium hydroxide dressings.
CHX is a positively charged hydrophobic and lipophilic molecule.24, 25 A few studies have indicated that its positively charged ions aid the adsorption of CHX into the dentine and generate
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residual effects on pathogens in the root canal.25–28 Furthermore, the positive charge of CHX also helps the CHX molecule bind to negatively charged phosphate groups on microbial cell walls and
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alter the cells’ osmotic equilibrium to further destroy bacteria.24 Therefore, after 1 min of pretreatment with CHX, although the nonadherent CHX molecules were rinsed away, the CHX
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molecules bound to the E. faecalis cells could continue to exert their activities. This residual effect
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might explain why E. faecalis did not grow in the subsequent pH 8 alkaline challenge, even though E. faecalis can generally grow in such low alkaline conditions. However, NaOCl did not exert such
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activity. The high concentration of 5.25% NaOCl effectively killed planktonic E. faecalis cells in 1 min, but the concentration of 1% did not. Similar to calcium hydroxide, NaOCl exerts its antibacterial effects via an amino acid chloramination reaction and high pH levels.23 Because alkalinity and a low concentration of NaOCl might create an adaptable environment for E. faecalis, the residual viable E. faecalis might have survived better during the subsequent alkaline challenge. Furthermore, OCl- is a negative ion and is not adsorbed into the bacteria. NaOCl has one advantage: the tissue dissolution capacity of NaOCl has been confirmed in a few studies.29,30 NaOCl irrigation might be inferior to CHX irrigation in improving the effects of subsequent alkaline challenge, but NaOCl is capable of dissolving organic matter. In our studies, we found that E. faecalis deposits were quickly dissolved during the 1-min treatment with NaOCl, which did not occur during the treatments with CHX. In the in vitro biofilm assay, NaOCl was superior to CHX in dissolving E. faecalis biofilm. Consequently, a high concentration of NaOCl is recommended for intracanal irrigation because it guarantees both good bactericidal activity and high Page 9 of 18
dissolution capacity and because a low concentration of NaOCl with a short duration of contact is unfavourable for both the subsequent alkaline challenge and necrotic tissue dissolution. The alkaline challenge of this study was intended to simulate the pH levels achieved by calcium hydroxide inside the root canal, and thus, different alkaline pH levels (pH 8, 9, 10, and 11)
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were used. A few studies have employed buffer solutions to maintain pH values.17,31 However, in the actual intracanal dressing, alkaline calcium hydroxide itself might be influenced by the
buffering effects of dentine and bacterial metabolites, and thus, the alkaline pH in a root canal might
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not be steadily maintained.32,33 Therefore, in our studies, buffer solutions were not used in the
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alkaline challenges because the effects of E. faecalis metabolites on the alkaline pH might more closely resemble the actual intracanal dressing, and the pH tests at each time point also indicated that the effect was negligible.
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The root canal system is very complex, and pathogens in the root canal might be confronted with various types of stress, for example, starvation.8,34 However, we did not perform alkaline
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challenges in starvation conditions. This study was conducted in a nutrient-rich condition. In the preliminary experiment, after 1 min of treatment with NaOCl and CHX, the E. faecalis survival
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rates were very low, and at such low cell concentrations, the subsequent alkaline challenges
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differentially altered the E. faecalis survival rates because E. faecalis was not grown under starvation conditions. Therefore, nutrition-rich conditions were used to establish the E. faecalis
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growth and inhibition curves to evaluate the possible effects of the use of these intracanal medicaments during root canal therapy procedures on planktonic and biofilm E. faecalis. Furthermore, E. faecalis usually exists in root canals as biofilms of a typical mushroom-shape, and endodontic bacteria often have a correlation between the capacity to form a biofilm and resistance to some antibiotics. 35,36 In our study, following pretreatment of NaOCl, the E. faecalis biofilm still regenerated a new biofilm in the subsequent alkaline challenge, but the E. faecalis biofilm pretreated with CHX did not. This indicated that the NaOCl-treated E. faecalis might have a high capacity for biofilm formation. In summary, CHX was effective in improving the subsequent antibacterial activities of alkaline intracanal dressings, and NaOCl did not exert such effects. This study implies that CHX might be more conducive to the desired effects of subsequent alkaline dressings in root canal therapy than NaOCl.
Funding Page 10 of 18
This research was supported by grants from the Guangdong Natural Science Foundation (No. S2013040014932) and the National Natural Science Foundation of China (No. 81200778). Competing interests The authors deny any conflicts of interest related to this study.
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Ethical approval Not required.
1. Zehnder M. Root canal irrigants. J Endod 2006;32(5):389-98.
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32. Wang JD, Hume WR. Diffusion of hydrogen ion and hydroxyl ion from various sources through dentine. Int Endod J 1988;21(1):17-26. 33. Haapasalo HK, Siren EK, Waltimo TM, Orstavik D, Haapasalo MP. Inactivation of local root canal medicaments by dentine: an in vitro study. Int Endod J 2000;33(2):126-31. 34. Portenier I, Waltimo T, Orstavik D, Haapasalo M. The susceptibility of starved, stationary phase, and growing cells of Enterococcus faecalis to endodontic medicaments. J Endod 2005;31(5):380-6. 35. Distel JW, Hatton JF, Gillespie MJ. Biofilm formation in medicated root canals. J Endod 2002;28(10):689-93. 36. Al-Ahmad A, Ameen H, Pelz K, Karygianni L, Wittmer A, Anderson AC, et al. Antibiotic resistance and capacity for biofilm formation of different bacteria isolated from endodontic infections associated with root-filled teeth. J Endod 2014;40(2):223-30. Page 13 of 18
Legend of figure Fig. 1 – The inhibitory effects of NaOCl, CHX and the subsequent alkaline challenges in planktonic E. faecalis. After 1-min pretreatments with 5.25% NaOCl, 1% NaOCl, 2% CHX, 0.2% CHX and the control solution, planktonic E. faecalis were challenged with different alkaline pH levels of 8 (a),
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9 (b), 10 (c) and 11 (d). Each test was performed three times on different days, and the means and the SDs are presented. The X-axes represent the exposure times, and the Y-axes represent the
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logarithmic E. faecalis counts.
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Fig. 2 – The inhibitory effects of NaOCl, CHX and the subsequent alkaline challenges on the E. faecalis biofilms. The E. faecalis biofilms were grown on the bottoms of 48-well plates for 1 d, and the viable cells in the E. faecalis biofilms were counted in alkaline pH levels of 8 (a), 9 (b), 10 (c)
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and 11 (d) following 1-min pretreatments with 5.25% NaOCl, 1% NaOCl, 2% CHX, 0.2% CHX, or the control. Each test was performed three times on different days, and the means and SDs are
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presented. The X-axes represent the exposure times, and the Y-axes represent the logarithmic E.
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faecalis counts.
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Fig. 3 – CLSM images of the E. faecalis biofilms following 1 min of treatment with NaOCl or CHX and the subsequent alkaline challenges. (a-e) The E. faecalis biofilm after 1-min treatments
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with 5.25% NaOCl, 1% NaOCl, 2% CHX, 0.2% CHX or the control, respectively. (f-j) The E. faecalis biofilms exposed to 24 h of the alkaline pH of 9 after the 1-min treatments with 5.25% NaOCl, 1% NaOCl, 2% CHX, 0.2% CHX and the control, respectively. (k-o) The E. faecalis biofilms exposed to 24 h of the alkaline pH of 11 following 1-min treatments with 5.25% NaOCl, 1% NaOCl, 2% CHX, 0.2% CHX or the control, respectively. The viable cells are stained fluorescent green, whereas the dead cells are stained fluorescent red. All images were magnified at 400 × using an Olympus CLSM.
Fig. 4 – The percentages of live cells in the E. faecalis biofilms following 1-min treatments with 5.25% NaOCl, 1% NaOCl, 2% CHX, 0.2% CHX or the control and the subsequent 24-h alkaline challenges. Five random areas (four angular vertexes and a central point in square) were captured from each group, and the percentages of live bacteria in the biofilm were analysed based on fluorescence intensity. “＊” represents significant differences between the percentages of live bacteria following the treatments with antimicrobial agents and the controls (p < 0.05). Page 14 of 18
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