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Extracellular Dextran and DNA Affect the Formation of Enterococcus faecalis Biofilms and Their Susceptibility to 2% Chlorhexidine
Basic Research—Biology
Extracellular Dextran and DNA Affect the Formation of Enterococcus faecalis Biofilms and Their Susceptibility to 2% Chlorhexidine Weilan Li, DDS,* Hongyan Liu, PhD,† and Qiong Xu, PhD† Abstract Introduction: Enterococcus faecalis is frequently recovered from root-filled teeth with refractory apical periodontitis. The ability of E. faecalis to form a matrix-encased biofilm contributes to its pathogenicity; however, the role of extracellular dextran and DNA in biofilm formation and its effect on the susceptibility of the biofilm to chlorhexidine remains poorly understood. Methods: E. faecalis biofilms were incubated on dentin blocks. The effect of a dextrandegrading enzyme (dextranase) and DNase I on the adhesion of E. faecalis to dentin was measured using the colony-forming unit (CFU) counting method. CFU assays and confocal laser scanning microscopy were used to investigate the influence of dextranase and DNase I on the antimicrobial activity of 2% chlorhexidine. Results: The CFU count assays indicated that the formation of biofilms by E. faecalis was reduced in cells treated with dextranase or DNase I compared with that in untreated cells (P < .05). In addition, we found that treating E. faecalis biofilms with dextranase or DNase I effectively sensitized the biofilms to 2% chlorhexidine (P < .05). Conclusions: Both dextranase and DNase I decrease the adhesion of E. faecalis to dentin and sensitized E. faecalis biofilms to 2% chlorhexidine. (J Endod 2012;38:894–898)
Key Words Biofilm, chlorhexidine, dextranase, DNase I, Enterococcus faecalis
From *Changsha Stomatological Hospital, Changsha, China; and †Guanghua School of Stomatology and Guangdong Provincial Key Laboratory of Stomatology, Sun Yat-sen University, Guangzhou, China. Weilan Li and Hongyan Liu contributed equally to this study. Supported by the Department of Science and Technology of Guangdong Province (2010B050700007, 2009B030801115). Address requests for reprints to Dr Qiong Xu, Guanghua School of Stomatology and Guangdong Provincial Key Laboratory of Stomatology, Sun Yat-sen University, 56 Ling Yuan Xi Road, Guangzhou, 510055, China. E-mail address: xqiong@ mail.sysu.edu.cn 0099-2399/$ - see front matter Copyright ª 2012 American Association of Endodontists. doi:10.1016/j.joen.2012.04.007
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nterococcus faecalis is the dominant species isolated from root canals with persistent endodontic infections (1, 2). It is responsible for many endodontic failures because of its inherent antibiotic resistance, adaptability to harsh environmental changes, and ability to penetrate deeply into dentinal tubules (3). E. faecalis can adhere to root canal walls where it can accumulate and form biofilms. Biofilm formation helps this species to rapidly acclimate to changing growth conditions and to survive in the presence of high concentrations of antimicrobial agents (4, 5). The bacteria in biofilms are up to 1,000-fold more resistant to antimicrobial agents than their planktonic form (6). Although some aspects of biofilm resistance are poorly understood, the dominant mechanisms are thought to involve extracellular polymeric substances (EPSs), which act as a barrier to both effectors of the immune system and antimicrobial agents (7). EPSs provide biofilms with mechanical stability, and it has been suggested that EPSs can interact with antibiotics in a manner that reduces antibacterial activity. In addition, EPSs are thought to be involved in drug tolerance (8, 9). Recently, dextran and extracellular DNA (eDNA) have been found in the matrix of E. faecalis biofilms, suggesting that dextran and eDNA are involved in the development of bacterial communities (10–12). Dextran is a class of extracellularly formed glucans produced by bacteria. The eDNA in E. faecalis biofilms is presumably derived from cell lysis. Some studies have suggested that the interplay of 2 secreted and coregulated proteases, GelE and SprE, is responsible for regulating the autolysis and release of eDNA (11, 13). However, little is known about the role of EPSs in E. faecalis biofilms attached to dentin. Irrigation is a crucial process for eliminating microorganisms from the root canal system. Because of its substantive antimicrobial activity, chlorhexidine (CHX) is used in irrigation solutions during root canal therapy (14, 15). In various in vivo and in vitro studies, CHX is effective in reducing or eliminating E. faecalis from the root canal space and dentinal tubules (16). However, CHX is less effective against biofilms compared with planktonic cultures (17). This appears to be related to the poor penetration of the irrigation solution through the matrix-encased biofilm (18). Therefore, to remove biofilms, it is necessary to develop strategies that not only counteract microorganisms but also affect the matrix. In the present study, we assessed the effect of 2 EPS-degrading enzymes (dextran and eDNA) on the formation of E. faecalis biofilms and the sensitivity of these biofilms to 2% CHX.
Materials and Methods Bacteria and Culture Conditions E. faecalis (American Type Culture Collection 29212; Guangdong Provincial Key Laboratory of Microbiol Culture Collection and Application, Guangdong Institute of Microbiology, Guangzhou, China) was used in this study. E. faecalis was removed from 80 C stocks, plated onto brain-heart infusion (BHI) plates that were supplemented with 1.5% agar, and incubated aerobically at 37 C for 24 hours. The expected colony, cell morphology, and Gram stain were verified. A single colony from a BHI agar plate was used to inoculate an overnight BHI liquid culture. The cultures were grown aerobically at 37 C for 16 hours. The density of the culture was measured spectrophotometrically and then diluted into fresh BHI medium at a density of 1 107 colony-forming units (CFUs) per milliliter (19).
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Basic Research—Biology Enzymes Dextranase (Sigma, St Louis, MO) with an activity of 25 U/mg and bovine pancreatic DNase I (Sigma) with an activity of 2,200 Kunitz units per milligram were used in the present study. Specimen Preparation The dentin block models used in this experiment were prepared as previously described (20) with slight modifications. In brief, healthy human third molars were extracted from 18- to 25-year-old adults at the Department of Oral and Maxillofacial Surgery, Guanghua School of Stomatology, SunYat-Sen University, Guangzhou, China. Informed consent was obtained from each subject, and the protocols were approved by the university’s ethics committee. The freshly obtained teeth were split longitudinally, and the coronal pulp chamber surfaces were cut into blocks (4 mm 4 mm 0.2 mm) without scraping using a hard-tissue cutting machine (Buehler, Chicago, IL). The dentin blocks were washed with distilled water, ultrasonically cleaned, autoclaved, stored in 10 mmol/L phosphate-buffered saline (PBS) at 4 C, and used within 1 week. The Effect of Dextranase and DNase I on Biofilm Formation Dentin blocks were placed in 24-well polystyrene cell culture plates with the pulpal surfaces facing upward. Each well was inoculated with a 1-mL suspension of E. faecalis (1 107 CFU/mL). For the experimental groups, the dentin blocks were treated with 1 mL BHI containing 40 mg/mL dextranase or 100 mg/mL DNase I. Control samples were treated with 1 mL BHI alone. The plates were left undisturbed in an anaerobic incubator at 37 C for 1, 12, 24, or 48 hours (n = 12). The culture media was replaced every 24 hours. At the end of the incubation, each block was aseptically removed and washed twice with PBS while shaking on a shaker (Unitwist RT; Uniequip Company, Laborgeratebau & Vertriebs GmbH, Martinsried, Germany) for 30 seconds to remove loosely adherent cells. The blocks were then incubated in 1 mL cysteine peptone water for 1 minute (21). Biofilm samples were harvested using sonication (42 kHz) for 5 minutes. The bacteria were serially diluted, and each dilution was plated onto a BHI plate. The plates were then incubated in an anaerobic atmosphere at 37 C for 48 hours, and the number of CFUs per square millimeter was calculated. The Effect of Dextranase and DNase I on the Susceptibility of 48-hour-old Biofilms to CHX The formation of E. faecalis biofilms on dentin was performed as described previously. To test the effect of dextranase and DNase I, 48-hour-old dentin biofilms were rinsed 3 times with 1 mL PBS buffer and then added to 1 mL BHI containing 40 mg/mL dextranase or 100 mg/mL DNase I. Control samples were treated with 1 mL BHI alone. After incubating for 1, 6, 10, 30, 60, or 180 minutes at 37 C (n = 12), 1 mL 2% CHX was added to each well, and the biofilms were incubated for an additional 5 minutes at room temperature. The control dentin biofilms were treated with 1 mL PBS. The biofilms were washed 3 times in PBS solution, 1 minute for each wash, to remove the CHX. The CFU assay was performed as previously described. Confocal Scanning Laser Microscopy Dentin biofilms were grown for 48 hours as described previously. The biofilms were incubated for 10 minutes with EPS-degrading enzymes before adding 2% CHX or BHI medium as a control. All confocal microscopy was performed with an LSM 710 Meta laser scanning confocal microscope (Carl Zeiss, Thornwood, NY) using a 63 oil JOE — Volume 38, Number 7, July 2012
immersion objective. The images were acquired, and 3-dimensional (3D) renditions of the biofilms were reconstructed using LSM Image Examiner software (Carl Zeiss). For the microscopy, washed dentin blocks were stained for 15 minutes in the dark at room temperature with 50 ml L-7012 LIVE/DEAD BacLight TM bacterial cell stains (Molecular Probes Inc, Eugene, OR). Live and dead cells in the biofilms were differentiated by staining with SYTO9 (green fluorescence) (Invitrogen, Carlsbad, CA) and propidium iodide (red fluorescence), respectively. Excess dye was removed by aspiration, and the biofilms were washed twice with PBS. The stained samples were mounted onto glass coverslips using an antifade solution before image acquisition. Confocal illumination was performed using an argon laser (488-nm laser excitation) fitted with a long-pass 515/30 filter for the green fluorescence signal and a 605/75 filter for the red fluorescence signal. Simultaneous dual-channel imaging was used to display green and red fluorescence. The fluorescence intensity profiles of dead and live bacteria were also analyzed using the LSM Image Examiner software. Confocal laser scanning microscopic images were processed to remove background fluorescence and quantify cells. The percentage of living cells was calculated to determine the spatial distribution of live/dead bacteria in the dentin biofilms. For each group, 12 dentin blocks were used for image analysis. The experiment was repeated at least 3 times.
Statistical Analysis Statistical analysis was performed using SPSS software (SPSS for Windows; SPSS Inc, Chicago, IL). To examine the effect of dextranase and DNase I on dentin biofilm formation, the CFU counts were analyzed using the analysis of variance method for a 4 3 factorial design. To examine the effect of dextranase and DNase I on 48-hour-old biofilms killed by CHX, the reduction in viable cells was calculated using a variance for a 6 3 factorial design. The image stacks acquired by confocal laser scanning microscopy were analyzed using 1-way analysis of variance and the Kruskal-Wallis test. P < .05 was considered to be statistically significant.
Results Dextranase and DNase I Decreased the Adhesion of E. faecalis to Dentin Figure 1 shows that both dextranase and DNase I decreased the adhesion of E. faecalis to dentin at all the selected time points (P < .05, n = 12). The formation of E. faecalis biofilms was significantly inhibited by dextranase and DNase I when compared with cells in an unsupplemented medium. The percentages of adherent bacteria after 1 hour of dextranase or DNase I treatment were 15.61% and 18.39%, respectively, which was lower than the control group. There was no significant difference between the dextranase and DNase I
Figure 1. DNase I and dextranase affect the adhesion of E. faecalis to dentin (n = 12). The formation of E. faecalis biofilms was significantly inhibited by dextranase and DNase I compared with cells in an unsupplemented medium (P < .05).
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Basic Research—Biology TABLE 1. The Effect of Dextranase aFnd DNase I on the Susceptibility of 48-hour-old Biofilms to CHX (x s; n = 12, l g [CFU/mm2]) Dex + CHX DNase I + CHX CHX Control
1 min
5 min
10 min
30 min
60 min
180 min
3.40 0.08 3.44 0.07 3.60 0.13 5.13 0.28
3.34 0.11 3.44 0.14 3.58 0.14 5.11 0.32
3.01 0.18 3.07 0.18 3.55 0.13 5.10 0.12
2.78 0.11 2.81 0.10 3.58 0.13 5.12 0.17
2.45 0.08 2.46 0.09 3.53 0.13 5.13 0.09
2.31 0.15 2.31 0.11 3.51 0.19 5.14 0.23
groups (P > .0167). During the 24 hours of incubation, the amount of bacteria on the dentin continued to increase in all groups. No significant difference was detected between biofilms incubated for 24 or 48 hours in the groups undergoing the same treatments (P > .0167).
Dextranase and DNase I Sensitized E. faecalis Biofilm to CHX The ability of dextranase and DNase I to render E. faecalis biofilm cells sensitive to CHX was tested (Table 1). The time-course studies revealed that pretreating cells with dextranase or DNase I increased the susceptibility of E. faecalis biofilms to CHX (P < .0167, n = 12) in a time-dependent manner. Dextranase and DNase I had identical effects on the susceptibility of E. faecalis biofilm cells to CHX (P > .0167). The results from the CFU assay indicated that pretreating E. faecalis biofilms with dextranase or DNase I for 10 minutes resulted in 0.54 or 0.48 logunit decreases in the number of CFU/mm2, respectively, compared with the control group that was treated with 2% CHX alone for 5 minutes. We used confocal scanning laser microscopy to monitor the 3D ultrastructure of biofilms of E. faecalis cells pretreated with an EPS-
degrading enzyme for 10 minutes and subsequently treated with 2% CHX. The biofilms formed by the cells incubated in the BHI medium alone were used as a control. The BacLight live/dead–stained biofilms were imaged and are as follows. The confocal laser scanning microscopic images (Fig. 2) indicated that the green fluorescent area was larger than the red fluorescent area in the control group. The percentage of green fluorescence intensity was approximately 62.70% (Table 2). In the CHX-treated group, the area of dead cells increased, and the area of live cells decreased (P < .0167, n = 12). The percentage of green fluorescence intensity was 37.49%. The biofilms treated with CHX were intensively damaged, and most cells were red in both the CHX + dextranase and CHX + DNase I groups in which the percentages of green fluorescence intensities were 25.88% and 26.34%, respectively.
Discussion The results of this study show that dextran and eDNA are important for maintaining biofilm stability, providing the framework into which microbial cells are inserted and protecting the dentin biofilm from CHX. When added to the growth medium, both dextranase and DNase
Figure 2. Confocal laser scanning microscopic images of E. faecalis–produced biofilms (n = 12). (A) Untreated, (B) treated with CHX, (C) Dex + CHX, and (D) DNase I + CHX. E. faecalis biofilms were stained with BacLight live/dead stain (600). E. faecalis biofilms were grown on dentin blocks for 48 hours and pretreated with 1 mL BHI containing 10 mmol/mL PBS and 40 mg/mL dextranase or 100 mg/mL DNase I for 10 minutes before being treated with 2% CHX. The BacLight live/dead-stained biofilms were imaged using confocal laser scanning microscopy.
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Basic Research—Biology TABLE 2. Confocal Laser Scanning Microscopic Analysis of the Effect of Dextranase and DNase I on 48-hour-old Biofilms Treated with CHX (x s; n = 12)
Dex + CHX DNase I + CHX CHX Control
GFI
RFI
Percentage of living cells
7.35 0.74 7.31 0.74 10.32 1.34 25.11 2.75
21.14 2.98 20.41 1.23 17.23 2.24 14.96 2.09
25.88 1.73 26.35 1.38 37.49 2.41 62.70 3.06
GFI, green fluorescence intensity; RFI, red fluorescence intensity.
I inhibited the adhesion of E. faecalis to dentin and sensitized the biofilm to chlorhexidine killing. We conclude that dextran and eDNA play structural roles in E. faecalis biofilms. The EPS matrix, which is composed of microbial substances derived from or found on the cell surface of E. faecalis, is encased by a complex mixture of proteins, polysaccharides, lipids, and DNA. Some studies report that the EPS matrix initiates the colonization of abiotic and biotic surfaces by planktonic cells and maintains the long-term attachment of whole biofilms to surfaces (22). The EPS matrix also provides biofilms with mechanical stability and a cohesive, 3D polymer network that interconnects and transiently immobilizes biofilm cells. In addition, the biofilm matrix acts as an external digestive system by trapping extracellular enzymes close to the cells. This enables the cells to metabolize dissolved, colloidal, and solid biopolymers (23). In this study, we investigated the effect of 2 major constituents of the EPS matrix, dextran and DNA, on E. faecalis biofilm formation. Our results indicate that the initial attachment of E. faecalis cells to dentin is an EPSdependent step. In the presence of dextranase or DNase I, bacteria were able to attach to the substratum but failed to form a large amount of structures compared with the control group. E. faecalis biofilms that were not treated with dextranase and DNase I exhibited better growth than the biofilms treated with the EPS-degrading enzymes. Taken together, our results suggest that dextran and eDNA directly participate in biofilm formation by E. faecalis by promoting bacterial adhesion. The exact mechanism through which this process occurs should be further investigated. CHX is a broad-spectrum antimicrobial agent. It is active against vegetative bacteria and mycobacteria, has moderate activity against fungi and viruses, and inhibits spore germination. CHX has been used in intracanal medications and in canal irrigation solutions because of its broad antimicrobial spectrum, its ability to remain active for prolonged periods when adhered to the hydroxyapatite component of dentin, and its ability to exhibit a slow release as its concentration decreases (24, 25). Although CHX is effective at killing the pathogens involved in endodontic infections, biofilms in root canals are resistant to CHX. In part, this resistance may be because of the inability of CHX to penetrate deeply into biofilms. Our data indicate that in the absence of dextran and eDNA, E. faecalis biofilms were less tolerant to CHX, which results in a statistically significant reduction in CFUs. The EPS matrix protects microbes against desiccation, oxidizing or charged biocides, some antibiotics, metallic cations, ultraviolet radiation, and host immune defenses. Therefore, in the presence of dextranase or DNase I, CHX may be able to act more directly on E. faecalis, thereby enhancing its bactericidal potential. The 2 EPS-degrading enzymes might act as biofilm-degrading agents to improve the efficiency of antimicrobial agents for the treatment of bacterial biofilms. However, their clinical applications, including the treatment of infected root canals, require further research. Numerous studies have shown that the bacteria in biofilms are embedded within an extracellular matrix that protects the bacteria JOE — Volume 38, Number 7, July 2012
from certain environmental factors (26, 27). Tetz and Tetz (28) reported that the cleavage of eDNA led to an increased penetration of antibiotics and decreased biofilm biomass and CFUs. This result suggests that eDNA plays an important role in the development of biofilm tolerance (29). Similar results have been observed in Staphylococcus epidermidis biofilms that were pretreated with dispersin B to reduce poly-N-acetylglucosamine surface polysaccharides. Izano et al (30) reported that dispersin B, a poly-N-acetylglucosamine surface polysaccharide (PNAG) degrading enzyme, sensitized S. epidermidis biofilms to cationic detergent cetylpyridinium chloride (CPC) killing, whereas DNase I sensitized S. aureus biofilms to CPC killing. It was concluded that PNAG and eDNA play fundamentally different structural roles in S. epidermidis and S. aureus biofilms. There are limitations of our study that must be acknowledged. Only 1 strain of 1 species of E. faecalis was used, and sodium hypochlorite was not used as a control. However, our findings still suggest that matrix polymers, such as dextran and eDNA, might act as general diffusion barriers to prevent CHX from accessing biofilm cells, which is consistent with previous studies (28, 31). In our study, we found that even when treated with CHX and EPS-degrading enzymes, some bacteria in the biofilms were not killed. This result might be related to the presence of other constituents in the biofilm matrix, such as heteroglycan, teichoic acid, or proteins, which might prevent CHX from killing the bacteria. It has also been suggested that different genes are activated and repressed in biofilms (32).
Conclusion The dextran-degrading enzyme (dextranase) and DNase I could decrease the adhesion of E. faecalis to human dentin, inhibit biofilm formation, and increase the susceptibility of E. faecalis biofilms to 2% CHX.
Acknowledgments The authors deny any conflicts of interest related to this study.
References 1. Siqueira JF, Rocas IN. Clinical implications and microbiology of bacterial persistence after treatment procedures. J Endod 2008;34:1291–301. 2. Zhu X, Wang Q, Zhang C, et al. Prevalence, phenotype, and genotype of Enterococcus faecalis isolated from saliva and root canals in patients with persistent apical periodontitis. J Endod 2010;36:1950–5. 3. Hancock HH, Sigurdsson A, Trope M, et al. Bacteria isolated after unsuccessful endodontic treatment in a North American population. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 2001;91:579–86. 4. Stuart CH, Schwartz SA, Beeson TJ, et al. Enterococcus faecalis: its role in root canal treatment failure and current concepts in retreatment. J Endod 2006;32:93–8. 5. Persoon IF, Hoogenkamp MA, Bury A, et al. Effect of vanadium chloroperoxidase on Enterococcus faecalis biofilms. J Endod 2012;38:72–4. 6. Simoes M. Antimicrobial strategies effective against infectious bacterial biofilms. Curr Med Chem 2011;18:2129–45. 7. Goodman SD, Obergfell KP, Jurcisek JA, et al. Biofilms can be dispersed by focusing the immune system on a common family of bacterial nucleoid- associated proteins. Mucosal Immunol 2011;4:625–37. 8. Baker-Austin C, Wright MS, Stepanauskas R, et al. Co-selection of antibiotic and metal resistance. Trends Microbiol 2006;14:176–82. 9. Chambless JD, Hunt SM, Stewart PS. A three-dimensional computer model of four hypothetical mechanisms protecting biofilms from antimicrobials. Appl Environ Microbiol 2006;72:2005–13. 10. Badel S, Laroche C, Gardarin C, et al. New method showing the influence of matrix components in Leuconostoc mesenteroides biofilm formation. Appl Biochem Biotechnol 2008;151:364–70. 11. Thomas VC, Thurlow LR, Boyle D, et al. Regulation of autolysis-dependent extracellular DNA release by Enterococcus faecalis extracellular proteases influences biofilm development. J Bacteriol 2008;190:5690–8.
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Basic Research—Biology 12. Thomas VC, Hiromasa Y, Harms N, et al. A fratricidal mechanism is responsible for eDNA release and contributes to biofilm development of Enterococcus faecalis. Mol Microbiol 2009;72:1022–36. 13. Wang L, Dong M, Zheng J, et al. Relationship of biofilm formation and gelE gene expression in Enterococcus faecalis recovered from root canals in patients requiring endodontic retreatment. J Endod 2011;37:631–6. 14. Mohammadi Z, Abbott PV. Antimicrobial substantivity of root canal irrigants and medicaments: a review. Aust Endod J 2009;35:131–9. 15. Rocas IN, Siqueira JF. Comparison of the in vivo antimicrobial effectiveness of sodium hypochlorite and chlorhexidine used as root canal irrigants: a molecular microbiology study. J Endod 2011;37:143–50. 16. Krithikadatta J, Indira R, Dorothykalyani AL. Disinfection of dentinal tubules with 2% chlorhexidine, 2% metronidazole, bioactive glass when compared with calcium hydroxide as intracanal medicaments. J Endod 2007;33:1473–6. 17. Abdullah M, Ng YL, Gulabivala K, et al. Susceptibilties of two Enterococcus faecalis phenotypes to root canal medications. J Endod 2005;31:30–6. 18. Arias-Moliz MT, Ferrer-Luque CM, Gonzalez-Rodriguez MP, et al. Eradication of Enterococcus faecalis biofilms by cetrimide and chlorhexidine. J Endod 2010; 36:87–90. 19. Guiton PS, Hung CS, Kline KA, et al. Contribution of autolysin and Sortase a during Enterococcus faecalis DNA-dependent biofilm development. Infect Immun 2009; 77:3626–38. 20. Liu H, Wei X, Ling J, et al. Biofilm formation capability of Enterococcus faecalis cells in starvation phase and its susceptibility to sodium hypochlorite. J Endod 2010;36:630–5. 21. Deng DM, Buijs MJ, ten Cate JM. The effects of substratum on the pH response of Streptococcus mutans biofilms and on the susceptibility to 0.2% chlorhexidine. Eur J Oral Sci 2004;112:42–7.
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22. Cerca N, Pier GB, Vilanova M, et al. Quantitative analysis of adhesion and biofilm formation on hydrophilic and hydrophobic surfaces of clinical isolates of Staphylococcus epidermidis. Res Microbiol 2005;156:506–14. 23. Flemming HC, Wingender J. The biofilm matrix. Nat Rev Microbiol 2010;8: 623–33. 24. Tervit C, Paquette L, Torneck CD, et al. Proportion of healed teeth with apical periodontitis medicated with two percent chlorhexidine gluconate liquid: a caseseries study. J Endod 2009;35:1182–5. 25. Lee Y, Han SH, Hong SH, et al. Antimicrobial efficacy of a polymeric chlorhexidine release device using in vitro model of Enterococcus faecalis dentinal tubule infection. J Endod 2008;34:855–8. 26. Hoiby N, Bjarnsholt T, Givskov M, et al. Antibiotic resistance of bacterial biofilms. Int J Antimicrob Agents 2010;35:322–32. 27. Flemming HC, Neu TR, Wozniak DJ. The EPS matrix: the ‘‘house of biofilm cells.’’. J Bacteriol 2007;189:7945–7. 28. Tetz VV, Tetz GV. Effect of extracellular DNA destruction by DNase I on characteristics of forming biofilms. DNA Cell Biol 2010;29:399–405. 29. Tetz GV, Artemenko NK, Tetz VV. Effect of DNase and antibiotics on biofilm characteristics. Antimicrob Agents Chemother 2009;53:1204–9. 30. Izano EA, Amarante MA, Kher WB, et al. Differential roles of poly-N- acetylglucosamine surface polysaccharide and extracellular DNA in Staphylococcus aureus and Staphylococcus epidermidis biofilms. Appl Environ Microbiol 2008;74: 470–6. 31. Chaignon P, Sadovskaya I, Ragunah Ch, et al. Susceptibility of staphylococcal biofilms to enzymatic treatments depends on their chemical composition. Appl Microbiol Biotechnol 2007;75:125–32. 32. Lo AW, Seers CA, Boyce JD, et al. Comparative transcriptomic analysis of Porphyromonas gingivalis biofilm and planktonic cells. BMC Microbiol 2009;9:18.
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Extracellular Dextran and DNA Affect the Formation of Enterococcus faecalis Biofilms and Their Susceptibility to 2% Chlorhexidine Weilan Li, DDS,* Hongyan Liu, PhD,† and Qiong Xu, PhD† Abstract Introduction: Enterococcus faecalis is frequently recovered from root-filled teeth with refractory apical periodontitis. The ability of E. faecalis to form a matrix-encased biofilm contributes to its pathogenicity; however, the role of extracellular dextran and DNA in biofilm formation and its effect on the susceptibility of the biofilm to chlorhexidine remains poorly understood. Methods: E. faecalis biofilms were incubated on dentin blocks. The effect of a dextrandegrading enzyme (dextranase) and DNase I on the adhesion of E. faecalis to dentin was measured using the colony-forming unit (CFU) counting method. CFU assays and confocal laser scanning microscopy were used to investigate the influence of dextranase and DNase I on the antimicrobial activity of 2% chlorhexidine. Results: The CFU count assays indicated that the formation of biofilms by E. faecalis was reduced in cells treated with dextranase or DNase I compared with that in untreated cells (P < .05). In addition, we found that treating E. faecalis biofilms with dextranase or DNase I effectively sensitized the biofilms to 2% chlorhexidine (P < .05). Conclusions: Both dextranase and DNase I decrease the adhesion of E. faecalis to dentin and sensitized E. faecalis biofilms to 2% chlorhexidine. (J Endod 2012;38:894–898)
Key Words Biofilm, chlorhexidine, dextranase, DNase I, Enterococcus faecalis
From *Changsha Stomatological Hospital, Changsha, China; and †Guanghua School of Stomatology and Guangdong Provincial Key Laboratory of Stomatology, Sun Yat-sen University, Guangzhou, China. Weilan Li and Hongyan Liu contributed equally to this study. Supported by the Department of Science and Technology of Guangdong Province (2010B050700007, 2009B030801115). Address requests for reprints to Dr Qiong Xu, Guanghua School of Stomatology and Guangdong Provincial Key Laboratory of Stomatology, Sun Yat-sen University, 56 Ling Yuan Xi Road, Guangzhou, 510055, China. E-mail address: xqiong@ mail.sysu.edu.cn 0099-2399/$ - see front matter Copyright ª 2012 American Association of Endodontists. doi:10.1016/j.joen.2012.04.007
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nterococcus faecalis is the dominant species isolated from root canals with persistent endodontic infections (1, 2). It is responsible for many endodontic failures because of its inherent antibiotic resistance, adaptability to harsh environmental changes, and ability to penetrate deeply into dentinal tubules (3). E. faecalis can adhere to root canal walls where it can accumulate and form biofilms. Biofilm formation helps this species to rapidly acclimate to changing growth conditions and to survive in the presence of high concentrations of antimicrobial agents (4, 5). The bacteria in biofilms are up to 1,000-fold more resistant to antimicrobial agents than their planktonic form (6). Although some aspects of biofilm resistance are poorly understood, the dominant mechanisms are thought to involve extracellular polymeric substances (EPSs), which act as a barrier to both effectors of the immune system and antimicrobial agents (7). EPSs provide biofilms with mechanical stability, and it has been suggested that EPSs can interact with antibiotics in a manner that reduces antibacterial activity. In addition, EPSs are thought to be involved in drug tolerance (8, 9). Recently, dextran and extracellular DNA (eDNA) have been found in the matrix of E. faecalis biofilms, suggesting that dextran and eDNA are involved in the development of bacterial communities (10–12). Dextran is a class of extracellularly formed glucans produced by bacteria. The eDNA in E. faecalis biofilms is presumably derived from cell lysis. Some studies have suggested that the interplay of 2 secreted and coregulated proteases, GelE and SprE, is responsible for regulating the autolysis and release of eDNA (11, 13). However, little is known about the role of EPSs in E. faecalis biofilms attached to dentin. Irrigation is a crucial process for eliminating microorganisms from the root canal system. Because of its substantive antimicrobial activity, chlorhexidine (CHX) is used in irrigation solutions during root canal therapy (14, 15). In various in vivo and in vitro studies, CHX is effective in reducing or eliminating E. faecalis from the root canal space and dentinal tubules (16). However, CHX is less effective against biofilms compared with planktonic cultures (17). This appears to be related to the poor penetration of the irrigation solution through the matrix-encased biofilm (18). Therefore, to remove biofilms, it is necessary to develop strategies that not only counteract microorganisms but also affect the matrix. In the present study, we assessed the effect of 2 EPS-degrading enzymes (dextran and eDNA) on the formation of E. faecalis biofilms and the sensitivity of these biofilms to 2% CHX.
Materials and Methods Bacteria and Culture Conditions E. faecalis (American Type Culture Collection 29212; Guangdong Provincial Key Laboratory of Microbiol Culture Collection and Application, Guangdong Institute of Microbiology, Guangzhou, China) was used in this study. E. faecalis was removed from 80 C stocks, plated onto brain-heart infusion (BHI) plates that were supplemented with 1.5% agar, and incubated aerobically at 37 C for 24 hours. The expected colony, cell morphology, and Gram stain were verified. A single colony from a BHI agar plate was used to inoculate an overnight BHI liquid culture. The cultures were grown aerobically at 37 C for 16 hours. The density of the culture was measured spectrophotometrically and then diluted into fresh BHI medium at a density of 1 107 colony-forming units (CFUs) per milliliter (19).
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Basic Research—Biology Enzymes Dextranase (Sigma, St Louis, MO) with an activity of 25 U/mg and bovine pancreatic DNase I (Sigma) with an activity of 2,200 Kunitz units per milligram were used in the present study. Specimen Preparation The dentin block models used in this experiment were prepared as previously described (20) with slight modifications. In brief, healthy human third molars were extracted from 18- to 25-year-old adults at the Department of Oral and Maxillofacial Surgery, Guanghua School of Stomatology, SunYat-Sen University, Guangzhou, China. Informed consent was obtained from each subject, and the protocols were approved by the university’s ethics committee. The freshly obtained teeth were split longitudinally, and the coronal pulp chamber surfaces were cut into blocks (4 mm 4 mm 0.2 mm) without scraping using a hard-tissue cutting machine (Buehler, Chicago, IL). The dentin blocks were washed with distilled water, ultrasonically cleaned, autoclaved, stored in 10 mmol/L phosphate-buffered saline (PBS) at 4 C, and used within 1 week. The Effect of Dextranase and DNase I on Biofilm Formation Dentin blocks were placed in 24-well polystyrene cell culture plates with the pulpal surfaces facing upward. Each well was inoculated with a 1-mL suspension of E. faecalis (1 107 CFU/mL). For the experimental groups, the dentin blocks were treated with 1 mL BHI containing 40 mg/mL dextranase or 100 mg/mL DNase I. Control samples were treated with 1 mL BHI alone. The plates were left undisturbed in an anaerobic incubator at 37 C for 1, 12, 24, or 48 hours (n = 12). The culture media was replaced every 24 hours. At the end of the incubation, each block was aseptically removed and washed twice with PBS while shaking on a shaker (Unitwist RT; Uniequip Company, Laborgeratebau & Vertriebs GmbH, Martinsried, Germany) for 30 seconds to remove loosely adherent cells. The blocks were then incubated in 1 mL cysteine peptone water for 1 minute (21). Biofilm samples were harvested using sonication (42 kHz) for 5 minutes. The bacteria were serially diluted, and each dilution was plated onto a BHI plate. The plates were then incubated in an anaerobic atmosphere at 37 C for 48 hours, and the number of CFUs per square millimeter was calculated. The Effect of Dextranase and DNase I on the Susceptibility of 48-hour-old Biofilms to CHX The formation of E. faecalis biofilms on dentin was performed as described previously. To test the effect of dextranase and DNase I, 48-hour-old dentin biofilms were rinsed 3 times with 1 mL PBS buffer and then added to 1 mL BHI containing 40 mg/mL dextranase or 100 mg/mL DNase I. Control samples were treated with 1 mL BHI alone. After incubating for 1, 6, 10, 30, 60, or 180 minutes at 37 C (n = 12), 1 mL 2% CHX was added to each well, and the biofilms were incubated for an additional 5 minutes at room temperature. The control dentin biofilms were treated with 1 mL PBS. The biofilms were washed 3 times in PBS solution, 1 minute for each wash, to remove the CHX. The CFU assay was performed as previously described. Confocal Scanning Laser Microscopy Dentin biofilms were grown for 48 hours as described previously. The biofilms were incubated for 10 minutes with EPS-degrading enzymes before adding 2% CHX or BHI medium as a control. All confocal microscopy was performed with an LSM 710 Meta laser scanning confocal microscope (Carl Zeiss, Thornwood, NY) using a 63 oil JOE — Volume 38, Number 7, July 2012
immersion objective. The images were acquired, and 3-dimensional (3D) renditions of the biofilms were reconstructed using LSM Image Examiner software (Carl Zeiss). For the microscopy, washed dentin blocks were stained for 15 minutes in the dark at room temperature with 50 ml L-7012 LIVE/DEAD BacLight TM bacterial cell stains (Molecular Probes Inc, Eugene, OR). Live and dead cells in the biofilms were differentiated by staining with SYTO9 (green fluorescence) (Invitrogen, Carlsbad, CA) and propidium iodide (red fluorescence), respectively. Excess dye was removed by aspiration, and the biofilms were washed twice with PBS. The stained samples were mounted onto glass coverslips using an antifade solution before image acquisition. Confocal illumination was performed using an argon laser (488-nm laser excitation) fitted with a long-pass 515/30 filter for the green fluorescence signal and a 605/75 filter for the red fluorescence signal. Simultaneous dual-channel imaging was used to display green and red fluorescence. The fluorescence intensity profiles of dead and live bacteria were also analyzed using the LSM Image Examiner software. Confocal laser scanning microscopic images were processed to remove background fluorescence and quantify cells. The percentage of living cells was calculated to determine the spatial distribution of live/dead bacteria in the dentin biofilms. For each group, 12 dentin blocks were used for image analysis. The experiment was repeated at least 3 times.
Statistical Analysis Statistical analysis was performed using SPSS software (SPSS for Windows; SPSS Inc, Chicago, IL). To examine the effect of dextranase and DNase I on dentin biofilm formation, the CFU counts were analyzed using the analysis of variance method for a 4 3 factorial design. To examine the effect of dextranase and DNase I on 48-hour-old biofilms killed by CHX, the reduction in viable cells was calculated using a variance for a 6 3 factorial design. The image stacks acquired by confocal laser scanning microscopy were analyzed using 1-way analysis of variance and the Kruskal-Wallis test. P < .05 was considered to be statistically significant.
Results Dextranase and DNase I Decreased the Adhesion of E. faecalis to Dentin Figure 1 shows that both dextranase and DNase I decreased the adhesion of E. faecalis to dentin at all the selected time points (P < .05, n = 12). The formation of E. faecalis biofilms was significantly inhibited by dextranase and DNase I when compared with cells in an unsupplemented medium. The percentages of adherent bacteria after 1 hour of dextranase or DNase I treatment were 15.61% and 18.39%, respectively, which was lower than the control group. There was no significant difference between the dextranase and DNase I
Figure 1. DNase I and dextranase affect the adhesion of E. faecalis to dentin (n = 12). The formation of E. faecalis biofilms was significantly inhibited by dextranase and DNase I compared with cells in an unsupplemented medium (P < .05).
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Basic Research—Biology TABLE 1. The Effect of Dextranase aFnd DNase I on the Susceptibility of 48-hour-old Biofilms to CHX (x s; n = 12, l g [CFU/mm2]) Dex + CHX DNase I + CHX CHX Control
1 min
5 min
10 min
30 min
60 min
180 min
3.40 0.08 3.44 0.07 3.60 0.13 5.13 0.28
3.34 0.11 3.44 0.14 3.58 0.14 5.11 0.32
3.01 0.18 3.07 0.18 3.55 0.13 5.10 0.12
2.78 0.11 2.81 0.10 3.58 0.13 5.12 0.17
2.45 0.08 2.46 0.09 3.53 0.13 5.13 0.09
2.31 0.15 2.31 0.11 3.51 0.19 5.14 0.23
groups (P > .0167). During the 24 hours of incubation, the amount of bacteria on the dentin continued to increase in all groups. No significant difference was detected between biofilms incubated for 24 or 48 hours in the groups undergoing the same treatments (P > .0167).
Dextranase and DNase I Sensitized E. faecalis Biofilm to CHX The ability of dextranase and DNase I to render E. faecalis biofilm cells sensitive to CHX was tested (Table 1). The time-course studies revealed that pretreating cells with dextranase or DNase I increased the susceptibility of E. faecalis biofilms to CHX (P < .0167, n = 12) in a time-dependent manner. Dextranase and DNase I had identical effects on the susceptibility of E. faecalis biofilm cells to CHX (P > .0167). The results from the CFU assay indicated that pretreating E. faecalis biofilms with dextranase or DNase I for 10 minutes resulted in 0.54 or 0.48 logunit decreases in the number of CFU/mm2, respectively, compared with the control group that was treated with 2% CHX alone for 5 minutes. We used confocal scanning laser microscopy to monitor the 3D ultrastructure of biofilms of E. faecalis cells pretreated with an EPS-
degrading enzyme for 10 minutes and subsequently treated with 2% CHX. The biofilms formed by the cells incubated in the BHI medium alone were used as a control. The BacLight live/dead–stained biofilms were imaged and are as follows. The confocal laser scanning microscopic images (Fig. 2) indicated that the green fluorescent area was larger than the red fluorescent area in the control group. The percentage of green fluorescence intensity was approximately 62.70% (Table 2). In the CHX-treated group, the area of dead cells increased, and the area of live cells decreased (P < .0167, n = 12). The percentage of green fluorescence intensity was 37.49%. The biofilms treated with CHX were intensively damaged, and most cells were red in both the CHX + dextranase and CHX + DNase I groups in which the percentages of green fluorescence intensities were 25.88% and 26.34%, respectively.
Discussion The results of this study show that dextran and eDNA are important for maintaining biofilm stability, providing the framework into which microbial cells are inserted and protecting the dentin biofilm from CHX. When added to the growth medium, both dextranase and DNase
Figure 2. Confocal laser scanning microscopic images of E. faecalis–produced biofilms (n = 12). (A) Untreated, (B) treated with CHX, (C) Dex + CHX, and (D) DNase I + CHX. E. faecalis biofilms were stained with BacLight live/dead stain (600). E. faecalis biofilms were grown on dentin blocks for 48 hours and pretreated with 1 mL BHI containing 10 mmol/mL PBS and 40 mg/mL dextranase or 100 mg/mL DNase I for 10 minutes before being treated with 2% CHX. The BacLight live/dead-stained biofilms were imaged using confocal laser scanning microscopy.
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Basic Research—Biology TABLE 2. Confocal Laser Scanning Microscopic Analysis of the Effect of Dextranase and DNase I on 48-hour-old Biofilms Treated with CHX (x s; n = 12)
Dex + CHX DNase I + CHX CHX Control
GFI
RFI
Percentage of living cells
7.35 0.74 7.31 0.74 10.32 1.34 25.11 2.75
21.14 2.98 20.41 1.23 17.23 2.24 14.96 2.09
25.88 1.73 26.35 1.38 37.49 2.41 62.70 3.06
GFI, green fluorescence intensity; RFI, red fluorescence intensity.
I inhibited the adhesion of E. faecalis to dentin and sensitized the biofilm to chlorhexidine killing. We conclude that dextran and eDNA play structural roles in E. faecalis biofilms. The EPS matrix, which is composed of microbial substances derived from or found on the cell surface of E. faecalis, is encased by a complex mixture of proteins, polysaccharides, lipids, and DNA. Some studies report that the EPS matrix initiates the colonization of abiotic and biotic surfaces by planktonic cells and maintains the long-term attachment of whole biofilms to surfaces (22). The EPS matrix also provides biofilms with mechanical stability and a cohesive, 3D polymer network that interconnects and transiently immobilizes biofilm cells. In addition, the biofilm matrix acts as an external digestive system by trapping extracellular enzymes close to the cells. This enables the cells to metabolize dissolved, colloidal, and solid biopolymers (23). In this study, we investigated the effect of 2 major constituents of the EPS matrix, dextran and DNA, on E. faecalis biofilm formation. Our results indicate that the initial attachment of E. faecalis cells to dentin is an EPSdependent step. In the presence of dextranase or DNase I, bacteria were able to attach to the substratum but failed to form a large amount of structures compared with the control group. E. faecalis biofilms that were not treated with dextranase and DNase I exhibited better growth than the biofilms treated with the EPS-degrading enzymes. Taken together, our results suggest that dextran and eDNA directly participate in biofilm formation by E. faecalis by promoting bacterial adhesion. The exact mechanism through which this process occurs should be further investigated. CHX is a broad-spectrum antimicrobial agent. It is active against vegetative bacteria and mycobacteria, has moderate activity against fungi and viruses, and inhibits spore germination. CHX has been used in intracanal medications and in canal irrigation solutions because of its broad antimicrobial spectrum, its ability to remain active for prolonged periods when adhered to the hydroxyapatite component of dentin, and its ability to exhibit a slow release as its concentration decreases (24, 25). Although CHX is effective at killing the pathogens involved in endodontic infections, biofilms in root canals are resistant to CHX. In part, this resistance may be because of the inability of CHX to penetrate deeply into biofilms. Our data indicate that in the absence of dextran and eDNA, E. faecalis biofilms were less tolerant to CHX, which results in a statistically significant reduction in CFUs. The EPS matrix protects microbes against desiccation, oxidizing or charged biocides, some antibiotics, metallic cations, ultraviolet radiation, and host immune defenses. Therefore, in the presence of dextranase or DNase I, CHX may be able to act more directly on E. faecalis, thereby enhancing its bactericidal potential. The 2 EPS-degrading enzymes might act as biofilm-degrading agents to improve the efficiency of antimicrobial agents for the treatment of bacterial biofilms. However, their clinical applications, including the treatment of infected root canals, require further research. Numerous studies have shown that the bacteria in biofilms are embedded within an extracellular matrix that protects the bacteria JOE — Volume 38, Number 7, July 2012
from certain environmental factors (26, 27). Tetz and Tetz (28) reported that the cleavage of eDNA led to an increased penetration of antibiotics and decreased biofilm biomass and CFUs. This result suggests that eDNA plays an important role in the development of biofilm tolerance (29). Similar results have been observed in Staphylococcus epidermidis biofilms that were pretreated with dispersin B to reduce poly-N-acetylglucosamine surface polysaccharides. Izano et al (30) reported that dispersin B, a poly-N-acetylglucosamine surface polysaccharide (PNAG) degrading enzyme, sensitized S. epidermidis biofilms to cationic detergent cetylpyridinium chloride (CPC) killing, whereas DNase I sensitized S. aureus biofilms to CPC killing. It was concluded that PNAG and eDNA play fundamentally different structural roles in S. epidermidis and S. aureus biofilms. There are limitations of our study that must be acknowledged. Only 1 strain of 1 species of E. faecalis was used, and sodium hypochlorite was not used as a control. However, our findings still suggest that matrix polymers, such as dextran and eDNA, might act as general diffusion barriers to prevent CHX from accessing biofilm cells, which is consistent with previous studies (28, 31). In our study, we found that even when treated with CHX and EPS-degrading enzymes, some bacteria in the biofilms were not killed. This result might be related to the presence of other constituents in the biofilm matrix, such as heteroglycan, teichoic acid, or proteins, which might prevent CHX from killing the bacteria. It has also been suggested that different genes are activated and repressed in biofilms (32).
Conclusion The dextran-degrading enzyme (dextranase) and DNase I could decrease the adhesion of E. faecalis to human dentin, inhibit biofilm formation, and increase the susceptibility of E. faecalis biofilms to 2% CHX.
Acknowledgments The authors deny any conflicts of interest related to this study.
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