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Collagen-binding Streptococcal Surface Proteins Influence the Susceptibility of Biofilm Cells to Endodontic Antimicrobial Solutions
Basic Research—Biology
Collagen-binding Streptococcal Surface Proteins Influence the Susceptibility of Biofilm Cells to Endodontic Antimicrobial Solutions Timothy L. Violante, MS,*† Elaine M. Haase, PhD,† and M. Margaret Vickerman, DMD, PhD*† Abstract Introduction: Previous studies have indicated that the antimicrobial efficacy of endodontic irrigants may be diminished in the presence of patient tissues and fluids. With Streptococcus gordonii as a model microorganism, we used a genetic approach to investigate the hypothesis that bacterial surface proteins with collagen-binding abilities may function to protect biofilm cells from antiseptics commonly used in root canal treatment. Methods: S. gordonii strain DL1 or isogenic mutant strains with deletions of genes encoding collagen-binding surface proteins were grown in microtiter plates to form 8-hour biofilms. Planktonic cells were aspirated, and the remaining biofilm cells were bufferwashed and then incubated with either pH-adjusted buffer or potentially protective solutions of type I collagen, serum, or saliva. Biofilms were rewashed, pulsed with sodium hypochlorite, chlorhexidine digluconate, or BioPure MTAD, and then rewashed. Fresh medium was added, and survivor cell growth was monitored for 24 hours. Results: Buffer-treated biofilm cells of all 3 strains were similarly killed by sodium hypochlorite, chlorhexidine digluconate, and MTAD. Collagen, serum, and saliva significantly protected strain DL1 from all 3 antiseptics compared with buffer-treated cells (P # .0004). However, preincubation with collagen, serum, or saliva left both mutant strain biofilms significantly more susceptible to all 3 antiseptics than were respectively treated strain DL1 biofilms (P # .005). Conclusions: Interactions of S. gordonii surface proteins with collagen or similar components in serum and saliva may play roles in protecting biofilm cells from endodontic antiseptics. Elucidating molecular mechanisms underlying bacterial resistance to antimicrobials may facilitate the development of more effective treatments. (J Endod 2013;39:370–374)
Key Words Antimicrobial, antiseptics, biofilm, collagen, endodontics, microbiology, streptococci
T
he bacteria that colonize the infected dental pulp often grow in complex multispecies communities as biofilms attached to dentin or tissue surfaces (1). Biofilm cells have different susceptibilities to antimicrobial compounds than do unattached planktonic bacteria. These differences have been attributed to multiple factors including metabolic differences between planktonic and biofilm cells, characteristics of the substratum to which the bacteria attach, and extracellular biofilm components that may act interact directly with the antimicrobial compound to decrease its efficacy or act as a physicochemical barrier that impedes penetration to the target bacterial cells (2, 3). Thus, studying susceptibility of endodontic bacteria to antimicrobial solutions may be most clinically relevant by using biofilm models (4, 5). To achieve the goal of eliminating or reducing the number of bacteria in infected root canals, irrigant solutions with antiseptic properties are commonly used in endodontic treatment. Previous studies have shown that the presence of collagen and other tissue components may diminish the antimicrobial efficacy of endodontic irrigants (5, 6). The ability of the endodontic pathogen Enterococcus faecalis to bind to dentin via its collagen-binding surface protein Ace (7, 8) has been suggested to contribute to the decreased susceptibility of this species to endodontic antiseptics in the presence of tissue components (9, 10). To determine whether this role is shared by collagen-binding proteins of other oral streptococci and gram-positive cocci that are more abundant in endodontic infections (11), we further investigated this hypothesis by using Streptococcus gordonii, which is found in both primary and persistent endodontic infections (11, 12), as a model microorganism. Biofilms of isogenic mutant strains with deletions of genes that encode the collagen-binding proteins SspA, SspB (13), and CbdA (14) were examined and compared with biofilms of their parental strain for their relative susceptibilities to endodontic irrigants in the presence of collagen, serum, and saliva. The results provide insights into the contribution of collagen-binding abilities of streptococci to their resistance to antimicrobial solutions used in endodontic treatment.
Materials and Methods Bacterial Strains and Growth Medium S. gordonii parental strain Challis DL1 and 2 isogenic derivative strains in which genes encoding proteins involved in collagen binding (SspA and SspB in strain UB1360 [13] or CbdA in strain BN1386 [14]) were replaced with the aad9 gene encoding a protein for spectinomycin resistance were maintained at 80 C in 50% glycerol stocks. Cultures were grown in Todd Hewitt (TH) medium (Becton Dickinson and Co, Sparks, MD); the 2 mutant strains were grown with 250 mg/mL spectinomycin for selection but were grown without antibiotic for all assays.
From the *Department of Periodontics and Endodontics and †Department of Oral Biology, School of Dental Medicine, University at Buffalo, Buffalo, New York. Supported by University at Buffalo School of Dental Medicine Department of Periodontics and Endodontics Student Research Funds. Address requests for reprints to Dr M. Margaret Vickerman, University at Buffalo School of Dental Medicine, 223 Foster Hall, Buffalo, NY 14214. E-mail address: [email protected] 0099-2399/$ - see front matter Copyright ª 2013 American Association of Endodontists. http://dx.doi.org/10.1016/j.joen.2012.10.019
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Basic Research—Biology Biofilm Model Late log phase cultures of each S. gordonii strain were diluted to approximately 5 105 cells/mL in fresh TH medium, and 200-mL aliquots were dispensed to wells of untreated polystyrene microtiter plates (Becton Dickinson and Co). Cells were grown without shaking for 8 hours aerobically with 5% CO2 at 36 C to allow biofilm formation. Planktonic cells were carefully aspirated, and the remaining biofilm cells were washed with phosphate-buffered saline (PBS), pH 7.0, and used for biofilm antiseptic susceptibility assays. Biofilm cells were incubated for 30 minutes with 250 mL of 1 of 3 potentially protective solutions: type I collagen, serum, or saliva, prepared as described, or pH-matched buffer controls. Biofilms were rewashed with PBS and then pulsed for 10 minutes with antimicrobial solution or pH-matched control buffer in volumes of 350 mL to ensure coverage of biofilm cells at the air-liquid interfaces because of potential differences of meniscus levels resulting from different surface tensions of the various solutions. After 10 minutes, the antiseptic solution was removed, biofilms were washed with PBS, and then 200 mL of fresh TH medium was added to each well. Each microtiter plate also had 3 wells of biofilms of each strain that were treated only with TH medium at each step of the experiment to provide baseline growth levels. Cells were then incubated aerobically at 36 C without shaking, and growth curves of biofilm cells that survived the antiseptic pulse were monitored spectrophotometrically by measuring optical density at 595 nm on a microtiter plate reader with internal statistical software (Beckman Coulter Model AD340, Brea, CA) for 24 hours. Assays were done in triplicate, and experiments were repeated 4 times.
scraping in fresh TH to remove the attached cells. Both planktonic and biofilm cells were then vortexed to disperse any cell aggregates, diluted, and plated on TH agar plates to determine the number of colony-forming units (CFUs). After 48 hours of incubation, the agar plates were examined for uniform colony morphology. To confirm the identities of the recovered cells, representative colonies were examined for alpha hemolysis on Columbia blood agar plates (Becton Dickinson and Co) and spectinomycin resistance or sensitivity. Differences between strains or conditions at various time points were compared to determine statistical significance (P < .05) by using a Student’s t test with Microsoft Excel 2011 version 14.2.3 (Microsoft Corp, Redmond, WA).
Protective Solutions Acid-soluble collagen type I (MP Biomedicals, Solon, OH) was solubilized to a concentration of 1 mg/mL in 0.01 mol/L acetic acid, pH 5.0, filter-sterilized, and stored at 4 C. Complement-inactivated, heat-fixed (56 C 30 minutes) horse serum (pH 7.3) (Sigma Aldrich Chemical Company, St Louis, MO) was frozen ( 20 C) in aliquots and thawed immediately before use. The University Human Subjects Institutional Review Board approved the use of de-identified pooled human saliva in this study. Whole unstimulated saliva was collected by expectoration on ice and clarified by centrifugation at 8000g for 15 minutes; the resulting supernatant was heated at 60 C for 30 minutes and diluted to 25% v/v in buffered KCl (2 mmol/L KH2PO4, 2 mmol/L K2HPO4, 1 mmol/L CaCl2, 50 mmol/L KCI, pH 6.8) (15) for use in experiments. To confirm sterility of the saliva, undiluted aliquots were plated on TH agar and incubated aerobically at 37 C for up to 1 week.
Establishment of Biofilm Model Initial 8-hour biofilm formation in the microtiter wells was similar for S. gordonii parental and mutant strains as indicated by viable counts and crystal violet staining of biofilm mass (Table 1). Control biofilms of each strain were precoated and pulsed with TH medium rather than protective and antiseptic solutions or their buffer controls and then subjected to the remaining experimental protocol; the resulting baseline growth curves of the biofilm cells for each strain showed similar doubling times (Fig. 1). Examination of similar growth curves for all protective and antibiotic solution combinations showed that culture turbidities of the surviving biofilm cells that were antiseptic-treated or buffer-treated had all reached stationary growth phase by 12 hours; CFUs recovered from representative wells confirmed the spectrophotometric readings (N $ 3, data not shown). Accordingly, culture turbidity as an indicator of growth of survivor cells at 12 hours after the antiseptic or buffer pulse was chosen as the time point for statistical comparison of experimental conditions.
Antiseptic Solutions Undiluted 5.25% sodium hypochlorite (NaOCl) (Austin Manufacturing, Mars, PA), aqueous 0.12% (v/v) chlorhexidine (CHX) (Sigma Aldrich Chemical Company), and 100% Biopure MTAD (Dentsply, Tulsa, OK), a proprietary liquid mix of doxycycline, citric acid, and Tween-80 detergent (16), were used as antimicrobial solutions to pulse biofilms. Buffers adjusted to pH 12.5, 6.1, and 1.9 were used as control solutions for NaOCl, CHX, and MTAD, respectively. The MTAD was prepared immediately before use according to the manufacturer’s directions. All solutions were filter sterilized. Enumeration of Viable Cells At the end of each experiment and at selected time points throughout the time course, representative wells were chosen for enumeration of viable planktonic and biofilm cells. Planktonic cells were aspirated from wells and dispensed into a duplicate microtiter plate, and the optical density at 595 nm for both the planktonic and remaining biofilm cells was measured. Biofilm cells were recovered by mechanical JOE — Volume 39, Number 3, March 2013
Determination of Biofilm Mass To measure biofilm mass, biofilms were washed twice with PBS to remove loosely attached biofilm cells and components, fixed with 100% methanol for 15 minutes, and air-dried for 25 minutes. Fixed biofilms were stained with a solution of 0.01% w/v crystal violet, 0.5% v/v isopropanol, and 0.5% v/v ethanol for 15 minutes at room temperature. The crystal violet solution was decanted, and biofilms were washed with PBS. The stain was then eluted from the biofilms with a solution of ethanolacetone (80:20 v/v) with gentle shaking for 1 hour, and the absorbance at 570 nm (A570) of the resolubilized stain in the eluate was measured spectrophotometrically on the microtiter plate reader. Values were expressed as the average A570 of at least 3 replicate wells for each condition standard deviation.
Results
Antiseptic Susceptibility of Unprotected Biofilms Biofilms of all 3 strains that were treated with buffer (unprotected) rather than with the potentially protective solutions of collagen, serum, or saliva were unable to survive and grow after 10-minute pulses with 5.25% NaOCl, 0.12% CHX, and MTAD (Fig. 2A). No significant increases from the initial optical densities were seen in the microtiter wells during the 24-hour time period (P $ .8 for all strains), and dilution and plating of representative wells confirmed the absence of CFUs at the 12- and 24-hour time points. Buffers that were pH-matched to NaOCl and CHX did not significantly diminish the survival and subsequent growth of the biofilm cells of all 3 strains compared with medium controls (P $ .2 for all strains, Fig. 2A). Although the pH 1.9 buffer control for MTAD did not significantly diminish survival and growth of strains DL1 and BN1386 (P # .12 for each strain compared with medium control), the acidic buffer did significantly inhibit strain UB1360 (P < .002 compared with medium control, Fig. 2A). Antiseptic Susceptibility of Streptococcal Biofilms
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Basic Research—Biology TABLE 1. Initial Eight-hour Biofilms* Strain
Biofilm CFU/well
Crystal violet stained biomass A570
DL1 BN1386 UB1360
2.1 105 + 1.2 104 2.0 105 + 1.3 104 2.2 105 + 1.1 104
0.198 0.014 0.191 0.021 0.201 0.019
*Average values standard deviation from 5 independent experiments.
Antiseptic Efficacy against S. gordonii Strain DL1 Biofilms Precoated with Collagen, Serum, or Saliva At the 12-hour time point, of the 3 antiseptics, CHX demonstrated the most effective killing of the parental strain DL1 cells, even when the biofilm cells were pretreated with collagen (P # .008, Fig. 2B), serum (P # .002, Fig. 2C), and saliva (P # .003, Fig. 2D) when compared with both NaOCl and MTAD. Growth at 12 hours after exposure to NaOCl and MTAD indicated that these 2 antiseptics had similar abilities to kill the DL1 biofilm cells whether protected with collagen, serum, or saliva (P > .05 for NaOCl compared with MTAD for all 3 protective solutions, Fig. 2B–D). Susceptibility of Collagen-binding Deficient Strains to Antiseptics in the Presence of Collagen, Serum, and Saliva When biofilms were incubated with type 1 collagen before being subjected to antiseptic pulses, strains BN1386 and UB1360 were significantly more susceptible to 5.25% NaOCl, 0.12% CHX, and MTAD than was strain DL1 (P < .001 for all 3 antiseptics) as indicated by decreased growth of survivor cells at 12 hours (Fig. 2B). Collagen-protected biofilms of strain UB1360 showed significantly less growth (P < .001) after exposure to both MTAD and its pH 1.9 buffer control than collagenprotected biofilms of strain BN1386, consistent with the acid sensitivity seen in the unprotected biofilms of this strain (Fig. 2A). Similarly, biofilms of strains BN1386 and UB1360 that were precoated with serum (Fig. 2C) or saliva (Fig. 2D) were significantly more susceptible to 5.25% NaOCl, 0.12% CHX, and MTAD than serum-treated or saliva-treated biofilms of strain DL1 (P < .001 for serum-coated and saliva-coated biofilms for all 3 antiseptics). Serum-coated and
Figure 1. Growth curves of 8-hour S. gordonii biofilm cells of strains DL1 (black circles), BN1386 (black triangles), and UB1360 (white squares) after control pulse and subsequent growth in TH medium. Average values from 4 independent experiments.
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saliva-coated strain BN1386 biofilms were significantly more susceptible (P < .01) to 5.25% NaOCl than were respectively precoated biofilms of strain UB1360, whereas strain UB1360 was significantly more susceptible (P < .001) to MTAD than was strain BN1386 (Fig. 2C and D) under the same conditions.
Discussion S. gordonii and other oral streptococcal species can colonize the pulpal tissues as pioneer members of endodontic infections (17). It has been hypothesized that the ability of enterococci and the genetically related but more numerous oral streptococci to persist in infected canals is due in part to the ability of these bacteria to bind collagen and colonize dentin via collagen-binding surface adhesins (7, 14, 18, 19). Genomic sequence data have clarified that gram-positive cocci share many determinants. One goal of this study was to determine whether the link between collagen-binding ability and protection from antiseptics found in irrigants was a phenotype exclusive to E. faecalis (9) or whether this property is shared by other species that could be members of polymicrobial endodontic infections. Accordingly, the common oral streptococcal species S. gordonii was used as a model. Removal of bacteria from infected canals is a primary goal of endodontic treatment and is accomplished by mechanical and chemical cleansing. In vitro and ex vivo studies have suggested that the presence of model tissue components such as collagen (5), bovine serum albumin (20), and dentin particles (6, 21) can interfere with the antimicrobial efficacy of root canal irrigants. The results of the present studies support those findings and indicated that S. gordonii biofilm cells precoated with collagen, serum, and saliva were less susceptible to the antimicrobial solutions tested than biofilms precoated with buffer. These putative protective solutions were chosen for testing in the biofilm model because of their clinical relevance and the likelihood that they would contain components with receptor sites recognized by the bacterial collagen-binding surface adhesins. Collagen and serum are normal root canal components; collagen type 1 is the main organic component of dentin (22), and collagen breakdown products and collagen-like peptides are found in serum (23, 24). Although not found in healthy canals, saliva can gain entry into the canal via carious lesions or defective restorations that allow direct communication between the pulp and oral cavity. The proline-rich proteins found in both saliva and serum (25) could potentially be recognized by collagen-binding adhesins. The S. gordonii SspA and SspB proteins differ from CbdA in their predicted functional domains and probably in their range of receptor specificity. SspA and SspB are members of the antigen I/II family of streptococcal proteins with conserved alanine-rich and proline-rich domains that bind multiple receptors in addition to collagen type 1 including molecules on other bacterial cells and salivary glycoproteins (26). In contrast, the CbdA protein has a conserved collagen-binding domain (CBD) and a stalk domain thought to hold the CBD in functional conformation similar to that seen in the Ace protein of E. faecalis and other members of this CBD protein family in Staphylococcus aureus and group A streptococci (14). Although Ace has been shown to bind the extracellular matrix components fibrinogen and fibronectin in addition to type 1 collagen (27), the range of specificity of CbdA for various receptors has not yet been determined. Recent studies have shown that both the CbdA-deficient strain BN1386 and the SspA/SspB-deficient strain UB1360 are less able to bind to immobilized type 1 collagen than the parental strain DL1, and that the collagen-binding ability of strain BN1386 is significantly less than that of strain UB1360 (14). These relative levels of collagen-binding ability are consistent with the levels of protection seen in the present study in that strain BN1386 was less JOE — Volume 39, Number 3, March 2013
Basic Research—Biology
Figure 2. Protective effects of collagen (B), serum (C), and saliva (D) as compared with unprotected buffer controls (A) on biofilm cells of S. gordonii strains DL1 (black bars), BN1386 (gray bars), and UB1360 (white bars). Bars indicate average values standard deviation at 12 hours after antiseptic or pH-matched buffer pulse. N = 4 independent experiments.
protected by the collagen from NaOCl than was strain UB1360; serum and saliva offered similar relative levels of protection. Unexpectedly, precoating with collagen, serum, or saliva protected the CbdA mutant more than the SspA/SspB mutant from the effects of MTAD. Control studies with pH-matched buffer suggested that this inconsistency between collagen protection and the strains’ relative collagen-binding abilities may be due to the acid sensitivity of strain UB1360. The basis for this increased acid sensitivity is unknown and may be due to a pleiotropic effect of the loss of the SspA and SspB surface proteins. The antiseptic solutions tested are currently used in clinical endodontics. Only biofilm cells that survived the 10-minute immersion in antibacterial solution were able to grow. Bacterial growth was monitored for 24 hours after the antibiotic pulse to increase the likelihood that cells that persisted but were in a state of metabolic stress after the antiseptic treatment would recover and grow (28). Comparisons were made at 12 hours after the pulse because growth curves confirmed that biofilm cells of all strains had recovered and were at maximum growth phase at this time point. Absence of CFUs after plating the well contents on fresh medium at the end of each experiment confirmed the killing efficacy of the antiseptics. CHX was the most effective against all the S. gordonii strains, even in the presence of the protective solutions, perhaps because of CHX substantivity properties (6). The molecular basis of the protective effects of collagen, serum, and saliva on the streptococcal biofilm cells is not known. Biofilms JOE — Volume 39, Number 3, March 2013
form a complex environment with microniches made up of bacterial cells, the products of bacterial growth, and the substratum on which they grow. In vivo, this environment is even more complex with the addition of host components. The present studies were done with early biofilms to minimize the effects of extrabacterial components that could interfere with interactions between the bacterial surface proteins and the protective molecules. Cells in more mature biofilms could differ metabolically and/or have additional biofilm architectural and structural components that could further influence antiseptic susceptibility (29). The increased quantity of extracellular DNA and carbohydrate material found in mature biofilms of E. faecalis has been suggested to contribute to the level of CHX resistance of this species (30). Adhesin-receptor interactions play an important role in biofilm architecture (2). Like all oral streptococci, S. gordonii have multiple cell wall surface proteins (31) with the potential to act as adhesins to facilitate colonization and persistence in the root canal by binding to receptor molecules in the dentin or pulp tissue substratum. Similarly, bacterial adhesins can bind to other bacteria directly or to molecules that act as bridges between bacterial cells, thereby facilitating the formation of microbial aggregates with subsequent effects on biofilm formation and composition (1). The present studies used a model system to examine a single variable and determine whether the collagen-binding ability of bacteria in a single species biofilm played a role in protecting the cells from antiseptics. In the polymicrobial biofilms of endodontic
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Basic Research—Biology infections in vivo, multiple adhesin-receptor interactions could contribute to biofilm architecture and integrity, thereby influencing bacterial susceptibility. Previous studies with an isogenic mutant strain suggested that the ability of E. faecalis to interact with type 1 collagen confers an increased resistance to endodontic antiseptics and that this resistance is in part mediated by the enterococcal surface protein Ace (9). The results of the present study suggest that similar protective effects may occur via S. gordonii SspA, SspB, and CbdA proteins because the strains with deletions of these proteins were more susceptible to all the antiseptics tested than the parental strain in the presence of collagen, serum, and saliva. It is possible that the protective substances bind to the biofilms via adhesin-receptor interactions, thereby creating a molecular sieve that limits the antiseptic penetration required to reach the bacterial cells (2). Deletion of bacterial adhesins in the mutant strains may have diminished binding to the protective substances and allowed increased antiseptic penetration. Alternatively, the deletion of SspA, SspB, and CbdA in the mutant strains may have made the bacterial cells more susceptible to the effects of the antiseptics via changes in overall cell surface charge or altered cell abilities to interact with neighboring bacterial cells or substratum components within the microenvironment of the biofilm. Finally, the mutant strains may have as-yet-unidentified metabolic changes that resulted from loss of the surface proteins and caused increased antiseptic sensitivity. Overall, the results of these studies support the hypothesis that the presence of collagen-binding proteins on the S. gordonii cell surface confers the ability to interact with protective tissue and fluid components that may be present in the infected root canal. These interactions may decrease the efficacy of antiseptics on biofilm bacteria. Additional studies will be necessary to further define the molecular mechanisms involved. Although the clinical situation is clearly much more complex, it is possible that other bacterial species in the polymicrobial biofilms of endodontic infections may share similar abilities to bind to collagen and/or tissue components and fluids that contribute to bacterial resistance to endodontic irrigants. Understanding how oral bacteria are protected from antimicrobial agents is essential for optimizing our clinical treatment and may provide crucial insights into the design of more effective clinical therapies that specifically target and disrupt such protective interactions.
Acknowledgments The authors thank Professor H. F. Jenkinson at the University of Bristol, United Kingdom for providing strain UB1360. The authors deny any conflicts of interest related to this study.
References 1. Svenst€ater G, Bergenholtz G. Biofilms in endodontic infections. Endod Topics 2004; 9:27–36. 2. Donlan RM, Costerton JW. Biofilms: survival mechanisms of clinically relevant microorganisms. Clin Microbiol Rev 2002;15:167–93. 3. Mah TF, O’Toole GA. Mechanisms of biofilm resistance to antimicrobial agents. Trends Microbiol 2001;9:34–9. 4. Chavez de Paz LE. Redefining the persistent infection in root canals: possible role of biofilm communities. J Endod 2007;33:652–62.
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5. Chavez de Paz LE, Bergenholtz G, Svens€ater G. The effects of antimicrobials on endodontic biofilm bacteria. J. Endod 2010;36:70–7. 6. Haapasalo M, Qian W, Portenier I, Waltimo T. Effects of dentin on the antimicrobial properties of endodontic medicaments. J Endod 2007;33:917–25. 7. Hubble TS, Hatton JF, Nallapareddy SR, et al. Influence of Enterococcus faecalis proteases and the collagen binding protein Ace, on adhesion to dentin. Oral Microbiol Immunol 2003;18:121–6. 8. Kowalski WJ, Kasper EL, Hatton JF, et al. Enterococcus faecalis adhesin, Ace, mediates attachment to particulate dentin. J Endod 2006;32:634–7. 9. Kayaoglu G, Erten H, Ørstavik D. Possible role of the adhesin ace and collagen adherence in conveying resistance to disinfectants on Enterococcus faecalis. Oral Microbiol Immunol 2008;23:449–54. 10. Kayaoglu G, Erten H, Bodrumlu E, Ørstavik D. The resistance of collagen associated, planktonic cells of Enterococcus faecalis to calcium hydroxide. J Endod 2009;35: 46–9. 11. Siqueira JF Jr, R^oc¸as IN. Diversity of endodontic microbiota revisited. J Dent Res 2009;88:969–81. 12. Chavez de Paz LE, Svens€ater G, Dahlen G, Bergenholtz G. Streptococci from root canals in teeth with apical periodontitis receiving endodontic treatment. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 2005;100:232–41. 13. Heddle C, Nobbs AH, Jakubovics NS, et al. Host collagen signal induces antigen I/II adhesin and invasin gene expression in oral Streptococcus gordonii. Mol Microbiol 2003;50:597–607. 14. Moses PJ, Power DA, Jesionowski AM, et al. Streptococcus gordonii collagenbinding domain protein CbdA may enhance bacterial survival in instrumented root canals ex vivo. J Endod 2013;39:39–43. 15. Appelbaum B, Golub E, Holt SC, Rosan B. In vitro studies of dental plaque formation: adsorption of oral streptococci to hydroxyapatite. Infect Immun 1979;25: 717–28. 16. Torabinejad M, Shabahang S, Aprecio RM, Kettering JD. The antimicrobial effect of MTAD: an in vitro investigation. J Endod 2003;29:400–3. 17. Fabricius L, Dahlen G, Holm SE, M€oller AJ. Influence of combinations of oral bacteria on periapical tissues of monkeys. Scand J Dent Res 1982;90:200–6. 18. Love RM. Enterococcus faecalis: a mechanism for its role in endodontic failure. Int Endod J 2001;34:399–405. 19. Love RM, Jenkinson HF. Invasion of dentinal tubules by oral bacteria. Crit Rev Oral Biol Med 2002;13:171–83. 20. Pappen FG, Qian W, Aleksejuniene J, et al. Inhibition of sodium hypochlorite antimicrobial activity in the presence of bovine serum albumin. J Endod 2010;36: 268–71. 21. Portenier I, Waltimo T, Ørstavik D, Haapasalo M. Killing of Enterococcus faecalis by MTAD and chlorhexidine digluconate with or without cetrimide in the presence or absence of dentine powder or BSA. J Endod 2006;32:138–41. 22. Mj€or IA. Human coronal dentine: structure and reactions. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 1972;33:810–23. 23. Matsushita M, Endo Y, Taira S, et al. A novel human serum lectin with collagen- and fibrinogen-like domains that functions as an opsonin. J Biol Chem 1996;271: 2448–54. 24. Risteli J, Risteli L. Serum-based test of the pathologic breakdown of type I collagen fibers. Clin Chem 2009;55:1032–3. 25. Loo JA, Yan W, Ramachandran P, Wong DT. Comparative human salivary and plasma proteomes. J Dent Res 2010;89:1016–23. 26. Jakubovics NS, Stromberg N, van Dolleweerd CJ, et al. Differential binding specificities of oral streptococcal antigen I/II family adhesins for human or bacterial ligands. Mol Microbiol 2005;55:1591–605. 27. Nallapareddy SR, Murray BE. Role played by serum, a biological cue, in the adherence of Enterococcus faecalis to extracellular matrix proteins, collagen, fibrinogen, and fibronectin. J Infect Dis 2008;97:1728–36. 28. Lewis K. Persister cells. Annu Rev Microbiol 2010;64:357–72. 29. Shen Y, Stojicic S, Haapasalo M. Antimicrobial efficacy of chlorhexidine against bacteria in biofilms at different stages of development. J Endod 2011;37:657–61. 30. Li W, Liu H, Xu Q. Extracellular dextran and DNA affect the formation of Enterococcus faecalis biofilms and their susceptibility to 2% chlorhexidine. J Endod 2012;38:894–8. 31. Davies JR, Svens€ater G, Herzberg MC. Identification of novel LPXTG-linked surface proteins from Streptococcus gordonii. Microbiology 2009;155:1977–88.
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Collagen-binding Streptococcal Surface Proteins Influence the Susceptibility of Biofilm Cells to Endodontic Antimicrobial Solutions Timothy L. Violante, MS,*† Elaine M. Haase, PhD,† and M. Margaret Vickerman, DMD, PhD*† Abstract Introduction: Previous studies have indicated that the antimicrobial efficacy of endodontic irrigants may be diminished in the presence of patient tissues and fluids. With Streptococcus gordonii as a model microorganism, we used a genetic approach to investigate the hypothesis that bacterial surface proteins with collagen-binding abilities may function to protect biofilm cells from antiseptics commonly used in root canal treatment. Methods: S. gordonii strain DL1 or isogenic mutant strains with deletions of genes encoding collagen-binding surface proteins were grown in microtiter plates to form 8-hour biofilms. Planktonic cells were aspirated, and the remaining biofilm cells were bufferwashed and then incubated with either pH-adjusted buffer or potentially protective solutions of type I collagen, serum, or saliva. Biofilms were rewashed, pulsed with sodium hypochlorite, chlorhexidine digluconate, or BioPure MTAD, and then rewashed. Fresh medium was added, and survivor cell growth was monitored for 24 hours. Results: Buffer-treated biofilm cells of all 3 strains were similarly killed by sodium hypochlorite, chlorhexidine digluconate, and MTAD. Collagen, serum, and saliva significantly protected strain DL1 from all 3 antiseptics compared with buffer-treated cells (P # .0004). However, preincubation with collagen, serum, or saliva left both mutant strain biofilms significantly more susceptible to all 3 antiseptics than were respectively treated strain DL1 biofilms (P # .005). Conclusions: Interactions of S. gordonii surface proteins with collagen or similar components in serum and saliva may play roles in protecting biofilm cells from endodontic antiseptics. Elucidating molecular mechanisms underlying bacterial resistance to antimicrobials may facilitate the development of more effective treatments. (J Endod 2013;39:370–374)
Key Words Antimicrobial, antiseptics, biofilm, collagen, endodontics, microbiology, streptococci
T
he bacteria that colonize the infected dental pulp often grow in complex multispecies communities as biofilms attached to dentin or tissue surfaces (1). Biofilm cells have different susceptibilities to antimicrobial compounds than do unattached planktonic bacteria. These differences have been attributed to multiple factors including metabolic differences between planktonic and biofilm cells, characteristics of the substratum to which the bacteria attach, and extracellular biofilm components that may act interact directly with the antimicrobial compound to decrease its efficacy or act as a physicochemical barrier that impedes penetration to the target bacterial cells (2, 3). Thus, studying susceptibility of endodontic bacteria to antimicrobial solutions may be most clinically relevant by using biofilm models (4, 5). To achieve the goal of eliminating or reducing the number of bacteria in infected root canals, irrigant solutions with antiseptic properties are commonly used in endodontic treatment. Previous studies have shown that the presence of collagen and other tissue components may diminish the antimicrobial efficacy of endodontic irrigants (5, 6). The ability of the endodontic pathogen Enterococcus faecalis to bind to dentin via its collagen-binding surface protein Ace (7, 8) has been suggested to contribute to the decreased susceptibility of this species to endodontic antiseptics in the presence of tissue components (9, 10). To determine whether this role is shared by collagen-binding proteins of other oral streptococci and gram-positive cocci that are more abundant in endodontic infections (11), we further investigated this hypothesis by using Streptococcus gordonii, which is found in both primary and persistent endodontic infections (11, 12), as a model microorganism. Biofilms of isogenic mutant strains with deletions of genes that encode the collagen-binding proteins SspA, SspB (13), and CbdA (14) were examined and compared with biofilms of their parental strain for their relative susceptibilities to endodontic irrigants in the presence of collagen, serum, and saliva. The results provide insights into the contribution of collagen-binding abilities of streptococci to their resistance to antimicrobial solutions used in endodontic treatment.
Materials and Methods Bacterial Strains and Growth Medium S. gordonii parental strain Challis DL1 and 2 isogenic derivative strains in which genes encoding proteins involved in collagen binding (SspA and SspB in strain UB1360 [13] or CbdA in strain BN1386 [14]) were replaced with the aad9 gene encoding a protein for spectinomycin resistance were maintained at 80 C in 50% glycerol stocks. Cultures were grown in Todd Hewitt (TH) medium (Becton Dickinson and Co, Sparks, MD); the 2 mutant strains were grown with 250 mg/mL spectinomycin for selection but were grown without antibiotic for all assays.
From the *Department of Periodontics and Endodontics and †Department of Oral Biology, School of Dental Medicine, University at Buffalo, Buffalo, New York. Supported by University at Buffalo School of Dental Medicine Department of Periodontics and Endodontics Student Research Funds. Address requests for reprints to Dr M. Margaret Vickerman, University at Buffalo School of Dental Medicine, 223 Foster Hall, Buffalo, NY 14214. E-mail address: [email protected] 0099-2399/$ - see front matter Copyright ª 2013 American Association of Endodontists. http://dx.doi.org/10.1016/j.joen.2012.10.019
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Basic Research—Biology Biofilm Model Late log phase cultures of each S. gordonii strain were diluted to approximately 5 105 cells/mL in fresh TH medium, and 200-mL aliquots were dispensed to wells of untreated polystyrene microtiter plates (Becton Dickinson and Co). Cells were grown without shaking for 8 hours aerobically with 5% CO2 at 36 C to allow biofilm formation. Planktonic cells were carefully aspirated, and the remaining biofilm cells were washed with phosphate-buffered saline (PBS), pH 7.0, and used for biofilm antiseptic susceptibility assays. Biofilm cells were incubated for 30 minutes with 250 mL of 1 of 3 potentially protective solutions: type I collagen, serum, or saliva, prepared as described, or pH-matched buffer controls. Biofilms were rewashed with PBS and then pulsed for 10 minutes with antimicrobial solution or pH-matched control buffer in volumes of 350 mL to ensure coverage of biofilm cells at the air-liquid interfaces because of potential differences of meniscus levels resulting from different surface tensions of the various solutions. After 10 minutes, the antiseptic solution was removed, biofilms were washed with PBS, and then 200 mL of fresh TH medium was added to each well. Each microtiter plate also had 3 wells of biofilms of each strain that were treated only with TH medium at each step of the experiment to provide baseline growth levels. Cells were then incubated aerobically at 36 C without shaking, and growth curves of biofilm cells that survived the antiseptic pulse were monitored spectrophotometrically by measuring optical density at 595 nm on a microtiter plate reader with internal statistical software (Beckman Coulter Model AD340, Brea, CA) for 24 hours. Assays were done in triplicate, and experiments were repeated 4 times.
scraping in fresh TH to remove the attached cells. Both planktonic and biofilm cells were then vortexed to disperse any cell aggregates, diluted, and plated on TH agar plates to determine the number of colony-forming units (CFUs). After 48 hours of incubation, the agar plates were examined for uniform colony morphology. To confirm the identities of the recovered cells, representative colonies were examined for alpha hemolysis on Columbia blood agar plates (Becton Dickinson and Co) and spectinomycin resistance or sensitivity. Differences between strains or conditions at various time points were compared to determine statistical significance (P < .05) by using a Student’s t test with Microsoft Excel 2011 version 14.2.3 (Microsoft Corp, Redmond, WA).
Protective Solutions Acid-soluble collagen type I (MP Biomedicals, Solon, OH) was solubilized to a concentration of 1 mg/mL in 0.01 mol/L acetic acid, pH 5.0, filter-sterilized, and stored at 4 C. Complement-inactivated, heat-fixed (56 C 30 minutes) horse serum (pH 7.3) (Sigma Aldrich Chemical Company, St Louis, MO) was frozen ( 20 C) in aliquots and thawed immediately before use. The University Human Subjects Institutional Review Board approved the use of de-identified pooled human saliva in this study. Whole unstimulated saliva was collected by expectoration on ice and clarified by centrifugation at 8000g for 15 minutes; the resulting supernatant was heated at 60 C for 30 minutes and diluted to 25% v/v in buffered KCl (2 mmol/L KH2PO4, 2 mmol/L K2HPO4, 1 mmol/L CaCl2, 50 mmol/L KCI, pH 6.8) (15) for use in experiments. To confirm sterility of the saliva, undiluted aliquots were plated on TH agar and incubated aerobically at 37 C for up to 1 week.
Establishment of Biofilm Model Initial 8-hour biofilm formation in the microtiter wells was similar for S. gordonii parental and mutant strains as indicated by viable counts and crystal violet staining of biofilm mass (Table 1). Control biofilms of each strain were precoated and pulsed with TH medium rather than protective and antiseptic solutions or their buffer controls and then subjected to the remaining experimental protocol; the resulting baseline growth curves of the biofilm cells for each strain showed similar doubling times (Fig. 1). Examination of similar growth curves for all protective and antibiotic solution combinations showed that culture turbidities of the surviving biofilm cells that were antiseptic-treated or buffer-treated had all reached stationary growth phase by 12 hours; CFUs recovered from representative wells confirmed the spectrophotometric readings (N $ 3, data not shown). Accordingly, culture turbidity as an indicator of growth of survivor cells at 12 hours after the antiseptic or buffer pulse was chosen as the time point for statistical comparison of experimental conditions.
Antiseptic Solutions Undiluted 5.25% sodium hypochlorite (NaOCl) (Austin Manufacturing, Mars, PA), aqueous 0.12% (v/v) chlorhexidine (CHX) (Sigma Aldrich Chemical Company), and 100% Biopure MTAD (Dentsply, Tulsa, OK), a proprietary liquid mix of doxycycline, citric acid, and Tween-80 detergent (16), were used as antimicrobial solutions to pulse biofilms. Buffers adjusted to pH 12.5, 6.1, and 1.9 were used as control solutions for NaOCl, CHX, and MTAD, respectively. The MTAD was prepared immediately before use according to the manufacturer’s directions. All solutions were filter sterilized. Enumeration of Viable Cells At the end of each experiment and at selected time points throughout the time course, representative wells were chosen for enumeration of viable planktonic and biofilm cells. Planktonic cells were aspirated from wells and dispensed into a duplicate microtiter plate, and the optical density at 595 nm for both the planktonic and remaining biofilm cells was measured. Biofilm cells were recovered by mechanical JOE — Volume 39, Number 3, March 2013
Determination of Biofilm Mass To measure biofilm mass, biofilms were washed twice with PBS to remove loosely attached biofilm cells and components, fixed with 100% methanol for 15 minutes, and air-dried for 25 minutes. Fixed biofilms were stained with a solution of 0.01% w/v crystal violet, 0.5% v/v isopropanol, and 0.5% v/v ethanol for 15 minutes at room temperature. The crystal violet solution was decanted, and biofilms were washed with PBS. The stain was then eluted from the biofilms with a solution of ethanolacetone (80:20 v/v) with gentle shaking for 1 hour, and the absorbance at 570 nm (A570) of the resolubilized stain in the eluate was measured spectrophotometrically on the microtiter plate reader. Values were expressed as the average A570 of at least 3 replicate wells for each condition standard deviation.
Results
Antiseptic Susceptibility of Unprotected Biofilms Biofilms of all 3 strains that were treated with buffer (unprotected) rather than with the potentially protective solutions of collagen, serum, or saliva were unable to survive and grow after 10-minute pulses with 5.25% NaOCl, 0.12% CHX, and MTAD (Fig. 2A). No significant increases from the initial optical densities were seen in the microtiter wells during the 24-hour time period (P $ .8 for all strains), and dilution and plating of representative wells confirmed the absence of CFUs at the 12- and 24-hour time points. Buffers that were pH-matched to NaOCl and CHX did not significantly diminish the survival and subsequent growth of the biofilm cells of all 3 strains compared with medium controls (P $ .2 for all strains, Fig. 2A). Although the pH 1.9 buffer control for MTAD did not significantly diminish survival and growth of strains DL1 and BN1386 (P # .12 for each strain compared with medium control), the acidic buffer did significantly inhibit strain UB1360 (P < .002 compared with medium control, Fig. 2A). Antiseptic Susceptibility of Streptococcal Biofilms
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Basic Research—Biology TABLE 1. Initial Eight-hour Biofilms* Strain
Biofilm CFU/well
Crystal violet stained biomass A570
DL1 BN1386 UB1360
2.1 105 + 1.2 104 2.0 105 + 1.3 104 2.2 105 + 1.1 104
0.198 0.014 0.191 0.021 0.201 0.019
*Average values standard deviation from 5 independent experiments.
Antiseptic Efficacy against S. gordonii Strain DL1 Biofilms Precoated with Collagen, Serum, or Saliva At the 12-hour time point, of the 3 antiseptics, CHX demonstrated the most effective killing of the parental strain DL1 cells, even when the biofilm cells were pretreated with collagen (P # .008, Fig. 2B), serum (P # .002, Fig. 2C), and saliva (P # .003, Fig. 2D) when compared with both NaOCl and MTAD. Growth at 12 hours after exposure to NaOCl and MTAD indicated that these 2 antiseptics had similar abilities to kill the DL1 biofilm cells whether protected with collagen, serum, or saliva (P > .05 for NaOCl compared with MTAD for all 3 protective solutions, Fig. 2B–D). Susceptibility of Collagen-binding Deficient Strains to Antiseptics in the Presence of Collagen, Serum, and Saliva When biofilms were incubated with type 1 collagen before being subjected to antiseptic pulses, strains BN1386 and UB1360 were significantly more susceptible to 5.25% NaOCl, 0.12% CHX, and MTAD than was strain DL1 (P < .001 for all 3 antiseptics) as indicated by decreased growth of survivor cells at 12 hours (Fig. 2B). Collagen-protected biofilms of strain UB1360 showed significantly less growth (P < .001) after exposure to both MTAD and its pH 1.9 buffer control than collagenprotected biofilms of strain BN1386, consistent with the acid sensitivity seen in the unprotected biofilms of this strain (Fig. 2A). Similarly, biofilms of strains BN1386 and UB1360 that were precoated with serum (Fig. 2C) or saliva (Fig. 2D) were significantly more susceptible to 5.25% NaOCl, 0.12% CHX, and MTAD than serum-treated or saliva-treated biofilms of strain DL1 (P < .001 for serum-coated and saliva-coated biofilms for all 3 antiseptics). Serum-coated and
Figure 1. Growth curves of 8-hour S. gordonii biofilm cells of strains DL1 (black circles), BN1386 (black triangles), and UB1360 (white squares) after control pulse and subsequent growth in TH medium. Average values from 4 independent experiments.
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saliva-coated strain BN1386 biofilms were significantly more susceptible (P < .01) to 5.25% NaOCl than were respectively precoated biofilms of strain UB1360, whereas strain UB1360 was significantly more susceptible (P < .001) to MTAD than was strain BN1386 (Fig. 2C and D) under the same conditions.
Discussion S. gordonii and other oral streptococcal species can colonize the pulpal tissues as pioneer members of endodontic infections (17). It has been hypothesized that the ability of enterococci and the genetically related but more numerous oral streptococci to persist in infected canals is due in part to the ability of these bacteria to bind collagen and colonize dentin via collagen-binding surface adhesins (7, 14, 18, 19). Genomic sequence data have clarified that gram-positive cocci share many determinants. One goal of this study was to determine whether the link between collagen-binding ability and protection from antiseptics found in irrigants was a phenotype exclusive to E. faecalis (9) or whether this property is shared by other species that could be members of polymicrobial endodontic infections. Accordingly, the common oral streptococcal species S. gordonii was used as a model. Removal of bacteria from infected canals is a primary goal of endodontic treatment and is accomplished by mechanical and chemical cleansing. In vitro and ex vivo studies have suggested that the presence of model tissue components such as collagen (5), bovine serum albumin (20), and dentin particles (6, 21) can interfere with the antimicrobial efficacy of root canal irrigants. The results of the present studies support those findings and indicated that S. gordonii biofilm cells precoated with collagen, serum, and saliva were less susceptible to the antimicrobial solutions tested than biofilms precoated with buffer. These putative protective solutions were chosen for testing in the biofilm model because of their clinical relevance and the likelihood that they would contain components with receptor sites recognized by the bacterial collagen-binding surface adhesins. Collagen and serum are normal root canal components; collagen type 1 is the main organic component of dentin (22), and collagen breakdown products and collagen-like peptides are found in serum (23, 24). Although not found in healthy canals, saliva can gain entry into the canal via carious lesions or defective restorations that allow direct communication between the pulp and oral cavity. The proline-rich proteins found in both saliva and serum (25) could potentially be recognized by collagen-binding adhesins. The S. gordonii SspA and SspB proteins differ from CbdA in their predicted functional domains and probably in their range of receptor specificity. SspA and SspB are members of the antigen I/II family of streptococcal proteins with conserved alanine-rich and proline-rich domains that bind multiple receptors in addition to collagen type 1 including molecules on other bacterial cells and salivary glycoproteins (26). In contrast, the CbdA protein has a conserved collagen-binding domain (CBD) and a stalk domain thought to hold the CBD in functional conformation similar to that seen in the Ace protein of E. faecalis and other members of this CBD protein family in Staphylococcus aureus and group A streptococci (14). Although Ace has been shown to bind the extracellular matrix components fibrinogen and fibronectin in addition to type 1 collagen (27), the range of specificity of CbdA for various receptors has not yet been determined. Recent studies have shown that both the CbdA-deficient strain BN1386 and the SspA/SspB-deficient strain UB1360 are less able to bind to immobilized type 1 collagen than the parental strain DL1, and that the collagen-binding ability of strain BN1386 is significantly less than that of strain UB1360 (14). These relative levels of collagen-binding ability are consistent with the levels of protection seen in the present study in that strain BN1386 was less JOE — Volume 39, Number 3, March 2013
Basic Research—Biology
Figure 2. Protective effects of collagen (B), serum (C), and saliva (D) as compared with unprotected buffer controls (A) on biofilm cells of S. gordonii strains DL1 (black bars), BN1386 (gray bars), and UB1360 (white bars). Bars indicate average values standard deviation at 12 hours after antiseptic or pH-matched buffer pulse. N = 4 independent experiments.
protected by the collagen from NaOCl than was strain UB1360; serum and saliva offered similar relative levels of protection. Unexpectedly, precoating with collagen, serum, or saliva protected the CbdA mutant more than the SspA/SspB mutant from the effects of MTAD. Control studies with pH-matched buffer suggested that this inconsistency between collagen protection and the strains’ relative collagen-binding abilities may be due to the acid sensitivity of strain UB1360. The basis for this increased acid sensitivity is unknown and may be due to a pleiotropic effect of the loss of the SspA and SspB surface proteins. The antiseptic solutions tested are currently used in clinical endodontics. Only biofilm cells that survived the 10-minute immersion in antibacterial solution were able to grow. Bacterial growth was monitored for 24 hours after the antibiotic pulse to increase the likelihood that cells that persisted but were in a state of metabolic stress after the antiseptic treatment would recover and grow (28). Comparisons were made at 12 hours after the pulse because growth curves confirmed that biofilm cells of all strains had recovered and were at maximum growth phase at this time point. Absence of CFUs after plating the well contents on fresh medium at the end of each experiment confirmed the killing efficacy of the antiseptics. CHX was the most effective against all the S. gordonii strains, even in the presence of the protective solutions, perhaps because of CHX substantivity properties (6). The molecular basis of the protective effects of collagen, serum, and saliva on the streptococcal biofilm cells is not known. Biofilms JOE — Volume 39, Number 3, March 2013
form a complex environment with microniches made up of bacterial cells, the products of bacterial growth, and the substratum on which they grow. In vivo, this environment is even more complex with the addition of host components. The present studies were done with early biofilms to minimize the effects of extrabacterial components that could interfere with interactions between the bacterial surface proteins and the protective molecules. Cells in more mature biofilms could differ metabolically and/or have additional biofilm architectural and structural components that could further influence antiseptic susceptibility (29). The increased quantity of extracellular DNA and carbohydrate material found in mature biofilms of E. faecalis has been suggested to contribute to the level of CHX resistance of this species (30). Adhesin-receptor interactions play an important role in biofilm architecture (2). Like all oral streptococci, S. gordonii have multiple cell wall surface proteins (31) with the potential to act as adhesins to facilitate colonization and persistence in the root canal by binding to receptor molecules in the dentin or pulp tissue substratum. Similarly, bacterial adhesins can bind to other bacteria directly or to molecules that act as bridges between bacterial cells, thereby facilitating the formation of microbial aggregates with subsequent effects on biofilm formation and composition (1). The present studies used a model system to examine a single variable and determine whether the collagen-binding ability of bacteria in a single species biofilm played a role in protecting the cells from antiseptics. In the polymicrobial biofilms of endodontic
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Basic Research—Biology infections in vivo, multiple adhesin-receptor interactions could contribute to biofilm architecture and integrity, thereby influencing bacterial susceptibility. Previous studies with an isogenic mutant strain suggested that the ability of E. faecalis to interact with type 1 collagen confers an increased resistance to endodontic antiseptics and that this resistance is in part mediated by the enterococcal surface protein Ace (9). The results of the present study suggest that similar protective effects may occur via S. gordonii SspA, SspB, and CbdA proteins because the strains with deletions of these proteins were more susceptible to all the antiseptics tested than the parental strain in the presence of collagen, serum, and saliva. It is possible that the protective substances bind to the biofilms via adhesin-receptor interactions, thereby creating a molecular sieve that limits the antiseptic penetration required to reach the bacterial cells (2). Deletion of bacterial adhesins in the mutant strains may have diminished binding to the protective substances and allowed increased antiseptic penetration. Alternatively, the deletion of SspA, SspB, and CbdA in the mutant strains may have made the bacterial cells more susceptible to the effects of the antiseptics via changes in overall cell surface charge or altered cell abilities to interact with neighboring bacterial cells or substratum components within the microenvironment of the biofilm. Finally, the mutant strains may have as-yet-unidentified metabolic changes that resulted from loss of the surface proteins and caused increased antiseptic sensitivity. Overall, the results of these studies support the hypothesis that the presence of collagen-binding proteins on the S. gordonii cell surface confers the ability to interact with protective tissue and fluid components that may be present in the infected root canal. These interactions may decrease the efficacy of antiseptics on biofilm bacteria. Additional studies will be necessary to further define the molecular mechanisms involved. Although the clinical situation is clearly much more complex, it is possible that other bacterial species in the polymicrobial biofilms of endodontic infections may share similar abilities to bind to collagen and/or tissue components and fluids that contribute to bacterial resistance to endodontic irrigants. Understanding how oral bacteria are protected from antimicrobial agents is essential for optimizing our clinical treatment and may provide crucial insights into the design of more effective clinical therapies that specifically target and disrupt such protective interactions.
Acknowledgments The authors thank Professor H. F. Jenkinson at the University of Bristol, United Kingdom for providing strain UB1360. The authors deny any conflicts of interest related to this study.
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