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Antimicrobial susceptibility of monoculture biofilms of a clinical isolate of enterococcus faecalis

Antimicrobial Susceptibility of Monoculture Biofilms of aClinical Isolate of Enterococcus faecalis Anne E. Williamson, DDS, MS, Jared W. Cardon, DDS, and David R. Drake, MS, PhD AbstractThe purpose of this study was to create a monoculturebiofilm of a clinical isolate of Enterococcus faecalis and The primary etiology of persistent periapical pathosis has been attributed to viable bacteria remaining in the undebrided areas of the root canal system Numerous to determine susceptibility against four antimicrobial authors have identified Enterococcus faecalis as a predominant species found in pre- irrigants. Biofilms were subjected to 1-, 3-, and viously treated root canals with persistent periapical disease The ability of E. 5-minute exposures to one of the following irrigants: faecalis to adapt to environmental changes in the root canal after endodontic treatment 6% sodium hypochlorite (NaOCl), 2% chlorhexidine and to remain as a pathogen in the root canal system makes the elimination of this gluconate (CHX) or one of two new products, Ͻ6% species very difficult E. faecalis has shown the ability to enter a viable but noncul- NaOCl with surface modifiers (Chlor-XTRA) or 2% CHX tivable state, a condition that protects the bacteria during extreme stress. Once the with surface modifiers (CHX-Plus™) (Vista Dental Prod- environmental stress has passed, the bacteria may return to normal function ucts, Racine, WI). It was hypothesized that NaOCl and E. faecalis has been shown to have a high affinity for biofilm formation CHX would be equally effective and that addition of Bacteria form biofilms as a means of defense and to facilitate physiologic processes.
surface modifiers would improve bactericidal activity of After a protein pellicle is deposited on the root surface, planktonic bacteria adhere and the respective irrigants compared to the original for- begin to multiply. Other planktonic bacteria adhere to the surface as the biofilm ma- mulations. Results indicate that 6% NaOCl and Chlor- tures. This layering organization of the biofilm protects the bacteria from changes in pH EXTRA™ were significantly superior against E. faecalis and other antimicrobial insults Furthermore, biofilm formation is enhanced with biolfilms compared to 2% CHX and CHX-Plus™ at all coaggregation, the adhesion of two or more bacterial species An important viru- time points except five minutes. (J Endod 2009;35: lence factor associated with E. faecalis is an aggregation substance that facilitates the ability of this species to form and remain viable in a biofilm Given that current root canal instrumentation techniques alone are unable to render root canals bacteria free, a chemical irrigant is necessary to assist in reducing Biofilms, endodontic irrigants, Enterococcus faecalis bacterial numbers and their toxic byproducts. Ideal irrigants should remove organicand inorganic debris; have low tissue toxicity and low surface tension; and lubricate,disinfect, and remove the smear layer Sodium hypochlorite possesses manyproperties of an ideal irrigant; however, it must be used cautiously to prevent extrusion From the Department of Endodontics, University of Iowa into the periapical tissues where it may cause severe inflammatory reactions College of Dentistry, Iowa City, Iowa.
In cases of concern (open apex, perforation, and so on), an alternate irrigant Address requests for reprints to Dr Anne E. Williamson, Department of Endodontics, University of Iowa College of showing low tissue toxicity is desirable. Chlorhexidine (CHX) has been shown to have Dentistry, 435 Dental Science Bldg-S, Iowa City, IA 52242.
low tissue toxicity while retaining other favorable properties including its bactericidal activity and has been recommended as an alternative or potentially preferred irrigant for endodontic treatment Based on previous studies, discrepancies exist regarding 2008 Published by Elsevier Inc. on behalf of the Amer- the efficacy of NaOCl and CHX with respect to bactericidal activity Two new ican Association of Endodontists.
doi:10.1016/j.joen.2008.09.004 products, Chlor-XTRA and CHX-Plus (Vista Dental Products, Racine, WI), have recentlybeen introduced to the market. According to the manufacturer, Chlor-XTRA contains awetting agent and proprietary surface modifiers. Additionally, to increase the electricalcapacity of the solution, alkylating agents have been incorporated into the solution.
CHX-Plus contains proprietary surface modifiers to lower viscosity. It is unknown whateffect the modification of NaOCl and CHX has on the bactericidal properties of theseirrigants. Therefore, the purpose of this study was to compare the bactericidal effect offour antimicrobial formulations: group 1, 6% NaOCl; group 2, Chlor-XTRA; group 3, 2%CHX; and group 4, CHX-Plus, against a monoculture biofilm of a clinical isolate of E.
faecalis
at 1-, 3-, and 5-minute intervals.
A clinical isolate of E. faecalis recovered from an asymptomatic, failing root canal was used in all of the biofilm assays. Strain (ER3/2s) was provided by CM Sedgley. Thestrain was recovered from the root canal of a healthy 45-year-old female patient whopresented for orthograde retreatment of a previously endodontically treated tooth (#7)with asymptomatic apical periodontitis. The procedure was approved by the Universityof Michigan Institutional Review Board, and patient written consent was obtained before JOE — Volume 35, Number 1, January 2009 Antimicrobial Susceptibility of a Clinical Isolate of E. faecalis Figure 1. The bactericial activity of antimicrobial formulations on biofilms of E. faecalis. treatment. Sample processing and species identification using culture bers were log transformed to normalize the data. Means of the trans- and polymerase chain reaction– utilized methods have been described formed data were obtained (geometric means), and, therefore, there elsewhere The organism was frozen down by quick freezing in were three independent measures from the three independent experi- liquid nitrogen and maintained at Ϫ80° C in cryovials.
ments. Data were analyzed by two-way repeated-measures analysis of For biofilm experiments, initial broth cultures were prepared in variance using Prism 5 software (GraphPad Software, La Jolla, CA). A brain heart infusion broth (BHIB) and incubated in a 5% CO incubator test for equal variances was performed. If significant differences (p Ͻ at 37°C overnight. Cells were then harvested by centrifugation and re- 0.05) were found between treatment groups, Tukey-Kramer post-tests suspended in fresh BHIB. Resulting suspensions were adjusted to a cell concentration of 108 colony forming units (cfu)/mL. Aliquots fromthese suspensions were spiral plated onto BHIB agar plates using an Autoplate 4000 (Spiral Biotech, Inc, Norwood, MA) to obtain the num- The analysis of the data revealed an interaction between the treat- ber of cells in the inocula described later.
ment groups and time of exposure. All treatment groups exhibited a The biofilm model system consisted of sterile glass microscope significant reduction in viable bacteria within the biofilms compared slides immersed in 30 mL of BHIB in sterile centrifuge tubes. These with the distilled water control (group 5) at all periods of exposure tubes were inoculated with 0.2 mL of the standardized suspensions (p Ͻ 0.001) Groups 1 and 2 (6% NaOCl and Chlor-XTRA, prepared as described earlier. Tubes were then incubated in 5% CO at respectively) reduced cfus in biofilms by 7 to 8 orders of magnitude.
37°C for 48 hours. This allows for ample biofilm development on the Conversely, groups 3 and 4 (2% CHX and CHX-Plus, respectively) re- slides. Pilot experiments showed stainable biofilms with crystal violet duced cfus in biofilms by 3 to 4 orders of magnitude. No statistically that could not be washed off but were recoverable upon scraping the significant difference was observed between the biofilms in groups 1 slides carefully (data not shown). We routinely conduct monoculture and 2. Likewise, no statistically significant difference was observed be- and polymicrobial biofilm studies in our laboratory and are confident tween groups 3 and 4 (p Ͼ 0.05). Statistically significant differences that we reproducibly obtained E. faecalis biofilms on the slides.
were observed between the biofilms in either group 1 or group 2 com- The exposure experiments were conducted as follows. Glass slides pared with group 3 (p Ͻ 0.001). However, when comparing group 1 or were removed from their media tubes and gently washed by repeated group 2 against group 4, a statistically significant difference was noted at dipping in sterile distilled water. The slides were then immersed in exposure intervals of 1 and 3 minutes (p Ͻ 0.001) but not at the centrifuge tubes containing 75 mL of each of the following treatments: group 1, 2% CHX; group 2, 2% CHX-Plus; group 3, 6% NaOCl; group 4,Chlor-XTRA; or group 5, sterile distilled water (control). The treatmentsolutions were in small beakers, with slow stirring achieved with small stir bars. Slides were exposed for different periods of time, 1, 3, and 5 This study evaluated the bactericidal efficacy of four endodontic minutes. After exposure, slides were removed and immediately im- irrigants, 6% NaOCl, Chlor-XTRA, 2% CHX, and CHX-Plus, against a mersed and dipped 10 times into tubes containing neutralizing broth monoculture biofilm established from a clinical isolate of E. faecalis (Benton, Dickinson, and Company, Sparks, MD) supplemented with (ER3/2s). We found that both NaOCl groups reduced numbers of bac- 0.5% Tween 80. Pilot experiments by others in the laboratory (data not teria within the biofilms significantly more than either CHX group. These shown) have shown that this indeed does stop the killing reaction findings are in agreement with some studies in the literature but are of the antimicrobials used in this study. After this treatment, slides with different that what was shown in others. Dunavant et al. compared the biofilms were immersed in 5 mL of sterile BHIB in Petri dishes and the efficacy of 1% or 6% NaOCl and 2% CHX, among other irrigants, the biofilms were scraped off the surfaces using a wire loop. The BHIB against E. faecalis biofilms in a novel in vitro model system. Their model was collected and repeatedly vortexed to create homogeneous suspen- consisted of biofilms grown in a flow cell system. Biofilms were im- sions. Suspensions were then diluted into sterile BHIB, and then the mersed in test irrigants for 1 or 5 minutes. Results indicated that both original suspension plus dilutions were spiral plated onto BHIB agar.
concentrations of NaOCl provided statistically significantly better bio- Numbers of viable bacteria in the biofilms were determined by sector film kill than any other of the tested agents. Although the results of the plate counts according to standard spiral-plating methodology.
Dunavant study are consistent with our findings, direct comparisons Each treatment group was done in triplicate per experiment, and cannot be made because of the differences in the biofilm model systems three independent experiments were performed. Raw bacterial num- themselves. Another study evaluated the effectiveness of varying concen- JOE — Volume 35, Number 1, January 2009 trations of NaOCl, 2% CHX, and 1% NaOCl followed by a final rinse of a experiment to experiment and were shown to be strongly bound mixture of tetracycline, acid and detergent (MTAD) on rendering bac- biofilms on the surfaces of the slides.
teria nonviable and physically removing a polymicrobial biofilm estab- 2. All of the antimicrobial formulations tested exhibited significant lished in an extracted tooth model Results indicated that 6% bactericidal activity against biofilms of E. faecalis. Sodium hypo- NaOCl was the only irrigant tested that provided the intended outcome of chlorite (6%) and Chlor-XTRA showed the highest levels of bac- bacterial kill and elimination of the biofilm. Although our results sup- tericidal activity across the spectrum of exposure times apart port their findings, our study used monoculture biofilms established on from biofilms exposed for 5 minutes.
smooth glass surfaces. As such, we did not deal with the confines of 3. Ongoing and future studies are focusing on polymicrobial bio- tooth structure, which could potentially affect physical access of the films and the efficacy of the various antimicrobial formulations on irrigant solutions to the biofilm. We chose this approach because it these complex communities of organisms.
would reduce variance among replicates and also provide a greatersurface area of the biofilms to be exposed to the irrigant solutions.
Conversely, a study conducted by Jeansonne and White used The authors wish to express sincere thanks to Deepjyoti Gadre, an in vitro root canal system comparing antimicrobial activity of 2% CHX MD, PhD, for her valuable technical support. and 5.25% NaOCl. They showed that although it is not statistically sig-nificant, the 2% CHX group rendered fewer positive cultures than the NaOCl group. Differences in results from their study could potentially be 1. Nair PN. On the causes of persistent apical periodontitis: a review. Int Endod J attributed to deficiencies in the methods if measures relating to the carryover effect of the irrigants were not performed.
2. Sundqvist G, Figdor D, Persson S, Sjogren U. Microbiologic analysis of teeth with There are limitations that have to be acknowledged in our study.
failed endodontic treatment and the outcome of conservative re-treatment. Oral SurgOral Med Oral Pathol Oral Radiol Endod 1998;85:86 –93.
The biofilm model system was chosen to be simple as to facilitate the 3. Siqueira JF, Rocas IN. Exploiting molecular methods to expolore endodontic infec- ease of preparation and maximal surface area of biofilms for the expo- tions? Part 2—Redefining the endodontic microbiota. J Endod 2005;31:488 –98.
sure experiments. Moreover, we have found in other studies that one 4. Hancock HH, Sigurdsson A, Trope M, Moiseiwitsch J. Bacteria isolated after unsuc- can obtain less variance in results in a more simple design as ours cessful endodontic treatment in a North American population. Oral Surg Oral Med described here than in complex models involving extracted teeth. How- Oral Pathol Oral Radiol Endod 2001;91:579 – 86.
5. Stuart CH, Schwartz SA, Beeson TJ, Owatz CB. Enterococcus faecalis: its role in root ever, keeping it simple can be a limitation in that we do not include canal treatment failure and current concepts in retreatment. J Endod 2006;32:93– 8.
variables associated with root canals in terms of physical access to the 6. Kayaoglu G, Orstavik D. Virulence factors of Enterococcus faecalis: relationship to biofilms and differences in the structure and development of biofilms in endodontic disease. Crit Rev Oral Biol Med 2004;15:308 –20.
tooth models because of inherent physic-chemical differences. Never- 7. Spratt DA, Pratten J, Wilson M, Gulabivala K. An in vitro evaluation of the antimicro- bial efficacy of irrigants on biofilms of root canal isolates. Int Endod J 2001;34: theless, we believe that a simple system as described here allows for the rapid assessment of the ability to reduce numbers of viable bacteria 8. Chavez de Paz LE. Redefining the persistent infection in root canals: possible role of colonizing a smooth surface. The data obtained, of course, cannot be biofilm communities. J Endod 2007;33:652– 62.
directly extrapolated to conditions of root canals in human patients, but 9. Johnson EM, Flannagan SE, Sedgley CM. Coaggregation interactions between oral and the data can serve as a guide for future investigations on potential dif- endodontic Enterococcus faecalis and bacterial species isolated from persistentapical periodontitis. J Endod 2006;32:946 –50.
ferences in antimicrobials used in endodontics.
10. Zehnder M. Root canal irrigants. J Endod 2006;32:389 –98.
The future direction for study involving specific irrigant effect on 11. Pashley EL, Birdsong NL, Bowman K, Pashley DH. Cytotoxic effects of NaOCl on vital biofilms should include standardization of biofilm models. Additionally, of interest would be the comparison of the depth of penetration into the 12. Gernhardt CR, Eppendorf K, Kozlowski A, Brandt M. Toxicity of concentrated sodium hypochlorite used as an endodontic irrigant. Int Endod J 2004;37:272– 80.
biofilm by specific irrigants and the resultant effect of penetration depth 13. Ercan E, Ozekinci T, Atakul F, Gul K. Antibacterial activity of 2% chlorhexidine gluconate and 5.25% sodium hypochlorite in infected root canal: in vivo study. JEndod 2004;30:84 –7.
14. Jeansonne MJ, White RR. A comparison of 2.0% chlorhexidine gluconate and 5.25% sodium hypochlorite as antimicrobial endodontic irrigants. J Endod 1994;20:276 – 8.
Within the limitations of this study, the following conclusions may 15. Sedgley C, Buck G, Appelbe O. Prevalence of Enterococcus faecalis at multiple oral sites in endodontic patients using culture and PCR. J Endod 2006;23:104 –9.
16. Dunavant TR, Regan JD, Glickman GN, Solomon ES, Honeyman AL. Comparative 1. We developed a model system for monoculture biofilms of E.
evaluation of endodontic irrigants against Enterococcus faecalis biofilms. J Endod2006;32:527–31.
faecalis that was adopted and revised from previously-published 17. Clegg MS, Vertucci FJ, Walker C, Belanger M, Britto LR. The effect of exposure to models. Reproducible levels of bacteria colonized the slides from irrigant solutionis on apical dentin biofilms in vitro. J Endod 2007;33:966 –9.
JOE — Volume 35, Number 1, January 2009 Antimicrobial Susceptibility of a Clinical Isolate of E. faecalis

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