Effectiveness of chlorhexidine and sodium hypochlorite to reduce Enterococcus faecalis biofilm biomass

Bacteria present in biofilm show an increased resistance to antimicrobial activity of endodontic irrigants, and the persistence of these microorganisms after chemomechanical preparation may lead to endodontic treatment failure. Thus, the aim of this study was to evaluate the effectiveness of sodium hypochlorite (NaOCl) and chlorhexidine (CHX) to reduce Enterococcus faecalis biofilm biomass. Biofilms built by E. faecalis ATCC 29212 in polystyrene 96-microtiter plate wells were exposed to different concentrations of NaOCl (1, 2.5 and 6%) and CHX (0.12, 0.2 and 2%) for three different time periods (1, 3 and 10 min). The reduction of the biofilm biomass was evaluated by measuring the optical density with a microplate absorbance reader. All the NaOCl solutions were significantly more effective than any of the CHX solutions tested, regardless of the time of exposure (P < 0.001). The three CHX solutions were not significantly different from the negative control group (PBS) in reducing the biofilm biomass (P > 0.05); by contrast, after 10 min of exposure to the NaOCl the biofilm was not significantly different from the positive control without biofilm (P > 0.05). Moreover, considering the period of times used in this study (1 to 10 min), no significant differences were observed among different concentrations for each desinfectant. In conclusion, none of the tested concentrations of CHX showed an ability to decrease the biofilm biomass. On the other hand, even the most diluted NaOCl solution (1%) reduced the biofilm biomass to a level that was compatible with the complete disruption of the biomass structure.


INTRODUCTION
The aim of endodontic treatment is to control and/or prevent the infection/reinfection of the root canal system (Zerella et al., 2005;Zehnder, 2006;Souza et al., 2010).This can be achieved by proper cleaning, shaping and filling of the root canal and coronal access using sterile instruments, antimicrobial solutions and proper sealing materials (Zehnder, 2006;Gillen et al., 2011).
Endodontists have been offered, over the last few years, an excess of instruments and systems to prepare root canals for cleaning and shaping; however, these new systems have not brought significant improvements regarding antimicrobial effectiveness during irrigation or disinfection of the root canal system (Brito et al., 2009;Pawar et al., 2012).In contrasts with that, studies have shown that the disinfection of the root canal system to levels compatible with apical tissue healing is one of the most predictable factors to achieve success in endodontic treatments (Zerella et al., 2005;Siqueira, 2001;Zehnder, 2006;Gillen et al., 2011).
Consequently, any eligible solution to be employed as an endodontic irrigant should exhibit a potent antimicrobial action against fungi, viruses and bacteria (including spore-forming species).Moreover, the ideal irrigant should also be able to promote soft-tissue dissolution without damaging periradicular host tissues (Zehnder, 2006).Although, hitherto, there is no chemical which fits all these requirements, more than 90% of the North American endodontists have elected sodium hypochlorite (NaOCl) as their first choice irrigant (Dutner et al., 2012).
NaOCl is a non-specific proteolytic, fungicide, and bactericidal agent.It is strongly alkaline and hypertonic, and although its action is more pronounced on necrotic tissues, NaOCl also exhibits toxicity on all living tissues, depending on the concentration used and time of exposure (Clarkson and Moule, 1998;Zehnder, 2006).NaOCl solutions used in endodontics vary in concentrations ranging from 0.5 to 6%, and although the majority of American endodontists prefer concentrations above 5% (Dutner et al., 2012), previous studies (in vitro and in vivo) have suggested that lower concentrations such as 2.5% are equally effective in disinfecting the root canal system (Zehnder, 2006;Rôças and Siqueira, 2011).
However, chlorhexidine (CHX) has been recommended as either an alternative or an adjunct root canal irrigant (Zehnder, 2006;Ryan, 2010).CHX is a cationic bisbiguanide that is stable as a salt (usually as chlorhexidine gluconate), and is often used as an endodontic irrigant at concentrations ranging from 0.2 to 2%.It exhibits an optimal antimicrobial activity at pH ranging from 5.5 to 7.0 (Zerella et al., 2005;Zehnder, 2006;Ryan, 2010).It is a broad-spectrum antimicrobial agent, and offers moderate activity against fungi and viruses (Zehnder, 2006;Ryan, 2010).It also shows some selective action against bacteria, exhibiting a more pronounced effectiveness against Gram-positive cocci, but being less active against Gram-positive and Gram-negative rods (Ryan, 2010).However, CHX has shown effectiveness against bacteria resistant to calcium hydroxide such as Enterococcus faecalis, and against Gram-negative bacteria commonly recovered from oral cavities (Stowe et al., 2004;Zerella et al., 2005;Mattigatti et al., 2012;Sreenivasan et al., 2013;Wang et al., 2012).
Bacteria participating in an endodontic infection consortium, such as E. faecalis, may be organized in a sessile structure as biofilm, or be suspended in fluids (planktonic phase) (Ricucci and Siqueira, 2010).Biofilms have a structure that provides a great protection for bacteria against antimicrobials (Ricucci and Siqueira, 2010), and to eliminate the biofilm containing the associated microorganisms is a challenge to any chemical irrigant.

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Thus, the aim of this study was to evaluate the effectiveness of NaOCl and CHX to reduce E. faecalis biofilm biomass.

MATERIALS AND METHODS
A standard strain of E. faecalis (ATCC 29212) was employed in this work.The Direct Contact Method was chosen to evaluate the effectiveness of each tested solution to reduce the biofilm biomass.
Eight wells of a polystyrene 96-microtiter flatten bottom plate (Nunclon, NUNC A/S, Roskilde, Denmark) were filled with 200 μl of sterile TSB (control).Two hundred microliters of the bacterial suspension in TSB were inoculated into each of the remaining wells.After filling the wells, the microtiter was covered with its lid, and left under incubation at 35°C for 24 h in an aerobic atmosphere to allow the building of biofilms in the wells with bacterial suspension.After confirming the presence of biofilm formation in the wells with the bacterial suspension, and the absence of turbidity in the wells only filled with sterile TSB, 100 μl of TSB was removed from the TSB wells by aspiration, and 100 μl of phosphate buffered saline (PBS) was added to each of these wells, which served as the positive control group for anti-biofilm effectiveness or absence of biofilm.The negative control group (ineffectiveness in biofilm biomass reduction) consisted of 4 wells where biofilms had been built up.In these negative control wells, 100 μl of TSB was carefully removed by aspiration and replaced by 100 μl of PBS, in order to halt the biofilm formation, but preserving it.The remaining 84 wells, presenting biofilms, were used in the disinfectant tests.The TSB was carefully removed from all wells by aspiration, and then they were refilled with one of the following solutions: four different dilutions of NaOCl solution (1, 2.5 and 6%), or three different dilutions of chlorhexidine gluconate solution (0.12, 0.2 and 2%).These test wells, containing the bacterial biofilms with the different solutions, were maintained for three different time intervals: 1, 3 and 10 min.All of these alternatives (different antimicrobial solutions at different concentrations, and at different time intervals) were quadruplicated (4 wells per test).All the groups with their results are shown in Figure 1.After the exposure to different time intervals, the wells with biofilms were rinsed with PBS, and the remaining biofilm biomass adhering to the well walls was stained with 0.1% crystal violet solution for 20 min.After that, the excess of dye was washed out with PBS, and the wells were then filled with 95% ethylic alcohol and placed in a iMark™ Microplate Absorbance Reader (model #168-1130XTU, Bio-Rad, Tokyo, Japan) to measure the biofilm biomass based on the optical density of each well at a wavelength of 590 nm.
Statistical analyses were performed using SPSS software (Statistical Package for the Social Sciences, version 17.0, IBM, Chicago, IL).The general linear model (GLM) for repeated measures was used to assess differences among the groups (different concentrations of sodium hypochlorite and chlorhexidine solutions, positive control group -PC, and negative control group -NC) for the E. faecalis ATCC 29212 biofilm biomass built up in the three different time intervals (1, 3, and 10 min).The analyses were adjusted by Tukey's post-hoc test for multiple comparisons.

RESULTS
The comparison among groups (different concentration of sodium hypochlorite and chlorhexidine solution) has shown that all the tested concentrations of NaOCl were significantly different from the negative control group -NC (wells with biofilm and PBS) (P < 0.001), and more effective than all of the tested concentrations of CHX, regardless of the time of exposure (P < 0.001).Even the lowest concentration of NaOCl solution (1%) produced a greater reduction in biofilm biomass than the highest concentration of CHX solution tested (2%), and was not significantly different from the positive control group -PC (without biofilm) after 10 min of exposure (P > 0.05).
Conversely, none of CHX the solutions were able to provide biofilm biomass reduction, and they were not significantly different from NC (P > 0.05) (Table 1).The analysis within each group regarding the time of exposure has shown no significant differences at any period of time, regardless of the solution concentration (Table 3).Considering 10 min of biofilm exposure to the disinfectants, there were no significant differences among the different NaOCl or CHX concentrations (P > 0.05), within their groups (NaOCl × NaOCl or CHX × CHX) (Table 2).

DISCUSSION
The current study compared the effectiveness of different concentration of NaOCl and CHX to reduce E. faecalis biofilm biomass.Only NaOCl solution (all concentration) reduced the biofilm biomass.
Microbial cells may survive within root canal systems after the chemomechanical preparation (Siqueira, 2001;Brito et al., 2009), and substances providing antimicrobial activity for longer periods of time may offer better outcomes.The cationic properties of CHX give it stability, which can bind to surfaces covered with acidic proteins, such as the hydroxyapatite of dentin, and it is then slowly released at therapeutic levels (Lenet et al., 2000).However, considering that bacteria organized as biofilm show greater resistance to antimicrobials than those in the planktonic phase, the antimicrobial activity of the irrigant may be impaired if this chemical is not able to dissolve tissues and biofilms.Sena et al. (2006) investigated the antimicrobial activity of 5.25% NaOCl and 2% CHX as endodontic-irrigating substances against selected single-species biofilms, and did not find any significant differences between them.Lima et al. (2001) employed three-day biofilms of E. faecalis induced on cellulose nitrate membrane filters and found that CHX was as effective as NaOCl in killing bacterial cells.Our results showed that NaOCl, regardless of the concentration employed, promoted greater reduction on biofilm biomass than CHX; even the lowest concentration of NaOCl solution (0.1%) exerted a greater reduction of the biofilm biomass than the highest concentration of CHX tested (2%).These results were corroborated by Ordinola-Zapata et al. (2012).These authors employed confocal microscopy to evaluate bacterial cell viability after the exposure of oral bacteria biofilm to NaOCl and CHX, and showed that NaOCl was pronouncedly more effective than CHX to kill bacteria cells organized in biofilm (Ordinola-Zapata et al., 2012).
Other studies that have compared the effectiveness of NaOCl and CHX against E. faecalis showed that CHX was as effective as, or even more effective than NaOCl, but these studies did not use a method based on antimicrobial activity against biofilms (Vianna and Gomes, 2009), or used unreliable methods such as the agar diffusion test (Poggio et al., 2012;Mattigatti et al., 2012;Sassone et al., 2008).However, it has been clearly shown that bacteria are usually organized in biofilm structures or within necrotic pulp tissue remnants in the root canal system (Baldasso et al., 2012;Vera et al., 2012;Ricucci and Siqueira, 2010;Ricucci et al., 2009), and these tissues/structures may protect bacterial cells against antimicrobial action.
CHX has been proposed as an alternative irrigant solution to NaOCl (Zehnder, 2006;Ryan, 2010).CHX is known to have a low toxicity to host tissues (Stowe et al., 2004;Zehnder, 2006;Ryan, 2010) exhibiting less cytotoxicity than NaOCl, although some issues have been raised regarding its supposed biocompatibility (Chang et al., 2001;Trevino et al., 2011). Indeed, Trevino et al. (2011) showed that CHX may cause an inflammatory response in periradicular tissues if extruded beyond the root canal apex, even when minimal CHX concentrations such as 0.1% are used.Other studies have also shown that even at low concentrations such as 0.12%, CHX induces inflammatory response in subcutaneous tissues of guinea pigs, ranging from mild/moderated reaction to foreign body granuloma formation or tissue death, and these levels of reactions are positively correlated with the concentration of CHX used (Zehnder, 2006;Faria et al., 2007).
The data reported in the present investigation should be interpreted with caution due to the limitations of an in vitro study to reproduce a normal biological condition.In addition, further studies should also evaluate if the addition of biofilm disrupting components to CHX solutions would provide a thorough removal of biofilm, equivalent to that provided by NaOCl solutions.

Conclusion
None of the tested concentrations of CHX showed an ability to decrease the biofilm biomass.On the other hand, even the most diluted NaOCl solution (0.5%) reduced the biofilm biomass to a level that was compatible with the complete disruption of the biomass structure.

Figure 1 .
Figure 1.Wells optical density (OD) average at a wavelength (WL) of 590 nm, considering different disinfectant solutions and periods of exposure time.

Table 1 .
Wells optical density average at a wavelength of 590 nm, considering different disinfectant solutions and periods of exposure time.

Table 2 .
Comparisons of the effects of different sodium hypochlorite and chlorhexidine solution on biofilm biomass originated by standard strain of E. faecalis ATCC 29212 over three times studied (GLM for repeated measures, standard error = 0.03).
H = Sodium hypochlorite; C = chlorhexidine; PC = positive control (saline solution on biofilm); NC = negative control (saline solution on medium without biofilm); CI = confidence interval; CI was adjusted by Tukey's post-hoc test for multiple comparisons.

Table 3 .
Comparisons of the effects over time on the biofilm biomass originated by E. faecalis ATCC 29212, within each group (GLM repeated measures)