Antimicrobial activity and enterococcus faecalis biofilm formation on chlorhexidine varnishes

Objective: To evaluate, in vitro, the antimicrobial activity and biofilm formation of three chlorhexidine varnishes in four Enterococcus faecalis strains: E. faecalis ATCC 29212, E. faecalis EF-D1 (from failed endodontic treatment), E. faecalis 072 (cheese) and E. faecalis U-1765 (nosocomial infection), and one Enterococcus durans strain (failed endodontic treatment). Study Design: The direct contact test was used to study the antimicrobial activity. Bacterial suspensions were exposed for one hour to EC40, Cervitec (CE) and Cervitec Plus (CEP) varnishes. “Eradication” was defined as 100% bacterial kill. The formation of enterococci biofilms was tested on the surface of the varnishes after 24 hours of incubation and expressed as percentage of biofilm reduction. Results: EC40 eradicated all strains except E. faecalis ATCC 29212, where 98.78% kill was achieved. CE and CEP showed antimicrobial activity against all the strains, but most clearly against E. durans and E. faecalis 072. EC40 completely inhibited the formation of biofilm of E. faecalis ATCC 29212, E. faecalis 072 and E. durans. CE and CEP led to over 92% of biofilm reduction, except in the case of E. faecalis U-1765 on CEP (76.42%). Conclusion: The three varnishes studied were seen to be effective in killing the tested strains of enterococci and in inhibiting the formation of biofilm, the best results being observed with EC40. Key words:Biofilm, chlorhexidine varnish, direct contact test, Enterococcus durans, Enterococcus faecalis, intracanal medication.


Introduction
An important aim of endodontic therapy is the elimination of microorganisms from root canal systems. This may be accomplished using mechanical instrumentation and chemical irrigation, and intracanal medication for persistent infections (1). Chlorhexidine (CHX) is a cationic molecule with a wide antimicrobial spectrum against both Gram positive and Gram negative bacteria (2). These would include Enterococcus faecalis (3), a microorganism frequently isolated from necrotic and failed endodontic treatments (4). Its antibacterial activity and substantivity (5) make CHX widely used as a root canal irrigant (6,7) and intracanal medication (8) and it is known to reduce and/or delay the entry of bacteria into the root canal system (9). Available in different formulations, CHX is commonly used in endodontics as a solution or gel (2). CHX has recently been incorporated into a variety of sustainedrelease systems, including varnishes, in order to prolong the period of active-agent delivery and achieve maximum antimicrobial effectiveness (10). Some of these slow-release systems have proven effective as intracanal medication against E. faecalis (11)(12)(13). The CHX varnishes apparently penetrate and seal tubules in dentin (14), reducing the level of mutans streptococci in exposed root surfaces (15) and it may help control established root caries lesions (16). However, its antimicrobial activity against bacteria involved in root canal infections is not known. The aim of this study was therefore to evaluate, in vitro, the antimicrobial activity and the ability to reduce biofilm formation of three CHX varnishes, two of them containing thymol, against four E. faecalis strains and one E. durans strain.

-Microorganisms and tested materials
The bacterial strains used in the study and their source are listed in table 1. Bacterial strains were taken from a 4ºC stock culture and streaked out twice on BHI (Scharlau Chemie S.A., Barcelona, Spain) agar plates for 24 hours at 37ºC. Colonies were suspended in BHI to obtain a 1 McFarland initial bacterial suspension of approximately 3×108 colony-forming units per mL (CFU/ mL ). All strain cultures were checked for purity by Gram stain and colony morphology, ( The DCT used to assess the antimicrobial activity of the CHX varnishes is based on previously reported methodology (17,18). While holding a 96-well microtiter plate (Nunclon Delta Surface; Nunc, Roskilde, Denmark) vertically, an area of established dimensions on one side of the wells was coated with an equal amount of each varnish using a cavity liner applicator. The varnishes were applied following the manufacturer´s instructions, and were allowed to dry for 30 minutes. A 10-μL aliquot of the initial bacterial suspension was placed on the surface of each varnish. Bacterial suspensions placed on the wall of uncoated wells served as the positive control. After incubation for 1 hour at 37ºC with 95% relative humidity to ensure direct contact between bacteria and tested materials, 240 μL of sterile BHI was added to each well. The bacterial suspension was mixed for 1 minute, diluted serially and plated for viable cell counting. Ten replicates per strain and varnish were performed.

-Biofilm Formation Test (BFT)
The biofilm model used was the MBEC high-throughput (HTP) device (Innovotech, Edmonton, Alberta, Canada) (3,19,20). This batch-culture apparatus has a lid with 96 pegs that fits over a standard 96-well microtiter plate (21). Ten pegs were coated with each varnish, following the manufacturer´s instructions; and 10 uncoated pegs served as the positive and sterility controls. Each assay was performed for a total of ten replicates per strain and CHX varnish in different devices. The wells of the microtiter plate were inoculated with 150 µL of a 1 in 30 dilution of the initial bacterial suspension, while 10 wells were inoculated with sterile BHI for the

Results
The results of the antibacterial effects of the CHX varnishes from the DCT are listed in Read vertically, same letters indicate non-significant differences. Read horizontally, same numbers indicate non-significant differences.

Discussion
The use of intracanal medication can help eliminate the bacteria remaining after chemo-mechanical instrumentation and may prevent microorganism invasion between treatment appointments (1). Our results suggest that all three varnishes studied exert antimicrobial activity against the five strains tested, and are capable of preventing or reducing the formation of biofilm.
Although the most frequently used intracanal medicament is calcium hydroxide, when CHX is used, E. faecalis is better eliminated from dentinal tubules (5). As inter-appointment medication, it may be applied in the form of a gel (8) or incorporated into sustained-release devices (13). The potential benefits of CHX varnishes in endodontics may stem from a greater and more prolonged release from dental tissue than with CHX gels (22), and their ability to penetrate and disinfect dentin tubules of the surface root (14). The bacteria selected for our study were enterococci strains, particularly E. faecalis, which are often isolated from necrotic or improperly filled root canal systems (23). E. faecalis ATCC 29212 is a strain of reference widely used in antimicrobial susceptibility studies (13). Moreover, two enterococci strains isolated from failed endodontic treatments were studied, E. faecalis EF-D1 and E. durans ED-C1, the latter reportedly present in root canals (24). The origin of enterococci found in the oral cavity is unclear; and whereas the most likely source would be food (25), nosocomial infections are another possibility. We therefore used strains pertaining to both sources: E. faecalis 072 (26) (27), who showed E. faecalis strains from endodontic sources to have a lower inherent capacity for biofilm formation.
In parallel to the results seen for DCT, EC40 was the most effective varnish, able to fully inhibit biofilm formation of three strains. This may be attributed to its high concentration of CHX, which would provide greater substantive antimicrobial activity (28). Lee et al (13) similarly obtained strong antimicrobial action on contaminated dentin blocks with E. faecalis using a polymeric CHX controlled device with 40% CHX. CE and CEP exhibited a lesser capacity to inhibit biofilm formation in our study, which also varied depending on the species involved. The lower concentration of CHX (1%), even though it included thymol in its composition (29), might explain these results. However, both these varnishes achieved a percentage of reduction over 92%; in contrast, CEP was not very effective against E. faecalis U-1765 (76.42%). There is no clear evidence pointing to one of these substances as better than the other, a point brought out previously in the context of cariogenic bacteria (30).
The study described here demonstrates that the CHX varnishes -and particularly EC40-exert effective antimicrobial action against the enterococci strains tested, and that they are able to inhibit or considerably reduce the formation of biofilms of these bacteria. Despite the limitations of this in vitro study, we may state that the results are promising, and encourage further studies to evaluate other properties of varnishes and new sustained-release systems.