Inhibitory activity of root canal irrigants against Candida albicans, Enterococcus faecalis and Staphylococcus aureus

MICROBIOLOGY   Inhibitory activity of root canal irrigants against Candida albicans, Enterococcus faecalis and Staphylococcus aureus     Tatiana Kelly da Silva FidalgoI; Roberta BarcelosII; Maristela Barbosa PortelaIII; Rosangela Maria de Araújo SoaresIV; Rogério GleiserIII; Fernando Costa e Silva-FilhoV ISchool of Dentistry of Rio de Janeiro, Federal University of Rio de Janeiro, Rio de Janeiro, RJ, Brazil IIDepartment of Specific Formation, School of Dentistry, Fluminense Federal University, Nova Friburgo, RJ, Brazil IIIDepartment of Pediatric Dentistry and Orthodontics, School of Dentistry, Federal University of Rio de Janeiro, Rio de Janeiro, RJ, Brazil IVProfessor Paulo de Goes Institute of Microbiology, University of Rio de Janeiro, Rio de Janeiro, RJ, Brazil VCarlos Chagas Filho Biophysics Institute, Federal University of Rio de Janeiro, Rio de Janeiro, RJ, Brazil Corresponding author     ABSTRACT The present study evaluated the antimicrobial activity of three root canal irrigants against Enterococcus faecalis, Candida albicans, and Staphylococcus aureus. These microorganisms were incubated in the presence of citric acid (6 and 10%), EDTA (17%), and NaOCl (0.5, 1.0, 2.5, and 5.25%). Agar diffusion tests were performed and redox indicator resazurin was used to evaluate the inhibitory effect of the irrigants on the metabolic activity of these microorganisms. The mean diameters of the inhibition zones for the C. albicans cultures were 11.6 mm (17% EDTA), 5.5 mm (0.5% NaOCl), 12.9 mm (1% NaOCl), 22.1 mm (2.5% NaOCl), and 28.5 mm (5.25% NaOCl). The mean diameters of the inhibition zones for E. faecalis were 2.8 mm (1% NaOCl), 5.4 mm (2.5% NaOCl), and 8.3 mm (5.25% NaOCl). For S. aureus, the mean values were 8.0 mm (17% EDTA), 3.0 mm (1% NaOCl), 8.8 mm (2.5% NaOCl), and 10.0 mm (5.25% NaOCl). Most of the irrigant solutions presented effective antimicrobial activity against C. albicans. A high inhibitory effect on the metabolic activity of E. faecalis was detected when the microorganisms were incubated with 17% EDTA. The same result was reached when S. aureus was incubated in the presence of > 2.5% NaOCl. Altogether, these results indicate that 2.5% and 5.25% NaOCl are microbicides against S. aureus while 0.5% and 1% NaOCl are only microbiostatic against the tested bacteria. The 6% and 10% citric acid as well as 17% EDTA did not affect the viability of any of the assayed microorganisms. Descriptors: Candida albicans; Enterococcus faecalis; Staphylococcus aureus; Root Canal Irrigants.     Introduction Microorganisms are the major causative factor associated with endodontic treatment failure.1,2 The success of endodontic treatment depends on the reduction or elimination of bacteria present in the root canal system. Residual pulpal tissue, bacteria, and dentine debris may persist in the irregularities of root canal systems, even after meticulous mechanical preparation.3 Therefore, irrigant solutions should be used in combination with canal preparation.4-6 Root canal irrigants are used during chemomechanical procedures not only as antimicrobial agents but also to flush out loose debris, to lubricate the dentinal walls, and to dissolve organic compounds in the canal.7, 8 Chemomechanical preparation is one of the most important phases of endodontic treatment and irrigants such as sodium hypochlorite (NaOCl), citric acid, and ethylene diamine tetra-acetic acid (EDTA) are commonly used. The efficacy of these procedures also depends upon the vulnerability of the species involved.9 Many in vitro studies relate the antimicrobial activity of root canal irrigants against microorganisms, but studies on the metabolic activity of microorganisms after contact with these irrigants was not found in the literature.5, 6 Therefore, this study first evaluated the effectiveness of NaOCl (0.5, 1, 2.5, 4, and 5.25%), EDTA (17%), and citric acid (6 and 10%) against Enterococcus faecalis, Candida albicans, and Staphylococcus aureus, and then assessed their metabolic activities under the same conditions.   Material and Methods Microorganisms The following microorganisms and their related ATCC strains were used throughout: Staphylococcus aureus (ATCC 6538), Enterococcus faecalis (ATCC 29212) and Candida albicans (ATCC 10231). All microorganisms were grown in BHIagar medium (Difco, Rio de Janeiro, RJ, Brazil) supplemented with 10% sheep blood (24 h, 37ºC, 5% CO2 atmosphere or in anaerobiosis). The microorganisms were then collected with a Drigalsky spatula for standardization of quantities collected and resuspended in sterile 0.01 M phosphate buffer pH 7.2, containing 0.15 M NaCl (PBS pH 7.2). The quantity of all microorganisms was adjusted using McFarland's scale (McFarland Standard no. 0.5) and spectrophotometrically adjusted to 105 CFU. ml-1 through optical density measurement at 600 nm (OD600). Root canal irrigants The root canal irrigants used were: citric acid (6 and 10% - VETEC, Rio de Janeiro, RJ, Brazil), EDTA (17% - Biodinámica, Rio de Janeiro, RJ, Brazil), and NaOCl (0.50, 1.00, 2.50, and 5.25% - Formula & Agáo, São Paulo, SP, Brazil). All of these compounds were diluted in sterile PBS pH 7.2. Agar Diffusion Test (ADT) For this test, nitrocellulose membranes (13.0 mm - Catalogue Nº GSWP04700; Millipore Co., Billerica, MA, USA) impregnated with the root canal irrigants (30 |xl each), described above, were placed on the top of S. aureus, E. faecalis, and C. albicans (0.1 ml, 105 CFU.ml-1, resuspended in PBS pH 7.2) homogeneously spread onto separated Petri dishes (10 cm diameter) containing BHI agar medium. The microorganism cultures were kept in an incubator (24 h, 37ºC, 5% CO2 atmosphere or in anaerobiosis) before measuring the growth inhibition zones. Cell viability (negative control) was determined by incubating the bacteria with Bactrin (Sulfametoxazol-trimetoprima - RJ 0401, Roche, Rio de Janeiro, RJ, Brazil) and the fungus with Fluconazole (Ache, Rio de Janeiro, RJ, Brazil); and the positive controls consisted in incubation of the same microorganisms in PBS pH 7.2. The inhibition zone limit was measured from the edge of the membrane disk to the end of the inhibition zone. A millimeter marking rule was used for this purpose.9 All assays were done in triplicate. Metabolic activity In order to collect a standard quantity of samples submitted to ADT, the authors developed the following method. Briefly, micropipette tips (S1111-0006; TipOne; Blakelands, MK, UK) with 15 mm cut off from the thinner end were used to collect a fixed (standard) quantity of sample from the inhibition zone. For each sample collection, the tip was inserted 1 mm from the nitrocellulose membrane, and then the collected sample was resuspended in PBS (300 Equivalent selection points to collect the microorganism are essential to maintain the same parameters for all microorganisms studied. To adjust the quantity of microorganisms to be (...truncated)