Antimicrobial evaluation of copper sulfate (II) on strains of Enterococcus faecalis. In vitro study

Marisol Sierra, Aldo Sanhueza, Rául Alcántara, Gabriela Sánchez


Abstract: Introduction: Controlling Enterococcus faecalis is of vital importance in endodontics, as this pathogen is associated with endodontic failure. Experimental evidence has shown that copper has antibacterial activity against other pathogens with similar characteristics. The objective of this study was to determine the antimicrobial activity of copper (II) or cupric (SC-II) sulfate on strains of Enterococcus faecalis and to compare it with the most commonly used antimicrobials. Methodology: We used 33 strains of Enterococcus faecalis isolated from different clinical pictures in different Chilean hospitals. The minimum inhibitory concentration (MIC) of SC-II, chlorhexidine and calcium hydroxide was determined by the broth microdilution technique, following the recommendations given by the Clinical and Laboratory Standards Institute. Results: The MIC for CHX varied in the range of 5-10 µg/ml; SC-II from 1.5 to 12 µg/ml, and HC was >32 mg/ml. The geometric mean of SC-II was 6 µg/ml, lower than that of CHX, which was 7.29 µg/ml. Conclusions: SC-II showed antimicrobial activity at lower concentrations than CHX. HC (which could have been affected by the buffer effect of the broth microdilution technique) showed high values, not comparable to other compounds. We suggest carrying out further studies on the properties of SC-II, such assessing its biocompatibility and reaction with other materials to be used clinically in endodontic therapy.

Keywords: “Enterococcus faecalis”[MeSH], “Copper sulfate”[MeSH], “Chlorhexidine”[MeSH], “Calcium Hydroxyde”[MeSH], “Endodontics”[MeSH].

Evaluación antimicrobiana del sulfato de cobre (II) sobre cepas de Enterococcus faecalis. Estudio in vitro.

Resumen: Introducción: El control de Enterococcus faecalis es de vital importancia en endodoncia, ya que este patógeno está asociado al fracaso endodóntico. Evidencias experimentales que han demostrado que el cobre presenta actividad antibacteriana en otros patógenos de similares características. El objetivo de este estudio es determinar la actividad antimicrobiana del sulfato de cobre (II) o cúprico (SC-II) sobre cepas de Enterococcus faecalis y compararla con los antimicrobianos más usados en la actualidad. Metodología: Estudio in vitro. Se utilizó la técnica de microdilución en caldo según lineamientos del Clinical and Laboratory Standards Institute, incluyendo 33 cepas de Enterococcus faecalis obtenidas desde hospitales chilenos, para cada una de las cuales se determinó las concentraciones mínimas inhibitorias (CMI) de: SC-II, Clorhexidina (CHX) e Hidróxido de calcio (HC). Resultados: La CMI para CHX tuvo un rango de 5-10 µg/ml, el SC-II de 1,5-12 mM y el HC fue >32 mg/ml. Estas diferencias fueron estadísticamente significativas entre los 3 antimicrobianos utilizados (p<0,001). Conclusiones: El SC-II mostró actividad antimicrobiana a bajas concentraciones, superiores a CHX, pero menores a HC (que pudo ser afectado por el efecto tampón de la técnica de microdilución en caldo). Se sugiere seguir con los estudios de las propiedades del SC-II, como evaluación de biocompatibilidad y reacción con otros materiales para ser utilizados clínicamente en la terapia endodóntica.

Palabras clave: Enterococcus faecalis, sulfato de cobre, clorhexidina, hidróxido de calcio, endodoncia.


Stuart CH, Schwartz SA, Beeson TJ, Owatz CB. Enterococcus faecalis: its role in root canal treatment failure and current concepts in retreatment. J Endod 2006; 32: 93–98.

Zoletti GO, Pereira EM, Schuenck RP, Teixeira LM, Siqueira JF Jr, dos Santos KR. Characterization of virulence factors and clonal diversity of enterococcus faecalis isolates from treated dental root canals. Res Microbiol 2011; 162: 151-158.

Zehnder M, Guggenheim B. The mysterious appearance of enterococci in filled root canals. Int Endod J 2009; 42: 277-287.

Warnes SL, Keevil CW. Mechanism of copper surface toxicity in vancomycin-resistant enterococci following wet or dry surface contact. Appl Environ Microbiol 2011; 77: 6049–6059.

Estrela C, Silva JA, de Alencar AH, Leles CR, Decurcio DA. Efficacy of sodium hypochlorite and chlorhexidine against Enterococcus faecalis – A ystematic review. J Appl Oral Sci 2008; 16: 364-368.

Lima R, Guerreiro J, Faria N, Tanomaru M. Effectiveness of calcium hydroxide-based intracanal medicaments against Enterococcus Faecalis. Int Endod J 2012; 45: 311-316.

Delgado RJ, Gasparoto TH, Sipert CR, Pinheiro CR, Moraes IG, Garcia RB, Bramante CM, Campanelli AP, Bernardineli N. Antimicrobial effects of calcium hydroxide and chlorhexidine on Enterococcus faecalis. J Endod 2010; 36: 1389-1393.

Okino LA, Siqueira EL, Santos M, Bombana AC, Figueiredo JA. Dissolution of pulp tissue by aqueous solution of chlorhexidine digluconate and chlorhexidine digluconate gel. Int Endod J 2004; 37: 38-41.

Athanassiadis B, Abbott PV, Walsh LJ. The use of calcium hydroxide, antibiotics and biocides as antimicrobial medicaments in endodontics. Aust Dent J 2007; 52: 64-82.

Mohammadi Z, Abbott PV. The properties and application of clorhexidine in endodontics. Int Endod J 2009. 42: 288-302.

Gould S, Fielders M, Kelly A, Morgan M, Kenny J, Naughton D. The antimicrobial properties of copper surfaces against a range of important nosocomial pathogens. Ann Microbiol 2009; 59: 151-156.

Grass G, Rensing G, Solioz M. Metallic copper as an antimicrobial surface. Appl Environ Microbiol 2011; 77: 1541–1547.

Gant VA, Wren MWD, Rollins MSM, Jeanes A, Hickok SS, Hall TJ. Three novel highly charged copper-based biocides: safety and efficacy against healthcare-associated organisms. J Antimicrob Chemother 2007; 60: 294-299.

Clinical and Laboratory Standards Institute. Methods for Dilution Antimicrobial Susceptibility Tests for Bacteria That Grow Aerobically; Approved Standard – Ninth Edition. CLSI document M07-A9. Pennsylvania: Clinical and Laboratory Standards Institute, 2010.

Ferreira FB, Torres SA, Rosa OP, Ferreira CM, Garcia RB, Marcucci MC, Gomes BP. Antimicrobial effect of propolis and other substances against selected endodontic pathogens. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 2007; 104: 709-716.

Pallotta RC, Ribeiro MS, de Lima Machado ME. Determination of the minimum inhibitory concentration of four medicaments used as intracanal medication. Aust Endod J 2007; 33: 107-111.

do Amorim CV, Aun CE, Mayer MP. Susceptibility of some oral microorganisms to chlorhexidine and paramonochlorophenol. Braz Oral Res 2004; 18: 242-246.

Siqueira JF Jr, Gonçalves RB. Antibacterial activities of root canal sealers against selected anaerobic bacteria. J Endod 2006; 22: 79-80.

Kõljalg S, Naaber P, Mikelsaar M. Antibiotic resistance as an indicator of bacterial chlorhexidine susceptibility. J Hosp Infect 2002; 51: 106-113.

Schäfer E, Bössmann K. Antimicrobial efficacy of chlorhexidine and two calcium hydroxide formulations against Enterococcus faecalis. J Endod 2005; 31: 53-56.

Delgado R, Gasparoto T, Sipert C, Pinheiro C, Moraes I, García R, Bramante C, Campanelli A, Bermardinelli N. Antimicrobial effect of calcium hydroxide and chlorhexidine on Enterococcus faecalis. J Endod 2010; 36: 1389-1393

Schäfer E, Bossmann K. Antimicrobial efficacy of chloroxylenol and chlorhexidine in the treatment of infected root canals. Am J Dent 2001; 14: 233-237.

Krithikadatta J, Indira R, Dorothykalyani AL. Disinfection of dentinal tubules with 2% chlorhexidine, 2% metronidazole, bioactive glass when compared with calcium hydroxide as intracanal medicaments. J Endod 2007; 33: 1473-1476.

D’Arcangelo C, Varvara, De Fazio P. An evaluation of the action of different root canal irrigants on facultative aerobic-anaerobic, oblígate anaerobic and microaerophilic bacteria. J Endod 1999; 25: 351-353.

Mortenson D, Sadilek M, Flake NM, Paranjpe A, Heling I, Johnson JD, Cohenca N. The effect of using an alternative irrigant between sodium hypochlorite and chlorhexidine to prevent the formation of para-chloroaniline within the root canal system. Int Endod J 2012; 45: 878-882.

Aarestrup FM, Hasman H. Susceptibility of different bacterial species isolated from food animals to copper sulphate, zinc chloride and antimicrobial substances used for disinfection. Vet Microbiol 2004; 100: 83-9.

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