Brief report| Volume 41, ISSUE 12, e119-e122, December 2013

Download started.


Chlorhexidine activity against bacterial biofilms

Published:August 05, 2013DOI:


      A biofilm is a complex microbiological ecosystem deposited on surfaces. Microorganisms in form of biofilms are of particular clinical concern because of the poor response to antimicrobial treatments. This study aimed to determine whether bacterial and fungal biofilms are able to resist the antimicrobial activity of chlorhexidine, a powerful antiseptic widely used in the hospital environment.


      Disk diffusion and susceptibility tests were conducted in accordance with Clinical and Laboratory Standards Institute standards for the determination of biofilm inhibitory concentration. Chlorhexidine was tested first at a minimum inhibitory concentration and then at higher concentrations when it was not able to destroy the biofilm. The plates were developed with a solution of 0.1% crystal violet, and readings were made at an optical density of 570 nm.


      Chlorhexidine demonstrated excellent antimicrobial activity for most microorganisms tested in their free form, but was less effective against biofilms of Acinetobacter baumannii, Escherichia coli, methicillin-resistant Staphylococcus aureus, and Pseudomonas aeruginosa.


      This study confirms that microorganisms in biofilms have greater resistance to chlorhexidine, likely owing to the mechanisms of resistance conferred to the structure of biofilms.

      Key Words

      To read this article in full you will need to make a payment

      Purchase one-time access:

      Academic & Personal: 24 hour online accessCorporate R&D Professionals: 24 hour online access
      One-time access price info
      • For academic or personal research use, select 'Academic and Personal'
      • For corporate R&D use, select 'Corporate R&D Professionals'


      Subscribe to American Journal of Infection Control
      Already a print subscriber? Claim online access
      Already an online subscriber? Sign in
      Institutional Access: Sign in to ScienceDirect


        • Nuñez L.
        • Moretto J.
        Disinfectant-resistant bacteria in Buenos Aires City Hospital wastewater.
        Braz J Microbiol. 2007; 38: 644-648
        • Vitalis G.S.
        Use of nanoparticles as an alternative to chlorhexidine dressing (master's dissertation).
        Franciscan University Center of Santa Maria, Santa Maria, Brazil2012 (Available from: Accessed October 2012.)
        • Duarte R.S.
        • Lawrence M.C.
        • Fonseca L.D.
        • Lion S.C.
        • Amorim E.D.
        Epidemic of postsurgical infections caused by Mycobacterium massiliense.
        J Clin Microbiol. 2009; 47: 2149-2155
        • Donlan R.M.
        Biofilm formation: a clinically relevant microbiological process.
        Healthc Epidemiol. 2001; 33: 1387-1392
        • Costerton J.W.
        • Stewart P.S.
        • Greenberg E.P.
        Bacterial biofilms: a common cause of persistent infections.
        Science. 1999; 284: 1318-1322
        • Hoiby N.
        • Ciofu O.
        • Johansen H.K.
        • Song Z.J.
        • Moser C.
        • Jensen P.O.
        • et al.
        The impact of bacterial biofilms.
        Int J Oral Sci. 2011; 3: 55-65
        • Davies D.G.
        • Parsek M.R.
        • Pearson J.P.
        • Iglewski B.H.
        • Costerton J.W.
        • Greenberg E.P.
        • et al.
        The involvement of cell-to-cell signals in the development of a bacterial biofilm.
        Science. 1998; 280: 295-298
      1. Silva RNP. The importance of Acinetobacter baumannii infection in acquired health care. Dissertation (Integrated Master Course in Medicine). University of Porto, 2009. Available from: Accessed November 2012.

        • Swindell K.
        Parenteral lipid emulsion induces germination of Candida albicans and increases biofilm formation on medical catheter surfaces.
        J Infect Dis. 2009; 200: 473-480
        • Hancock V.
        • Ferrières L.
        • Klemm P.
        Biofilm formation by asymptomatic and virulent urinary tract infectious Escherichia coli strains.
        FEMS Microbiol Lett. 2007; 267: 30-37
        • O’Toole G.A.
        • Kolter R.
        Flagellar and twitching motility are necessary for Pseudomonas aeruginosa biofilm development.
        Mol Microbiol. 1998; 30: 295-304
        • Clinical and Laboratory Standards Institute
        Methods for dilution antimicrobial susceptibility tests for bacteria that grow aerobically: approved standard. 7th ed. CLSI document M7-A7.
        Clinical and Laboratory Standards Institute, Wayne [PA]2006
        • Clinical and Laboratory Standards Institute
        Reference method for broth dilution antifungal susceptibility testing of yeasts: approved standard. 3rd ed. CLSI document M27-MA2.
        Clinical and Laboratory Standards Institute, Wayne [PA]2008
        • Merritt J.H.
        • Kadouri D.E.
        • O'Toole G.A.
        Growing and analyzing static biofilms.
        Curr Protoc Microbiol. 2005; Chapter 1 (Unit 1B.1)
        • Mah T.C.
        • O'Toole G.A.
        Mechanisms of biofilm resistance to antimicrobial agents.
        Trends Microbiol. 2001; 9: 34-39
        • Shen Y.
        • Stojicic S.
        • Haapasalo M.
        Antimicrobial efficacy of chlorhexidine against bacteria in biofilms at different stages of development.
        J Endodont. 2011; 37: 657-661
        • Totè K.
        • Horemans T.
        • Vanden Berghe D.
        • Maes L.
        • Cos P.
        Inhibitory effect of biocides on the viable masses and matrices of Staphylococcus aureus and Pseudomonas aeruginosa biofilms.
        Appl Environ Microbiol. 2010; 76: 3135-3142
        • Jass J.
        • Lappin-Scott H.M.
        The efficacy of antibiotics enhanced by electrical currents against Pseudomonas aeruginosa biofilms.
        J Antimicrob Chemother. 1996; 38: 987-1000
        • Wroblewska M.M.
        • Sawicka-Grzelak A.
        • Marchel H.
        • Luczak M.
        • Sivan A.
        Biofilm production by clinical strains of Acinetobacter baumannii isolated from hospitalized patients in two tertiary care hospitals.
        FEMS Immunol Med Microbiol. 2008; 53: 140-144
        • Lamfon H.
        • Porter S.R.
        • McCullough M.
        • Pratten J.
        Susceptibility of Candida albicans biofilms grown in the constant depth film fermentor to chlorhexidine, miconazole and fluconazole: a longitudinal study.
        J Antimicrob Chemother. 2004; 53: 383-385
        • Zanatta F.B.
        • Rosing C.K.
        Chlorhexidine's action mechanisms and recent evidence of its efficacy over supragingival biofilm context.
        Sci-A. 2007; 1 (Available from: Accessed December 2012.): 35-43
        • Murtough S.M.
        • Hiom S.J.
        • Palmer M.
        • Russell A.D.
        Biocide rotation in the healthcare setting: is there a case for policy implementation?.
        J Hosp Infect. 2001; 8: 1-6