Abstract
A new, quick method, using the resazurin dye test as a bacterial respiration indicator, has been developed to assay the antibacterial activity of various substances used as disinfectants against bacterial biofilm growth on clinical devices. Resazurin was used to measure the presence of active biofilm bacteria, after adding disinfectant, in relation to a standard curve generated from inocula in suspension of the same organism used to grow the biofilm. The biofilm was quantified indirectly by measuring the fluorescent, water-soluble resorufin product produced when resazurin is reduced by reactions associated with respiration. Four products used as disinfectants and the biofilm growth of five bacterial species on carriers made of materials commonly found in clinical devices were studied. Under test conditions, chlorhexidine, NaOCl, ethanol, and Perasafe at concentrations of 0.2, 0.01, 350, and 0.16 mg/ml, respectively, all produced 5-log reductions in biofilm cell numbers on the three different carriers. The redox-driven test depends on bacterial catabolism, for which reason resazurin reduction produces an analytic signal of the bacterial activity in whole cells, and therefore could be used for determining disinfectant efficacy in an assay based on the metabolic activity of microorganisms grown as biofilm or in suspension.
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References
Abbondanzi F, Cachada A, Campisi T, Guerra R, Raccagni M, Iacondini A (2003) Optimisation of a microbial bioassay for contaminated soil monitoring: bacterial inoculum standardisation and comparison with Microtox® assay. Chemosphere 53:889–897
AFNOR. (1995) Antiseptiques et désinfectants. NF T 72-150
Buckingham-Meyer K, Goeres D, Hamilton M (2007) Comparative evaluation of biofilm disinfectant efficacy tests. J Microbiol Methods 70:236–244
Carpentier B, Cerf O (1993) Biofilms and their consequences, with particular reference to hygiene in the food industry. J Appl Bacteriol 75:499–511
Ceri H, Olson ME, Stremick C, Read RR, Morck D, Buret A (1999) The calgary biofilm device: new technology for rapid determination of antibiotic susceptibilities of bacterial biofilms. J Clin Microbiol 37:1771–1776
Christensen GD, Simpson WA, Younger JJ (1985) Adherence of coagulase-negative staphylococci to plastic tissue culture plates: a quantitative model for the adherence of staphylococci to medical devices. J Clin Microbiol 22:996–1006
Costerton JW (1999) Introduction to biofilm. Int J Antimicrob Agents 11:217–221
Djordjevic D, Wiedmann M, McLandsborough LA (2002) Microtiter plate assay for assessment of Listeria monocytogenes biofilm formation. Appl Environ Microbiol 68:2950–2958 doi:https://doi.org/10.1128/AEM.68.6.2950-2958.2002
Donlan RM, Costerton JW (2002) Biofilms: survival mechanisms of clinically relevant microorganisms. Clin Microbiol Rev 15:167–193 doi:https://doi.org/10.1128/CMR.15.2.167-193.2002
European Standardization Committee (1997) Norma española UNE—EN 1040. Antisépticos y desinfectantes químicos. Actividad bactericida básica. Método de ensayo y requisitos (fase 1). AENOR, Madrid
Guerin TF, Mondido M, McClenn B, Peasley B (2001) Application of resazurin for estimating abundance of contaminant-degrading microorganisms. Lett Appl Microbiol 32:340–345
Jackson G, Beyenal H, Rees WM, Lewandowski Z (2001) Growing reproducible biofilms with respect to structure and viable cell counts. J Microbiol Methods 47:1–10
Jessen B, Lammert L (2003) Biofilm and disinfection in meat processing plants. Int Biodeterior Biodegrad 51:265–269
Luppens SBI, Reij MW, Van der Heijden RWL, Rombouts FM, Abee T (2002) Development of a standard test to assess the resistance of Staphylococcus aureus biofilm cells to disinfectants. Appl Environ Microbiol 68:4194–4200
Maillard J (2007/6) Bacterial resistance to biocides in the healthcare environment: should it be of genuine concern? J Hosp Infect 65:60–72
Marion K, Freney J, James G, Bergeron E, Renaud FNR, Costerton JW (2006) Using an efficient biofilm detaching agent: an essential step for the improvement of endoscope reprocessing protocols. J Hosp Infect 64:136–142
Mariscal A, Carnero-Varo M, Gutierrez-Bedmar M, Garcia-Rodriguez A, Fernandez-Crehuet J (2007) A fluorescent method for assessing the antimicrobial efficacy of disinfectant against Escherichia coli ATCC 35218 biofilm. Appl Microbiol Biotechnol 77:233–240
O’Brien J, Wilson I, Orton T, Pognan F (2000) Investigation of the Alamar Blue (resazurin) fluorescent dye for the assessment of mammalian cell cytotoxicity. Eur J Biochem 267:5421–5426
O’Toole GA, Kolter R (1998) Initiation of biofilm formation in Pseudomonas fluorescens WCS365 proceeds via multiple, convergent signalling pathways: a genetic analysis. Mol Microbiol 28:449–461
Peeters E, Nelis HJ, Coenye T (2008a) Comparison of multiple methods for quantification of microbial biofilms grown in microtiter plates. J Microbiol Methods 72:157–165
Peeters E, Nelis HJ, Coenye T (2008b) Evaluation of the efficacy of disinfection procedures against Burkholderia cenocepacia biofilms. J Hosp Infect 70:361–368 doi:https://doi.org/10.1016/j.jhin.2008.08.015
Perret-Vivancos C, Marion K, Renaud FNR, Freney J (2008) Efficient removal of attached biofilm in a naturally contaminated colonoscope using detachment-promoting agents. J Hosp Infect 68:277–278
Pitts B, Hamilton MA, Zelver N, Stewart PS (2003) A microtiter-plate screening method for biofilm disinfection and removal. J Microbiol Methods 54:269–276
Reid G (1999) Biofilms in infectious disease and on medical devices. Int J Antimicrob Agents 11:223–226
Sarker SD, Nahar L, Kumarasamy Y (2007) Microtitre plate-based antibacterial assay incorporating resazurin as an indicator of cell growth, and its application in the in vitro antibacterial screening of phytochemicals. Methods 42:321–324
Shi Z, Neoh KG, Kang ET, Wang W (2006) Antibacterial and mechanical properties of bone cement impregnated with chitosan nanoparticles. Biomaterials 27:2440–2449
Shiloh M, Ruan J, Nathan C (1997) Evaluation of bacterial survival and phagocyte function with a fluorescence-based microplate assay. Infect Immun 65:3193–3198
Singh R, Paul D, Jain RK (2006) Biofilms: implications in bioremediation. Trends Microbiol 14:389–397 doi:https://doi.org/10.1016/j.tim.2006.07.001
Stepanovic S, Vukovic D, Dakic I, Savic B, Svabic-Vlahovic M (2000) A modified microtiter-plate test for quantification of staphylococcal biofilm formation. J Microbiol Methods 40:175–179
Surdeau N, Laurent-Maquin D, Bouthors S, Gelle MP (2006/4) Sensitivity of bacterial biofilms and planktonic cells to a new antimicrobial agent, Oxsil(R) 320N. J Hosp Infect 62:487–493
Tizzard AC, Bergsma JH, Lloyd-Jones G (2006) A resazurin-based biosensor for organic pollutants. Biosens Bioelectron 22:759–763
Twigg RS (1945) Oxidation-reduction aspects of Resazurin. Nature 155:401–402
van der Wende E, Characklis WG, Smith DB (1989) Biofilms and bacterial drinking water quality. Water Res 23:1313–1322
Yamashoji S, Asakawa A, Kawasaki S, Kawamoto S (2004) Chemiluminescent assay for detection of viable microorganisms. Anal Biochem 333:303–308
Zelver N, Hamilton M, Goeres D, Heersink J (2001) Development of a standardized antibiofilm test. Methods Enzymol 337:363–376
Acknowledgments
This work was supported by the Consejería de Educación of the Junta de Andalucía and the Ministerio de Ciencia y Tecnología in the Programa General del Conocimiento. The authors wish to thank T. MacFarlane for his helpful suggestions while translating the manuscript.
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Mariscal, A., Lopez-Gigosos, R.M., Carnero-Varo, M. et al. Fluorescent assay based on resazurin for detection of activity of disinfectants against bacterial biofilm. Appl Microbiol Biotechnol 82, 773–783 (2009). https://doi.org/10.1007/s00253-009-1879-x
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DOI: https://doi.org/10.1007/s00253-009-1879-x