Microplate Alamar Blue Assay for Staphylococcus epidermidis Biofilm Susceptibility Testing

Abstract

Biofilms are at the root of many infections largely because they are much more antibiotic resistant than their planktonic counterparts. Antibiotics that target the biofilm phenotype are desperately needed, but there is still no standard method to assess biofilm drug susceptibility. Staphylococcus epidermidis ATCC 35984 biofilms treated with eight different approved antibiotics and five different experimental compounds were exposed to the oxidation reduction indicator Alamar blue for 60 min, and reduction relative to untreated controls was determined visually and spectrophotometrically. The minimum biofilm inhibitory concentration was defined as <50% reduction and a purplish well 60 min after the addition of Alamar blue. All of the approved antibiotics had biofilm MICs (MBICs) of >512 g/ml (most >4,096 g/ml), and four of the experimental compounds had MBICs of <128 g/ml. The experimental aaptamine derivative hystatin 3 was used to correlate Alamar blue reduction with 2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide (XTT) reduction and viable counts (CFU/ml) for S. epidermidis ATCC 35984, ATCC 12228, and two clinical isolates. For all four strains, Alamar blue results correlated well with XTT (r ‫؍‬ 0.83 to 0.97) and with CFU/ml results (r ‫؍‬ 0.85 to 0.94). Alamar blue's stability and lack of toxicity allowed CFU/ml to be determined from the same wells as Alamar blue absorbances. If the described method of microplate Alamar blue biofilm susceptibility testing, which is simple, reproducible, cost-effective, nontoxic, and amenable to high throughput, is applicable to other important biofilm forming species, it should greatly facilitate the discovery of biofilm specific agents.Given the tremendous clinical importance of biofilms, it is somewhat surprising that there is no standard method for investigating the drug susceptibility of bacterial biofilms. Several methods are available but are limited by long processing times, incompatibility with high throughput, expensive reagents or equipment, or the fact that the method measures mass instead of viability (2,4,7,13,14,24,25). For bacteria, a common method of assessing susceptibility is to quantitate the mass of biofilms by crystal violet or safranin staining, followed by extraction of bound dye with a solvent and measurement of absorption (6,24). This method provides no information about viability. Another common method of assessing bacterial biofilm susceptibility is to disrupt the biofilm by sonication, vortexing, or scraping, followed by dilution plating for determination of CFU/ml (27,28,31). This method has serious limitations; biofilm clumps can be difficult to dissociate into single-cell suspensions for plate counting, it is extremely laborious, and antibiotic carryover is a concern. For fungi, the most common method is an XTT [2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide] reduction assay (1,8,25), and this method has also been used for bacterial biofilms (1). While XTT reduction does measure metabolic activity,...

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