Abstract
Rapid and simple method for the determination of minimum biofilm eradication concentration (MBEC) using a colorimetric microbial viability assay based on reduction of a tetrazolium salt WST-8 and the biofilm formation method on 96-pegs on the lid of a microtiter plate was developed. The biofilms formed on the 96 pegs were challenged by antibiotics, and the MBEC was then determined from the microbial viability of the biofilms formed on the 96 pegs, assessed by the WST-8 colorimetric assay. The MBECs obtained by the proposed and conventional methods favorably agreed. The most effective inhibitors of S. aureus and P. aeruginosa biofilms were vancomycin and ciprofloxacin, respectively. In addition, we clarified that Staphylococcus aureus biofilm was maximally suppressed by a combination of vancomycin, daptomycin, and teicoplanin. The proposed method yields similar performance to conventional methods, but is faster and more easily implemented. Therefore, the proposed method alleviates the tediousness and time-consuming nature of conventional biofilm susceptibility assay.
Highlights
Biofilms are formed when microorganisms aggregate with each other or adhere to a solid surface and encase themselves in a self-produced matrix of extracellular polysaccharides and proteins
In our colorimetric microbial viability assay, biofilms are formed on 96 pegs on the lid of a microtiter plate, and the minimum biofilm eradication concentrations (MBECs) is evaluated from the reduction of WST-8 to formazan
For S. aureus, 180 μl of bacterial inoculum grown to approximately 107 CFU ml-1 in Mueller–Hinton broth (MHB) was added to each well of a 96-well microtiter plate
Summary
Biofilms are formed when microorganisms aggregate with each other or adhere to a solid surface and encase themselves in a self-produced matrix of extracellular polysaccharides and proteins. A general procedure for the determination of these biofilm parameters is extremely complicated because it is difficult to wash the biofilms cultivated directly on the bottom of wells in microtiter plates and to collect the supernatant on the biofilms with pipetting (Nett et al, 2011; Qu et al, 2010). In our colorimetric microbial viability assay, biofilms are formed on 96 pegs on the lid of a microtiter plate, and the MBEC is evaluated from the reduction of WST-8 to formazan. We applied our approach to the biofilm susceptibility of Staphylococcus aureus and Pseudomonas aeruginosa exposed to plural antibiotics
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