Robust biofilm assay for quantification and high throughput screening applications.

Area of Science:

• Microbiology
• Biochemistry
• Drug Discovery

Background:

• Bacterial biofilms exhibit high resistance to antimicrobial treatments.
• Current biofilm quantification methods lack dynamic range, throughput, and species adaptability.
• A need exists for a versatile, high-throughput biofilm quantification assay.

Purpose of the Study:

• To develop and validate a broad-utility, high-throughput method for bacterial biofilm quantification.
• To demonstrate the assay's applicability across diverse bacterial pathogens.
• To screen compounds for biofilm inhibition using the new assay.

Main Methods:

• Biofilm staining with FM1-43 fluorescent dye.
• Solvent extraction of the stained biofilm.
• Quantification using a fluorescent plate reader.
• Screening of 1200 compounds for biofilm inhibition.

Main Results:

• The FM1-43 assay demonstrated a wider dynamic range (8-146 fold) than crystal violet staining (2-22 fold).
• The method proved effective for eight different bacterial pathogens.
• Rifabutin and ethavarine were identified as potential inhibitors of *Burkholderia pseudomallei* and *Acinetobacter baumannii* biofilms, respectively.

Conclusions:

• The validated FM1-43 assay is a robust, sensitive, and high-throughput method for bacterial biofilm quantification.
• This assay can be readily adapted for antibiofilm screening campaigns.
• The method offers a superior alternative to existing, less sensitive, and lower-throughput techniques.