High-throughput screening for antimicrobial compounds using a 96-well format bacterial motility absorbance assay

Area of Science:

• Microbiology
• Drug Discovery
• Biotechnology

Background:

• Increasing antibiotic resistance necessitates novel antimicrobial drug development.
• Bacterial motility is a key factor in pathogenesis and a potential drug target.
• Existing screening methods may not be efficient for identifying motility inhibitors.

Purpose of the Study:

• To develop and validate a novel high-throughput screening (HTS) methodology.
• To screen for both antimicrobial compounds and inhibitors of bacterial motility.
• To adapt the assay for compatibility with standard laboratory automation.

Main Methods:

• Utilized a bacterial motility swarming agar assay in 96-well plates.
• Employed offset inoculation for rapid compound screening.
• Used a standard absorbance microplate reader for data acquisition.
• Validated the assay by screening a library of 960 diverse compounds against Salmonella typhimurium.

Main Results:

• The assay demonstrated robust performance with an average Z' value of 0.67 +/- 0.14.
• A significant signal-to-baseline ratio (5.9 +/- 1.1) was achieved.
• Successfully identified 70 known antimicrobial compounds from the screened library.
• The methodology proved compatible with 96-well automation.

Conclusions:

• The developed HTS methodology is effective for identifying antimicrobial and antimotility compounds.
• This assay is suitable for drug discovery and chemical biology applications.
• The method offers a rapid, automatable approach to combat antibiotic resistance.