A high-throughput assay identifies molecules with antimicrobial activity against persister cells

Area of Science:

• Microbiology and Infectious Diseases
• Drug Discovery and Development
• Antimicrobial Resistance

Background:

• Persister cells are dormant, non-growing bacteria that tolerate antibiotics, leading to persistent infections and treatment failures.
• Existing high-throughput drug discovery assays primarily identify growth inhibitors, neglecting compounds effective against non-growing persister cells.
• A critical need exists for novel assays capable of identifying antimicrobial agents that specifically target and eliminate persister bacteria.

Purpose of the Study:

• To develop a simple, high-throughput assay for identifying compounds with antimicrobial activity against *Staphylococcus aureus* persister cells.
• To screen for molecular motifs exhibiting potent activity against antibiotic-tolerant persister bacteria.
• To establish a reliable method for discovering novel biocidal antibiotics effective against recalcitrant infections.

Main Methods:

• Quantification of *Staphylococcus aureus* persister cells using colony-forming unit enumeration after ciprofloxacin treatment.
• Optimization of parameters including cell concentration, antibiotic concentration, growth phase, and nutrient availability for persister cell generation.
• Screening of compound fragments for antimicrobial activity against persister cells using a carbon-free minimal medium starvation protocol.

Main Results:

• A protocol involving starvation in carbon-free minimal medium effectively generated high concentrations of *S. aureus* persister cells tolerant to 50× MIC ciprofloxacin.
• Seven compounds from four distinct structural clusters demonstrated activity against antibiotic-tolerant *S. aureus*.
• Two identified compounds exhibited moderate cytotoxicity, while the remaining compounds displayed high cytotoxicity, necessitating further optimization.

Conclusions:

• Transferring stationary-phase cultures to a carbon-free minimal medium is a facile and effective strategy for high-throughput screening of persister cell-killing antibiotics.
• The study successfully identified molecular fragments with activity against persister cells.
• Further research is required to identify drug motifs with potent antimicrobial activity and reduced general cytotoxicity.