A cell-based infection assay identifies efflux pump modulators that reduce bacterial intracellular load

Area of Science:

• Microbiology
• Pharmacology
• Infectious Diseases

Background:

• Bacterial efflux pumps are crucial for multidrug resistance (MDR) in pathogens.
• Inhibiting efflux pumps is a promising strategy for developing new antibiotics.

Purpose of the Study:

• To identify novel efflux pump modulators (EPMs) using an in-cell screening approach.
• To evaluate the antibacterial activity and synergistic potential of identified EPMs.

Main Methods:

• An in-cell screen using macrophages infected with Salmonella enterica identified EPM candidates.
• Secondary screening assessed Hoechst 33342 accumulation to confirm efflux pump inhibition.
• Antibacterial activity, synergy with antimicrobials, and efflux pump binding were evaluated.

Main Results:

• Three EPMs were identified with activity comparable to PAβN.
• EPMs showed antibacterial effects against Salmonella within host cells but not in standard media.
• Compounds inhibited energy-dependent efflux, bound the AcrB subunit, and synergized with antimicrobial peptides and antibiotics.
• EPMs inhibited efflux in MDR Gram-negative ESKAPE pathogens.

Conclusions:

• In-cell screening successfully identified novel EPMs.
• These EPMs enhance innate immunity and antibiotic efficacy against MDR bacteria.
• EPMs represent a potential new class of adjuvants for antibiotic therapy.