| Summary: | <i>Pseudomonas aeruginosa</i> infection is considered a high-risk nosocomial infection and is very difficult to eradicate because of its tolerance to antibiotic treatment. A new compound, autoinducer analog-1 (AIA-1), has been demonstrated to reduce antibiotic tolerance, but its mechanisms remain unknown. This study aimed to investigate the mechanisms of AIA-1 in the antibiotic tolerance of <i>P. aeruginosa</i>. A transposon mutant library was constructed using miniTn5pro, and screening was performed to isolate high tolerant mutants upon exposure to biapenem and AIA-1. We constructed a deletion mutant and complementation strain of the genes detected in transposon insertion site determination, <i>pruR</i> and PA0066-65-64, and performed killing assays with antibiotics and AIA-1. Gene expression upon exposure to biapenem and AIA-1 and their relationship to stress response genes were analyzed. High antibiotic tolerance was observed in Tn5-<i>pruR</i> and Tn5-PA0065 transposon mutants and their deletion mutants, Δ<i>pruR</i> and ΔPA0066-65-64. Complemented strains of <i>pruR</i> and PA0066-65-64 with their respective deletion mutants exhibited suppressed antibiotic tolerance. It was determined that deletion of PA0066-65-64 increased <i>rpoS</i> expression, and PA0066-65-64 affects antibiotic tolerance via the <i>rpoS</i> pathway. Additionally, antibiotics and AIA-1 were found to inhibit <i>pruR</i> and PA0066-65-64. This study proposed that <i>pruR</i> and PA0066-65-64 are members of the antibiotic tolerance suppressors.
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