Repurposed Drugs and Efflux Pump Inhibitors Against Gram-Negative Urinary Tract Pathogenic Bacteria

<b>Background/Objectives</b>: Urinary tract infections (UTIs) represent a major healthcare challenge due to antimicrobial resistance and biofilm formation. Our aim was to evaluate whether repurposed drugs and efflux pump inhibitors (EPIs) could provide alternative strategies by investigating their antibacterial, anti-biofilm, and resistance-modifying properties against Gram-negative uropathogens under varying pH conditions. <b>Methods</b>: Clinical isolates of <i>Escherichia coli</i>, <i>Klebsiella pneumoniae</i>, and <i>Proteus mirabilis</i> were tested. Minimum inhibitory concentrations (MICs) of thioridazine (TZ), promethazine (PMZ), fluoxetine (Fx), sertraline (Sr), phenylalanine arginine β-naphthylamide (PAβN), carbonyl cyanide m-chlorophenyl hydrazone (CCCP), and the glutamine uptake inhibitor V9302 were determined at pH 5-8. Biofilm inhibition was assessed by crystal violet staining, while MIC reduction assays tested antibiotic combinations. Efflux pump inhibition was examined using an ethidium bromide accumulation assay. <b>Results</b>: TZ reduced biofilm formation in sensitive <i>K. pneumoniae</i> at all pH levels and enhanced ciprofloxacin (CIP) activity, whereas PMZ showed a weaker effect, limited mainly to neutral pH. Fx and Sr exhibited pH-dependent anti-biofilm activity, with Fx particularly effective against <i>P. mirabilis</i> at alkaline pH. PAβN consistently decreased biofilm biomass in both sensitive and resistant <i>K. pneumoniae</i> and, at pH 7-8, potentiated CIP activity with a 16-fold MIC reduction in the sensitive strain. CCCP showed pH-dependent activity, with stronger effects under acidic conditions, notably in <i>E. coli</i> and <i>P. mirabilis</i>. V9302 was a potent biofilm inhibitor in <i>K. pneumoniae</i> and resistant <i>E. coli</i> and interfered with efflux activity, showing strong effects in acidic environments. <b>Conclusions</b>: Repurposed drugs and EPIs may be useful as antibiotic adjuvants or biofilm inhibitors in treating resistant UTIs.