Background/Objectives: Antimicrobial resistance (AMR) is an escalating global threat driven by antibiotic misuse and bacterial adaptation. Efflux pumps are major contributors to multidrug resistance in Escherichia coli, as they expel antibiotics and reduce their intracellular activity. This study examined efflux-mediated resistance in extended-spectrum beta-lactamase (ESBL)-producing E. coli and evaluated the potential of several efflux pump inhibitors (EPIs)—promethazine (PMZ), thioridazine (TZ), carbonyl cyanide m-chlorophenyl hydrazine (CCCP), reserpine (RES), and phenyl-arginine-β-naphthylamide (PAβN)—as therapeutic adjuncts. Methods: Antibacterial and anti-biofilm activities of EPIs were tested using broth microdilution, real-time fluorimetry, and crystal violet assays, while ceftriaxone–PMZ interactions were assessed by checkerboard analysis. Results: TZ and CCCP showed strain-specific antibacterial activity, whereas PMZ, RES, and PAβN did not exert any effect. PMZ, TZ, and especially CCCP effectively inhibited efflux pump function, while RES and PAβN were less active. Biofilm inhibition varied between strains, with PMZ and TZ producing moderate reductions. We observed a quite weak synergism between ciprofloxacin, ceftriaxone, and PMZ; however, the result was not significant. Conclusions: Overall, the results highlight the central role of efflux pumps in ESBL-producing E. coli and indicate that EPIs can reverse resistance (e.g., PMZ) and exhibit potent anti-biofilm activity and show additive interactions with antibiotics. However, further studies are needed to optimize their safety, pharmacokinetics, and antibiotic pairing for potential clinical use.

Background/Objectives: 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. Methods: Clinical isolates of Escherichia coli, Klebsiella pneumoniae, and Proteus mirabilis 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. Results: TZ reduced biofilm formation in sensitive K. pneumoniae 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 P. mirabilis at alkaline pH. PAβN consistently decreased biofilm biomass in both sensitive and resistant K. pneumoniae 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 E. coli and P. mirabilis. V9302 was a potent biofilm inhibitor in K. pneumoniae and resistant E. coli and interfered with efflux activity, showing strong effects in acidic environments. Conclusions: Repurposed drugs and EPIs may be useful as antibiotic adjuvants or biofilm inhibitors in treating resistant UTIs.

Background: 8-Hydroxyquinoline and phthalimide are two significant heterocyclic scaffolds in medicinal chemistry due to their pharmacological profiles. Hybridizing these pharmacophores and further modifying them via modified Mannich reactions provides a strategy to improve their physicochemical parameters and selectivity toward multidrug-resistant (MDR) cancer cells. Objectives: To synthesize a series of 8-hydroxyquinoline–phthalimide hybrids and their Mannich base derivatives and evaluate their cytotoxic activity and resistance-selective properties against sensitive Colo205 and resistant Colo320 cancer cell lines. Methods: Four hybrid compounds were synthesized by reacting 5-amino-8-hydroxyquinoline with different phthalic anhydride derivatives. Twelve fine-tuned derivatives were prepared by using the modified Mannich reaction. Cytotoxic activity was measured using the MTT assay, and relative resistance (RR) was calculated to determine selectivity toward the resistant cell line. P-glycoprotein (Pgp) ATPase activity was evaluated for the most active compounds. Results: All derivatives displayed cytotoxic activity, with higher potency toward the resistant Colo320 cell line. Compounds 2 and 4 showed the strongest activity against both cell lines (IC₅₀ down to 4.88 µM). Compounds 5, 8a, 9a, and 9c retained potent activity against Colo320 (IC₅₀ = 9.89–22.79 µM). Incorporating a CH₂–N group at position C7 substantially enhanced the selectivity for MDR cells. Compounds 9c, 9a, and 8a exhibited the highest selectivity, with RR values of 0.29, 0.33, and 0.35, respectively. Compounds 2, 4, 5, 8a, and 9a showed inhibitory effects on Pgp ATPase activity. Conclusions: The newly synthesized HQ–phthalimide hybrids represent promising candidates for targeting MDR in colorectal cancer, with Mannich modification enhancing the selectivity toward resistant cells.