Overcoming Multidrug Resistance by Bacterial Efflux Pump Inhibitors in Clinical <i>Escherichia coli</i> Strains

<b>Background/Objectives</b>: Antimicrobial resistance (AMR) is an escalating global threat driven by antibiotic misuse and bacterial adaptation. Efflux pumps are major contributors to multidrug resistance in <i>Escherichia coli</i>, as they expel antibiotics and reduce their intracellular activity. This study examined efflux-mediated resistance in extended-spectrum beta-lactamase (ESBL)-producing <i>E. coli</i> and evaluated the potential of several efflux pump inhibitors (EPIs)—promethazine (PMZ), thioridazine (TZ), carbonyl cyanide <i>m</i>-chlorophenyl hydrazine (CCCP), reserpine (RES), and phenyl-arginine-β-naphthylamide (PAβN)—as therapeutic adjuncts. <b>Methods</b>: 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. <b>Results:</b> 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. <b>Conclusions:</b> Overall, the results highlight the central role of efflux pumps in ESBL-producing <i>E. coli</i> 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.