Nitazoxanide synergizes polymyxin B against Escherichia coli by depleting cellular energy

The rapid expansion of antibiotic-resistant bacterial diseases is a global burden on public health. It makes sense to repurpose and reposition already-approved medications for use as supplementary agents in synergistic combinations with existing antibiotics. Here, we demonstrate that the anthelmintic drug nitazoxanide (NTZ) synergistically enhances the effectiveness of the lipopeptide antibiotic polymyxin B in inhibiting gram-negative bacteria, including those resistant to polymyxin B. Mechanistic investigations revealed that nitazoxanide inhibited calcium influx and cell membrane depolarization, enhanced the affinity between polymyxin B and the extracellular membrane, and promoted intracellular ATP depletion and an increase in reactive oxygen species (ROS), thus enhancing the penetration and disruption of the Escherichia coli cell membrane by polymyxin B. The transcriptomic analysis revealed that the combination resulted in energy depletion by inhibiting both aerobic and anaerobic respiration patterns in bacterial cells. The increased bactericidal effect of polymyxin B on the E. coli ∆nuoC strain further indicates that NuoC could be a promising target for nitazoxanide. Furthermore, the combination of nitazoxanide and polymyxin B showed promising therapeutic effects in a mouse infection model infected with E. coli. Taken together, these results demonstrate the potential of nitazoxanide as a novel adjuvant to polymyxin B, to overcome antibiotic resistance and improve therapeutic outcomes in refractory infections.IMPORTANCEThe rapid spread of antibiotic-resistant bacteria poses a serious threat to public health. The search for potential compounds that can increase the antibacterial activity of existing antibiotics is a promising strategy for addressing this issue. Here, the synergistic activity of the FDA-approved agent nitazoxanide (NTZ) combined with polymyxin B was investigated in vitro using checkerboard assays and time-kill curves. The synergistic mechanisms of the combination of nitazoxanide and polymyxin B were explored by fluorescent dye, transmission electron microscopy (TEM), and transcriptomic analysis. The synergistic efficacy was evaluated in vivo by the Escherichia coli and mouse sepsis models. These results suggested that nitazoxanide, as a promising antibiotic adjuvant, can effectively enhance polymyxin B activity, providing a potential strategy for treating multidrug-resistant bacteria.