ABSTRACTThe increasing occurrence of extensively drug-resistant and pan-drug-resistantK. pneumoniaehas posed a serious threat to global public health. Therefore, new antimicrobial strategies are urgently needed to combat these resistantK. pneumoniae-related infections. Drug repurposing and combination are two effective strategies to solve this problem. By a high-throughput screening assay of FDA-approved drugs, we found that the potential small molecule 9-aminoacridine (9-AA) could be used as an antimicrobial alone or synergistically with rifampin (RIF) against extensively/pan-drug-resistantK. pneumoniae. In addition, 9-AA could overcome the shortcomings of RIF by reducing the occurrence of resistance. Mechanistic studies revealed that 9-AA interacted with bacterial DNA and disrupted the proton motive force inK. pneumoniae. Through liposomeization and combination with RIF, the cytotoxicity of 9-AA was significantly reduced without affecting its antimicrobial activity. In addition, we demonstrated thein vivoantimicrobial activity of 9-AA combined with RIF without detectable toxicity. In summary, 9-AA has the potential to be an antimicrobial agent or a RIF adjuvant for the treatment of multidrug-resistantK. pneumoniaeinfections.IMPORTANCEKlebsiella pneumoniaeis a leading cause of clinically acquired infections. The increasing occurrence of drug-resistantK. pneumoniaehas posed a serious threat to global public health. We found that the potential small molecule 9-AA could be used as an antimicrobial alone or synergistically with RIF against drug-resistantK. pneumoniaein vitroand with low resistance occurrence. The combination of 9-AA or 9-AA liposomes with RIF possesses effective antimicrobial activityin vivowithout detected toxicity. 9-AA exerted its antimicrobial activity by interacting with specific bacterial DNA and disrupting the proton motive force inK. pneumoniae. In summary, we found that 9-AA has the potential to be developed as a new antibacterial agent and adjuvant for RIF. Therefore, our study can reduce the risk of antimicrobial resistance and provide an option for the exploitation of new clinical drugs and a theoretical basis for the research on a new antimicrobial agent.