NorA, an extensively studied efflux pump in Staphylococcus aureus , has been connected to fluoroquinolone, antiseptic, and disinfection resistance. Several studies have also emphasized how efflux pumps, including NorA, function as the first line of defense of S. aureus against antibiotics. In this study, we have screened some chemically synthesized indole derivatives for their activity as efflux pump inhibitors (EPIs). The derivative SMJ-5 was found to be a potent NorA efflux pump inhibitor among the screened indole derivatives, owing to increased ethidium bromide and norfloxacin accumulation in norA over-expressing S. aureus . The combination of SMJ-5 and ciprofloxacin demonstrated the eradication of S. aureus biofilm and prolonged the post-antibiotic effect more than ciprofloxacin alone. SMJ-5 was able to inhibit staphyloxanthin virulence. In in vitro time-kill trials and in vivo efficacy investigations, the combination enhanced the bactericidal activity of ciprofloxacin against S. aureus . Additionally, reverse transcription PCR results revealed that SMJ-5 also inhibits the NorA efflux pump indirectly at the transcriptional level. IMPORTANCE: The NorA efflux pump is the most effective resistance mechanism in S. aureus . The clinical importance of NorA efflux pumps is demonstrated by the expression of pump genes in S. aureus strains in response to fluoroquinolones and biocides. Along with the repercussions of decreased fluoroquinolone sensitivity, increasing expression of efflux pump genes by their substrate necessitates the importance of efflux pump inhibitors. Reserpine and verapamil are clinically used to treat ailments and have proven NorA inhibitors, but, unfortunately, the concentration needed for these drugs to inhibit the pump is not safe in clinical settings. In the current study, we have screened some indole derivatives, and among them, SMJ-5 was reported to potentiate norfloxacin and ciprofloxacin at their sub-inhibitory concentration by inhibiting the norA gene transcriptionally. Here we highlight the promising points of this study, which could serve as a model to design a therapeutic EPI candidate against norA over-expressing S. aureus .

Background: Staphylococcus aureus is a highly lethal Gram-positive bacterium that is responsible for over one million deaths annually. As a member of the ESKAPE pathogens, its methicillin-resistant strains (MRSA) are prevalent worldwide and exhibit significant antimicrobial resistance (AMR). Bacterial efflux pumps play a pivotal role in the development of AMR by facilitating the expulsion of a range of antimicrobial agents. Methods: The S. aureus strain SA-1199B, which overexpresses NorA and carries a GrlA mutation, was utilized to comprehensively profile the mechanism of the compounds PQQ16P and PQK4F. To assess the toxicity and genotoxicity of these compounds, RAW macrophages, HEK 293T, and HepG2 cell lines were utilized. Female BALB/c mice were utilized to assess the in vivo synergism of EPIs with CPX, Results: NorA efflux pump inhibitors (EPIs), PQQ16P and PQK4F, enhanced the efficacy of the antibacterial ciprofloxacin (CPX) against resistant S. aureus strains. The mechanism of EPIs involved the inhibition of NorA efflux pump, without compromising bacterial membrane permeability, ATP levels, or mammalian calcium channels. Moreover, the EPIs significantly augmented the bactericidal and post-antibiotic effects of CPX, elevating its mutation prevention concentration without manifesting substantial toxicity to human cells. Furthermore, the EPIs reduced S. aureus invasiveness in macrophages, indicating a role for NorA in bacterial virulence. Notably, the in vivo synergism of these EPIs with CPX was observed in a mouse infection model. Conclusions: This study provides substantial evidence for the potential of employing EPIs in a combination with CPX to counteract AMR, both in vitro and in vivo.

Background: Staphylococcus aureus is a highly lethal Gram-positive bacterium that is responsible for over one million deaths annually. As a member of the ESKAPE pathogens, its methicillin-resistant strains (MRSA) are prevalent worldwide and exhibit significant antimicrobial resistance (AMR). Bacterial efflux pumps play a pivotal role in the development of AMR by facilitating the expulsion of a range of antimicrobial agents; Methods: The S. aureus strain SA-1199B, which overexpresses norA and carries a GrlA mutation, was utilized to comprehensively profile the mechanism of the compounds PQQ16P and PQK4F. To assess the toxicity and genotoxicity of these compounds, RAW macrophages, HEK 293T, and HepG2 cell lines were utilized. Female BALB/c mice were utilized to assess the in vivo synergism of EPIs with CPX; Results: NorA efflux pump inhibitors (EPIs), PQQ16P and PQK4F, enhanced the efficacy of the antibacterial ciprofloxacin (CPX) against resistant S. aureus strains. The mechanism of EPIs involved the inhibition of NorA efflux pump, without compromising bacterial membrane permeability, ATP levels, or mammalian calcium channels. Moreover, the EPIs significantly augmented the bactericidal and post-antibiotic effects of CPX, elevating its mutation prevention concentration without manifesting substantial toxicity to human cells. Furthermore, the EPIs reduced S. aureus invasiveness in macrophages, indicating a role for NorA in bacterial virulence. Notably, the in vivo synergism of these EPIs with CPX was observed in a mouse infection model; Conclusions: This study provides substantial evidence for the potential of employing EPIs in a combination with CPX to counteract AMR, both in vitro and in vivo.