Doxofylline Protects Gram-Negative Pathogens against Antibiotic-Mediated Killing

Given the growing rate of Gram-negative bacterial infections, antibiotics are now frequently prescribed for various respiratory diseases. Doxofylline is a newer generation xanthine with both bronchodilating and anti-inflammatory activities, but its influence on antibiotics remains poorly understood. Here, we first report the discovery of doxofylline-induced high minimum inhibitory concentrations of antibiotics. We also showed that doxofylline blocked antimicrobial-mediated killing for Gram-negative pathogens in vitro and in murine lung infection models in vivo. By combining efflux pump inhibition tests, gene expression analyses, and using the gene tolC knockout strain, we found that doxofylline positively regulated gene expression of the AcrAB-TolC efflux pump and attenuated the effect of doxofylline on antibacterial activities in ΔtolC mutants. Notably, doxofylline-mediated protection correlated with decreased reactive oxygen species (ROS) production. Collectively, our study indicates that doxofylline protects Gram-negative bacteria from antimicrobial lethality by regulating the AcrAB-TolC efflux pump in a TolC-dependent manner and suppressing antibiotic-induced ROS accumulation. These results suggest caution when using antibiotics alongside doxofylline in clinical treatment.