PFK-158 enhances colistin efficacy against resistant Edwardsiella piscicida through synergistic mechanisms

<i>Edwardsiella piscicida</i> (<i>E. piscicida</i>) is a well-known bacterial pathogen that causes severe diseases in various cultured fish species, posing a significant threat to the global aquaculture industry. The increasing incidence of multidrug resistant (MDR) <i>E. piscicida</i> has greatly limited the efficacy of conventional antibiotics, highlighting the urgent need for new and effective therapeutic strategies. In the current study, several potential non-antibiotic adjuvants were screened, and PFK-158 was identified as promising compound that synergistically decreased the minimum inhibitory concentration (MIC) of colistin resistant <i>E. piscicida</i>, and significantly enhanced colistin's bactericidal activity against <i>E. piscicida</i> and other fish pathogens (e.g., <i>Vibrio parahaemolyticus</i>). Mechanistic characterization revealed that the combined treatment with PFK-158 and colistin increased bacterial membrane permeability, inhibited efflux pump activity and biofilm formation, promoted colistin accumulation, and induced the overproduction of reactive oxygen species (ROS). Transcriptomic analysis further demonstrated that PFK-158 in combination with colistin significantly downregulated genes associated with bacterial secretion systems, virulence, and lipopolysaccharide modification. Moreover, the co-administration of colistin and PFK-158 efficiently reduced bacterial loads <i>in vivo</i> and improved survival rates in infected fish. These results indicate that PFK-158 may serve as a safe and effective colistin adjuvant to synergistically combat MDR <i>E. piscicida</i> infections. This study provides valuable insights for the development of non-antibiotic adjuvants to manage diseases caused by <i>Edwardsiella</i> and other colistin-resistant pathogens in aquaculture.