Biosynthesis of H₂S and Siderophores Targeting Gram-Negative Bacterial Resistance to Reactive Oxygen Species

Reactive oxygen species (ROS) are a promising alternative bactericide. However, it is questioned that bacteria can potentially develop resistance to ROS, similar to their resistance against antibiotics and silver. Herein, it is reported that Gram-negative bacteria, including Pseudomonas aeruginosa, Escherichia coli, and Klebsiella pneumoniae, develop resistance to ROS after six repeated exposures. Notably, ROS minimum inhibitory concentration of Pseudomonas aeruginosa significantly increases to 256-fold after ten passages. The resistance mechanism predominantly originates from the intensified biosynthesis of the highly reductive hydrogen sulfide (H₂S) and pyoverdine (PVD) siderophores, effectively neutralizing ROS. Simultaneously, PVD transports Fe³⁺ from the extracellular space into the bacteria, releasing H₂S bound to Fe³⁺ and enhancing ROS scavenging. Additionally, the enhanced outer membrane (OM) biogenesis establishes a robust OM barrier, impeding ROS penetration. The acquired resistance to ROS can be significantly reduced by incorporating additional Fe³⁺ into the culture medium or disrupting the H₂S biosynthetic gene. These observations suggest that careful consideration is required when utilizing ROS against Gram-negative bacteria. It is anticipated that understanding this resistance mechanism can inform the development of future antimicrobial agents, particularly for Gram-negative bacteria.