Biosynthesis of H ₂ S and Siderophores Targeting Gram‐Negative Bacterial Resistance to Reactive Oxygen Species

Abstract 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 (H ₂ S) and pyoverdine (PVD) siderophores, effectively neutralizing ROS. Simultaneously, PVD transports Fe ³⁺ from the extracellular space into the bacteria, releasing H ₂ S bound to Fe ³⁺ 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 Fe ³⁺ into the culture medium or disrupting the H ₂ S 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.