Nitrite Promotes ROS Production to Potentiate Cefoperazone-Sulbactam-Mediated Elimination to Lab-Evolved and Clinical-Evolved Pseudomonas aeruginosa

Cefoperazone-sulbactam (SCF)-resistant Pseudomonas aeruginosa poses a big challenge in the use of SCF to treat infection caused by the pathogen. We have recently shown exogenous nitrite-enabled killing of naturally and artificially evolved Pseudomonas aeruginosa strains (AP-R_CLIN-EVO and AP-R_LAB-EVO, respectively) by SCF. However, the underlying mechanism is unknown. Here, reprogramming metabolomics was adopted to investigate how nitrite enhanced the SCF-mediated killing efficacy. Nitrite-reprogrammed metabolome displayed an activated pyruvate cycle (P cycle), which was confirmed by elevated activity of pyruvate dehydrogenase (PDH), α-ketoglutarate dehydrogenase, succinate dehydrogenase, and malate dehydrogenase. The activated P cycle provided NADH for the electron transport chain and thereby increased reactive oxygen species (ROS), which potentiated SCF to kill AP-R_CLIN-EVO and AP-R_LAB-EVO. The nitrite-enabled killing of AP-R_CLIN-EVO and AP-R_LAB-EVO by SCF was inhibited by PDH inhibitor furfural and ROS scavenger N-Acetyl-L-cysteine but promoted by ROS promoter Fe³⁺. SCF alone could not induce ROS, but SCF-mediated killing efficacy was enhanced by ROS. In addition, the present study demonstrated that nitrite repressed antioxidants, which were partly responsible for the elevated ROS. These results reveal a nitrite-reprogrammed metabolome mechanism by which AP-R_CLIN-EVO and AP-R_LAB-EVO sensitivity to SCF is elevated. IMPORTANCE Antibiotic-resistant Pseudomonas aeruginosa has become a real concern in hospital-acquired infections, especially in critically ill and immunocompromised patients. Understanding antibiotic resistance mechanisms and developing novel control measures are highly appreciated. We have recently shown that a reduced nitrite-dependent NO biosynthesis contributes to cefoperazone-sulbactam (SCF) resistance, which is reverted by exogenous nitrite, in both naturally and artificially evolved P. aeruginosa strains (AP-R_CLIN-EVO and AP-R_LAB-EVO, respectively). However, the mechanism is unknown. The present study reports that the nitrite-enabled killing of AP-R_CLIN-EVO and AP-R_LAB-EVO by SCF is attributed to the promoted production of reactive oxygen species (ROS). Nitrite activates the pyruvate cycle to generate NADH for the electron transport chain, which in turn promotes ROS generation. Nitrite-potentiated SCF-mediated killing is decreased by pyruvate dehydrogenase inhibitor furfural and ROS scavenger N-Acetyl-L-cysteine but increased by ROS promoter Fe³⁺. Furthermore, SCF-mediated killing is promoted by H₂O₂ in a dose-dependent manner. In addition, the combination of nitrite and H₂O₂ greatly enhances SCF-mediated killing. These results not only disclose a nitrite-ROS-potentiated SCF-mediated killing, but also show SCF-mediated killing is dependent upon ROS.