Production of Reactive Oxygen Species Induced by Peracetic Acid Enhances Bactericidal Activity and Mutagenesis in <i>Salmonella</i> Typhimurium

Peracetic acid (PAA) is extensively used in poultry processing, but its bactericidal mechanism remains poorly understood. Reactive oxygen species (ROS) are linked to bacterial cell death by many bactericidal agents. This study investigated the role of ROS in PAA against <i>Salmonella</i> Typhimurium 14028 using bacterial suspensions treated with PAA alone (20 ppm), PAA combined with 2,2'-dipyridyl (Dip; iron chelator) or with thiourea (ROS scavenger) followed by incubation at 37°C for 1 h. Post-incubation, serial dilutions were plated on Luria-Bertani agar to determine colony-forming units per milliliter. Additionally, eight single-deletion mutants and the wild-type (control) of <i>Salmonella</i> Typhimurium 14028 were tested for survivability after exposure to PAA (treatment; 20 ppm) and hydrogen peroxide (H₂O₂; control). The mutants were categorized by predicted ROS impact: increase in ROS production (<i>atpC</i>, <i>gnd</i>, <i>nuoG</i>, <i>pta, sdhC</i>, and <i>zwf</i>) and no change in ROS production (negative control; <i>edd</i> and <i>pykA</i>). Finally, rifampicin-based selection assay was utilized to evaluate the mutation rate of the wild-type strain in the presence of PAA (60 ppm), H₂O₂ (control), and PAA + thiourea. Treatment with 20 ppm PAA significantly (<i>p</i> < 0.05) reduced the viability of <i>Salmonella</i> Typhimurium 14028 by a log₁₀ reduction of 0.34, while co-treatment with thiourea restored counts to control level. The survival of the mutants predicted to increase ROS production was significantly reduced compared with the control mutants and wild-type strain (<i>p</i> < 0.05). Exposure to sublethal PAA led to a 28-fold increase in mutation rate suggesting the production of ROS by PAA was responsible for the observed increase. ROS production is a significant component of PAA's bactericidal activity against <i>Salmonella</i> Typhimurium, and increasing ROS production might be exploited to enhance PAA-mediated killing of <i>Salmonella</i> Typhimurium. ROS production by PAA can lead to the development of antibiotic resistance in <i>Salmonella</i> Typhimurium when cells manage to escape cell death.