Ciprofloxacin resistance in Salmonella drives cross-protection to UV-C and HHP via rpoD mutation.

Antimicrobial resistance (AMR) remains a major public health concern, being the agri-food chain crucial in the emergence and dissemination of resistant pathogens such as Salmonella Typhimurium. Effective hygienization is key to prevent access of AMR bacteria into the agri-food chain. In this work, we investigated the consequences of ciprofloxacin (CIP) resistance acquisition in S. Typhimurium on tolerance to emerging non-thermal preservation technologies, including pulsed electric fields (PEF), ultraviolet C irradiation (UV-C) and high hydrostatic pressure (HHP). We focused on a CIP-resistant S. Typhimurium variant (SeTRV1) previously shown to exhibit cross-protection to thermal treatments. While no significant differences were observed following PEF treatments, UV-C and HHP susceptibility was significantly reduced, resulting in survival increases ranging from approximately 32- to 2000-fold. We confirmed that a mutation in rpoD was the primary contributor to these phenotypes and associated with transcriptional changes in selected stress- and AMR-related genes, as determined by RT-qPCR. Virulence assessment using Galleria mellonella larvae revealed compensatory and divergent phenotypes associated with different mutations, ranging from reduced to enhanced virulence. Additional CIP-resistant variants harboring distinct mutations were subsequently evaluated, revealing that although PEF tolerance remained unaffected, several variants exhibited decreased susceptibility to UV-C, potentially linked to optimized DNA topology and enhanced oxidative stress responses. Overall, this study identifies rpoD as key driver of cross-protection to UV-C and HHP and highlights mutation-specific roles of ramR and cyaA in modulating UV-C tolerance and virulence, as well as gyrA/gyrB in increasing UV-C tolerance, with implications for food safety risk assessment in minimally processed foods.