Effects of <scp>NarX</scp> / <scp>NarL</scp> two‐component system on stress tolerance and biofilm formation in <i>Cronobacter malonaticus</i>

Background Cronobacter malonaticus is an opportunistic foodborne pathogen capable of tolerating multiple stress conditions, thereby posing potential safety risks to dairy products. The NarX/NarL two‐component system plays important regulatory roles in bacterial perception and response to external stresses. Aim(s) This study investigated the impact of the NarX/NarL system on environmental adaptation and biofilm formation of C. malonaticus . Methods Using molecular biology techniques, we constructed knockout and complementation strains of C. malonaticus for the NarX/NarL system. The survival rates of C. malonaticus and its derivatives under various abiotic stresses were assessed using the colony counting method. Additionally, crystal violet staining was employed for quantitative evaluation, while scanning electron microscopy (SEM) was utilised for qualitative assessment of biofilm formation. Major Findings Cronobacter malonaticus demonstrates significant tolerance to both desiccation and osmotic stress, while the bacterium is sensitive to heat stress. Compared with the WT strain, the mutant strains exhibited substantially reduced survival rates under bile salt, gastric acid, desiccation, hyperosmolarity, high temperature or oxidative stressful growth conditions. Concurrently, in comparison with Δ narX , Δ narL caused a more pronounced reduction in the strain's tolerance to environmental stress. Survival of C. malonaticus and its derivatives showed the same trend during simulation of dairy processing. Furthermore, C. malonaticus exhibited a robust capacity for biofilm formation, which was reduced by the absence of NarX/NarL system. The diminished ability to form biofilms may be a key factor contributing to the significantly lower survival rates of Δ narX / narL strains under various environmental stresses. Scientific or industrial implications This paper advances the understanding of the in vivo function of the NarX/NarL system in C. malonaticus YE‐01 from the phenotypic differences under various simulated environmental pressures. Furthermore, the data provide a valuable reference for optimising temperature–time parameters to ensure the effective elimination of Cronobacter during milk powder production processes.