Freeze-thaw resilience of Shigella flexneri NCCP 10852 in co-culture with Listeria monocytogenes: Implications for biofilm-mediated survival in cold environment

Shigella flexneri is an enteric pathogen traditionally associated with poor survival under freezing conditions. However, recent studies have reported its persistence even in frozen foods, raising concerns about its environmental resilience. In real-world food processing environments, multiple microbial species frequently coexist, yet the survival dynamics of S. flexneri in dual-species biofilms, particularly with psychrotolerant Listeria monocytogenes, remain largely unexplored. This study aimed to investigate how co-cultivation with L. monocytogenes influences the freeze-thaw (FT) resilience and biofilm formation of S. flexneri under food-relevant conditions. A series of experiments were conducted, including injured rate analysis, membrane permeability assays, extracellular polymeric substances(EPS) quantification, field emission-scanning electron microscopy(FE-SEM) imaging, and reverse transcription-quantitative polymeric chain reaction(RT-qPCR). Results showed that dual-species biofilms significantly reduced S. flexneri injured rate under FT conditions and enhanced its biofilm formation at 4 °C. Increased membrane damage, higher EPS production, and upregulation of stress-related genes were observed in co-cultures, suggesting a synergistic survival mechanism. These findings demonstrate that S. flexneri can survive under FT and form biofilms when coexisting with L. monocytogenes, likely through biofilm-mediated protection and interspecies interactions. These findings highlight the importance of understanding multi-species biofilms in frozen food environments.