Induction of a viable but non-culturable state by stringent response in Escherichia coli O157:H7 during macrophage interaction enables immune evasion

Enterohemorrhagic Escherichia coli (EHEC) O157:H7 is a major foodborne pathogen. It can survive harsh conditions in the gastrointestinal tract and macrophages, contributing to its pathogenicity. This study aimed to investigate how E. coli O157:H7 enters the Viable But Non-Culturable (VBNC) state within macrophages to evade immune recognition. First, we assessed bacterial survival under simulated gastrointestinal conditions to exclude potential confounding effects. Next, we examined bacterial viability, growth, and gene expression in macrophages using wild-type and relA -deficient strains. Results showed that macrophage ingestion triggers reactive oxygen species (ROS), activating the stringent response and inducing the VBNC state. VBNC state E.coli O157:H7 significantly reduced expression of pathogen-associated molecular patterns (PAMPs), helping them evade immune detection. Furthermore, VBNC state E.coli O157:H7 exhibited enhanced resistance to ROS stress. This study provides insights into bacterial survival strategies in host environments and underscores the health risks associated with VBNC pathogens. • E. coli O157:H7 enters a VBNC state within macrophages through the stringent response, and thus making it more resistant to intracellular stresses. • ROS in macrophages triggers the VBNC state in bacteria, whereas acidic stress does not induce VBNC state. • E. coli O157:H7 in a VBNC state evade macrophage recognition by downregulating the expression of PAMPs.