RelA-Mediated Stringent Response Drives Pathogen-Specific Chlorine Resistance and VBNC Survival via Ribosomal Hibernation (rmf) and Efflux Pump Activation (tolC)

Pathogenic Enterobacteriaceae can enter a viable but non-culturable (VBNC) state under disinfectant stress. Disregarding species-specific differences in VBNC behavior likely leads to ineffective disinfection and underestimated contamination risks. Here, we compared VBNC dynamics in wild-type Escherichia coli O157:H7, Salmonella Typhimurium, Cronobacter sakazakii, and an E. coli O157:H7 ΔrelA mutant (constructed by homologous recombination) exposed to 1 mg/L free chlorine. All strains entered the VBNC state, with E. coli O157:H7 showing the highest incidence rate (56.53%), followed by C. sakazakii (29.44%) and S. Typhimurium (25.35%). In contrast, the ΔrelA mutant exhibited minimal VBNC incidence rate (10.84%). VBNC E. coli O157:H7 displayed a 5.15-fold increase in catalase activity and a 30.96% survival rate under ampicillin, indicating enhanced stress resistance. Notably, resuscitation time varied significantly: C. sakazakii recovered within 6 h, whereas E. coli O157:H7 and S. Typhimurium required 9 h; the ΔrelA mutant needed 10 h. Further analysis revealed that key stringent response regulators (SRRs), particularly RelA, drove bacterial chlorine resistance through a multipathway regulatory network. The RelA (encoded by relA) mediated species-specific survival by regulating antioxidant defense (oxyR, soxR), stress adaptation (rpoS), ribosome hibernation (rmf, hpf), cell division arrest (sulA), and efflux/porin systems (tolC, acrA/B/D, ompF). Notably, rmf and tolC showed maximal induction (6.4-fold and 10.0-fold, respectively) in E. coli O157:H7, significantly exceeding those of their orthologs in C. sakazakii and S. Typhimurium (P < 0.05). Collectively, our results reveal the enhanced chlorine resistance of E. coli O157:H7 through SRR-mediated networks and provide practical strategies for pathogen-specific disinfection.