ClO₂-induced VBNC state in Enterococcus faecalis poses a health risk in drinking water systems

E. faecalis is widely distributed in water and poses a serious threat to human health. Although ClO₂ is commonly used for drinking water disinfection, its risk of inducing E. faecalis into the viable but non-culturable (VBNC) state remains unclear. This study collected actual water samples throughout the entire drinking water treatment process (source water, pre-filtered water, filtered water, and pipeline network water) to simulate a disinfection stress environment. It systematically investigated the VBNC conversion characteristics of E. faecalis and its potential health risks. Flow cytometry results showed that 0.6-2.0 mg/L ClO₂ stress E. faecalis to form the VBNC state in all four types of water. VBNC cells maintained membrane integrity but exhibited altered metabolism: increased ATP, decreased esterase activity and respiration intensity. Crucially, VBNC bacteria maintained infectivity by adhering to intestinal (Caco-2) and blood-brain barrier cells (HBMEC) (49.02-68.76% of control), induce the release of inflammatory cytokines (IL-1β, IL-6, IL-8, TNF-α), and survive and recover in macrophages and sheep red blood cells (reaching 7.0-8.0 Log CFU/mL). Transcriptomics revealed downregulated energy metabolism (amino acid transport, TCA cycle) and ATP hydrolysis genes (uvrA, ftsH) during VBNC entry, whereas resuscitation involved upregulated pyruvate metabolism (pdhA, pyk), carbohydrate transport (ABC transporters), and nucleotide synthesis (pnp) genes with ribosomal reactivation. Water quality (e.g., Ca²⁺ deficiency) critically influenced VBNC formation and resuscitation. This study provides new insights into the formation of the VBNC state of pathogenic bacteria under ClO₂ disinfection stress in drinking water systems and emphasizes the need to strengthen disinfection strategies in water treatment systems.