Abstract
This study systematically compared the induction and resuscitation characteristics of the viable but non-culturable (VBNC) state in Klebsiella pneumoniae FY170-1 following sublethal exposure to sodium hypochlorite (NaClO) or glutaraldehyde (GA). Treatment with 30 mg/L NaClO or 60 mg/L GA for 60 min reduced culturability to below the detection limit (<1 CFU/mL). However, CTC staining showed that 50.80% and 63.44% of cells, respectively, retained respiratory activity, while SYTO 9/PI staining indicated that membrane integrity was largely preserved, consistent with induction of the VBNC state. Scanning electron microscopy revealed distinct morphological alterations in the two groups. NaClO-induced VBNC cells showed surface depressions and wrinkling, consistent with oxidative damage, whereas GA-induced cells exhibited filamentous and net-like surface structures, consistent with aldehyde-mediated cross-linking. Among the tested additives, sodium succinate showed the strongest resuscitation-promoting effect under the experimental conditions, with OD600 increasing after approximately 2 h of incubation. Post-resuscitation analysis further revealed marked differences between the two VBNC states. In resuscitated NaClO-induced VBNC cells, ATP partially recovered, but reactive oxygen species remained elevated and catalase activity showed little recovery. In contrast, resuscitated GA-induced VBNC cells exhibited lower ATP recovery but more rapid normalization of ROS and better recovery of oxidative stress-related parameters. Total protein analysis and SDS-PAGE further supported distinct patterns of protein-level alteration between the two treatments. Overall, these findings suggest that NaClO and GA induce phenotypically distinct VBNC states in K. pneumoniae, with different recovery behaviors and stress response profiles. Sodium succinate was identified as the most effective recovery-promoting additive under the tested conditions. These results highlight the risk of underestimating bacterial survival when culturability is used as the sole indicator of disinfection efficacy and support the need for more comprehensive viability assessment.