Repeat Prolonged Chlorination at Low Dose Induces Chlorine Tolerance in Legionella pneumophila via Viable but Non-culturable State

Chlorine-resistant Legionella pneumophila, the causative agent of fatal pneumonia, poses a major global public health threat. However, the mechanistic basis for this pathogen's resistance evolution remains elusive. Our study demonstrates that a minimally dormant or the early stages of viable but non-culturable (VBNC) state of bacteria is critical to chlorine tolerance development, representing an initial stage of resistance acquisition. Prolonged low-dose chlorination (12 h exposure to 2 mg/L chloramine-T) induced VBNC transformation in L. pneumophila, with a resuscitation lag time ∼10-fold longer than that of parallel nonchlorinated but starved bacteria. Upon resuscitation, secondary chlorination (3 or 12 h) of stationary-phase cells maintained growth rates similar to those of untreated cells, but with significantly shortened lag times (to ∼26 and ∼41 h, vs ∼40 and ∼47 h for single-chlorinated groups, with p < 0.01 for the 3 h treatment). Flow cytometry showed that rechlorinated groups had a higher proportion of active cells and a lower proportion of death-analogous cells than single-chlorinated counterparts, indicating the emergence of a chlorine-tolerant subpopulation. These findings directly link the VBNC state to bacterial resistance evolution, underscoring the urgent need to investigate dormant bacteria to control antimicrobial resistance spread in water systems.