Legionella’s 'Dormancy Trick' to Evade Chlorination: New Water Disinfection Target to Tackle Chlorine-resistant Bacteria
Chlorination stands as one of the most successful public health interventions in human history, acting as a cornerstone for controlling pathogens in global water systems. Yet a critical puzzle persists: Legionella pneumophila (L. pneumophila)—the causative agent of fatal Legionnaires' disease—continues to drive a global rise in infection rates, even though the bacterium is theoretically sensitive to chlorine. For years, the academic community has suspected that genetic mutations might explain its growing chlorine resistance, but the mechanistic basis for this pathogen's resistance evolution has remained elusive.
A research team led by Chair Professor Yan Zheng from the School of Environmental Science and Engineering at Southern University of Science and Technology (SUSTech) has recently solved this mystery. Their study has revealed that when L. pneumophila evades chlorine damage by entering a minimally dormant or early-stage viable but non-culturable (VBNC) state—a biological "hiding mode"—it emerges with enhanced chlorine tolerance after revival. This breakthrough not only links the VBNC state directly to bacterial resistance evolution but also offers a novel target for optimizing water disinfection and curbing the spread of antimicrobial resistance in water systems.
Their findings, titled “Repeat Prolonged Chlorination at Low Dose Induces Chlorine Tolerance in Legionella pneumophila via Viable but Non-culturable State”, have been published in the authoritative environmental health journal Environment & Health.
To mimic real-world conditions—where water pipelines are continuously exposed to low-concentration chlorine—the team designed controlled experiments using chloramine-T (CAT), a stable chlorine-based disinfectant. Their key findings, rooted in rigorous laboratory testing, uncover the step-by-step process of Legionella's tolerance acquisition.
The team exposed the bacteria to 2 mg/L CAT for 12 h—a scenario mirroring long-term low-dose chlorination in real water systems. This treatment successfully pushed the bacteria into the VBNC state: while the cells retained viability, they could no longer form colonies on routine nutrient agar plates (a trait that often leads to misclassification as "dead" in standard microbial detection assays) (Figure 1B).
The VBNC L. pneumophila took approximately 47 h to recover from dormancy and resume active growth—nearly 10-fold longer than their parallel non-chlorinated but starved bacteria (which regrew in just 4–6 h). This extended lag time reflects the metabolic reactivation and damage repair the bacteria undergo during dormancy, laying the groundwork for tolerance (Figure 1A).
Figure 1. (A, B) Growth of L. pneumophila after starvation (in deionized water, i.e., DI-H2O) and chlorination. (C) Proportion of active subpopulation P1 (see Figure 3) in differently treated cells.
After resuscitating the VBNC bacteria in a nutrient-rich medium, the team subjected the bacteria (now in stationary phase) to a second round of chlorination (3 or 12 h, the same 2 mg/L CAT concentration), followed by a second round of post-treatment culture. They found that the re-chlorinated cells maintained growth rates nearly identical to those of non-treated L. pneumophila, but with significantly shortened lag time: ~26 h for 3 h re-chlorination (vs. ~40 h for single-chlorinated groups, with p < 0.01) and ~41 h for 12 h re-chlorination (vs. ~47 h for single-chlorinated groups) (Figure 2).
Figure 2. Growth behavior characterization. Lag times (A) and growth rates (B) of L. pneumophila in liquid nutrient culture medium (BYE-Culture) after four treatments with varying durations.
The team then used flow cytometry to visualize the bacterial subpopulation composition. Results showed that resuscitated L. pneumophila has a distinctly different subpopulation composition from non-treated original bacteria (Figure 1C). In parallel, the non-chlorinated but starved bacteria have a similar subpopulation composition to their parental strain after regrowth. Moreover, re-chlorinated groups (Figure 3, the third plot from the left in each panel) had a higher proportion of active cells and a lower proportion of death-analogous cells (compared with those before the treatment, i.e., the second plot here) than single-chlorinated counterparts, indicating the emergence of a chlorine-tolerant subpopulation. These clear shifts in subpopulation structure directly confirm the emergence of a chlorine-tolerant bacterial subset—one that originates from the VBNC state—which may account for the shortened lag time after re-chlorination.
Figure 3. Subpopulation dynamics of L. pneumophila under sequential treatments.
A key insight from the study is that the VBNC state acts as an "initial stage of resistance acquisition"—a stepping stone for further evolution. Combined with existing evidence, the researchers note that these tolerant cells are likely to eventually evolve into fully chlorine-resistant bacteria (CRB) with repeated exposure to chlorination. This explains why global Legionnaires’ disease rates continue to rise despite widespread chlorination: standard disinfection practices may inadvertently promote the VBNC state, creating a "breeding ground" for resistant strains.
Beyond scientific significance, the findings translate directly to improved real-world water disinfection, addressing the urgent need to control dormant bacteria in water systems: 1. Developing VBNC-specific detection tools (e.g., targeted molecular assays) will enable accurate risk assessment; 2. Refining chlorination strategies by re-evaluating dose cycles to reduce VBNC formation; 3. Targeting VBNC pathways to block the bacterial ability to enter/exit this state—cutting off tolerance acquisition at its source.
Dr. Xiaofei Yuan from Zhejiang Lab (formerly of SUSTech), is the first author of the paper. Chair Professor Yan Zheng is the corresponding author, and SUSTech is the corresponding institution.
Paper Link: https://doi.org/10.1021/envhealth.5c00360
