Ozonation Eliminates Viable but Not Culturable Legionella pneumophila More Effectively than Chlorination

Legionella pneumophila, the causative agent of Legionnaires' disease, remains a major public health concern in water systems. Chlorination is the most widely used disinfection strategy but presents important limitations, including reduced stability in hot water systems and the generation of carcinogenic by-products. This study evaluated ozonation as an alternative disinfection method, comparing its efficacy with chlorination against different physiological states of L. pneumophila, including culturable and viable but non-culturable (VBNC) cells, as well as biofilm-associated bacteria and amoebal hosts. The biocidal effect of ozone and chlorination on planktonic L. pneumophila was assessed by culture and viability-qPCR. Biofilm viability was evaluated using fluorescein diacetate staining, whereas amoeba viability was determined by flow cytometry. Ozonation achieved complete inactivation of both culturable and VBNC L. pneumophila within 5 min. In contrast, chlorination achieved complete loss of culturability only at high concentrations and was associated with the persistence of VBNC cells. Neither ozonation nor chlorination significantly reduced established biofilms or amoebae. These findings indicate that ozonation is a promising strategy for rapid inactivation of planktonic L. pneumophila and may overcome important limitations associated with chlorine-based treatments.