García Esteban, María TeresaJones, SnakePelaz, CarmenMillar, Richard D.Kwaik, Yousef Abu2025-10-292025-10-292007-02-12García, M. T., Jones, S., Pelaz, C., Millar, R. D., & Abu Kwaik, Y. (2007). Acanthamoeba polyphaga resuscitates viable non-culturable Legionella pneumophila after disinfection. Environmental Microbiology, 9(5), 1267-1277. https://doi.org/10.1111/J.1462-2920.2007.01245.X1462-291210.1111/j.1462-2920.2007.01245.xhttps://hdl.handle.net/20.500.14352/125487M.T. García is supported by a grant from the Ramon Areces Foundation from Spain. Y.A.K. is supported by the Commonwealth of Kentucky Research Challenge Trust Fund. We thank Maëlle Molmeret and Rexford Asare and all the members of the Abu Kwaik laboratory for technical advice and helpful discussions.Amoebae are the natural hosts for Legionella pneumophila and play essential roles in bacterial ecology and infectivity to humans. When L. pneumophila colonizes an aquatic installation, it can persist for years despite repeated treatments with disinfectants. We hypothesized that freshwater amoebae play an important role in bacterial resistance to disinfectants, and in subsequent resuscitation of viable non-culturable (VNC) L. pneumophila that results in re-emergence of the disease-causing strain in the disinfected water source. Our work showed that in the absence of Acanthamoeba polyphaga, seven L. pneumophila strains became non-culturable after treatment by 256 p.p.m. of sodium hypochlorite (NaOCl). In contrast, intracellular L. pneumophila within A. polyphaga was resistant to 1024 p.p.m. of NaOCl. In addition, L. pneumophila-infected A. polyphaga exhibited increased resistance to NaOCl. When chlorine-sterilized water samples were co-cultured with A. polyphaga, the non-culturable L. pneumophila were resuscitated and proliferated robustly within A. polyphaga. Upon treatment by NaOCl, uninfected amoebae differentiated into cysts within 48 h. In contrast, L. pneumophila-infected A. polyphaga failed to differentiate into cysts, and L. pneumophila was never detected in cysts of A. polyphaga. We conclude that amoebic trophozoites protect intracellular L. pneumophila from eradication by NaOCl, and play an essential role in resuscitation of VNC L. pneumophila in NaOCl-disinfected water sources. Intracellular L. pneumophila within trophozoites of A. polyphaga block encystation of the amoebae, and the resistance of both organisms to NaOCl is enhanced. To ensure long-term eradication and complete loss of the VNC state of L. pneumophila, we recommend that Legionella-protozoa co-culture should be an important tool to ensure complete loss of the VNC state of L. pneumophila.engjournal article1462-2920https://doi.org/10.1111/j.1462-2920.2007.01245.xhttps://enviromicro-journals.onlinelibrary.wiley.com/doi/10.1111/j.1462-2920.2007.01245.xrestricted access579.61616.9615.28Microbiología (Biología)Enfermedades infecciosas2414 Microbiología3205.05 Enfermedades Infecciosas2302.20 Química Microbiológica