Protozoan-Priming and Magnesium Conditioning Enhance Legionella pneumophila Dissemination and Monochloramine...

Opportunistic pathogens (OPs) are of concern in drinking water distribution systems because they persist despite disinfectant residuals. While many OPs garner protection from disinfectants via a biofilm lifestyle, Legionella pneumophila (Lp) also gains disinfection resistance by being harbored within free-living amoebae (FLA). It has been long established, but poorly understood, that Lp grown within FLA show increased infectivity toward subsequent FLA or human cells (i.e., macrophage), via a process we previously coined "protozoan-priming". The objectives of this study are (i) to identify in Lp a key genetic determinant of how protozoan-priming increases its infectivity, (ii) to determine the chemical stimulus within FLA to which Lp responds during protozoan-priming, and (iii) to determine if more infectious forms of Lp also exhibit enhanced disinfectant resistance. Using Acanthamoeba castellanii as a FLA host, the priming effect was isolated to Lp's sidGV locus, which is activated upon sensing elevated magnesium concentrations. Supplementing growth medium with 8 mM magnesium is sufficient to produce Lp grown in vitro with an infectivity equivalent to that of Lp grown via the protozoan-primed route. Both Lp forms with increased infectivity (FLA-grown and Mg²⁺-supplemented) exhibit greater monochloramine resistance than Lp grown in standard media, indicating that passage through FLA not only increases Lp's infectivity but also enhances its monochloramine resistance. Therefore, laboratory-based testing of disinfection strategies should employ conditions that simulate or replicate intracellular growth to accurately assess disinfectant resistance.