Protozoan-Priming and Magnesium Conditioning Enhance <i>Legionella pneumophila</i> Dissemination and Monochloramine Resistance

Abstract

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, <i>Legionella pneumophila</i> (<i>Lp</i>) also gains disinfection resistance by being harbored within free-living amoebae (FLA). It has been long established, but poorly understood, that <i>Lp</i> 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 <i>Lp</i> a key genetic determinant of how protozoan-priming increases its infectivity, (ii) to determine the chemical stimulus within FLA to which <i>Lp</i> responds during protozoan-priming, and (iii) to determine if more infectious forms of <i>Lp</i> also exhibit enhanced disinfectant resistance. Using <i>Acanthamoeba castellanii</i> as a FLA host, the priming effect was isolated to <i>Lp</i>'s <i>sidGV</i> locus, which is activated upon sensing elevated magnesium concentrations. Supplementing growth medium with 8 mM magnesium is sufficient to produce <i>Lp</i> grown <i>in vitro</i> with an infectivity equivalent to that of <i>Lp</i> grown via the protozoan-primed route. Both <i>Lp</i> forms with increased infectivity (FLA-grown and Mg²⁺-supplemented) exhibit greater monochloramine resistance than <i>Lp</i> grown in standard media, indicating that passage through FLA not only increases <i>Lp</i>'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.