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Water conservation efforts have focused on gray water (GW) usage, especially for applications that do not require potable water quality. However, there is a need to better understand environmental pathogens and their free-living amoeba (FLA) hosts within GW, given their growth potential in stored gray water. Using synthetic gray water (sGW) we examined three strains of the water-based pathogenLegionella pneumophila and its FLA hostsAcanthamoeba polyphaga ,A. castellanii , andVermamoeba vermiformis . Exposure to sGW for 72 h resulted in significant inhibition (P &lt; 0.0001) of amoebal encystation versus control-treated cells, with the following percentages of cysts in sGW versus controls:A. polyphaga (0.6 versus 6%),A. castellanii (2 versus 62%), andV. vermiformis (1 versus 92%), suggesting sGW induced maintenance of the actively feeding trophozoite form. During sGW exposure,L. pneumophila culturability decreased as early as 5 h (1.3 to 2.9 log10 CFU,P &lt; 0.001) compared to controls (Δ0 to 0.1 log10 CFU) with flow cytometric analysis revealing immediate changes in membrane permeability. Furthermore, reverse transcription-quantitative PCR was performed on total RNA isolated fromL. pneumophila cells at 0 to 48 h after sGW incubation, and genes associated with virulence (gacA ,lirR ,csrA ,pla , andsidF ), the type IV secretion system (lvrB andlvrE ), and metabolism (ccmF andlolA ) were all shown to be differentially expressed. These results suggest that conditions within GW may promote interactions between water-based pathogens and FLA hosts, through amoebal encystment inhibition and alteration of bacterial gene expression, thus warranting further exploration into FLA andL. pneumophila behavior in GW systems.

Exposure to Synthetic Gray Water Inhibits Amoeba Encystation and Alters Expression of Legionella pneumophila Virulence Genes