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

Abstract

bWater 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 waterbased pathogen Legionella pneumophila and its FLA hosts Acanthamoeba polyphaga, A. castellanii, and Vermamoeba vermiformis. Exposure to sGW for 72 h resulted in significant inhibition (P < 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%), and V. 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 log 10 CFU, P < 0.001) compared to controls (⌬0 to 0.1 log 10 CFU) with flow cytometric analysis revealing immediate changes in membrane permeability. Furthermore, reverse transcription-quantitative PCR was performed on total RNA isolated from L. pneumophila cells at 0 to 48 h after sGW incubation, and genes associated with virulence (gacA, lirR, csrA, pla, and sidF), the type IV secretion system (lvrB and lvrE), and metabolism (ccmF and lolA) 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 and L. pneumophila behavior in GW systems. G ray water (GW) usage has proven to be both an economically and a logistically suitable solution to alleviate the stresses from increasing potable water demand and climate/demographic change (1), when used (following adequate treatment) in largeand small-scale irrigation, toilet flushing, and car washing (2-4). Defined as household wastewater that is not contaminated with toilet waste, GW is further categorized as light (derived from bathroom showers, tubs, and washbasins) and dark (derived from laundry machines, dishwashers, and kitchen sinks) gray water (5). In households surveyed internationally, light GW constitutes about 47% of total GW, with the remaining 27 and 26% originating from kitchen sinks/dishwashers and laundry washing machines, respectively (reviewed in reference 5). The composition of GW varies greatly with light GW containing mostly personal care products and dark GW composed mostly of dish and laundry detergents, food residue, and fats, oils, and grease.Few studies report on the densities of microbial contaminants within treated GW, and of these, most present fecal indicator data, with less information on pathogens (6). When reusing GW there is concern for fecally borne pathogens (such as species in the bacterial genera Campylobacter, Salmonella, and Vibrio [7-10]), as well as an emerg...

Short-term effects of atmospheric pressure, temperature, and rainfall on notification rate of community-acquired Legionnaires' disease in four European countries

“…Unusually high temperature may be associated with long-lasting dryness not compensated for by rainfall in seasons with intense evaporation. The effect of the studied environmental factors on the protozoan hosts of Legionella may also play a role that should be investigated further [26, 27]. It may be that Legionella or its protozoan hosts have lower optimum temperature growth and range in some regions or under certain climates.…”

Legionella pneumophila: From potable water to treated greywater; quantification and removal during treatment

“…Thus, the increase in bulk levels of L. pneumophila might be explained by Legionella growth inside its natural hosts. In a recent study about the behavior of amoebas in synthetic GW, Buse et al (2015) concluded that GW conditions may promote interactions between water-based pathogens such as Legionella and amoeba hosts. However, no data is available about the amoeba concentrations in the studied greywater systems.…”

Occurrence of Legionella in Nonpotable Reclaimed Water

“…Despite the increased awareness of the risk of waterborne legionellosis, there have not been any reported outbreaks resulting from the use of reclaimed water. Legionella is known to occur in reclaimed water, and several studies have reported detection of the organism in a variety of systems (Buse et al , Ajibode et al , Jjemba et al , Birks et al ). Birks et al () detected Legionella pneumophila serotypes 2–14 in the three raw graywater samples, but found no significant microbiological growth in the Millennium Dome building water system, presumably because of the high level of reverse osmosis treatment and the maintenance of a sodium hypochlorite disinfectant residual.…”

“…No outbreaks have been attributed to the use of reclaimed water, although a number of studies have identified Legionella sp. in recycled water (Buse et al , Ajibode et al , Jjemba et al , Birks et al ). Water used for unrestricted urban reuse (USEPA ) is not regulated to control risks from Legionella spp., and no requirements exist for monitoring or control after the treatment process.…”