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Exposure to synthetic greywater inhibits amoebae encystation and alters expression of Legionella pneumophila virulence genes
Buse, H., J. Lu, AND N. Ashbolt. Exposure to synthetic greywater inhibits amoebae encystation and alters expression of Legionella pneumophila virulence genes. APPLIED AND ENVIRONMENTAL MICROBIOLOGY. American Society for Microbiology, Washington, DC, 81(2):630-639, (2015).
The aim of this study was to examine the behavior of the FLA Acanthamoeba polyphaga (Ap), A. castellanii (Ac), and Vermamoeba vermiformis (Vv) and the water-based pathogen, L. pneumophila (Lp) within GW. For consistency and control, synthetic GW (sGW) was utilized to examined the survival and encystation rates of Ap, Ac, and Vv as well as Lp culturability, membrane permeability status, and gene expression during sGW exposure, so as to reveal potential human health risk from GW use.
Water conservation efforts have focused on greywater (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 amoebae (FLA) hosts within GW. Using synthetic greywater (sGW) we examined three strains of the water-based pathogen Legionella pneumophila (Lp) and its FLA hosts Acanthamoeba polyphaga (Ap), A. castellanii (Ac) and Vermamoeba vermiformis (Vv). Exposure to sGW for 72 h resulted in significant inhibition (P < 0.0001) of amoebal encystation versus control-treated cells, with the percentage of cysts in sGW v controls: Ap (0.6 v. 6 %), Ac (2 v. 62 %), and Vv (1 v. 92%); suggesting sGW-induced maintenance of the actively feeding trophozite form. During sGW exposure, Lp culturability decreased as early as 5 h (1.3-2.9 log10 CFU, P < 0.001) compared to controls (Δ0-0.1 log10 CFU) with flow cytometric analysis revealing immediate changes in membrane permeability. Furthermore, reverse transcription/qPCR was performed on total RNA isolated from Lp cells, at 0-48 h post-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 environmental pathogens and FLA hosts, through amoebal encystment inhibition, and alteration of bacterial virulence gene expression. Thus their behavior in GW needs to be further explored to assess potential health risks associated with greywater usage, particularly if aerosolized.
Record Details:Record Type: DOCUMENT (JOURNAL/PEER REVIEWED JOURNAL)
Organization:U.S. ENVIRONMENTAL PROTECTION AGENCY
OFFICE OF RESEARCH AND DEVELOPMENT
NATIONAL EXPOSURE RESEARCH LABORATORY
MICROBIOLOGICAL AND CHEMICAL EXPOSURE ASSESSMENT DIVISION
BIOHAZARD ASSESSMENT RESEARCH BRANCH