Particle-associated and free-living Legionella spp. in drinking water.

The Legionella genus houses several opportunistic pathogens that cause legionellosis, which ranges from mild (Pontiac Fever) to severe (Legionnaires' disease). Legionella pneumophila (Lp), the leading observed cause of legionellosis, persists in drinking water distribution systems (DWDSs) and building plumbing systems due to factors such as intrinsic disinfectant resistance and shielding by biofilms and free-living amoebae. Remediation efforts, therefore, might be hindered if Lp is primarily associated with biofilms, free-living amoebae, or other particles. However, the extent to which Lp and other Legionella spp. in drinking water occur as free-living organisms versus associated with free-living amoebae or other particles is unclear. In this study, we quantified free-living and particle-associated Lp and Legionella spp. in first-draw and flushed drinking water samples. Citizen scientists collected 1-L cold tap samples (first-draw and flushed) at 19 sites across the U.S. and across three seasons. Free-living and particle-associated microorganisms were size fractionated via membrane filtration, and Legionella spp. and Lp were quantified by digital polymerase chain reaction. Further, amplicon sequencing targeting the Legionella genus was used to explore the diversity of Legionella spp. at each site. The concentration of free-living Legionella spp. was significantly higher than that of particle-associated Legionella spp. (p=2.86×10-9) in paired samples at each site, and significantly higher concentrations of total Legionella spp. were found in surface water than in groundwater systems (p=0.020). Lp detection was geographically and temporally sporadic, with detections occurring at seven sites. Sequencing results showed that the site strongly influenced the specific Legionella spp. present. These results emphasize the influence of source water type and site on Legionella spp. in DWDSs and building plumbing. Further, this study suggests that consumers are more frequently exposed to free-living than to particle-associated Legionella spp. at the tap.