ABSTRACT: In this study, droplet digital PCRTM (ddPCRTM) was used to characterize total Legionella spp. and five specific Legionella species from source (groundwater) to exposure sites (taps and cooling towers). A total of 42-10 L volume water samples were analyzed during this study: 12 from a reservoir (untreated groundwater and treated water storage tanks), 24 from two buildings (influents and taps), and six from cooling towers, all part of the same water system. The approximate water age (time in the system) for all sample locations are as follows: ~4.5, 3.4, 9.2, 20.8, and 23.2 h (h) for the groundwater to the reservoir influent, reservoir influent to the reservoir effluent, reservoir effluent to building Fa (building names are abbreviated to protect the privacy of site location), building ERC and the cooling towers, respectively. Results demonstrated that gene copies of Legionella spp. (23S rRNA) were significantly higher in the cooling towers and ERC building (p < 0.05) relative to the reservoir and building Fa (closest to reservoir). Legionella spp. (23S rRNA) were found in 100% (42/42) of water samples at concentrations ranging from 2.2 to 4.5 Log10 GC/100 mL. More specifically, L. pneumophila was found in 57% (24/42) of the water samples, followed by L. bozemanii 52% (22/42), L. longbeachae 36% (15/42), L. micdadei 23% (10/42) and L. anisa 21% (9/42) with geometric mean concentrations of 1.7, 1.7, 1.4, 1.6 and 1.7 Log10 GC/100 mL, respectively. Based on this study, it is hypothesized that water age in the distribution system and the premise-plumbing system as well as building management plays a major role in the increase of Legionella spp., (23S rRNA) and the diversity of pathogenic species found as seen in the influent, and at the taps in the ERC building-where the building water quality was most comparable to the industrial cooling towers. Other pathogenic Legionella species besides L.pneumophila are also likely amplifying in the system; thus, it is important to consider other disease relevant species in the whole water supply system-to subsequently control the growth of pathogenic Legionella in the built water environment.
