**1. Introduction**

Various *Legionella* species, such as *L. anisa*, *L. feeleii*, *L. longbeachae*, *L. pneumophila*, and *L. micdadei*, can cause legionellosis, which are bacterial infections resulting in either a mild flu-like illness (Pontiac Fever) or a potentially fatal form of pneumonia (Legionnaires' Disease (LD)) [1]. These infections are primarily caused by the inhalation of *Legionella*-contaminated aerosols generated from engineered water systems [2,3]. Of the 74 drinking water-associated outbreaks reported in the US between 2011 and 2014, *Legionella* was responsible for 61% of those outbreaks causing 17% (241/1437) of the illness cases, 88% (200/226) of hospitalizations, and 100% of the outbreak deaths (27/27) [4,5]. *Legionella* presence in the building water systems (BWSs) was cited as the main deficiency leading to those outbreaks underscoring the need to control and prevent *Legionella* growth within these BWSs.

Over the past 40 years, *Legionella* occurrence in drinking water distribution systems (DWDSs) has been well-studied since the first speculation that exposure to aerosols, derived from contaminated water chillers, caused the deadly 1976 American Legion convention outbreak in Philadelphia [6]. *Legionella* has been shown to occur at low levels in drinking water treatment plants and distribution systems and at higher levels in both cold and hot water within BWSs [7–9]. *Legionella* occurrence has been documented in both antiquated (>100 years old) and newly constructed buildings [10,11]. Moreover, numerous studies have reported stable colonization of, and continuing infections caused by, *L. pneumophila* within BWSs over a ≥ 15 year period, despite repeated cycles of shock chlorination, superheating and thermal inactivation, and flushing [12–16].

*Legionella* occurrence in diverse environments and conditions (e.g., in sea-, fresh-, rain-, and treated water; in soil; at temperatures between 4 and 60 ◦C and a pH range of 2.7–8.3) [1] suggests the presence of heterogeneous survival mechanisms acquired through e ffective evolutionary processes. Indeed, *L. pneumophila* is genetically well-equipped due to their (1) high recombination rate and DNA exchange among strains and di fferent *Legionella* species [17] and (2) large repertoire of e ffector proteins allowing exploitation of conserved cellular pathways in various eukaryotic hosts [18,19].

Due to their stringent nutrient requirements, it is hypothesized that *Legionella* growth within DWDSs is largely dependent on their parasitization of drinking water-associated eukaryotic hosts, specifically free-living amoebae (FLA). FLA, such as *Acanthamoeba* spp. and *Vermamoeba vermiformis*, are problematic in drinking waters systems as they can cause diseases like keratitis and can also amplify other human pathogens such as *L. pneumophila* and mycobacteria, including *Mycobacterium intracellulare* [20,21]. *M. intracellulare*, a member of the non-tuberculous mycobacteria (NTM) group, is a significant cause of pulmonary NTM infections [22] and has been isolated in higher frequencies and concentrations in both bulk water and biofilms from drinking water distribution systems compared to *Mycobacterium avium* [23].

Numerous studies have demonstrated extracellular growth of *L. pneumophila* in drinking water. Specifically, four di fferent types of non-*Legionella* drinking water bacteria [24,25]; heat-inactivated cooling tower biofilms, *Escherichia coli*, and *Pseudomonas putida* [26]; extracellular cyanobacterial components [27]; and filtered sterilized drinking water [28] were capable of supporting *L. pneumophila* growth, with the latter also supporting their colonization and growth within biofilms for prolonged periods. Collectively, *Legionella* associations with, and dependencies on, other microbes can explain their vast environmental distribution, ability to survive in DWDSs, and human pathogenicity, especially in engineered environments where confined, close proximity to humans have increased disease risk from this pathogen.

Thus, current approaches to *Legionella* exposure prevention, e.g., through environmental monitoring [29] and/or implementation of building water managemen<sup>t</sup> plans [30,31], require a thorough understanding of their persistence and transmission mechanisms in premise plumbing systems. In this study, to better understand BWS conditions supporting *Legionella* survival, microbial water quality parameters, such as the occurrence of *Legionella* spp., *L. pneumophila*, and *M. intracellulare*; and the FLA hosts, *Acanthamoeba* spp. and *Vermamoeba vermiformis*, and heterotrophic plate count levels, were monitored, along with chemical water quality, engineering, and operational parameters. Determining how various physical, chemical, and microbial BWS characteristics influence *Legionella* occurrence may elucidate ways to minimize and eliminate their growth within these systems.
