**1. Introduction**

*Legionella pneumophila* is an opportunistic pathogen associated with community-acquired and nosocomial infections. It is the causative agen<sup>t</sup> of legionellosis, which includes Legionnaires' disease (LD), a severe atypical pneumonia infection, and Pontiac fever, an acute "flu-like" illness [1]. Globally, the incidence of LD has been increasing. In Europe, the number of notified cases increased from 4921 in 2011 to 11,343 in 2018 [2]. In the US, the number of notified LD cases has increased from 2301 in 2005 [3] to 7104 in 2018 [4], a 300% increase. Globally, the fatality rate of LD ranges from 2.2–10.3%, with the lowest in Singapore and the highest in European countries [5]. In nosocomial outbreaks the fatality rate can reach up to 48% [6–8].

The genus *Legionella* is comprised of 60 species and 80 distinct serogroups [9]. Globally, *L. pneumophila* is the primary aetiological agen<sup>t</sup> of LD. In Europe and the US, *L. pneumophila* serogroup (SG1) is responsible for 70–92% reported cases [8]. According to WHO, 20–30% infections are caused by other *L. pneumophila* serogroups and only 5–10% are caused by other *Legionella* species (*L. micdadei*, *L. bozemanii*, *L. dumo*ffi*i* and *L. longbeachae*) [10]. However, unlike rest of the world, in Australia and New Zealand, *L. longbeachae* is associated with ≈ 50% reported cases of legionellosis [11,12].

*L. pneumophila* is ubiquitous in manufactured water systems [10] and in the USA has been identified as the primary cause of all potable water related outbreaks [13]. Manufactured water systems, building plumbing systems, recreational water, cooling towers and humidifiers are major sources of *L. pneumophila* [10]. Inside these plumbing structures, *Legionella* and protozoan hosts are incorporated within biofilms. Factors like water stagnation, higher levels of organic carbon and moderate temperatures can increase the rate of biofilm formation [14,15]. Transmission occurs through inhalation or aspiration of contaminated aerosols or water [16]. *L. pneumophila* maintains long term contamination of manufactured water systems through its growth within protozoan hosts, association with biofilms and disinfectant resistance or tolerance [17,18]. Freshwater amoebae are the natural eukaryotic hosts of *Legionella*; whereas, humans are considered accidental hosts [19]. In the human body, *Legionella*–contaminated aerosols are inhaled into the lungs and phagocytosed by alveolar macrophages. The alveolar macrophages behave like amoebae hosts and facilitate the intracellular division and propagation of *Legionella*, resulting in LD [20,21].

Understanding the interactions between *L. pneumophila* and protozoan hosts is essential to inform water treatment and risk managemen<sup>t</sup> strategies for the prevention of LD. Protozoan hosts play an important role in the ability of *L. pneumophila* to survive exposure to physiochemical and environmental stresses. Protozoans facilitate the intracellular replication and packaging of live bacterial cells in the stress resistant membrane bound infectious export vesicles [22,23]. The cysts of cyst-forming amoebae provide a protective shelter from prolonged environmental stress [24]. There are numerous reports describing existence of *L. pneumophila* harboring within protozoans from thermally-, chemically-, and UV radiation-treated potable water supplies and storage reservoirs [25]. Protozoan hosts and environmental stress may facilitate the genesis of highly resistant and potentially infectious viable but non-culturable (VBNC) *L. pneumophila* [26,27]. Importantly, water storage facilities and distribution networks of many countries have been shown to be highly contaminated with protozoans that may act as hosts for *L. pneumophila* (>0–4500 cell/L cell density) [28].

This systematicliterature review collated studieswhich detected*L. pneumophila* in association/connection with protozoan hosts from hospital or municipal potable water distribution systems and discusses this relationship under diverse environmental conditions. The effectiveness of different physical and chemical water treatment methods to control the *L. pneumophila* and its protozoan hosts is described and implications for the control and managemen<sup>t</sup> of these water distribution systems is explored.
