**1. Introduction**

*Legionella pneumophila* is an aerobic, Gram-negative bacterium that causes Legionellosis, a severe form of pneumonia, following inoculation with contaminated aerosol [1]. This bacterial infection manifests as two clinical forms that include Legionnaires' disease, which is a life-threatening respiratory disease, and Pontiac fever, a milder self-limiting illness [2,3]. Among the sixteen currently identified serogroups of *L. pneumophila*, serogroup 1 is involved in the majority of infections [4,5]. This microorganism is ubiquitous throughout natural and artificial aquatic environments [6]. Legionellosis outbreaks are frequently related to contaminated water systems that produce aerosols, which occurs primarily within cooling towers [7]. Indeed, cooling towers provide ideal conditions for pathogen growth, as they frequently possess temperatures above 20 ◦C, at which *L. pneumophila* can proliferate [8–10].

Free-living amoebae (FLA) are ubiquitous protozoa that inhabit common aquatic environments and are frequently co-isolated with *L. pneumophila* in water cooling towers [11,12]. FLA are predatory

and consume bacteria to facilitate their growth [13,14]; however, some bacteria such as *L. pneumophila* have evolved to avoid the phagolysosome fusion and can multiply within FLA, ultimately killing these amoebae before disseminating into the environment [9,15–17]. Furthermore, amoeba cysts can provide *L. pneumophila* with protection against unfavourable conditions and chemical treatments. Therefore, the association between FLA and this pathogen makes the control and monitoring of water-cooling towers difficult and makes eradication of *L. pneumophila* almost impossible [18,19].

Previous studies, however, have demonstrated that all FLAs do not exhibit the same behaviours when they come into contact with *L. pneumophila* strains. While *Acanthamoeba* sp. and *Vermamoeba* (formerly *Hartmannella*) *vermiformis* support the intracellular growth of *L. pneumophila,* the *Willaertia magna* strain C2c Maky has been demonstrated to eliminate the *L. pneumophila* serogroup 1 strain Paris ATCC 33152 [20], which is a virulent pathogen strain responsible for severe legionellosis epidemics in France [21]. *W. magna* C2c Maky is a free-living amoeba that is a member of the Vahlkampfiidae family [22]. This amoeba is a thermophilic FLA that is isolated from the water of thermal swimming pools (http://www.amoeba-biocide.com/en/page/learn-more-about-willaertia-magna-c2c-maky), and it has the capacity to grow at high temperatures (up to 44 ◦C) in xenic or axenic media. The living forms of this amoeba include a large trophozoite (50–100 μm) and a cys<sup>t</sup> (18–21 μm) form, and it can produce temporary flagella [22,23]. The lack of pathogenicity of this amoeba was demonstrated by cytotoxicity testing on human cells and was confirmed by genomic analysis [24]. According to these findings, the Amoéba company developed a natural biocide using *W. magna* C2c Maky to eliminate *L. pneumophila* as an alternative to chemical biocides (http://www.amoeba-biocide.com/en/page/revolutionary-biocide). The present study is performed to verify the elimination and the absence of the reservoir effect. Specifically, the decrease in the number of internal *L. pneumophila* and the absence of internal *L. pneumophila* multiplication within *W. magna* C2c Maky, when both microorganisms are co-cultured, is confirmed. The assay is performed by examining adhesion (the usual way of life for free-living amoeba) with three strains of *L. pneumophila* to assess the consistency of amoeba behaviour toward legionella strains. The assay lasts for one week and includes a daily count of intracellular *L. pneumophila* and amoebas by culture and Trypan blue staining, respectively. The behaviour of *W. magna* C2c Maky is compared to that of *W. magna* Z503 to determine if two amoeba strains of the same species have the same behavior. Moreover, it is compared to *A. castellanii*, an amoeba known to multiply amoeba-resistant bacteria such as the three *L. pneumophila* strains studied.
