**2. Results**

#### *2.1. L. pneumophila Survival in Coculture Medium*

The survival of the three *L. pneumophila* strains in the calf serum-casein-yeast extract medium (SCYEM) was evaluated at 22 ◦C and 37 ◦C (Figure 1a,b). The survival of *L. pneumophila* Lens decreased to 2 × 10<sup>4</sup> CFU/mL and to 40 CFU/mL in SCYEM medium within 96 h at 22 ◦C and 37 ◦C, respectively. The survival of *L. pneumophila* Paris decreased to 7 × 10<sup>3</sup> CFU/mL and to 1 CFU/mL in SCYEM medium within 96 h at 22 ◦C and 37 ◦C, respectively. Finally, the survival of *L. pneumophila* Philadelphia decreased to 3 × 10<sup>3</sup> CFU/mL and to 2 CFU/mL in SCYEM medium within 96 h at 22 ◦C and 37 ◦C, respectively.

**Figure 1.** *L. pneumophila* survival in coculture medium at 22 ◦C (**a**) and 37 ◦C (**b**). Results are expressed as the mean +/− 95% CI (Confidence Interval based on the standard error of the mean).

#### *2.2. Amoeba Survival in coculture Medium*

Survival of the three amoebas in the presence or in absence of bacteria was evaluated over 96 h at 22 ◦C and 37 ◦C in coculture medium (Figure 2a,b). The three amoeba strains could be maintained in SCYEM medium for 96 h in the presence or absence of bacteria at 22 ◦C and 37 ◦C with the exception of *A. castellanii* when co-cultivated with *L. pneumophila* strains. Found at the end of the experiment, the control condition of *A. castellanii* in the absence of bacteria was maintained at 2 × 10<sup>5</sup> cells/mL, while in the presence of *L. pneumophila* Lens, Paris, and Philadelphia, the amoeba number decreased to 556, 444 and 2333 cells/mL, respectively (Figure 2b). *A. castellanii* could not survive in the presence of the three *L. pneumophila* strains at 37 ◦C.

**Figure 2.** Amoeba survival at 22 ◦C (**a**) and 37 ◦C (**b**) in coculture medium in the presence or absence of the three *L. pneumophila* strains (Lens, Paris, and Philadelphia). The red bar is the detection limit of the Malassez cell counting. Results are expressed as the mean +/− 95% CI (Confidence Interval based on the standard error of the mean).
