<i>L</i>. <i>pneumophila</i> sg. 1 growth in axenic conditions and in co-culture with the two <i>Acanthamoeba</i> strains in PYG liquid media.

<p>Samples were taken at different time points and plated on BCYE agar plates. Data are presented as means ± SD (error bars; n = 3).</p

Calculated time for a 4-log reduction of <i>L</i>. <i>pneumophila</i> sg. 1 env. associated with <i>A</i>.<i>castellanii</i> CCAP 1534/2 and <i>Acanthamoeba</i> sp. 155 after the exposure to different concentrations of free chlorine and temperatures.

<p>Inactivation kinetics adjusted to first-order models. R² values showed the robustness of the models.</p

Effect of thermal treatments on the inactivation of 5 <i>Legionella</i> strains.

<p>Bacterial inactivation was determined using viable counts on BCYE agar medium. Data are presented as means ± SD (columns and error bars; n = 3). Statistical differences between means within each time point were represented assigning different letters to the bar plot. The same letter was assigned to bars with no significant differences between them. Statistical analyses were performed by ANOVA and pairwise Fisher’s LSD test (<i>p</i><0.05).</p

Effect of free chlorine 0.2 mg L^-1 and 0.5 mg L^-1, on the inactivation of 5 <i>Legionella</i> strains.

<p>Bacterial inactivation was determined using viable counts on BCYE agar medium. Data are presented as means ± SD (columns and error bars; n = 3). Statistical differences between means within each time point were represented assigning different letters to the bar plot. The same letter was assigned to bars with no significant differences between them. Statistical analyses were performed by ANOVA and pairwise Fisher’s LSD test (<i>p</i><0.05).</p

Effect of free chlorine, 1.2 mg L^-1 and 2.5 mg L^-1, on the inactivation of 2 <i>Acanthamoeba</i> strains treating separately trophozoites and cysts.

<p>Amoebal inactivation was determined using an adaptation of the MPN method. Data are presented as means ± SD (columns and error bars; n = 3). Statistical differences between means within each time point were represented assigning different letters to the bar plot. The same letter was assigned to bars with no significant differences between them. Statistical analyses were performed by ANOVA and pairwise Fisher’s LSD test (<i>p</i><0.05).</p

Effect of free chlorine, on the inactivation of <i>L</i>. <i>pneumophila</i> sg. 1 env. associated to two <i>Acanthamoeba</i> strains, <i>A</i>.<i>castellanii</i> CCAP 1534/2 and <i>Acanthamoeba</i> sp. 155 strains.

<p>Bacterial inactivation was determined using viable counts on BCYE medium. Data are presented as means ± SD (columns and error bars; n = 3). Statistical differences between means within each time point were represented assigning different letters to the bar plot. The same letter was assigned to bars with no significant differences between them. Statistical analyses were performed by ANOVA and pairwise Fisher’s LSD test (<i>p</i><0.05).</p

Effect of thermal treatments on the inactivation of <i>L</i>. <i>pneumophila</i> sg. 1 env. associated to two <i>Acanthamoeba</i> strains, <i>A</i>.<i>castellanii</i> CCAP 1534/2 and <i>Acanthamoeba</i> sp. 155 strains.

<p>Bacterial inactivation was determined using viable counts on BCYE medium. Data are presented as means ± SD (columns and error bars; n = 3). Statistical differences between means within each time point were represented assigning different letters to the bar plot. The same letter was assigned to bars with no significant differences between them. Statistical analyses were performed by ANOVA and pairwise Fisher’s LSD test (<i>p</i><0.05).</p

Pictures obtained by FISH using an epifluorescence microscope to monitor the intracellular presence of <i>L</i>. <i>pneumophila</i> sg. 1 (env.) within the two amoeba strains, <i>A</i>. <i>castellanii</i> CCAP 1534/2 (first column) and <i>Acanthamoeba</i> sp. 155 (env.) (second column).

<p>Negative controls of pure cultures are shown in the first line (<b>A</b>, <b>B</b> and <b>C</b>). Then, the presence of <i>L</i>. <i>pneumophila</i> was analysed at different time points: just after the co-culture protocol (<b>T0</b>) and after 24 h, 40 h and 48 h (<b>T24</b>, <b>T40</b> and <b>T48</b>, respectively). All samples, including the controls, were simultaneously hybridized with the LEGPNE1 probe (FITC, green) and the probe EUK 516 (Cy3, red). Pictures were taken at 1000X magnification, bar scale = 9.2 µm.</p

Additional file 1: of Persistent presence of outer membrane epitopes during short- and long-term starvation of five Legionella pneumophila strains

Figure S1. Culturability of five L. pneumophila strains during starvation [18]. Table S1. Spearman’s rank correlation results of the IF-FCM-data and the viability data (taken from the parallel investigations [18]). Figure S2. Example plots for IF-FCM analysis (mAb 8/4 and mAb MOMP) of starved Legionella cells and gating strategy after IF staining. Figure S3. Viability indicator data for the five L. pneumophila strains examined during starvation in ultrapure water at 45 °C for up to 400 days [18]. (DOCX 868 kb