Fig 1.
Effect of free chlorine 0.2 mg L-1 and 0.5 mg L-1, on the inactivation of 5 Legionella strains.
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 (p<0.05).
Table 1.
Calculated time for a 4-log reduction of five Legionella strains at 0.2 mg L-1 and 0.5 mg L-1 of free chlorine.
Inactivation kinetics adjusted to a biphasic decay model (*) and to a first-order (straight line) model. R2 values showed the robustness of the model. NA (not achieved).
Fig 2.
Effect of free chlorine, 1.2 mg L-1 and 2.5 mg L-1, on the inactivation of 2 Acanthamoeba strains treating separately trophozoites and cysts.
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 (p<0.05).
Table 2.
Calculated time for a 3-log reduction of two Acanthamoeba strains treated separately by its life stage, cyst or trophozoite at 1.2 mg L-1 and 2.5 mg L-1 of free chlorine.
Inactivation kinetics of amoeba strains adjusted to a first-order model and to a biphasic decay model (*). R2 values showed the robustness of the model. NA (not achieved).
Fig 3.
Effect of thermal treatments on the inactivation of 5 Legionella strains.
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 (p<0.05).
Table 3.
Calculated time for a 4-log reduction of five Legionella strains at five different temperatures.
Inactivation kinetics adjusted to first-order models (straight line). R2 values showed the robustness of the models.
Fig 4.
L. pneumophila sg. 1 growth in axenic conditions and in co-culture with the two Acanthamoeba strains in PYG liquid media.
Samples were taken at different time points and plated on BCYE agar plates. Data are presented as means ± SD (error bars; n = 3).
Fig 5.
Pictures obtained by FISH using an epifluorescence microscope to monitor the intracellular presence of L. pneumophila sg. 1 (env.) within the two amoeba strains, A. castellanii CCAP 1534/2 (first column) and Acanthamoeba sp. 155 (env.) (second column).
Negative controls of pure cultures are shown in the first line (A, B and C). Then, the presence of L. pneumophila was analysed at different time points: just after the co-culture protocol (T0) and after 24 h, 40 h and 48 h (T24, T40 and T48, 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.
Fig 6.
Effect of free chlorine, on the inactivation of L. pneumophila sg. 1 env. associated to two Acanthamoeba strains, A.castellanii CCAP 1534/2 and Acanthamoeba sp. 155 strains.
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 (p<0.05).
Table 4.
Calculated time for a 4-log reduction of L. pneumophila sg. 1 env. associated with A.castellanii CCAP 1534/2 and Acanthamoeba sp. 155 after the exposure to different concentrations of free chlorine and temperatures.
Inactivation kinetics adjusted to first-order models. R2 values showed the robustness of the models.
Fig 7.
Effect of thermal treatments on the inactivation of L. pneumophila sg. 1 env. associated to two Acanthamoeba strains, A.castellanii CCAP 1534/2 and Acanthamoeba sp. 155 strains.
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 (p<0.05).