Effect of Common Drinking Water Disinfectants, Chlorine and Heat, on Free Legionella and Amoebae-Associated Legionella

Abstract

Chlorine and thermal treatments are the most commonly used procedures to control and prevent Legionella proliferation in drinking water systems of large buildings. However, cases of legionellosis still occur in facilities with treated water. The purpose of this work was to model the effect of temperature and free chlorine applied in similar exposure conditions as in drinking water systems on five Legionella spp. strains and two amoebal strains of the genera Acanthamoeba. Inactivation models obtained were used to determine the effectiveness of the treatments applied which resulted more effective against Legionella than Acanthamoeba, especially those in cystic stages. Furthermore, to determine the influence of the relationship between L. pneumophila and Acanthamoeba spp. on the treatment effectiveness, inactivation models of the bacteria-associated amoeba were also constructed and compared to the models obtained for the free living bacteria state. The Legionella-amoeba association did not change the inactivation models, but it reduced the effectiveness of the treatments applied. Remarkably, at the lowest free chlorine concentration, 0.5 mg L-1, as well as at the lowest temperatures, 50°C and 55°C, the influence of the Legionella-amoeba associate state was the strongest in reducing the effectiveness of the treatments compared to the free Legionella state. Therefore, the association established between L. pneumophila and amoebae in the water systems indicate an increased health risk in proximal areas of the system (close to the tap) where lower free chlorine concentrations and lower temperatures are commonly observed.

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).

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).

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).

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).

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).