Amoebae in domestic water systems: resistance to disinfection treatments and implication in Legionella persistence

Aims: Monitoring of microbial changes during and after application of various disinfection treatments in a model domestic water system. Methods and Results: A pilot-scale domestic water system consisting of seven galvanized steel re-circulation loops and copper dead legs was constructed. Culture techniques, confocal laser scanning microscopy after fluorescent in situ hybridization and viability staining with the BacLight LIVE/DEAD kit were used for planktonic and biofilm flora monitoring. Before starting the treatments, the system was highly contaminated with Legionella pneumophila and biofilm populations mainly consisted of beta-proteobacteria. In the water and the biofilm of the loops, continuous application of chlorine dioxide (0.5 mg l-1), or chlorine (2.5 mg l-1) were very effective in reducing the microbial flora, including L. pneumophila. Heterotrophic bacteria, although strongly reduced, were still detectable after ozone application (0.5 mg l-1), whereas with monochloramine (0.5 mg l-1) and copper-silver ionization (0.8/0.02 mg l-1), the contamination remained significantly higher. Monochloramine and copper-silver did not remove the biofilm. During copper-silver application, Legionella re-growth was observed. Only chlorine dioxide led to detectable effects in the dead leg. Amoebae could not be eliminated, and after interrupting the treatments, L. pneumophila quickly recovered their initial levels, in all cases. Conclusions: Chlorine dioxide, applied as a continuous treatment, was identified in this study as the most efficient for controlling L. pneumophila in a domestic water system. Chlorine dioxide showed a longer residual activity, leading to improved performance in the dead leg. Amoebae resisted to all the treatments applied and probably acted as reservoirs for L. pneumophila, allowing a quick re-colonization of the system once the treatments were interrupted. Significance and Impact of the Study: Control of microbial contamination requires maintenance of a constant disinfectant residual throughout the water system. Treatment strategies targeting free-living amoebae should lead to improved control of L. pneumophila. Such treatment strategies still have to be investigated.