Incorporation of natural uncultivable Legionella pneumophila into potable water biofilms provides a protective niche against chlorination stress

Legionella pneumophila is a waterborne pathogen that has been isolated sporadically from drinking water distribution systems (DWDS). Resistance to disinfectants is mainly attributed to the association of cells with amoebae, but biofilms are also thought to provide some degree of protection. In the present work, a two-stage chemostat was used to form heterotrophic biofilms from drinking water to study the influence of chlorine on the presence of naturally occurring L. pneumophila. The pathogen was tracked in planktonic and sessile biofilm phases using standard culture recovery techniques for cultivable cells and a peptide nucleic acid fluorescence in situ hybridisation assay for total cells. The results showed that the total number of L. pneumophila cells in biofilms was not affected by the concentrations of chlorine tested, and the presence of L. pneumophila could not be detected by culturing. To restrict the outbreaks of disease caused by this bacterium, efforts need to be concentrated on preventing L. pneumophila from re-entering an infectious state by maintaining residual disinfectant levels through the entire DWDS network so that the resuscitation of cells via contact with amoebae is prevented.