Spatial arrangement of Legionella colonies in model cooling tower systems

Legionella have been shown to multiply within biofilms in both anthropogenic and natural water sources, either in association with bacteria and algae or as an intracellular parasite of free living amoebae. To investigate these interactions visually, laboratory derived biofilms grown in simulated cooling tower systems were tagged using fluorescent in-situ hybridisation (FISH). These tagged films were then imaged using confocal laser scanning microscopy (CSLM) and 3D representations of each sample were generated. Within these films, although the overall architecture of the film was not uniformly flat, the distribution of microorganisms throughout the thickness of the film was mostly homogenous. In contrast to this relatively even distribution, Legionella appeared as small, separate microcolonies, with distinct boundaries, suggesting that their formation is mediated by specific environmental conditions. This may be a representation of Legionella’s adaptation to growth in acidified waters and hot springs in which colonies form in microclimates of more favourable (higher) pH. Alternatively these cloud-like colonies may be a product of Legionella’s parasitic capability, in which the bacteria multiplies within a trophozoite until lysis occurs. This process leaves a significantly higher concentration of Legionella within the nutrient rich remnants of their host than in the surrounding film. This work represents the first time that Legionella has been fluorescently tagged within a complex, multispecies biofilm and the distinct special arrangement and occurrence of cloud-like colonies noted.