Enrichment of free-living amoebae in biofilms developed at upper water levels in drinking water storage towers: An inter- and intra-seasonal study

Free-living amoebae (FLA) are ubiquitous organisms present in various natural and artificial environments, such as drinking water storage towers (DWST). Some FLA, such as Acanthamoeba sp., Naegleria fowleri, and Balamuthia mandrillaris, can cause severe infections at ocular or cerebral level in addition to being potential reservoirs of other pathogens. In this work, the abundance and diversity of FLA was evaluated in two sampling campaigns: one performed over five seasons in three DWST at three different levels (surface, middle and bottom) in water and biofilm using microscopy and PCR, and one based on the kinetics analysis in phase contrast and confocal microscopy of biofilm samples collected every two weeks during a 3-month period at the surface and at the bottom of a DWST. In the seasonal study, the FLA were detected in each DWST water in densities of ~20 to 25amoebaeL^-1. A seasonal variation of amoeba distribution was observed in water samples, with maximal densities in summer at ~30amoebaeL^-1 and minimal densities in winter at ~16amoebaeL^-1. The FLA belonging to the genus Acanthamoeba were detected in two spring sampling campaigns, suggesting a possible seasonal appearance of this potentially pathogenic amoeba. Interestingly, a 1 log increase of amoebae density was observed in biofilm samples collected at the surface of all DWST compared to the middle and the bottom where FLA were at 0.1-0.2amoebae/cm². In the kinetics study, an increase of amoebae density, total cell density, and biofilm thickness was observed as a function of time at the surface of the DWST, but not at the bottom. To our knowledge, this study describes for the first time a marked higher FLA density in biofilms collected at upper water levels in DWST, constituting a potential source of pathogenic micro-organisms.