Biodiversity of FLA and associated bacteria Targeted Locus. freshwater metagenome

Free-living amoebae could be found in water networks where they prey on bacteria within biofilms. Most bacteria are digested rapidly by phagocytosis, however some are able to survive within amoebae and some are even able to multiply, as it is the case for Legionella pneumophila. These resisting bacteria are a potential health problem as they could also resist to macrophage phagocytosis. Several publications already reported intra-amoebal bacteria but the method of identification did not allow metagenomic analysis. The aim of our study was to conduct a metagenomic analysis on amoebae and intra-amoebal bacteria found in water networks, to give the first FLA microbiome in environmental strains. Drinking water networks in Paris were sampled during four months. Cultivating amoebae were isolated and total DNA was prepared from them, allowing purification of both amoebal and bacterial DNA. Metagenomic studies were then conducted by 18S or 16S sequencing. Hartmannella was by far the most represented genus of FLA. Regarding intra-amoebal bacteria 54 genera were identified, among which 24 were newly described intra-amoebal bacteria. There were high differences in bacterial diversity between the three sample location. Several genera were highly represented and/or found at least two networks, underlying that this bacteria could be able to multiply within FLA. This method is therefore useful to identify the whole FLA microbiome and could be apply to other networks to better compare their microbiome.