Determination of the bacterial microbiome of free-living amoebae isolated from wastewater by 16S rRNA amplicon-based sequencing.

Free-living amoebae (FLA) are ubiquitous protozoa commonly found in water. FLA are well-established hosts for amoeba-resistant bacteria, most of which are pathogenic, and offer them shelter from adverse environmental conditions or water treatments. Since there is very little knowledge about the complete bacterial microbiome of FLA, in this work the bacterial microbiome of FLA isolated from wastewater both after secondary and tertiary treatments was studied by amplicon-based sequencing. FLA were detected in 87.5% and 50.0% of wastewater samples taken after secondary and tertiary disinfection treatments, respectively. The most abundant bacterial phyla were Proteobacteria, Planctomycetes, Bacteroidetes and Firmicutes, which represented 83.77% of the total bacterial FLA microbiome. The most abundant class of bacteria was Gammaproteobacteria, which contains an important number of relevant pathogenic bacteria. The bacteria of public health concern Aeromonas, Arcobacter, Campylobacter, Helicobacter, Klebsiella, Legionella, Mycobacterium, Pseudomonas and Salmonella were detected as part of the FLA microbiome. Although different microbial communities were identified in each sample, there is no correlation between the microbiome of FLA and the extent of wastewater treatment. To our knowledge, this is the first work in which the bacterial microbiome of FLA isolated from wastewater is studied. Obtained results indicate that FLA are hosts of potentially pathogenic bacteria in treated wastewater used for irrigation, which may pose a public health threat.