Characterization of the natural bacterial microbiota of pathogenic free-living amoebae (Acanthamoeba spp and Naegleria fowleri) isolated from rivers and tap water in Guadeloupe

Free-living amoebae (FLA) are ubiquitous protist mainly found in water and feeding on bacteria. While most FLA are harmless, Acanthamoeba spp and Naegleria fowleri can cause keratitis and/or meningitis. FLA can harbor amoeba-resistant bacteria (ARB), but the natural bacterial microbiota of these pathogenic FLA remains largely unknown. Our work aimed at characterizing the natural bacterial microbiota of Naegleria fowleri, Acanthamoeba castellanii, Acanthamoeba lenticulata and Acanthamoeba sp (T17 genotype) recently isolated from rivers and tap water in Guadeloupe.The whole bacterial microbiota of the water source and the FLA grown with E. coli and under axenic culture conditions, during successive passages, were characterized using 16S rRNA gene metabarcoding. The culturable subset of ARB was analyzed by mass spectrometry (MALDI-TOF MS), conventional 16S PCR, and disk diffusion method. Transmission electron microscopy was used to locate the ARB inside the amoebae.The metabarcoding analyses revealed that while Enterobacter, Klebsiella and Salmonella genera were detected in water, the most common ARB belonged to Bosea, Escherichia/Shigella, Microbacterium and Pseudomonas genera. The amoebal hosts showed temporary and permanent associations with different bacterial genera (including the genera Legionella and Bordetella in Ac. T17 and A. lenticulata, respectively), depending on the number of passages and culture conditions. The Pseudomonas species isolated from distinct FLA exhibited resistance to key antibiotics. The ARB were detected in the cytoplasm of the trophozoites.The presence of pathogenic FLA and ARB in treated and non-treated waters in drinking water distribution systems in Guadeloupe poses a potential health risk. Our results emphasize the need to regularly control the FLA and their associated ARB to ensure water safety and improve knowledge on amoebae-bacteria interactions to establish better water management protocols. The natural presence of ARB in pathogenic FLA also raises key questions regarding the host immune response during amoeba infection.