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 protists found in water, feeding mainly on bacteria. While most FLA are harmless, Acanthamoeba spp. and Naegleria fowleri can cause keratitis and/or meningitis. FLA can host amoeba-resistant bacteria (ARB), but their natural bacterial microbiota is largely unknown. This study aimed to identify the natural bacterial microbiota of Naegleria fowleri, Acanthamoeba castellanii, Acanthamoeba lenticulata, and Acanthamoeba sp. T17, isolated from untreated (rivers) and treated (tap) waters 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 identified by mass spectrometry (MALDI-TOF MS) followed by conventional 16S PCR, and bacterial antibiotic resistance was analyzed using the disk diffusion method. Transmission electron microscopy was used to locate ARB within the amoebae. The metabarcoding analyses identified Salmonella, Enterobacter and Klebsiella genera as the most abundant bacteria in untreated and treated waters. However, the most frequently detected amoeba-resistant bacteria (ARB) were from the Bosea, Escherichia-Shigella, Microbacterium, and Pseudomonas genera. Our findings revealed, for the first time, the natural occurrence of several bacteria within N. fowleri, including Pseudomonas spp. and Escherichia coli. Additionally, we detected Legionella in A. castellanii and Bordetella in A. lenticulata. The four pathogenic FLA showed both temporary and permanent associations with various bacterial genera, depending on the number of passages and culture conditions. Pseudomonas species isolated from distinct FLA exhibited resistance to different antibiotics. ARB were detected within the cytoplasm of trophozoites. The presence of pathogenic FLA and ARB in untreated and treated water in Guadeloupe's drinking systems pose health risks. Our results highlight the need for regular monitoring to ensure water safety and understanding amoebae-bacteria interactions for better management. The natural presence of ARB in pathogenic FLA also questions the host immune response during amoeba infection.