Zooming in on the intracellular microbiome composition of bacterivorous isolates.

, a free-living amoeba in water and soil, is an emerging pathogen causing severe eye infection known as keratitis. In its natural environment, performs a dual function as an environmental heterotrophic predator and host for a range of microorganisms that resist digestion. Our objective was to characterize the intracellular microorganisms of phylogenetically distinct spp. isolated in Australia and India through directly sequencing 16S rRNA amplicons from the amoebae. The presence of intracellular bacteria was further confirmed by hybridization and electron microscopy. Among the 51 isolates assessed, 41% harboured intracellular bacteria which were clustered into four major phyla: Pseudomonadota (previously known as Proteobacteria), Bacteroidota (previously known as Bacteroidetes), Actinomycetota (previously known as Actinobacteria), and Bacillota (previously known as Firmicutes). The linear discriminate analysis effect size analysis identified distinct microbial abundance patterns among the sample types; species was abundant in Australian corneal isolates ( < 0.007), Enterobacteriales showed higher abundance in Indian corneal isolates ( < 0.017), and Bacteroidota was abundant in Australian water isolates ( < 0.019). The bacterial beta diversity of isolates from keratitis patients in India and Australia significantly differed ( < 0.05), while alpha diversity did not vary based on the country of origin or source of isolation ( > 0.05). More diverse intracellular bacteria were identified in water isolates as compared with clinical isolates. Confocal and electron microscopy confirmed the bacterial cells undergoing binary fission within the amoebal host, indicating the presence of viable bacteria. This study sheds light on the possibility of a sympatric lifestyle within , thereby emphasizing its crucial role as a bunker and carrier of potential human pathogens.