First Identification of Holosporales Endosymbionts in Acanthamoeba spp. Isolated from Keratitis Patients in Taiwan

Background Acanthamoeba spp. are free-living amoebas commonly found in various environmental settings and are recognized as opportunistic pathogens in humans. Endosymbiosis within Acanthamoeba , particularly involving bacteria, can enhance the pathogenic potential of both the host and symbiont, influencing human health risks. This study aims to characterize the clinical Acanthamoeba isolate and to identify and localize the Acanthamoeba endosymbiont. Methods Environmental and clinical Acanthamoeba isolates were collected and identified through morphological analysis and 18S rRNA gene sequencing. The presence of bacterial endosymbionts was determined using PCR amplification targeting 16S rRNA and glt A genes. Phylogenetic analysis characterized Acanthamoeba and the bacterial endosymbiont. Results PCR and cultivation methods identified Acanthamoeba in 40% of clinical samples, with phylogenetic analysis revealing that all isolated clinical strains belonged to the T4 genotype, while environmental strains were classified as T3, T4, and T5. Phylogenetic analysis of the DF3 region in Acanthamoeba 18S rDNA classified clinical and environmental isolates into six T4 sub-genotypes. Two clinical isolates harbored bacterial endosymbionts from the order Holosporales, as supported by 16S rRNA and glt A gene analyses. Fluorescence microscopy revealed the intracellular DNA-containing structures consistent with endosymbiont localization. Conclusion Our study reveals the genetic diversity of Acanthamoeba isolates from clinical and environmental sources and highlights the widespread presence of bacterial endosymbionts. These findings underscore the need for continued surveillance and advanced genomic studies to understand endosymbiont- Acanthamoeba interactions and their potential impact on public health.