Core gene-based molecular detection and identification of Acanthamoeba species

Acanthamoeba spp. are predominant free-living amoebae of water and soil. They have been used as tools for the isolation and culture of microbes that resist after their phagocytosis, such as Legionella-like bacteria, and, more recently giant viruses for which differences in permissiveness have been reported. However, problems have been reported regarding their identification at the species level. The present work implemented specific PCR systems for the detection and identification of Acanthamoeba species through comparison of sequences and phylogenetic analyses. Thirty-three Acanthamoeba isolates were studied, including 20 reference strains and 13 isolates retrieved from water, soil or clinical samples. Previous delineation of a core genome encompassing 826 genes based on draft genome sequences from 14 Acanthamoeba species allowed designing PCR systems for one of these core genes that encodes an alanine-tRNA ligase. These primers allowed an efficient and specific screening to detect Acanthamoeba presence. In addition, they identified all 20 reference strains, while partial and complete sequences coding for 18S ribosomal RNA identified only 11 (55%). We found that four isolates may be considered as new Acanthamoeba species. Consistent with previous studies, we demonstrated that some Acanthamoeba isolates were incorrectly assigned to species using the 18S rDNA sequences. Our implemented tool may help determining which Acanthamoeba strains are the most efficient for the isolation of associated microorganisms.