Molecular identification and genotype distribution of Acanthamoeba in environmental water

Acanthamoeba is a free-living amoeba and opportunistic amoeba commonly found in various water sources and poses a potential risk to human health.

The aim of this study was to investigate the presence of the pathogenic Acanthamoeba, which poses a risk to humans, in various environmental water sources and to determine the pathogenic genotypes.

A total of 50 samples were collected from various water sources, including rivers, canals, streams, natural springs, lakes, waterfalls, ponds, and tap water. The presence of Acanthamoeba was determined using culture, PCR, sequencing assays, and phylogenetic analysis of pathogen genotypes. Thermo- and osmotolerance assays were performed to evaluate the pathogenic potential of culture-positive isolates.

Free-living amoebae were detected in 56% (28/50) of samples by culture, while Acanthamoeba DNA was identified in 46% (23/50) by real-time PCR. Sequence analysis of 14 isolates revealed predominance of genotype T4 (71.4%, 10/14), followed by T2 (14.2%, 2/14) and T11 (14.2%, 2/14). Genotype T4 isolates clustered robustly with reference pathogenic strains and exhibited 98-100% sequence similarity. Thermo- and osmotolerance assays demonstrated that 44.4% (4/9) of culture-positive isolates tolerated 42 °C and 1 M mannitol, indicating enhanced pathogenic potential.

The high detection rate, predominance of the clinically relevant T4 genotype, and identification of potentially virulent strains highlight a substantial environmental reservoir with epidemiological significance. These findings underscore the need for routine molecular surveillance of environmental waters within a One Health framework to mitigate the risk of severe infections such as Acanthamoeba keratitis and granulomatous amoebic encephalitis.