First Molecular and Physiological Characterization of Free-Living Amoebae from Environmental Water Sources in Honduras

Free-living amoebae (FLA) are ubiquitous protozoa with pathogenic potential that also serve as environmental reservoirs for bacteria of public health importance. Owing to the lack of information on their occurrence in Honduras, this study aimed to isolate and characterize FLA from environmental water sources using morphological, molecular, and physiological approaches. A total of 87 water samples collected from domestic tap water, rivers, and outdoor washbasins were analyzed by non-nutrient agar culture, polymerase chain reaction, and 18S rRNA gene sequencing. Overall, 43 samples (49.4%) were positive for FLA, with the highest detection rate observed in domestic tap water (38/69; 55.1%). Molecular characterization of these 43 positive samples yielded 45 isolates in total, as two samples (codes 18.4 and 34.4) each produced two co-occurring but independently culturable isolates belonging to different genera. Of these 45 isolates, Acanthamoeba spp. accounted for 26 isolates (57.8%), predominantly belonging to genotype T4; Vermamoeba vermiformis accounted for 9 isolates (20.0%); and mixed cultures containing both genera accounted for 10 isolates (22.2%). A subset of Acanthamoeba isolates exhibited thermotolerance at 42 °C (7/26; 26.9%), with three isolates remaining viable at 45 °C, while most tolerated 0.5 M mannitol, and 57.7% retained viability at 1 M, characteristics commonly associated with pathogenic potential. To the best of our knowledge, this study constitutes the first report of free-living amoebae in environmental water sources in Honduras and provides an important baseline to strengthen water quality surveillance to mitigate potential public health risks.