Molecular and Culture-Based Surveillance of Free-Living Amoebae in Human Related Sources in an Outermost Region

In this study, we investigated the presence and diversity of FLA in 62 environmental samples collected across Tenerife, Canary Islands, Spain including agricultural and playground soils, and on double treated water from public refrigerated fountains. Amoebae were isolated by culturing processed samples onto 2% Non-Nutrient Agar plates (NNA) which were checked daily for further processing up to molecular characterization. In this case, two approaches for molecular identification were assessed: direct multiplex qPCR targeting four potentially pathogenic FLA (Acanthamoeba spp., Vermamoeba vermiformis, Naegleria fowleri, and Balamuthia mandrillaris) DNA, and culture-based isolation followed by standard PCR and sequence analysis. Regarding qPCR results, 72.6% (45/62) of the samples were positive for at least one FLA, with V. vermiformis (37/62) and Acanthamoeba spp. (34/62) being the most frequent. Moreover, B. mandrillaris was detected for the first time in the Canary Islands in 6 out of 62 samples. Results from standard PCR from cultured isolates confirmed the presence of Acanthamoeba (mainly genotype T4) and Vermamoeba and also allowed the identification of Vahlkampfia and Vannella genera, as well as the genus Rhogostoma-its first report in the Canary Islands. Thermotolerance and osmotolerance assays were performed on Acanthamoeba spp. and, innovatively, on V. vermiformis isolates. Both were capable of surviving at 37 °C and during incubation with 0.5 M mannitol, suggesting potential pathogenicity. However, growth was significantly impaired under harsher conditions (42 °C and 1 M mannitol). These findings underscore the widespread occurrence of FLA in public and agricultural environments in Tenerife and highlight their potential risk to public health. Their ability to act as carriers of pathogenic bacteria/viruses further reinforces the need for routine surveillance and preventive measures in the environment.