Molecular Species Identification and Genotyping of Free-Living Amoebae in Soil of Recreational Mountain Areas in the Babiogórski National Park and Surroundings, Southern Poland

Free-living amoebae (FLAs) are widely present in the environment and may be pathogenic for animals and humans. Studies on the prevalence of FLAs in European soils are few in number. This study aimed to molecularly identify the species and genotypes of FLAs occurring in soil from Southern Poland. Forty soil samples were collected in June 2024 in the Babiogórski National Park. Amoebae cultures and a thermal-tolerance test were conducted, and all strains grew at 37 °C. Five PCR protocols were applied for the amplification of FLA <i>SSU rRNA</i> fragments. The following FLAs, including potentially pathogenic ones, were detected: <i>Acanthamoeba</i> T4 and T13 genotypes in 79.1% of positive samples, <i>Naegleria gruberi</i> and <i>Naegleria galeacystis</i> in 25%, <i>Vermamoeba vermiformis</i> in 12.5%, and <i>Paravahlkampfia</i> sp. and <i>Ptolemeba bulliensis</i> in 8.3%. Species and genotype identification were determined by sequence comparison and phylogenetic analysis. This study reports, for the first time, the isolation of <i>N. galeacystis</i> from soil and <i>N. gruberi</i> and <i>V</i>. <i>vermiformis</i> from soil collected in Europe. The used primer sets have different usefulness for <i>Naegleria</i> species identification and their phylogenetic analysis. The primers applied in this study may not reveal the full diversity of amoebae in soil; therefore, it is necessary to design new primers for this purpose.