Molecular identification of free-living amoebae isolated from artificial water bodies located in Poland

Free living amoebae (FLA) are amphizoic protozoa that are widely found in various environmental sources. They are known to cause serious human infections, including a fatal encephalitis, a blinding keratitis, and pneumonia. The main aim of the study was detection and molecular identification of Acanthamoeba spp., Naegleria fowleri, Balamuthia mandrillaris, Sappinia pedata, and Vermoamoeba vermiformis (formerly Hartmannella vermiformis) in artificial water bodies in North-Western Poland. We examined 86 water samples collected during 2-year period from 43 water bodies, including outdoor and indoor swimming pools, firefighting reservoirs, fountains, as well as water network. The samples were filtrated using Filta-Max® membrane filters (IDEXX Laboratories, USA) and, in order to select potentially pathogenic, thermophilic strains and to limit the number of PCR examined samples, the thermal tolerance test was carried out. Obtained filtrates were transferred to non-nutrient agar plates with E. coli. The agar plates were incubated at 37°C and then proliferated amoebae were passaged at 42°C. DNA was extracted from the thermophilic trophozoites and then polymerase chain reactions and sequence analysis were performed for molecular identification of FLA. From the 86 collected water samples 57 strains of FLA were able to proliferate at 37°C and 7 of them showed ability to proliferate at 42°C. For molecular identification of Acanthamoeba spp. and V. vermiformis, regions of 18S rDNA were amplified. In order to detect B. mandrillaris DNA, we used mitochondrial 16S rDNA as a marker, and for detection of N. fowleri and S. pedata – ITS regions. Based on molecular analysis, isolates were classified to the genus Acanthamoeba (T4 and T11 genotypes, as well as the new genotypes detected earlier in clinical samples and named T16) and V. vermiformis species. Detected strains were highly similar or identical to pathogenic strains detected earlier in patients. Our results show a wide distribution of potential pathogenic FLA, as Acanthamoeba T4, T11, T16 genotypes, and V. vermiformis species in various artificial water bodies located in North-Western Poland and suggest a potential threat to health of humans in this part of the country.