Molecular identification and phylogenetic analysis of free-living amoeba (Naegleria and Acanthamoeba) from treated and untreated drinking water

Abstract

Free-living amoebas (FLAs) are a group of protozoa, which are frequently reported in environmental and clinical samples worldwide. The current study aimed to investigate the prevalence of FLAs in treated/untreated drinking water in a touristic region, Khorasan Razavi province. Sixty samples of treated and untreated drinking waters were collected and were filtered. The filters were cultivated onto non-nutrient agar. The presence of FLAs was screened based on morphological criteria. Total DNA was extracted and the presence of Acanthamoeba spp., and Naegleria spp., was investigated using specific primers. To identify species/genotypes, PCR products were sequenced. An approximately 450-bp of the 18S rRNA region was amplified for Acanthamoeba sp., and all isolates were the genotype T4. The 500-bp fragment of the ITS region was amplified for Naegleria spp. Sequencing results revealed N. americana, N. clarki, N. pagei, and N. australiensis. The phylogenetic tree showed that Acanthamoeba and Naegleria species were clearly separated. All Acanthamoeba isolates were the genotype T4, indicating the wide distribution of this genotype in environmental samples. This study also confirmed the low prevalence of high pathogenic Naegleria species, N. fowleri, in Iran. This study signifies the public health concern due to FLA in touristic province, Khorasan Razavi.