Pathogenic Naegleria fowleri and Acanthamoeba spp., free-living amoebae exist in the natural environment, are causing agents of an acute and lethal primary amoebic meningoencephalitis (PAM) and amoebic keratitis (AK) in humans, respectively. To ascertain the existence of free-living amoebae in Korea, in late August 2015, water samples of eight sites were collected in Korean hydrosphere where water skiing and recreation have been actively performed, and then the non-nutrient agar culture and PCR-based detection technique were carried out. The surface waters were collected and filtered, and then final samples were cultured on non-nutrient agar medium with inactivated E. coli and subjected to PCR with various primer pairs (amplify mainly the 18S-small ribosomal RNA). Free-living amoebae were intensively detected by PCR in two collection regions (Yeoju and Yangpeong city around Southern Han-River). PCR products obtained from water samples of Yeoju and Yangpeong city were subjected to gene sequencing. The similarity of 18S-rRNA sequences were compared with various reference amoebae in GeneBank, and they showed 86-99% homology with N. gruberi, N. philippinensis, N. clarki, Acanthamoeba polyphaga and Hartmannella (Vermamoeba) vermiformis. A Korean isolate (confirmed by PCR as A. polyphaga) was isolated from Yeoju sample and have been subcultured in Nelson's and PYG liquid medium with 10% FBS at 30 ℃ incubator. In the in vitro cytotoxicity test, Korean isolate (tentative A. polyphaga) showed high cytotoxicity as much as reference amoebae, A. polyphaga and A. castellanii. This study will be useful, in the further study, for the detailed seasonal detection of free-living amoebae from Korean hydrosphere. ABSTRACT 1 Department of Microbiology, Ajou University School of Medicine 2 Department of Biomedical Science, Graduate School of Ajou University, Suwon 443-721, Republic of Korea 3 Department of Biomedical Laboratory Science, Molecular Diagnostics Research Institute, School of Health and Medicine, Namseoul University, Cheonan 31020, Republic of Korea 4 Division of Malaria and Parasitic Disease, Korea National Institute of Health, Osong 363-951, Republic of Korea Hee-kyoung Kang 1,2 , Gi-Sang Seong 1,2 , Hae-jin Sohn 1,2 , Suk-Yul Jung 3 , Sang-Eun Lee 4 , Mi-Yeoun Park 4 , Ho-Joon Shin 1,2 Pathogenic Naegleria fowleri and Acanthamoeba spp., free-living amoebae (FLA) in the natural environments, also cause devastating diseases in humans leading to death. Acanthamoeba spp. cause granulomatous amebic encephalitis (GAE) and amoebic keratitis (AK) in animals and human. Pathogenic Naegleria fowleri causing an acute primary amoebic menigoencephalitis (PAM) in the central nerve system (CNS) has a worldwide distribution. More than 300 cases of PAM have been reported internationally mostly in the United States, Australia and Europe. Recently, Increasing PAM cases are becoming a serious issue in the subtropical and tropical countries as the Neglected Tropical Diseases (NTD). Especially young PAM-patients have been with a history of swimming and recreational activities in contaminated warm fresh water as a result of global warming. PAM is a rare disease but almost always fatal. In this study, to establish the distribution of free-living amoebae in Korea, the non-nutrient agar culture and PCR-based detection technique from samples of variable hydrosphere were carried out. INTRODUCTION 1. Sample collection and filteration The surface water were collected from eight sites of Korean hydrosphere in late August, 2015. 2. PCR Amplification and sequencing Three kinds of primer pairs were selected and prepared as follows: Pfla-F 5’-CGCGGTAATTCCAGCTCCAATAGC-3’ Pfla-R 5’-CAGGTTAAGGTCTCGTTCGTTAAC-3’ Nf-ITS1 5’-GAACCTGCGTAGGGATCATTT-3’ Nf-ITS2 5’-TTTCTTTTCCTCCCCTTATTA-3’ Hart-NA1 5’-AGAAAGAGCTATCAATCTGT-3’ Hart-NA2 5’-GCTCCAATAGCGTATATTAA-3’ 3. Phylogenetic analysis Similarity of DNA sequencing compared with various reference amoebae in GeneBank. 4. In vitro cytotoxicity of an isolated sample (A. polyphaga) MATERIALS & METHODS ▶ Water samples from eight sites of Korean hydrosphere were subjected to PCR and DNA sequencing. Their similarity of DNA sequencing showed 86-99% homology with non-pathogenic N. gruberi, N. philippinensis, N. clarki, Hartmannella (Vermamoeba) vermiformis and pathogenetic A. polyphaga. ▶ Free-living amoeba was isolated from Yeoju sample (Korean isolate) by subculture in Nelson's and PYG medium with 10% FBS at 30℃ incubator. ▶ Isolated A. polyphaga (Yeoju amoeba) induced in vitro cytotoxicity against target CHO cells. CONCLUSIONS RESULTS Isolation of free-living amoebae from Southern Han-River in Korea 1. Detection of free-living amoebae by PCR 4. Phylogenetic analysis 3. Nucleotide sequencing alignments FLA amplified from water samples 5. Culture for morphological identification Fig. 5. Morphological observation of free- living amoebae cultured on non-nutrient agar medium. (Bar, 10 μm). CHO + Yeoju amoeba 6h 12h 24h 0 50 100 Yeoju 0.1:1 Yeoju 0.5:1 Yeoju 1:1 A.polyphaga 0.1:1 A.polyphaga 0.5:1 A.polyphaga 1:1 A.castellanii 0.1:1 A.castellanii 0.5:1 A.castellanii 1:1 % Cytotoxiciy (LDH release) 2. Nucleotide sequencing of amplified FLA DNAs from water samples Fig. 1. PCR products of free-living amoebae DNA amplified with P-FLA primer (a), ITS primer (b) and Hart NA primer (c). N.f, N. fowleri; Ac, A. castellanii; Ap, A. polyphaga; Ht, Hartmannella (Vermamoeba) vermiformis; Lane 1-5, environmental samples. Fig. 2. 18S-rRNA Sequence of free-living amoeba (Yeoju sample) amplified with P-FLA primer showed 96-99% homology with N. clarki. (a), 5.8S-rRNA Sequence of FLA (Yeoju sample) amplified with ITS primer showed 96-99% homology with N. australiensis. (b), 18S-rRNA Sequence of FLA (Yeoju sample) amplified with Hart NA primer showed 96-99% homology with A. polyphaga and A. castellanii. (c). 6. Axenic culture of amoebae from water sample Fig. 3. Sequence alignment of free-living amoebae DNA amplified with ITS primer. Sequencing and alignment analysis has revealed that this sequence belonged to Naegleria spp. (a) (b) (c) Fig. 7. Yeoju amoeba, A. polyphaga and A. castellanii induced cytotoxicity on target CHO cells. CHO cells were incubated for 6, 12, 24h with or without amoeba trophozoites at a ratio of 0.1:1, 0.5:1, 1:1 for the cytotoxicity analysis, the level of lactate dehydrogenase from the culture supernatants was estimated. Fig. 4. Neighbour-joining tree depicting the relationships between environment samples with reference strains of Naegleria spp., Acanthamoeba spp. (a,b) P-FLAprimer, (c) ITS primer. Fig. 6. Morphological observation of free-living amoebae cultured on Nelson’s and PYG medium. Box, magnified cysts. (Bar, 10 ㎛). Ref) J Eukaryot Microbiol., 2000;47(2):116-21, Parasitol Res.2004;92(5):405-13, Iran J Parasitol., 2012;7(2):47-52. 7. Measure of in vitro cytotoxicity