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

1Department of Microbiology, Ajou University School of Medicine2Department of Biomedical Science, Graduate School of Ajou University, Suwon 443-721, Republic of Korea

3Department of Biomedical Laboratory Science, Molecular Diagnostics Research Institute, School of Health and Medicine, Namseoul University, Cheonan 31020, Republic of Korea4Division of Malaria and Parasitic Disease, Korea National Institute of Health, Osong 363-951, Republic of Korea

Hee-kyoung Kang1,2, Gi-Sang Seong1,2, Hae-jin Sohn1,2, Suk-Yul Jung3, Sang-Eun Lee4, Mi-Yeoun Park4, Ho-Joon Shin1,2

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 filterationThe 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 analysisSimilarity 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

% C

yto

toxic

iy

(LD

H r

ele

ase)

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-FLA primer, (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