Molecular detection of free-living amoebae from Namhangang (southern Han River) in Korea

The free-living amoebae Naegleria spp. and Acanthamoeba spp. exist in the natural environment and are sometimes causal agents of lethal primary amoebic meningoencephalitis (PAM), amoebic keratitis (AK) and granulomatous amebic encephalitis (GAE) in humans, respectively. To ascertain the existence of free-living amoebae in Korea, water samples were collected from the Korean hydrosphere, Namhangang (southern Han River), an active location for water skiing and recreation. Samples underwent two-step filtration and were cultured on non-nutrient agar medium with inactivated E. coli. The remaining samples were subjected to PCR for primarily the 18S small ribosomal RNA gene and gene sequencing. Similarities in 18S rDNA sequences, in comparison with various reference amoebae in GenBank, showed 86~99% homology with N. gruberi, N. philippinensis, N. clarki, A. polyphaga, A. castellannii, and Hartmannella (Vermamoeba) vermiformis. Therefore, this study will be useful for seasonal detection of free-living amoebae from various Korean hydrospheres in future studies.

The occurrence of PAM cases continues to increase in the tropics and subtropics, with increasing risk worldwide due to global warming. In addition, as many people enjoy water-related sports, PAM occurrence has been a social issue through the broadcast media. While it has been reported in many countries, there has not yet been reported in South Korea. There, we wanted to find out what kinds of free living amoeba, especially N. fowleri, exist in South Korea. In addition, various FLAs are expected to reside in the Korean hydrosphere. Therefore, a distribution survey was conducted to identify Naegleria and Acanthamoeba species in southern Han River, an active location for water skiing and recreation.

Materials and Methods
Water sample collection. To investigate the distribution of free living amoeba in Yeoju city and Yangpyeong-gun, near southern Han River (Namhangang), which are geographically close to Seoul, we collected 1-L surface water samples with a 20-cm diameter scoop from 10 sites periodically from August 2015 to August 2016 (over 120 water samples) (Fig. 1). Sample collection sites where carries out water recreational activities were shown in Fig. 2. The atmospheric temperature at the water sampling sites was between −11 °C and 35 °C, and the water temperature ranged from 0.1 °C to 29 °C, especially 26-29 °C in August ( Table 1).

Harvesting of FLA.
To concentrate FLA from water samples, we used a two-step filtration system. The collected surface water was first filtered using a Whatman filter, followed by filtration (with a 0.4 μm pore-size bottle top filter (Thermo Fisher Scientific Inc., USA) (Fig. 3). After leaving about 30 mL of solution and thorough washing of the filter, the mixture was centrifuged at 1,500 rpm for 5 min. A portion of the pellet and filter paper were placed on a non-nutrient agar (NN-agar) plate for the amoeba culture, and the remaining pellet was subjected to PCR for amplification of the 18S RNA gene.

Culturing of FLA.
To culture FLA, the NN-agar plate was prepared with NN-agar medium. Briefly, 15 g of nutrient agar medium and 0.1 g of yeast extract were dissolved in 1,000 mL of distilled water. After sterilization and solidification, the agar medium was poured into 10 petri dishes. Escherichia coli was killed at 60 °C for 30 min, and then evenly coated on the surface of the prepared agar plate. The prepared specimen was dropped onto the medium, following which the culture dish was covered and incubated at 27 °C for 2-4 days. The amoebae were sub-cultured on fresh NN-agar plates two or three times. Further, N. fowleri, A. castellanii, and A. polyphaga used as positive control amoebae were cultured on Nelson's and PYG medium according to previous reports 29,30 . The morphological identification of cultured amoebae was observed with an inverted microscope (Olympus CKX 31, Japan). www.nature.com/scientificreports www.nature.com/scientificreports/ PCR identification of FLA. DNA was extracted from environmental sample according to the manufacturer's protocol (Qiagen, USA). Next, molecular identification of FLA has been performed as described below.
Briefly, 2 uL of the extracted DNA was used as a template, and mixed with 10 μL of Noblezyme ™ PCR Plus Premix (Noble Bioscience Inc., Korea) in a PCR tube. Primer pairs to amplify the 18S rRNA gene of various FLAs, called pan primers (P-FLA) (2pi, Bioneer Inc., Korea), were according to previous studies 31,32 . The recommended amplicon sizes were as follows: Acanthamoeba spp., 1080-1500 bp; Vahlkampfia spp., 950 bp; N. fowleri, 900 bp; and Vermamoeba (Hartmannella) vermiformis, 800 bp. Amplification was performed with an initial polymerase activation step (5 min at 95 °C), followed by 35 cycles of denaturation (1 min s at 94 °C), primers hybridisation (1 min at 60 °C), and extension (3.5 min at 74 °C) in a G-Storm thermocycler (Genetechnologie, UK). All PCR experiments were performed with the inclusion of positive controls (DNA extracted from N. fowleri, A. polyphaga, and A. castellanii) to ensure correct functionality of the reaction.
After completion of the reaction, the PCR products were electrophoresed at 120 V for 20 min using a 1% agarose gel stained with ethidium bromide (0.005%) and then analyzed by Gel-doc (Bio-rad, USA).
18S rDNA sequencing and phylogenetic analysis. The FLA nucleotide sequences of the PCR-amplified 18S rRNA gene were obtained from the direct sequencing (Genotech, Daejeon, Korea), and homology against registered FLAs in GenBank was analyzed. Based on this, we conducted FLA phylogenetic analysis by estimating the neighbor-joining distance using the MEGA6 program 33 .

Results
Morphology of cultured FLA. FLA cultured from water samples showed a resemblance to the presumed genus Naegleria or Acanthamoeba, as trophozoites showing round pseudopodia or acanthopodia; cysts were also observed in the colony (Fig. 4).
Phylogenetics of FLA 18S rDNA sequences. A neighbor-joining distance tree was constructed based on phylogenetic analysis of the DNA sequences from the amplified FLA 18S rRNA gene. Many Yeoju specimens clustered mainly with N. clarki and N. gruberi, whereas Yangpyeong samples were closely related to N. gruberi and N. australiensis (Fig. 8). However, one of the Yeoju samples clustered with A. castellanii and A. polyphaga (Fig. 8).  Table 1. Summary of free-living amoebae and other microorganisms from Yeoju and Yangpyeong water samples identified by the homology analysis with 18s-rRNA gene sequence.

Discussion
Epidemiological studies of PAM have mainly occurred in the southeastern part of the United States, through swimming and watering activities in N. fowleri-contaminated freshwater, such as lakes and ponds, during the summer months 20,34,35 . Subsequently, in other countries, various survey have been conducted on ponds, lakes, rivers, swimming pools, hot springs, and contaminated sewage, which are known as FLA habitats.  www.nature.com/scientificreports www.nature.com/scientificreports/ In Thailand and Japan, Naegleria and Acanthamoeba species were detected in hot springs frequented by tourists and local residents 36,37 . In Italy, New Zealand, and California of the United States, N. fowleri was detected in a river area used for swimming and in a swimming pool [38][39][40] . In Taiwan, the nearest Asian country, N. fowleri was isolated from hot springs, and Acanthamoeba and Naegleria spp. from recreational water 41 . Acanthamoeba spp. was detected in water in neighboring China 42 . In Korea, Acanthamoeba spp. has been detected in amoeba-contaminated tap water in damaged water pipes and in several AK patients 43,44 .
We investigated only water samples where water sports or leisure activities were active, because we have the interest in public health which is caused by pathogenic free-living amoebae, especially Naegleria spp. and Acanthamoeba spp. Based on this survey on FLA distribution in the Korean hydrosphere, especially Namhangang, various species of Naegleria sand Acanthamoeba were found in Yeoju and Yangpyeong samples, especially in August. Notably, the highly virulent N. fowleri (which favors high temperatures) was not found in this survey, possibly because the temperature of the water system was inadequate (measured as 26-29 °C). A future detailed and extensive survey will be required to determine whether virulent N. fowleri inhabits this area. We conducted the survey over the course of a year to observe seasonal variations as well. However, the temperature in Korea