Evaluation of molecular characterization and phylogeny for quantification of Acanthamoeba and Naegleria fowleri in various water sources, Turkey.

Free-living amoeba (FLA) is widely distributed in the natural environment. Since these amoebae are widely found in various waters, they pose an important public health problem. The aim of this study was to detect the presence of Acanthamoeba, B. mandrillaris, and N. fowleri in various water resources by qPCR in Izmir, Turkey. A total of (n = 27) 18.24% Acanthamoeba and (n = 4) 2.7% N. fowleri positives were detected in six different water sources using qPCR with ITS regions (ITS1) specific primers. The resulting concentrations varied in various water samples for Acanthamoeba in the range of 3.2x105-1.4x102 plasmid copies/l and for N. fowleri in the range of 8x103-11x102 plasmid copies/l. The highest concentration of Acanthamoeba and N. fowleri was found in seawater and damp samples respectively. All 27 Acanthamoeba isolates were identified in genotype level based on the 18S rRNA gene as T4 (51.85%), T5 (22.22%), T2 (14.81%) and T15 (11.11%). The four positive N. fowleri isolate was confirmed by sequencing the ITS1, ITS2 and 5.8S rRNA regions using specific primers. Four N. fowleri isolates were genotyped (three isolate as type 2 and one isolate as type 5) and detected for the first time from water sources in Turkey. Acanthamoeba and N. fowleri genotypes found in many natural environments are straightly related to human populations to have pathogenic potentials that may pose a risk to human health. Public health professionals should raise awareness on this issue, and public awareness education should be provided by the assistance of civil authorities. To the best of our knowledge, this is the first study on the quantitative detection and distribution of Acanthamoeba and N. fowleri genotypes in various water sources in Turkey.

Introduction

Free-living amoeba (FLA) are unicellular protozoa that commonly find in soil and water throughout the world. Free-living amoeba could be found in tap water, well water, seawater, streams, river, swimming pools, dams, lakes, and air-conditioning systems [1]. Among the numerous FLA species present in nature, the most common species are Acanthamoeba, Balamuthia mandrillaris (B. mandrillaris), and Naegleria species, which play a role in human and animal infections [2]. Acanthamoeba spp. and B. mandrillaris may cause granulomatous amoebic encephalitis (GAE), cutaneous lesions, lung infections, and also Acanthamoeba keratitis (AK) in immunocompetent persons. Acanthamoeba genus is divided into 22 different genotypes based on the 18S rRNA gene, and genotype T4 is one of the most common in the environment and the most common genotype causing human infection [3, 4]. N. fowleri causes primary amoebic meningoencephalitis (PAM) in immunocompetent children and young adults [1, 5].

This pathogenic FLA enters the body via nasal mucosa and/or the skin lesions and then disseminate along the olfactory neuroepithelial route or by following the hematogenous spread route they gain entry into the brain to occur infection [6]. Quantitative screening of these amoebae in various water sources is crucial since they pose risk to human health. To date, in vitro culture methods were used to quantitatively assessment of aquatic FLA, but there are some limitations such as time-consuming procedure, precision, and accuracy [7]. Quantitative real-time PCR (qPCR) assay is a method with high specificity and sensitivity useful for detecting the presence of the amoebae in water resources [8, 9].

The aim of this study was to identify rapidly and accurately the presence of Acanthamoeba spp., B. mandrillaris, and N. fowleri in various water sources by qPCR assay. Moreover, the sensitivity, specificity, and efficiency of the qPCR were also evaluated. Finally, the water quality parameters were measured and Acanthamoeba culture-positive samples were subjected to osmo/thermo-tolerance test to measure their pathogenicity. In the light of the results obtained, identified isolates were evaluated as potential risks for humans.

Material and methods

The geographical location of the study area

The study is conducted in the province of Izmir, which is the third-largest city in western Turkey. Izmir is located between the northern latitudes 37° 45’ and 39° 15’ and 26° 15’ and the east longitudes 28° 20’ and have a surface area of 12.012 km2. Izmir city is in Mediterranean climate zone and summers are also hot and dry and followed by mild and wet winters. According to National Meteorological Service average temperature in summertime was higher than 30°C and highest temperature might be higher than 40°C. While the approximate population for 2020 was 4.394.694, this number increases even more during the summer months, since the region is one of the important touristic areas in Turkey. There are many irrigation dams, lakes, and ponds in this region.

Water sample collection and processing

A total of 148 water samples were collected from places within the boundaries of Izmir that could pose a risk to people and where human contact was high. Tap water (TW), pool water (PW), well water (WW), lake water (LW), dam water (DW), stream water (StW), seawater (SeW), and thermal spring water (TsW) were collected from various water sources and the geographic coordinates were shown (S1 Table). Water samples were collected in approximately one liter (lt) of the sterile glass bottles and stored at 4°C for subsequent analyses within 24 hr.

For culture and DNA isolation one liter of water sample was concentrated by filtration using a nitrocellulose membrane with a pore size of 0.22 μm (Sartorius Stedim Biotech, Göttingen, Germany). The filter membrane was divided into two equal parts with the help of a sterile scalpel and forceps, then half of the filter was transferred into the center of the NNA plate [10].

Analysis of water quality parameters

Water quality parameters, including total dissolved solids content (TDS), electrical conductivity (EC), and temperature were measured in situ using the portable thermometer (TDS&EC meter hold). The temperature, TDS, and EC of the parameters indicate sensitivity in degrees between 0.1 and 80.0°C (Celsius), 0 and 5000 ppm (parts per million), 0 and 9990 μs/cm, respectively. The chlorine level of the water samples was evaluated in situ using the chlorine test kit (Sutest Liquid Test Kit). The pH of the collected water samples was determined using a pH-meter (HI 2211–02, Hanna Instruments Inc., Woonsocket, MA, USA) in Ege University Parasitology Department Laboratory.

Culture of free-living amoeba

After filtration of each water sample, the half of the cut filter was placed in the center of 2% non-nutrient agar (NNA) plates previously seeded with heat killed 100 μl Escherichia coli (ATCC 25922) bacterial suspension and the edges of the plates were sealed around with parafilm (Heathrow Scientific, Vernon Hills, IL, USA). The plates were incubated in the inverted position at 30–32°C and exanimated daily with the inverted microscope for 10 days. Plates without proliferation were considered negative after a check of at least two weeks. FLA-positive plates were then sub-cultured by cutting off small pieces circled with a pen under the microscope and transferring them to new fresh NNA plates to purifying them from other organisms, especially fungi and yeasts. The grown of Acanthamoeba trophozoites and cysts were characterized from other free-living amoebas. Besides, the cyst shape has been easily identified by the double wall of the cyst and typical star shape [11, 12].

Tolerance assays for Acanthamoeba positive samples

Pathogenicity tests were repeated three times for each positive sample. The pathogen strains of Acanthamoeba castellanii and Acanthamoeba spp. (EU266547 –T4) from Cumhuriyet and Dokuz Eylül Universities were used as reference strains.

Osmo-tolerance assay

To investigate the effect of osmolarity of each isolate on the trophozoites of Acanthamoeba (approximately 103 trophozoites/plate), trophozoites were coated with mannitol-free E. coli and transferred to the center of NNA plates (as a control). Positive isolates (approximately 103 trophozoites/plate) were transferred to the center of the plates by coating the NNA plate with E. coli suspension prepared at 0.5 M and 1 M mannitol concentration. The plates were then incubated at 30°C for 10 days and the growth of amoebae at 24, 48, and 72 hours was evaluated. Trophozoites or cysts were counted at a microscopic magnification at x100 of five microscopic areas of approximately 20 mm from the center of each plate. The presence of proliferation was evaluated as (+) positive, and the absence of growth (-) as negative [13, 14].

Thermo-tolerance assay

For the thermo-tolerance assay, the trophozoites of Acanthamoeba spp. (approximately 103 trophozoites/plate) were transferred to the center of E. coli coated NNA plates. These plates were incubated at 30°C (as a control), 37°C, and 42°C for 10 days; It was evaluated after 24, 48, and 72 hours of the incubation. During this period, proliferation was evaluated under the microscope as mentioned in the osmo-tolerance test [13, 14].

DNA extraction from culture and filter membranes

For the DNA isolation of the amoeba from the NNA plates, which were identified as Acanthamoeba spp. by microscopy, 2 ml of 1xPBS (Thermo Fisher Scientific, Phosphate-Buffered Saline (PBS), pH: 7.4) buffer solution was dropped onto the plate. The amoebas from agar plates were collected into the tube using the sterile swab after waiting for approximately 5 minutes. The tubes were centrifuged at 2500 rpm for 10 min and washed with PBS buffer solution. Acanthamoeba genomic DNA was extracted with the QIAamp DNA mini kit (Qiagen GmbH, Germany) according to the manufacturer’s recommendations.

Half of the 0.22 μm diameter membrane of the filtered water sample was cut into several pieces with sterile scissors and transferred to the bead tube. Total genomic DNA was extracted using the Norgen Biotek Water RNA/DNA Purification Kit (Water RNA / DNA Norgen Biotek Corp., Canada) following the manufacturer’s protocol. Briefly, after placing the filter into the tube with beads, 500 μl Lysis buffer E was added. The tubes were vortexed for 30 sec using FastPrep®-24 instrument (MP Biomedical). The tubes were then incubated in the thermal block at 65°C for 10 min. and then centrifuged at 20 000 g for 1 min. After centrifugation, 600 μl ethanol was added to the mixture and transferred to filter tubes. The filter tubes were then washed twice with 400 μl of wash solution A and genomic DNA sample was obtained by adding 100 μl of elution buffer H. DNA concentration and purity were measured using the NanoDrop® 1000 spectrophotometer (NanoDrop Technologies, Wilmington, DE, USA). DNA samples were kept at -20°C until the PCR experiments.

Positive control plasmid for PCR

The positive control of Acanthamoeba spp. was obtained from a reference strain, which was isolated from a human case with Acanthamoeba keratitis (GenBank No: EU266547 –T4). DNA samples of N. fowleri and B. mandrillaris strains were obtained from the Center for Disease Control and Prevention (CDC).

The 18S rRNA gene for Acanthamoeba spp., and B. mandrillaris and the 5.8S rRNA and ITS (ITS1 and ITS2) regions for N. fowleri were selected as targets to determine the presence of the plasmid copy quantification in water samples [15-17]. The primers and conditions were used for amplification are the same as those used for the LightCycler 480 PCR test described below. A plasmid containing the PCR amplified product was commercially synthesized by Letgen Biotechnology Laboratory (Letgen Biotechnology, İzmir, Turkey) using the pGEM-T Vector cloning kit (Promega Corporation, Madison, WI) following the manufacturer’s instructions. The number of copies in the plasmid solution was calculated using a NanoDrop ND1000 spectrophotometer (NanoDrop Technologies, Wilmington, DE, USA). Serial dilutions (plasmid controls ranging from 1x109 to 1x100 copies plasmid/μl) on the order of 10-fold of 18S rRNA gene and ITS region fragment were used to generate the standard curve of concentrations expressed in log units (log10) versus the values obtained in amplification cycles. Quantification analysis for each plasmid control was performed with the Light Cycler 480 II® Thermal Cycler (Roche Diagnostic) on 96-well white LightCycler 480® multiwell plates (Roche Diagnostics Ltd, Switzerland).

Quantitative real-time PCR (qPCR) assay

The qPCR was performed using DNA obtained from cultures and direct water filters. The quantification of Acanthamoeba spp., B. mandrillaris, and N. fowleri DNA was performed by using a LightCycler 480 II (Roche Diagnostics, Mannheim, Germany) Real-Time PCR Systems. The targets sequence of the 180-bp (Acanthamoeba spp), 171-bp (B. mandrillaris), and 123-bp (N. fowleri) fragments of the 18S rRNA and ITS gene regions were amplified. The primers and the respective probes used in this study were described in S2 Table [15, 17]. Reaction was performed in a final volume of a 20 μl in 96-well plates, containing 5 μl DNA template or controls, 5 x TaqMan Master Mix (Roche), 0.25 μM each of primer and 0.2 μM of each probe and then centrifuged at 1500 g for 2 min at 4°C. The PCR conditions were conducted using the following calculated control protocol: 10 min pre-incubation step at 95°C, followed by 45 cycles of 10 sec at 95°C, 1 min at 60°C and 1 sec at 72°C and followed by a final cooling step at 40°C for 30 sec.

All qPCR reactions were run using positive control (plasmid DNA), negative control DNA (using double distilled water) and were tested in triplicate in each reaction. The Ct value (cycle threshold) was defined as the number of cycles required for the fluorescence signal to cross the threshold. The Ct value is inversely correlated with the quantity of DNA. A standard curve was constructed using a series of dilutions with a known quantity of plasmid DNA standards. The slope (S) of the standard curve was adopted as an indication of the efficiency of the real-time PCR amplification. The efficiency (E) of the qPCR amplification was calculated according to the equation of E = 10 (-1 /Slope)-1.

Assessment of possible the qPCR inhibition in water samples

Duplicate PCR reactions were performed to eliminate possible PCR inhibition that might arise from water samples. One of the reactions contained only purified water sample DNA, while the other reaction contained ten plasmid copies with the spiked into the purified water DNA sample. To generate a cycle threshold value, three reactions containing ten plasmid control DNA copies in distilled water were performed. The Ct values observed for the spiked water sample with a positive control plasmid differed from the mean >40 Ct value were considered to be indicative of PCR inhibition. The inhibited samples were diluted 10-fold and PCR was analyzed again as described above.

DNA sequencing and phylogenetic analysis

To genotype qPCR positive samples, conventional PCR was performed for Acanthamoeba spp. and Naegleria spp., with primers specific for 18S rRNA gene (JDP1-JDP2) and ITS region (FW2-RV2), respectively (S2 Table) [18, 19]. PCR was performed in a total volume of 50 μl including 25 μl of 2x PCR Master Mix, 10 pmol of each primer and 5 μl template DNA. reaction was carried out in a Techne TC-3000 Thermal Cycle (Techne, Staffordshire, UK) by following program: an initial denaturation at 95°C for 2 min followed by 35 cycles 95°C for 35 s, annealing step at 60°C and 58°C for 40 s for Acanthamoeba spp. and Naegleria spp. respectively and final extension 3 min at 72°C. All PCR products were separated by electrophoresis on 2% agarose gel, stained with Safeview Classic (Applied Biological Materials Inc., Richmond, Canada) and were photographed using an Alpha Imager HP (Alpha Innotech), on a UV transilluminator. All the PCR products were purified with the QIAquick PCR purification kit (QIAGEN, Germany) according to the manufacturer’s instructions. Sequencing was performed by a commercial company using an ABI automated sequencing system (Microsynth, Balgach, Switzerland). The resulting sequences were alimented using ClustalW based on sequence analysis of DF3 and ITS region as previously described [20, 21] by comparing to the available Acanthamoeba and N. fowleri DNA sequences in GenBank database (Department of Molecular Genetics, The Ohio State University, OH, USA). Phylogenetic relationship among the sequences was made using the neighbor-joining with molecular distances under the Kimura two-parameter distance model with the Molecular Evolutionary Genetics Analysis (MEGA X) software program [22]. The accuracy of the phylogenetic tree was assessed by 1000 bootstrap replicate data sets. The root of the tree was established using an outgroup (Saccamoeba lacustris GenBank No: JN112797.1). Sequence data obtained for Acanthamoeba and N. fowleri isolates were deposited in the GenBank database under the accession numbers MW689474–MW689500 and MW677627-MW677629, MW676178, respectively.

Statistical analysis

The data obtained were analyzed using Mac OSX SPSS 25.0 version (SPSS Inc, Chicago, IL, USA) software. Numerical variables were summarized using the mean and standard error of the mean. Kolmogorov-Smirnov test was used to determine the importance of normality. The Mann-Whitney U test was used to compare the relationship between water quality parameters and the presence/absence of Acanthamoeba spp., and N. fowleri in environmental water samples. A value of p≤0.05 was considered statistically significant.

Results

Isolation of Acanthamoeba spp. in culture

A total of 148 water samples were collected from 19 different districts of Izmir including tap water (n = 44), well water (n = 31), pool water (n = 26), lake water (n = 18), dam water (n = 10), stream (n = 9), seawater (n = 8) and thermal spring water (n = 2). From the total of samples, 71 (47.97%) were found to be positive for FLA. Eighteen out of 148 water samples (12.16%) included in this study were found positive for Acanthamoeba spp. according to Page’s morphological analysis criteria (Fig 1). Acanthamoeba were detected in various water sources including 12.9% (4/31) WW, 44.44% (4/9) StW, 6.81% (3/44) TW, 11.53% (3/26) PW, 11.11% (2/18) LW, and 25% (2/8) SeW (Table 1).

Fig 1

Acanthamoeba cysts and trophozoites in non-nutrient agar plate found in various water sources collected from in İzmir, Turkey.

A) Acanthamoeba cysts, B) Acanthamoeba trophozoites.

Table 1

Result of the qPCR assay and culture for Acanthamoeba spp. in various waters sources.

Water sources	Sample No.	Culture		qPCR		Mean Ct value	Acanthamoeba spp. (plasmid copies/l)
		N	(%)	N	(%)
StW	9	4	44.44	4	44.44	32.9	8.8x104-7.3x102
LW	18	2	11.11	5	27.77	37.2	1.2x103-1.4x102
SeW	8	2	25	2	25	31.2	3.2x105-5.3x103
PW	26	3	11.53	6	23.07	36.8	5.3x103-1.6x102
WW	31	4	12.9	6	19.35	34.4	1.6x104-1.4x103
TW	44	3	6.81	4	9.09	36.1	1.9x104-1.8x102
Total	136	18	13.23	27	19.85

TW: Tap water, PW: Pool water, WW: Well water, LW: Lake water, StW: Stream water, SeW: Seawater

Pathogenic potential of Acanthamoeba spp. in positive water samples

The results of the tolerance assay of 18 Acanthamoeba isolates grown in culture were shown in Table 2. All of the 18 Acanthamoeba isolates investigated were grown at 37°C and 0.5 M mannitol. Ten (55.5%) of the isolates were grown both at 42°C, and 1 M mannitol. Only eight (44.4%) of the isolates were not grown at 42°C and 1 M mannitol. Therefore, ten isolates (55.5%) were considered potentially pathogenic, and the rest (44.4%) were classified as low pathogenic potential.

Table 2

Thermo/osmo-tolerance assay of Acanthamoeba positive strains isolated from water samples in different districts of İzmir province.

								Tolerance Assay
Strain No.	Sampling area	Locality	NNA	qPCR	PCR (JDP)	Species	Genotype	Osmo-tolerance (M, mannitol)	Thermo-tolerance (°C)
								0.5 / 1	37 / 42
IWS3	Tap water	Torbalı	+	+	+	Acanthamoeba sp.	T4	+/+	+/+
IWS24	Tap water	Torbalı	+	+	+	Acanthamoeba sp.	T4	+/+	+/+
IWS30	Tap water	Çeşme	+	+	+	Acanthamoeba sp.	T5	+/+	+/+
IWS47	Pool water	Bornova	+	+	+	Acanthamoeba sp.	T2	+/-	+/-
IWS56	Pool water	Bornova	+	+	+	Acanthamoeba sp.	T5	+/-	+/-
IWS60	Pool water	Bornova	+	+	+	Acanthamoeba sp.	T4	+/+	+/+
IWS78	Well water	Bayraklı	+	+	+	Acanthamoeba sp.	T4	+/+	+/+
IWS80	Well water	Dikili	+	+	+	Acanthamoeba sp.	T15	+/-	+/-
IWS93	Well water	Bayraklı	+	+	+	Acanthamoeba sp.	T4	+/+	+/+
IWS95	Well water	Dikili	+	+	+	Acanthamoeba sp.	T2	+/+	+/+
IWS103	Lake water	Menderes	+	+	+	Acanthamoeba sp.	T5	+/+	+/+
IWS114	Lake water	Ödemiş	+	+	+	Acanthamoeba sp.	T4	+/-	+/-
IWS130	Stream water	Menderes	+	+	+	Acanthamoeba sp.	T2	+/-	+/-
IWS132	Stream water	Menderes	+	+	+	Acanthamoeba sp.	T5	+/+	+/+
IWS134	Stream water	Menderes	+	+	+	Acanthamoeba sp.	T4	+/+	+/+
IWS136	Stream water	Menderes	+	+	+	Acanthamoeba sp.	T4	+/-	+/-
IWS142	Seawater	Çeşme	+	+	+	Acanthamoeba sp.	T15	+/-	+/-
IWS146	Seawater	Çeşme	+	+	+	Acanthamoeba sp.	T4	+/-	+/-

(+): Positive samples, (-): Negative samples

The growth ability was evaluated by exposure at different temperatures (37°-42°C) and osmolarity ranges (0.5-1M mannitol) to test the pathogenicity potential of 18 Acanthamoeba positive samples in NNA plate culture. The isolates that can grow at high temperature (at 42°C) and high osmolarity (1 M mannitol) were considered as potential pathogenic strains. However, the other isolates that were able to grow at 37°C temperature and 0.5 M osmolarity were considered as low pathogens.

PCR amplification efficiency (E) and standard curve

A standard curve was developed by serial dilutions of known amounts of plasmid DNA ranging 109 to 100 plasmids copy from Acanthamoeba, N. fowleri, and B. mandrillaris. The standard curve of the known plasmid concentrations and Ct values of Acanthamoeba, N. fowleri, and B. mandrillaris was shown in Fig 2. A slope (S) of -3.37, -3.58, and -3.35 equals a PCR amplification efficiency (E) of 97.8%, 90.2% and 98.5% for Acanthamoeba, N. fowleri and B. mandrillaris, respectively. Moreover, the determination coefficient (R) of 0,99, 0,99, and 0,98 were observed for Acanthamoeba, N. fowleri, and B. mandrillaris, respectively. The lowest detection limit for Acanthamoeba, and N. fowleri of the qPCR assay was determined as one plasmid copy per reaction. However, the lowest detection limit for B. mandrillaris was set at 10 plasmid control DNA copies.

Fig 2

Standard curves were generated by linear regression of the cycle threshold (Ct) versus (A) Acanthamoeba, (B) N. fowleri, and (C) B. mandrillaris plasmid control DNAs.

Quantification of Acanthamoeba and N. fowleri from various water

A total of 27/148 (18.24%) Acanthamoeba spp. and 4/148 (2.7%) N. fowleri positives were detected in six different water sources by the qPCR assay. However, all water samples were found negative for B. mandrillaris. The qPCR assay was applied to confirm positive samples in terms of FLA in culture, and 18 (12.16%) Acanthamoeba and four (2.7%) N. fowleri were found to be positive. The overall presence of Acanthamoeba spp. in various water sources were 4/9 (44.44%) in StW, 5/18 (27.77%) in LW, 2/8 (25%) in SeW, 6/26 (23.07%) in PW, 6/31 (19.35%) in WW, and 4/44 (9.09%) in TW (Table 1).

Acanthamoeba spp. were found more frequent in streams samples than tap waters. However, it could not detect in dam water and thermal spring water. The Ct value of PCR amplification of Acanthamoeba in StW, LW, SeW, PW, WW, and TW in the ranges from 28.2 to 39.4, and also plasmid copies concentrations of Acanthamoeba 18S rRNA gene were detected in the range of 3.2x105-1.4x102 plasmid copies/l. In our study, the highest concentration of Acanthamoeba was found quantitatively in seawater samples, while the lowest concentration was found in lake water (Fig 3).

Fig 3

DNA quantity of Acanthamoeba plasmid copies determined by qPCR assay in various water sources from İzmir, Turkey.

N. fowleri was detected in various water sources, 2/26 (7.69%) in PW, 1/18 (5.5%) in LW, and 1/10 (10%) in DW samples. For various water sources in PW, LW, and DW, means of Ct values was ranged from 35.13 to 38.13, and plasmid copies of N. fowleri were between 8x103 and 11x102 plasmid copies/l. The average highest concentration of N. fowleri was shown in dam water (Table 3). Two water samples which sample code PW12 and LW4 were detected positive both Acanthamoeba and N. fowleri.

Table 3

Result of the qPCR assay for Naegleria fowleri in various waters.

Strain No.	Sample code	Mean Ct value	N. fowleri (plasmid copies/l)
IWS52	PW8	38.13	11 x102
IWS56	PW12	37.71	15 x102
IWS105	LW4	36.68	3 x103
IWS120	DW1	35.13	8 x103

IWS: Izmir water sample, PW: Pool water, LW: Lake water, DW: Dam water

Genotyping of Acanthamoeba spp. and N. fowleri-positives in water samples

All 27 Acanthamoeba isolates detected positive by qPCR were subjected for PCR amplification using JDP primer sets, which is specific for the DF3 region of the 18S rRNA gene sequences. The sequence data obtained from Acanthamoeba isolates were aligned using Clustal W software and were used to construct the phylogenetic tree to illustrate the relationships between the isolates obtained and reference sequences of Acanthamoeba genotypes retrieved from GenBank (A. palestinensis genotype T2 accession nos: U07411 and L09599; A. castellanii genotype T4 accession nos: MF806034, KT892904, MF139789,MH620482, MH620483, MK192795 and MG969963; A. lenticulate genotype T5 accession nos: KX018036, U94730, EU377584, U94740, U94737 and U94736; A. jacobsi genotype T15 accession nos:KX870203, KT892847, GQ905495 and MH790995; Acanthamoeba genotype T17 accession no:GU808277; Acanthamoeba genotype T18 accession no:KC822461). All samples showed nucleotide identity between 98% and 100% with reference strains deposited in GenBank (BLASTn) (www.ncbi.nlm.nih.gov/BLAST). In this study, four different genotypes (T2, T4, T5, and T15) were detected in water samples. Fourteen (51.85%) of 27 Acanthamoeba isolates were belonged to T4 genotype. The remaining isolates were belonging to T5 genotype 6/27 (22.22%), T2 genotype 4/27 (14.81%) and T15 genotype 3/27 (11.11%). The distribution of Acanthamoeba genotypes according to different districts of İzmir province and various water resources was showed in Fig 4 and Table 4. According to the phylogenetic tree, Acanthamoeba isolates T4 obtained from various water sources were grouped within the clade including the other sequences of Acanthamoeba castellanii complex available from Genbank. Six isolates (IWS_132, IWS_79, IWS_30, IWS_56, IWS_68, IWS_103) were found to be Acanthamoeba genotype T5 revealing 98% sequence identity to various T5 reference strain. Phylogenetic tree showed that three isolates (IWS_61, IWS_80 and IWS_142) were strictly related with Acanthamoeba T15 genotype chosen as references with 100% of identity, four isolates (IWS_14, IWS_47, IWS_95 and IWS_130) T2 genotype with 98% of identity with the T2 sequence references (Fig 5).

Fig 4

The distribution of Acanthamoeba genotypes according to different districts of İzmir province and various water resources.

Table 4

Distribution of Acanthamoeba genotype in various water sources.

Genotype	Total		LW	WW	TW	StW	PW	SeW
	%	n
T4	51.85	14	4	3	2	2	2	1
T5	22.22	6	1	1	1	1	1
T2	14.81	4		1	1	1	1
T15	11.11	3		1			1	1
Total	100	27	5	6	4	4	5	2

TW: Tap water, PW: Pool water, WW: Well water, LW: Lake water, StW: Stream water, SeW: Seawater

Fig 5

Phylogenetic tree inferred using neighbor-joining models of the 18S rRNA gene DF3 region sequence data for the Acanthamoeba genotypes by MEGA X.

Bootstrap values are based on 1000 replicates and the root of the tree evaluated by an outgroup Saccamoeba lacustris.

Four positive samples detected N. fowleri by qPCR were shown to be 99–100% nucleotide identity with references isolates in GenBank (N. fowleri accession nos: AJ132028, X96565, AJ132019 and X96564). Genotypic differences of N. fowleri can be distinguished based on a one bp transition in 5.8S rRNA and the length of the internally transcribed spacer (1) [23]. Among the N. fowleri positive water samples (DW1, LW1and PW12), three of them do have T at position 31 in the 5.8S rRNA, and the ITS1 length was 42 bp, so these were identified as belonging to type 2. However, in one N. fowleri (PW8) sample, the sequence C has not changed to T at position 31 in 5.8S rRNA and the ITS1 length was found to be 84 bp with a two bp deletion in the sequence of repeat. According to obtained data this sample was identified as type 5 (Table 5). The phylogenetic tree was shown using neighbor-joining models of 5.8S rRNA, ITS1, and ITS2 sequence data for Naegleria spp. (Fig 6). According to the phylogenetic tree, N. fowleri isolates obtained from various water sources were grouped within the clade including the other sequences of N. fowleri genotypes available from GenBank. Three isolates (LW1, DW1 and PW12) were found to be N. fowleri type 2 revealing 100% sequence identity to various N. fowleri type 2 reference strains (N. fowleri type 2 accession nos: AJ132019 and X96564). However, phylogenetic tree showed that one isolates (PW8) were strictly related with N. fowleri type 5 chosen as references (N. fowleri type 5 accession nos: AJ132028 and X96565) with 99% of identity. In the present study, N. fowleri type 2 and type 5 were identified in water samples for the first in Turkey.

Table 5

Base length and position of ITS1, ITS2 and 5.8S rRNA sequences of references strains and all the positive isolates in this study.

Isolate	Genotype	ITS1	5.8S	Position 31 in 5.8S	ITS2	Total length	Accession number	Reference
7853	T1	42	175	C	106	323	AY376149	[1]
AR12	T2	42	175	T	106	323	X96564	[2]
LEE	T3	86	175	T	106	367	X96562	[2]
Ch2-1-f2	T4	86	175	T	106	367	AJ132030	[3]
Na 420c	T5	84	175	C	106	365	AJ132028	[3]
J2B2	T6	114	175	C	106	395	FR875287	[4]
M4E	T7	142	175	T	106	423	X96563	[2]
C0504	T8	130	175	T	106	411	FR875288	[4]
DW1	T2	42	175	T	106	323	MW677629	This study
LW1	T2	42	175	T	106	323	MW677627	This study
PW12	T2	42	175	T	106	323	MW676178	This study
PW8	T5	84	175	C	106	365	MW677628	This study

[1] Zhou et al., 2003.

[2] De Jonckheere, 1998.

[3] Pélandakis et al., 2000.

[4] De Jonckheere, unpublished.

Fig 6

Phylogenetic tree of Naegleria isolates for the ITS sequences.

Phylogenetic tree inferred using neighbor-joining cluster analysis of the sequence obtained and the sequences from ITS1, 5.8S rRNA and ITS2 sequences of various Naegleria strains, with special reference to N. fowleri produced in MEGA X. Bootstrap values are based on 1000 replicates.

Associations between Acanthamoeba and water quality parameter variables

A non-parametric test was conducted to determine the relationship between five different water quality parameters and the presence/absence of Acanthamoeba in various water sources. The result of the non-parametric statistical test was shown in Table 6. A significant relationship with Mann-Whitney U test was found between the presence/absence of Acanthamoeba spp. and pH values from well water and also EC-TDS values from pool water, lake water, and sea waters (p<0.05).

Table 6

Result of non-parametric test showing the relationship between five different water quality parameters and the presence/absence of Acanthamoeba spp. in various water sources.

Water quality parameters	Mann-Whitney U test
	TW	PW	WW	LW	StW	SeW
pH value	P = 0,390	P = 0,223	P = 0,004 *	P = 0,692	P = 0,135	P = 0,495
Temperature (°C)	P = 0,744	P = 0,271	P = 0,515	P = 0,235	P = 0,618	P = 0,495
Free Cl (ppm (mg/l))	P = 0,163	P = 1,000	P = 0,118	P = 1,000	P = 0,091	P = 1,000
a EC (0–9990 μs/cm)	P = 0,624	P = 0,002 *	P = 0,146	P = 0,002 *	P = 0,135	P = 0,008 *
b TDS (0–9990 ppm)	P = 0,540	P = 0,002 *	P = 0,051	P = 0,002 *	P = 0,618	P = 0,040 *

a Electrical conductivity,

b Total dissolved solids

* Statistically significant p < 0.05

TW: Tap water, PW: Pool water, WW: Well water, LW: Lake water, StW: Stream water, SeW: Seawater

Discussion

Among the FLA, Acanthamoeba spp., N. fowleri, and B. mandrillaris are eukaryotic protists widely found in many places of the world. These FLAs can potentially cause opportunistic/non-opportunistic infections in humans and animals [1, 24]. Recently, they have received increasing attention in the medical and scientific world due to the serious fatal infections in humans. The impact of Acanthamoeba and N. fowleri on human health is associated with the genotypes of these pathogens and their reproduction in water and soil resources, a natural reservoir location. Therefore, preventive, and investigational monitoring programs for measuring the density of Acanthamoeba and N. fowleri are important in aquatic environments with human exposure, which can be achieved with real-time qPCR [3]. This study was occurred the quantify presence of Acanthamoeba and N. fowleri from various water sources in province İzmir, Turkey. Acanthamoeba spp. were found positive ranging from 4.4% to 50% in various water sources such as tap water, ponds, rivers, streams, and water wells in Turkey [25-29]. Acanthamoeba was detected positively 17.3%, 28.8%, 15.9% and 42.9% in Jamaica, Iran, Thailand, and Uganda in tap water sources, respectively [30-33]. The results of this study which indicate that the Acanthamoeba spp. occurrence in various water sources (18.24%) are similar to those results obtained in the America, Brazil, and Japan Jamaica, Iran, Thailand, Turkey. In previous reports, Naegleria spp. was found in various water sources worldwide at 0.6%–60.9% all over the world, and 0.7–10% in Turkey. [26, 34, 35]. We conclude that Naegleria spp. and Acanthamoeba spp. are free-living amoebas that have suitable growth in various water sources worldwide, but detection rates at different regions may be influenced by water types and geographical conditions.

The qPCR assay is a method that can effectively detect and quantify amoeba even it was failed to culture due to low sample. However, standard curves characterizing the relationship between plasmid copy number and qPCR data can facilitate the quantification of microorganisms in environmental water samples [8, 17]. In this study, the lowest detection limit (one plasmid copy) was detected for Acanthamoeba and N. fowleri for each reaction by qPCR method. In previous reports, similar results was identified the lowest detection limit of one plasmid copy DNA [9]. In this study, the quantitative amount of Acanthamoeba spp. DNA obtained from direct water samples was ranged from 3.2x105 to 1.4x102 plasmid copies/l. In previous studies performed in Germany and Taiwan, Acanthamoeba spp. was reported to range from 2.0–3.0×103 and 2.0×102–9.0×104 and 3.4–5.0×103−2.2–1.4×103 amoebae/l, respectively [8, 36]. We thought that might be the reason why Acanthamoeba is detected in high concentrations in seawater samples is due to the mixing of sewage wastes or industrial wastewater to the beaches. Moreover, The World Health Organization (WHO 2006) reported the presence of Acanthamoeba in seawater to be associated with sewage and waste effluent outlets. Lorenzo-Morales et al. reported the high rate of isolation (49.6% and 64.0%) for Acanthamoeba in seawater due to the sewage-waste and industrial effluent [30, 37]. In our study, the quantitative amount of N. fowleri in dam, lake, and pool water samples was determined as between 11x102 and 8.0x103 plasmid copies/l. Recent study performed in Belgium was reported that Naegleria spp. in cooling water samples was 6.3x102–4.1x103 cells/l [7]. In Australia, Naegleria spp. have been reported in the range of 4–3.4×102 cells/l in the drinking water sample [38]. Naegleria spp. concentration has been reported in the range of 1.1–24.2 cells/l in hot spring and drinking water samples in Taiwan [39].

Up to date, 22 genotypes (T1-22) of Acanthamoeba have been identified as a result of the sequence analysis of the DF3 region of the 18S rRNA gene, but this classification containing both pathogens and non-pathogens genotypes [3, 40]. Among these genotypes, it has been reported that the T4 genotype was found the most common in environmental and clinical samples and is the pathogen of different diseases (AK, GAE, skin lesions) [5, 41]. Furthermore, the other genotypes including T2, T3, T5, T6, T10, T11, T12, T15, and T18 was reported the association with human infections [1, 42, 43]. In our study, genotype T4 (51.85%) was determined as the most common genotype in water samples. Apart from that, T5 (22.22%), T2 (14.81%), and T15 (11.11%) genotypes were detected in water samples. In earlier studies, genotypes T1, T2, T3, T4, and T7 of Acanthamoeba spp. were detected in freshwater resources in Egypt [44]. In another study, the genotype T4 (51%), as well as T14 (18%), T5 (11%), T3, T15, T16 and T10 (4% per each), T11 (3%) and T7, T9 (1% per each) was reported the most common genotype in water samples in Tunisia [45]. The most common genotypes, T4 (93.7%) and T2 (6.25%) were determined in the geothermal river in the southwest of Iran by Niyyati et al. [46]. The most common T4 and T2, T3, T5, T11, T15 genotypes of Acanthamoeba were detected in environmental water samples in Turkey [34, 47–50]. In the previous study T4 and T5 genotypes were reported in keratitis wild birds in İzmir [51]. Moreover, Acanthamoeba T4 genotypes were detected in corneal scraping samples taken from patients with suspected Acanthamoeba keratitis in Turkey [52, 53]. Acanthamoeba genotypes T4 and T5 commonly found in environmental samples pose a greater risk to humans and animals.

N. fowleri, which is the pathogen type of the Naegleria genus for humans, causes fatal PAM. N. fowleri is also a protist pathogen widely found in the environment including rivers, lakes, dams, hot springs, geothermal springs, untreated and treated domestic water sources, and swimming pools [54, 55]. In this study, N. fowleri was found positive for the first time in environmental water resources collected in İzmir province, Turkey. N. fowleri was found positively in various water sources including in 7.69% (2/26) pool water, 10% (1/10) dam water and 5.5% (1/18) lake water. The presence of Naegleria species has been reported to be positively detected in environmental water samples at a rate of 13.2–60.9% in Europe, 3–46% in the USA and 0.6–56.9% in Asia [7, 56–58]. Up to date, there are 47 species in the genus of Naegleria and also eight genotype of N. fowleri [58, 59] were identified. N. fowleri genotypes are characterized that difference 1, 2, 3, and 4 types are the only C to T transition at position 31 in the 5.8S rRNA sequence, because of the equal of the ITS lengths. However, N. fowleri type 5 has not identical the ITS1 lengths with the T at position 31 in the 5.8S rRNA sequence [23, 60, 61]. In this study N. fowleri type 2 and type 5 were isolated for the first time from water sources in Turkey. N. fowleri type 2 and 3 have been reported in many patients and water samples worldwide. Type 2, 3, 4, 5, 6, 7, and 8 in Europe, types 1, 2, and 3 in the USA, types 2 and 3 in Asia and only one type of type 5 in the Western Pacific (Oceania and Japan) has been found in human specimens and water samples [18, 23, 62]. Since there are a limited number of studies typing N. fowleri, the information about the pathogenicity of the types is insufficient. However, there is not yet conclusive evidence of any difference in virulence for any of the detected N. fowleri types. It is likely to be detected in humans, as types 2, 3, and 5 are the most common in waters.

Conclusions

In conclusion, the present study reports both the presence and the concentration of Acanthamoeba and N. fowleri in various water sources and demonstrates their rapidly determination by qPCR. Although the presence of Acanthamoeba was found higher in stream samples, the quantitative value of Acanthamoeba was detected higher in seawater samples. Therefore, it should be kept in mind that it may pose a risk for people during sea activities in summer in Izmir, which is a holiday region. Acanthamoeba T4 and T5 genotypes, which are commonly detected as causal agents of AK and GAE infection, were found at a high rate in various water sources in our study. In this study, N. fowleri type 2 and type 5 were isolated the first time from water sources in Turkey. Since the genotypes of Acanthamoeba spp. and N. fowleri types can be detected in many environments, they have pathogenic potentials that may pose a risk to human health. For this reason, a mandatory inspection is necessary, especially in potable waters, as it may pose a risk to people in swimming and recreational waters. In this study, the presence of pathogenic potential of the identified strains has been revealed. However, further studies needs to be done using environmental samples across Turkey. Also, clinicians and public health professionals should increase awareness about these issues by the help of civilian authorities.

Various water samples collected from İzmir region and their geographic coordination.

(DOCX)

Click here for additional data file.

The set of primers and probes in 18S rRNA and ITS gene amplification for Acanthamoeba spp., B. mandrillaris and N. fowleri.

(DOCX)

Click here for additional data file.

14 Jun 2021

PONE-D-21-09475

Evaluation of molecular characterization and phylogeny for quantification of Acanthamoeba and Naegleria fowleri in various water sources, Turkey

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Additional Editor Comments:

Line 43: It is not clear which ITS region was amplified by cPCR. only ITS1 and 5.8S rRNA were mentioned; in the text it was  described that also ITS2 was also analysed. See lines 186 and 371.

Lines 73- 76: change, for example, into “Quantitative real-time PCR (qPCR) assay is a method with high specificity and sensitivity useful for detecting the presence of the amoebae in water resources (8,9).

Lines 77-83: delete the sentences or summarize this part being the usefulness of the qPCR is well known.

Lines 86- 88: delete the  sentence; it belongs to M&M section

Line 167: please check the reference (i.e., 17)

Lines 181-182: delete the sentence “In this study, positive …. and B. mandrillaris.”

Line 186: specify “ITS”, the authors refer to ITS1, or ITS2 and 5.8S; see line 43

Line 273: change into “Table 1”, being the first table mentioned in the text

Line 281: change into “Table 2”

Lines 273 and 281: check the order of tables 1 and 2 throughout the text

Line 304: delete “in water samples collected from different districts of Izmir”

Line 334: delete “27”

Lines 335 and 362: change “homology” into “nucleotide identity”

Lines 337 and 363: add the accession number of the reference strains

Lines 341-344: phylogenetic analyses are poorly described; The phylogeny does not show homology among sequence but their phylogenetic relationship. Please modify the sentence accordingly. In the results only a comment about T4, what about the other genotypes detected?; Acanthamoeba castellanii has not been indicated in the tree; modify the figure adding for example “T4 A. castellanii complex”. I would suggest changing the sentence for example as: “According to the phylogenetic tree, Acanthamoeba isolates T4 obtained from various water sources were grouped within the clade including the other sequences of Acanthamoeba castellanii complex available from Genbank”

Lines 370- 372: the phylogenetic relationship of N. fowleri is not well define and described; the authors have to explain why the strain DW1 is included in a different clade respective to the other sequences having the genotype T2. The same is for the strain LW1. I suggest to use in the Mega X software, the best fit model program for the selection of the analysis model; I would suggest including an outgroup for both pathogens in the phylogenetic analyses.

Line 352-361: check this part. It could be included in another paragraph.

Change 5.8S rDNA into “5.8S rRNA”  throughout the ms.

The discussion needs to be improved by not including the results, but simply commenting on them, see for example lines 461-465.

Line 410: delete “by qPCR”

Figure:

Delete fig. 1, being superfluous.

Table

Table 2: please add the average of the CT value for Acanthamoeba

Table S2: specify which ITS region has been amplified in qPCR.

Reviewers' comments:

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**********

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Reviewer #1: Abstract

Line 44-45: "and that was the first-time detection in water sources, Turkey", reformulate this sentence

Line 49: To "the" best of our knowledge...

Introduction

Line 57: "are unicellular protozoa that find commonly in soil and water" commonly find

Line 67: "in particularly children and young adults" inmunocompetents childern and young adults

Material and methods

Lines 110-112: reformulate this sentence

Line 115: "It was filtrate in 0.22 μm pore-..."; write "through" instead of "in"

Line 130: Are the bacteria (E. coli) inactivated?

Line 136: "from" other organisms

Line 137: The grown "of" Acanthamoeba...was diferenciated from / Acanthamoeba trophs and cysts were diferenciated from

Osmo-tolerance assay: have you axenified the Acanthamoeba samples? were they in liquid culture? Why did you add E. coli in the tolerance assay plates? The tolerance asssay could not be trustable if the authors use a bacteria suspension

Line 181: Do you know the Acanthamoeba reference strain specie?

Line 184: Center(without s) for Disease Control and Prevention

Line 187: "quantification"

Results

Line 268: From the total of samples / From the 148 samples

Lines 282/283: ...were grown...

Table 1: positive / negative SAMPLES

As you could detect N.fowleri by qPCR, why you could not isolated it by NNA culture?

Discussion

Line 396: Reformulate this sentence

Lines398-406: the authors are presenting the results again. It is necessary to develope this paragraph by a comparison with other authors and reason the obtained results.

Line 453: including rivers...

Lines 455-457: Reformulate this sentence

It could be interesting if the authors talk about the pathogenicity of the different N.fowleri genotypes.

Conclusions

Lines 474-475: ...,THE present study reports both the presence and THE concentration" or "presence and concentration"..."and DEMONSTRATES THEIR RAPIDILY DETERMINATION by qPCR."

Line 479: "...which are commonly detected AS CAUSAL AGENTS OF AK..."

Line 488: "...should increase..."

**********

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7 Jul 2021

Authors’ Responses to the Review Comments:

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Response to Editor Office: As Editorial Office suggested, the manuscript was arranged in the PLOS ONE's style requirements

Editorial Office: Thank you for stating the following in the Acknowledgments Section of your manuscript:

"The research was supported by a grant from the Budget of the Academic Staff Training Program by The Council of Higher

Education and the Scientific Research Projects Branch Directorate of Ege University, Turkey (Project No: 18-TIP-025)."

We note that you have provided funding information that is not currently declared in your Funding Statement. However,

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Please include your amended statements within your cover letter; we will change the online submission form on your behalf.

Response to Editor Office: As Editorial Office suggested, all funding-related text from the manuscript have been removed from the manuscript. Additionally, the amended Finance Statement has been attached to the cover letter.

Additional Editor Comments:

Editorial Office: Line 43: It is not clear which ITS region was amplified by cPCR. only ITS1 and 5.8S rRNA were mentioned; in the text it was described that also ITS2 was also analysed. See lines 186 and 371.

Response to Editor Office: As Editorial Office suggested, line 41, 47 and line 192-193 it explained which ITS region was amplified by qPCR. The statement was added to article as follows.

Line 41: "N. fowleri positives were detected in six different water sources using qPCR which 5.8S rRNA gene and ITS regions (ITS1 and ITS2) specific primers".

Line 47:"The four positive N. fowleri isolate was confirmed by sequencing the ITS1, ITS2 and 5.8S rRNA regions using specific primers"

Line 211-212: "the 5.8S rRNA and ITS (ITS1 and ITS2) regions for N. fowleri were selected".

Editorial Office: Lines 73- 76: change, for example, into “Quantitative real-time PCR (qPCR) assay is a method with high specificity and sensitivity useful for detecting the presence of the amoebae in water resources (8,9).

Response to Editor Office: As Editorial Office suggested, the statement in line 82-83 was changed as “Quantitative real-time PCR (qPCR) assay is a method with high specificity and sensitivity useful for detecting the presence of the amoebae in water resources (8,9)”.

Editorial Office: Lines 77-83: delete the sentences or summarize this part being the usefulness of the qPCR is well known.

Response to Editor Office: As Editorial Office suggested, the paragraph on line 77-83 has deleted.

Editorial Office: Lines 86- 88: delete the sentence; it belongs to M&M section

Response to Editor Office: As Editorial Office suggested, the sentence on line 86-88 has deleted.

Editorial Office: Line 167: please check the reference (i.e., 17)

Response to Editor Office: As Editorial Office suggested, the reference on line 193 (i.e., 17) has checked and removed.

Editorial Office: Lines 181-182: delete the sentence “In this study, positive …. and B. mandrillaris.”

Response to Editor Office: As Editorial Office suggested, the sentence on line 207-208 has deleted.

Editorial Office: Line 186: specify “ITS”, the authors refer to ITS1, or ITS2 and 5.8S; see line 43

Response to Editor Office: As Editorial Office suggested, on line 211-212 "ITS" was indicated as ""the 5.8S rRNA and ITS (ITS1 and ITS2) regions for ". Also, references 15,16 and 17 were added.

Editorial Office: Line 273: change into “Table 1”, being the first table mentioned in the text

Response to Editor Office: As Editorial Office suggested, “Table 2” on line 306 was changed as “Table 1”

Editorial Office: Line 281: change into “Table 2”

Response to Editor Office: As Editorial Office suggested, “Table 1” on line 321 was changed as “Table 2”

Editorial Office: Lines 273 and 281: check the order of tables 1 and 2 throughout the text

Response to Editor Office: As Editorial Office suggested, on line 306 and line 321 checked the order of tables 1 and 2 throughout the text.

Editorial Office: Line 304: delete “in water samples collected from different districts of Izmir”

Response to Editor Office: As Editorial Office suggested, on line 361 the sentence “in water samples collected from different districts of Izmir” was deleted.

Editorial Office: Line 334: delete “27”

Response to Editor Office: As Editorial Office suggested, on line 403 “27” was deleted.

Editorial Office: Lines 335 and 362: change “homology” into “nucleotide identity”

Response to Editor Office: As Editorial Office suggested, on line 412 and 440 ““homology” was changed as “nucleotide identity”.

Editorial Office: Lines 337 and 363: add the accession number of the reference strains

Response to Editor Office: As Editorial Office suggested, on line 337 and 363 was added to article as follows the accession number of the reference strains.

Line 405-411: added the sentence “reference sequences of Acanthamoeba genotypes retrieved from GenBank (A. palestinensis genotype T2 accession nos: U07411 and L09599; A. castellanii genotype T4 accession nos: MF806034, KT892904, MF139789,MH620482, MH620483, MK192795 and MG969963; A. lenticulate genotype T5 accession nos: KX018036, U94730, EU377584, U94740, U94737 and U94736; A. jacobsi genotype T15 accession nos:KX870203, KT892847, GQ905495 and MH790995; Acanthamoeba genotype T17 accession no:GU808277; Acanthamoeba genotype T18 accession no:KC822461)”.

Line 440-441: added the sentence “references isolate in GenBank (N. fowleri accession nos: AJ132028, X96565, AJ132019 and X96564)”

Editorial Office: Lines 341-344: phylogenetic analyses are poorly described; The phylogeny does not show homology among sequence but their phylogenetic relationship. Please modify the sentence accordingly. In the results only a comment about T4, what about the other genotypes detected?; Acanthamoeba castellanii has not been indicated in the tree; modify the figure

adding for example “T4 A. castellanii complex”. I would suggest changing the sentence for example as: “According to the phylogenetic tree, Acanthamoeba isolates T4 obtained from various water sources were grouped within the clade including the other sequences of Acanthamoeba castellanii complex available from Genbank”

Response to Editor Office: As Editorial Office suggested, phylogenetic analysis was explained in detail and phylogenetic relationships between sequences were demonstrated. Also added comments about other genotypes and to article as follows.

Line 404-406: added the sentence “The sequence data obtained from Acanthamoeba isolates were aligned using Clustal W software and were used to construct the phylogenetic tree to illustrate the relationships between the isolates obtained and reference sequences of Acanthamoeba genotypes retrieved from GenBank”.

Line 420-431: added the sentence “Six isolates (IWS_132, IWS_79, IWS_30, IWS_56, IWS_68, IWS_103) were found to be Acanthamoeba genotype T5 revealing 98% sequence identity to various T5 reference strain. Phylogenetic tree showed that three isolates (IWS_61, IWS_80 and IWS_142) were strictly related with Acanthamoeba T15 genotype chosen as references with 100% of identity, four isolates (IWS_14, IWS_47, IWS_95 and IWS_130) T2 genotype with 98% of identity with the T2 sequence references.”

As Editorial Office suggested, the figure of the phylogenetic tree was changed to “T4 A. castellanii complex”. Also, the sentence was changed as you suggested “According to the phylogenetic tree, Acanthamoeba isolates T4 obtained from various water sources were grouped within the clade including the other sequences of Acanthamoeba castellanii complex available from Genbank”.

Editorial Office: Lines 370- 372: the phylogenetic relationship of N. fowleri is not well define and described; the authors have to explain why the strain DW1 is included in a different clade respective to the other sequences having the genotype T2. The same is for the strain LW1. I suggest to use in the Mega X software, the best fit model program for the selection of the analysis model; I would suggest including an outgroup for both pathogens in the phylogenetic analyses.

Response to Editor Office: As Editorial Office suggested, phylogenetic analysis was explained in detail and phylogenetic relationships between sequences were demonstrated. Also added comments about other genotypes and to article as follows.

Line 475-482: added the sentence “According to the phylogenetic tree, N. fowleri isolates obtained from various water sources were grouped within the clade including the other sequences of N. fowleri genotypes available from GenBank. Three isolates (LW1, DW1 and PW12) were found to be N. fowleri type 2 revealing 98% sequence identity to various N. fowleri type 2 reference strains (N. fowleri type 2 accession nos: AJ132019 and X96564). However, phylogenetic tree showed that one isolates (PW8) were strictly related with N. fowleri type 5 chosen as references (N. fowleri type 5 accession nos: AJ132028 and X96565) with 99% of identity.”

Moreover, As Editorial Office suggested, the phylogenetic tree was reconstructed using the Mega X software program and the optimal model, and an outgroup was added for the phylogenetic tree (Figure 7).

Editorial Office: Line 352-361: check this part. It could be included in another paragraph. Change 5.8S rDNA into “5.8S rRNA” throughout the ms.

Response to Editor Office: As Editorial Office suggested, the paragraph on lines 352-361 has been moved to lines 326-335. Also, "5.8S rDNA" in the entire manuscript was changed to "5.8S rRNA".

Editorial Office: The discussion needs to be improved by not including the results, but simply commenting on them, see for example lines 461-465.

Response to Editor Office: As Editorial Office suggested, it has been developed by making comments where necessary in the discussion section.

Editorial Office: Line 410: delete “by qPCR”

Response to Editor Office: As Editorial Office suggested, “by qPCR” on line 547 was deleted.

Editorial Office: Figure: Delete fig. 1, being superfluous.

Response to Editor Office: As Editorial Office suggested, figure 1 has been removed.

Editorial Office: Table 2: please add the average of the CT value for Acanthamoeba

Response to Editor Office: As Editorial Office suggested, table 2 was added the average of the Ct value for Acanthamoeba.

Editorial Office: Table S2: specify which ITS region has been amplified in qPCR.

Response to Editor Office: As Editorial Office suggested, Table S2 was changed as “the 5.8S rRNA and ITS (ITS1 and ITS2) regions”

Reviewer #1:

Editorial Office: Line 44-45: "and that was the first-time detection in water sources, Turkey", reformulate this sentence

Response to Editor Office: As Editorial Office suggested, the sentence "and that was the first-time detection in water sources, Turkey" was edited as “and detected for the first time from water sources in Turkey.”

Editorial Office: Line 49: To "the" best of our knowledge...

Response to Editor Office: As Editorial Office suggested, the word "the" was edited as “the best of our knowledge”

Editorial Office: Line 57: "are unicellular protozoa that find commonly in soil and water" commonly find

Response to Editor Office: As Editorial Office suggested, the sentence "are unicellular protozoa that find commonly in soil and water" was edited as "are unicellular protozoa that commonly find in soil and water".

Editorial Office: Line 67: "in particularly children and young adults" immunocompetent children and young adults

Response to Editor Office: As Editorial Office suggested, the sentence "in particularly children and young adults" was edited as "in immunocompetent children and young adults”.

Editorial Office: Lines 110-112: reformulate this sentence

Response to Editor Office: As Editorial Office suggested, this sentence was reformulated as following:

“For culture and DNA isolation one liter of water sample was concentrated by filtration using a nitrocellulose membrane with a pore size of 0.22 μm.”

Editorial Office: Line 115: "It was filtrate in 0.22 μm pore-..."; write "through" instead of "in"

Response to Editor Office: As Editorial Office suggested, since figure 1 was removed upon the suggestion of other referees, the figure explain mentioned is not found in the manuscript.

Editorial Office: Line 130: Are the bacteria (E. coli) inactivated?

Response to Editor Office: As Editorial Office suggested, Yes, bacteria inactivated and added to sentence “with heat killed”.

Editorial Office: Line 136: "from" other organisms

Response to Editor Office: As Editorial Office suggested, the word “from” was added as “from other organism”

Editorial Office: Line 137: The grown "of" Acanthamoeba...was diferenciated from / Acanthamoeba trophs and cysts were diferenciated from Osmo-tolerance assay: have you axenified the Acanthamoeba samples? were they in liquid culture? Why did you add E. coli in the tolerance assay plates? The tolerance asssay could not be trustable if the authors use a bacteria suspension

Response to Editor Office: Acanthamoeba can be distinguished in NNA plates by looking at the morphological features of trophozoites and cysts. It is reported in reference sources (“Pussard M, Pons R. Morphology of cystic wall and taxonomy of genus Acanthamoeba (Protozoa, Amoebida). Protistologica. 1977;13(4):557-98 and Page FC. A new key to freshwater and soil gymnamoebae: with instructions for culture 542 1988.”).

Acanthamoeba samples were grown on xenic culture non-nutrient agar plates by coating with heat-inactivated E. coli. Liquid PYG medium, which is axenic culture, was not used. There are no studies using liquid media in the tolerance methods of Acanthamoeba. As in many studies, the necessary nutrient E. coli is used for the proliferation of amoebae on non-nutrient agar plates. For example, reference resources: (Todd CD, Reyes-Batlle M, Martin-Navarro CM, Dorta-Gorrin A, Lopez-Arencibia A, Martinez-Carretero E, et al. Isolation and Genotyping of Acanthamoeba Strains from Soil Sources from Jamaica, West Indies. Journal of Eukaryotic Microbiology. 2015;62(3):416-21 and Caumo K, Frasson AP, Pens CJ, Panatieri LF, Frazzon AP, Rott MB. Potentially pathogenic Acanthamoeba in swimming pools: a survey in the southern Brazilian city of Porto Alegre. Annals of tropical medicine and parasitology. 2009;103(6):477-85.)

Editorial Office: Line 181: Do you know the Acanthamoeba reference strain specie?

Response to Editor Office: No, Acanthamoeba species was not identified in the study, only T4 is reported as genotype.

Editorial Office: Line 184: Center(without s) for Disease Control and Prevention

Response to Editor Office: As Editorial Office suggested, the sentence was edited as “Center for Disease Control and Prevention”.

Editorial Office: Line 187: "quantification"

Response to Editor Office: As Editorial Office suggested, the word "quantitation" was edited as “quantification”.

Editorial Office: Line 268: From the total of samples / From the 148 samples

Response to Editor Office: As Editorial Office suggested, the sentence was edited as “From the total of samples ”.

Editorial Office: Lines 282/283: ...were grown...

Response to Editor Office: As Editorial Office suggested, the word was changed as “were grown”.

Editorial Office: Table 1: positive / negative SAMPLES

Response to Editor Office: As Editorial Office suggested, table 2 was added the word “samples”

Editorial Office: As you could detect N. fowleri by qPCR, why you could not isolated it by NNA culture?

Response to Editor Office: Since Naegleria species do not differ morphologically from N. fowleri, N. fowleri was detected by qPCR from other FLA that we found positive in culture. In fact, although free-living amoebae such as Naegleria and Vermamoeba etc. have been isolated in culture, it is very difficult to diagnose directly microscopically. Therefore, the diagnosis is made with species-specific primers by PCR assay and sequence.

Editorial Office: Line 396: Reformulate this sentence

Response to Editor Office: As Editorial Office suggested, this sentence was reformulated as following:

Line 513-515 added the sentence: “This study was occurred the quantify presence of Acanthamoeba and N. fowleri from various water sources in province İzmir, Turkey.”

Editorial Office: Lines398-406: the authors are presenting the results again. It is necessary to develope this paragraph by a comparison with other authors and reason the obtained results.

Response to Editor Office: As Editorial Office suggested, this paragraph was developed in comparison with other authors and the results are justified as follows.

Line 518-525 added the sentence: “The results of this study which indicate that the Acanthamoeba spp. occurrence in various water sources (18.24%) are similar to those results obtained in the America, Brazil, and Japan Jamaica, Iran, Thailand, Turkey. In previous reports, Naegleria spp. was found in various water sources worldwide at 0.6%–60.9% all over the world, and 0.7–10% in Turkey. [26, 35, 36]. We conclude that Naegleria spp. and Acanthamoeba spp. are free-living amoebas that have suitable growth in various water sources worldwide, but detection rates at different regions may be influenced by water types and geographical conditions.”

Editorial Office: Line 453: including rivers...

Response to Editor Office: As Editorial Office suggested, the sentence was edited as “including rivers”

Editorial Office: Lines 455-457: Reformulate this sentence

Response to Editor Office: As Editorial Office suggested, this sentence was reformulated as following:

Line 593-594 added the sentence “In this study, N. fowleri, a human pathogen, was found positive for the first time in environmental water resources collected in İzmir province, Turkey.”

Editorial Office: It could be interesting if the authors talk about the pathogenicity of the different N. fowleri genotypes.

Response to Editor Office: As Editorial Office suggested, a paragraph has been added about the effect on the pathogenicity of N. fowleri types.

Line 614-617 added the paragraph “Since there are a limited number of studies typing N. fowleri, the information about the pathogenicity of the types is insufficient. However, there is not yet conclusive evidence of any difference in virulence for any of the detected N. fowleri types. It is likely to be detected in humans, as types 2, 3, and 5 are the most common in waters.”

Editorial Office: Lines 474-475: ...,THE present study reports both the presence and THE concentration" or "presence and

concentration"..."and DEMONSTRATES THEIR RAPIDILY DETERMINATION by qPCR."

Response to Editor Office: As Editorial Office suggested, the sentence was added.

Editorial Office: Line 479: "...which are commonly detected AS CAUSAL AGENTS OF AK..."

Response to Editor Office: As Editorial Office suggested, the sentence was added as “which are commonly detected as causal agents of AK”.

Editorial Office: Line 488: "...should increase..."

Response to Editor Office: As Editorial Office suggested, the sentence was edited as “should increase"

Submitted filename: Response to Reviewers.docx

Click here for additional data file.

28 Jul 2021

PONE-D-21-09475R1

Evaluation of molecular characterization and phylogeny for quantification of Acanthamoeba and Naegleria fowleri in various water sources, Turkey

PLOS ONE

Dear Dr. Aykur,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

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Academic Editor

PLOS ONE

Journal Requirements:

Please review your reference list to ensure that it is complete and correct. If you have cited papers that have been retracted, please include the rationale for doing so in the manuscript text, or remove these references and replace them with relevant current references. Any changes to the reference list should be mentioned in the rebuttal letter that accompanies your revised manuscript. If you need to cite a retracted article, indicate the article’s retracted status in the References list and also include a citation and full reference for the retraction notice.

Additional Editor Comments:

In my opinion the article has been improved and deserves to be published pending few suggestions

Line 36: change “which” in “with”

Lines 36-37: check the sentence; indeed the qPCR usually works on small fragments, the region that includes both 5.8S and ITS1-ITS2 could be a large fragment; so specify exactly which target gene/regions the primers are targeting.

Lines 40-41: specify which target gene has been used for the identification of Acanthamoeba isolate, see for example lines 64 and 172.

Line 78: delete “in water samples.”

Lines 277-278 and 281-282: I suggest deleting what concerns on genotypes, and possibly including this information in the specific paragraph on this topic.

Line 466: delete "a human pathogen"

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While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email PLOS at figures@plos.org. Please note that Supporting Information files do not need this step.

9 Aug 2021

Authors’ Responses to the Review Comments

Editorial Office: Please review your reference list to ensure that it is complete and correct. If you have cited papers that have been retracted, please include the rationale for doing so in the manuscript text, or remove these references and replace them with relevant current references. Any changes to the reference list should be mentioned in the rebuttal letter that accompanies your revised manuscript. If you need to cite a retracted article, indicate the article’s retracted status in the References list and also include a citation and full reference for the retraction notice.

Response to Editor Office: As Editorial Office suggested, all references have been reviewed. Some of the references from the retracted articles were removed as follows and replaced with relevant current references.

" Kolören Z, Taş B. Molecular Characterization of Acanthamoeba species in Water Resources of Ordu Province in Turkey 2017" which is a reference in the reference list [27] has been removed.

Erdogan D.D., et al., 2020, unpublished reference has been replaced with relevant current references [52 and 53]. “[52] Ertabaklar H, Turk M, Dayanir V, Ertug S, Walochnik J. Acanthamoeba keratitis due to Acanthamoeba genotype T4 in a non-contact-lens wearer in Turkey. Parasitology research. 2007;100(2):241-6. doi: 10.1007/s00436-006-0274-0. PubMed PMID: 17013653.”

“[53]. Ozkoc S, Tuncay S, Delibas SB, Akisu C, Ozbek Z, Durak I, et al. Identification of Acanthamoeba genotype T4 and Paravahlkampfia sp. from two clinical samples. Journal of medical microbiology. 2008;57(Pt 3):392-6. doi: 10.1099/jmm.0.47650-0. PubMed PMID: 18287307.”

The following sentence and cite have been removed from the manuscript.

"Acanthamoeba T4 genotype was detected in the CSF sample of a patient with unknown cause of encephalitis in İzmir, Turkey (Aykur M., et. al., 2021, unpublished)"

“Also in previous study, we have reported the presence of N. fowleri genotype 2 from one patient in a cerebrospinal fluid sample (Aykur M., et. al., unpublished).”

Additional Editor Comments:

Editorial Office: In my opinion the article has been improved and deserves to be published pending few suggestions

Response to Editor Office: Many thanks for your time and interest in handling our manuscript. Many thanks for your insightful comments concerning our manuscript. They really helped our manuscript appear stronger. Specifically, we have revised our manuscript according to all your comments.

Editorial Office: Line 36: change “which” in “with”

Response to Editor Office: As Editorial Office suggested, line 36 “which” was changed as “with”.

Editorial Office: Lines 36-37: check the sentence; indeed the qPCR usually works on small fragments, the region that includes both 5.8S and ITS1-ITS2 could be a large fragment; so specify exactly which target gene/regions the primers are targeting.

Response to Editor Office: As Editorial Office suggested, qPCR specific primers target the ITS1 region and It has been corrected as follows. “N. fowleri positives were detected in six different water sources using qPCR with ITS regions (ITS1) specific primers”.

Editorial Office: Lines 40-41: specify which target gene has been used for the identification of Acanthamoeba isolate, see for example lines 64 and 172.

Response to Editor Office: As Editorial Office suggested, see example lines 64-172, 18S rRNA target gene has been used for the identification of Acanthamoeba isolate.

Editorial Office: Line 78: delete “in water samples.”

Response to Editor Office: As Editorial Office suggested, in line 78 “in water samples” was deleted.

Editorial Office: Lines 277-278 and 281-282: I suggest deleting what concerns on genotypes, and possibly including this information in the specific paragraph on this topic.

Response to Editor Office: As Editorial Office suggested, in lines 277-278 and 281-282 was deleted what concerns on genotypes.

Editorial Office: Line 466: delete "a human pathogen"

Response to Editor Office: As Editorial Office suggested, in line 466 “a human pathogen” was deleted.

Finally, we are sincerely grateful to both reviewers and editors for your time and interest in reviewing and accepting to publish our manuscript. We look forward to having our manuscript accepted and published with Plos one.

Submitted filename: Response to Reviewers-090821.docx

Click here for additional data file.

12 Aug 2021

Evaluation of molecular characterization and phylogeny for quantification of Acanthamoeba and Naegleria fowleri in various water sources, Turkey

PONE-D-21-09475R2

Dear Dr. Aykur,

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Kind regards,

Maria Stefania Latrofa

Academic Editor

PLOS ONE

13 Aug 2021

PONE-D-21-09475R2

Evaluation of molecular characterization and phylogeny for quantification of Acanthamoeba and Naegleria fowleri in various water sources, Turkey

Dear Dr. Aykur:

I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department.

If your institution or institutions have a press office, please let them know about your upcoming paper now to help maximize its impact. If they'll be preparing press materials, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information please contact onepress@plos.org.

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on behalf of

Dr. Maria Stefania Latrofa

Academic Editor

PLOS ONE