Detection and Molecular Characterization of Potentially Pathogenic Free-Living Amoebae from Recreational and Public Soils in Mazandaran, Northern Iran.

BACKGROUND: Free-living amoeba (FLA) belonging to Acanthamoeba spp., Naegleria, and Balamuthia mandrillaris are the soil-born protozoa. This study aimed to survey the occurrence of FLA, including Acanthamoeba spp., B. mandrillaris, Vermamoeba spp., and Naegleria spp., in soil samples collected from various districts of Mazandaran Province (Northern Iran) from July to December 2018.
METHODS: Overall, 118 soil samples from the recreational and public places were surveyed for the existence of Acanthamoeba spp., Vermamoeba, Naegleria, and B. mandrillaris using both morphological key and molecular tools with genus-specific primers of JDP1, NA, ITS1, and Bal, respectively. To verify the taxonomic status of isolated amoeba, the phylogenetic tree was made based on sequences of 18S rRNA by MEGA (5.05) software with the maximum likelihood model.
RESULTS: Overall, 61/118 samples (51.6%) were contaminated with FLA, and based on the sequencing data, 29 isolates were successfully sequenced. Among the samples, all isolated Acanthamoeba (52.4%) belonged to the T4 genotype with amplification of the DF3 region (18S rRNA gene). Internal transcribed spacer (ITS) sequencing revealed the presence of one strain of Naegleria americana. Twenty-eight V. vermiformis were also confirmed based on Nuclear SSU rDNA. Morphological survey and PCR assay did not show any positive samples for B. mandrillaris.
CONCLUSION: The present study indicates the occurrence of FLA in soil sources of the recreational and public places in Mazandaran province that it can be a severe risk to human health. Thus, more studies are expected to survey the infection source in patients with FLA-related diseases.

Introduction

Free-living amoebae (FLA) including several genera, Acanthamoeba spp., Balamuthia mandrillaris (B. mandrillaris), and Naegleria spp. can be found in the environment (1). Potentially pathogenic FLA able to cause severe and fatal diseases with poor prognoses such as the central nervous system (CNS), amoebic keratitis (AK), and cutaneous ulcers (2, 3).

The prevalence of Acanthamoeba in different water sources such as tap water, freshwater, hot springs, and bottled mineral water is estimated to be 42.6% in Iran (1, 4). Only a few studies are surveying the contamination with FLA in soil sources of Iran (42.1%), and in most studies, Acanthamoeba has been identified according to the morphological characteristics, and the distribution of other FLA has not thoroughly evaluated.

There are several studies on AK caused by Acanthamoeba spp., Vermamoebae, and Valkampfia from Iran as well as a case report of primary amoebic meningoencephalitis (5). Moreover, B. mandrillaris has been reported in three studies from this country. For the first time, B. mandrillaris strain (ID-19) was isolated from dust particles of a hospital in Tehran (6); the second study reported B. mandrillaris in 5 of 55 soil samples collected from recreational areas in East Azerbaijan (7); and the third study showed that 2 of 66 water samples collected from hot-spring in northern Iran were positive for B. mandrillaris (8, 9).

Only some Naegleria species including Naegleria americana (N. americana), N. australiensis, N. dobsoni, N. polaris, N. fultoni, and N. pagei were isolated from hot-spring sources located in Iran. However, so far no report of N. fowleri has been found in the environmental sources of this country (9). Studies focusing on the farmlands soils are missing or empty in Iran.

On the other hand, the irrigation of agricultural lands and farmlands with untreated urban waste-water, which are potential sources for FLA colonization, is particularly the concern that agricultural and environmental officials and experts have been focusing on this problem for years as this is an essential and intensely vital issue for the national health insurance system (5).

This study aimed to survey the occurrence of FLA, including Acanthamoeba spp., B. mandrillaris, Vermamoeba spp., and Naegleria spp., in soil samples collected from various districts of Mazandaran province (Northern Iran) by using both morphological and molecular-based approaches. Besides, the phylogenetic tree was constructed for isolated stains.

Materials and Methods

The geographical location of the study area Mazandaran Province is one of the most densely populated provinces in Iran and is divided into 20 counties. This province is located in the central-northern of Iran, along the southern coast of the Caspian Sea and the Central Alborz mountain range. It has various climates, including the mild and humid climate of the Caspian shoreline and the moderate and cold climate of mountainous regions with 700 mm rainfall annually (Fig. 1).

Fig. 1:

Map showing the location of the study area in Iran (The map has been given from Wikipedia)

Samples Collection and Processing

Overall, 118 soil samples from the recreational and public places, including parks, hospitals, surrounding spaces of swimming pools, and school campuses, were collected from 14 cities in Mazandaran Province, Northern Iran from July to December 2018.

Briefly, 50 g of soil samples were placed in the sterile plastic bags and transferred immediately to the laboratory of the Department of Parasitology, Mazandaran University of Medical Sciences, Sari, Iran, for further processing. The soil samples were dissolved in 10 ml sterile distilled water; remained for about an hour, and the suspension of samples filtered using a pumping machine with a 0.45-μm cellulose nitrate membrane (Millipore, SA).

The filters were inoculated on 1.5% non-nutrient agar (NNA) plates covered with a layer of heat-killed Escherichia coli. Then, all the plates were incubated at 37 °C, and amoebae were morphologically identified to detect FLA plaques and the presence of cysts and trophozoites, daily for three weeks, according to taxonomic criteria using an inverted microscope (10, 11).

Positive samples were purified to harvest amoebae. Plates were stored for up to 2 months, and positive plates (plates exhibiting amoeba growth) were subjected to the following processes.

DNA Extraction, Polymerase Chain Reaction (PCR), and Phylogenetic Analysis

The genomic DNA from the positive samples was extracted using the phenol-chloroform method base on our previous study (12).

PCR assay was done using genus-specific primers, including JDP1 and 2 for Acanthamoeba spp. (12), NA1 and NA2 for Vermamoeba vermiformis (13), ITS1 and ITS2 for Naegleria spp. (14), and Bal1 and 2 for B. mandrillaris (15) (Table 1). The PCR re-action was carried out in a volume of 25μl, containing 12.5μl PCR Master Mix (Pishgam Co., Iran), 1μl forward primer, 1μl reverse primer, 8.5μl of distilled water, and 2μl DNA template.

Table 1:

Primers and amplification conditions for the identification of free-living amoebae in the pre-sent study

Free-living amoebae	Primer name	Primers	Amplicon size (bp)	Target gene	Amplification conditions	Ref.
Acanthamoeba spp.	JDP1	5-GGC CCA	∼ 500	Nuclear SSUrDNA	95°C (1 min) followed by 45 cycles of 95 °C (15 sec), 60 °C (1 min) and 72 °C (40 sec)	(4, 16)
	JDP2	GAT
		CGT TTA
		CCG
		TGAA-3
		5-TCT CAC
		AAG
		CTG CTA
		GGG
		AGT CA-3
V. vermiformis	NA1	5-GCT CCA	∼ 700	Nuclear SSUrDNA	95 °C (3 min) followed by 45 cycles of 95 °C (20 sec), 58 °C (30 s) and 72 °C (40 sec)	(17)
	NA2	ATA
		GCG TAT
		ATT
		AA-3
		5-AGA AAG
		AGC TAT
		CAA
		TCT
		GT-3
Naegleria spp.	ITS1	5-GAA CCT	400	InternalTranscribed Spacer (ITS)	94 °C (1 min) followed by 35 cycles of 94 °C (45 sec), 56 °C (45 sec) and 72 °C (1 min)	(15)
	ITS2	GCG
		TAG GGA
		TCA
		TTT-3
		5-TTT CTT
		TTC
		CTC CCC
		TTA
		TTA-3
B. mandrillaris	Bal1	5-CGC ATG	∼ 1000	Mitochondrial SSU rDNA	94 °C (1 min) followed by 40 cycles of 94 °C (45 sec), 48 °C (45 sec) and 72 °C (3 min)	(18)
	Bal2	TAT
		GAA GAA
		GAC
		CA-3
		5-TTA CCT
		ATA
		TAA TTG
		TCG
		ATA CCA -3

The PCR cycles were set up using a thermocycler (BIO-RAD, Hercules, California, USA) with the programs listed for Acanthamoeba, Vermamoeba, Naegleria, and Balamuthia. Amplified DNA was detected on 1.5% agarose gel electrophoresis, and the DNA was visualized by the solution of safe stain under a UV gel documentation system.

PCR products were submitted for sequencing using the ABI 3130X automatic sequencer. For homology analysis, the sequences were assembled and analyzed with Basic Local Alignment Search Tool (BLAST) from the National Center for Biotechnology Information homepage (NCBI) for comparing the identified sequences with other sequences available in the GenBank database.

The multiple sequence alignment was done using the SequencherTmv.4.1.4 software based on the ClustalW method version 7.2.5. To identify genetic associations and taxonomic status of isolated FLA from soil sources, phylogenetic trees were displayed using the MEGA 5 software and maximum likelihood algorithm by Kimura 2-parameter model.

Results

Out of 118 soil samples in thirteen cities of Mazandaran Province, 61 (51.6%) were positive for FLA in cultivation based on morphological criteria of Page (10). Approximately 500 bp bands were identified as Acanthamoeba spp., using JDP1 and JDP2 primers based on the PCR technique.

Overall, 32 out of 61 (52.4%) soil samples were positive for Acanthamoeba. Besides, using the PCR method, approximately 750 bp bands using specific primers were considered as V. vermiformis, and 28 from 61 (45.9%) soil samples were positive for V. vermiformis.

In soil samples of all 13 cities surveyed in the present study, were found Acanthamoeba and V. vermiformis. Moreover, about 400 bp bands with ITS1 and ITS2 primers were identified as Naegleria spp., just observed in 1 out of 61 (1.7%) samples in Galougah city. Whereas, morphological survey and PCR assay using B. mandrillaris specific primers failed to report any positive results, but four samples had a mixed infection (Acanthamoeba and vermamoebae) (Table 2).

Table 2:

Data regarding the source and the isolated genus of free-living amoebae from recreational and public places in Mazandaran, Northern Iran

City	Total No. of samples	No. Acanthamoeba positive samples	No. Vermamoeba positive samples	No. Naegleria positive samples	No. Mix positive samples
Galougah	9	3	1	1	-
Behshar	12	2	2	-	-
Neka	10	4	1	-	1(Achanthamoeb a+ Vermamoeba )
Sari	16	5	5	-	3 (Achanthamoeb a+ Vermamoeba)
Qaemshahr	12	1	1	-	-
Babol	6	2	1	-	-
Amol	8	4	3	-	-
Mahmudabad	4	2	1	-	-
Babolsar	11	3	3	-	-
Chaloos	6	1	4	-	-
Noshahr	10	2	3	-	-
Feredonkenar	4	1	1	-	-
Noor	6	1	1	-	-
Joybar	4	1	1	-	-
Total	118	32	28	1	4

All sequenced Vermamoeba isolates (n=20) belonged to V. vermiformis, and all sequenced Acanthamoeba isolates (n=8) were identified as T4 genotype. ITS sequencing showed that one strain belonged to N. americana (Table 3). Phylogenetic analysis confirmed that all sequenced isolates had high homology (95%–100%) with reference isolates from Gene Bank (Figs. 2 and 3). Data sequences of 29 isolated strains were deposited in the GenBank under accession numbers present in Table 3.

Table 3:

Free-living amoebae genotypes in the present study

Code	sequenced FLAs	Genotype/spicese	Gene Bank accession no.
MGS961109	Acanthamoeba	T4	MG722753
MGS961107	Acanthamoeba	T4	MG890344
MGS961108	Acanthamoeba	T4	MG890345
Acn8F	Acanthamoeba	T4	MG969816
Acn5F	Acanthamoeba	T4	MG969817
Acn2F	Acanthamoeba	T4	MG969830
Acn9F	Acanthamoeba	T4	MG969818
Acn10F	Acanthamoeba	T4	MG969831
Hart1HviF	Vermamoeba	vermiformis	MG969819
Hart8HviR	Vermamoeba	vermiformis	MG969820
Hart16HviR	Vermamoeba	vermiformis	MG969823
Hart6HviR	Vermamoeba	vermiformis	MG969821
Hart15HviR	Vermamoeba	vermiformis	MG969826
Hart1HviF	Vermamoeba	vermiformis	MG969829
Hart8HviR	Vermamoeba	vermiformis	MG969833
Hart14HviR	Vermamoeba	vermiformis	MG969828
Hart11HviR	Vermamoeba	vermiformis	MG969825
Hart13HviR	Vermamoeba	vermiformis	MG969827
Hart5HviF	Vermamoeba	vermiformis	MG969835
Hart3HviF	Vermamoeba	vermiformis	MG969836
Hart17HviF	Vermamoeba	vermiformis	MG969838
Hart16HviF	Vermamoeba	vermiformis	MG969840
Hart4HviF	Vermamoeba	vermiformis	MG969837
Hart2HviF	Vermamoeba	vermiformis	MG969839
Hart9HviR	Vermamoeba	vermiformis	MG969824
Hart12HviR	Vermamoeba	vermiformis	MG969832
MGS961109	Vermamoeba	vermiformis	MG969863
Hart12HviR	Vermamoeba	vermiformis	MG969832
NEG1	Naegleria	americana	MH000323

Fig. 2:

Phylogenetic analysis of Acanthamoeba isolates. Neighbor-joining tree based on 18S rDNA sequences, with 1000 bootstrap replicates. The sequences from these isolates were aligned by MEGA 5 software using reference isolates from Gene Bank. The bar is an index of evolutionary distances (0.02) among the different sequences

Fig. 3:

Phylogenetic analysis of Vermamoeba isolates. Neighbor-joining tree based on 18S rDNA se-quences, with 1000 bootstrap replicates. The sequenced isolates were aligned by MEGA 5 software using reference isolates from Gene Bank. The bar is an index of evolutionary distances (0.05) among the different sequences

Discussion

Among soil-born free-living amoebae, commonly Acanthamoeba spp., Vahlkampfiids, and B. mandrillaris cause severe amoebic keratitis and encephalitis. The current research showed that 51.6% of recreational and public places soil sources are contaminated with FLA. The present study was the first to survey the recreational and public soils in northern Iran.

Moreover, primary, recreational, and tourism counties such as Amol and Mahmudabad, showed a high pollution rate with FLA (50%), which is a significant threat to public health.

During the period between 1997 and 2000, overall 136 corneal scrapings of clinically-suspected Acanthamoeba ulcers were screened and tested for the existence of Acanthamoeba. Eleven out of 136 patients were positive for Acanthamoeba that all of these patients were agricultural workers and did not wear contact lenses (19).

Another study collected soil and tap water samples from several locations in Yanji, China. Acanthamoeba species identified in environmental samples belonged to T4, T5, and T16 genotypes (20). In another research, sixty soil samples were collected from public and recreational sides in East Azerbaijan, Iran, and examined the presence of Acanthamoeba spp. using morphological and PCR techniques. Acanthamoeba spp. was found in 41.6% of soil samples and led to the identification of four of the genotypic clades (T3, T4, T5, and T11) (21).

Moreover, 47.8% of hot spring samples in Mazandaran Province were contaminated with Acanthamoeba, and the genotype of 100% isolates belonged to the T4 (12). The difference in the abundance of FLA in soil samples of different areas of the world could be due to the moisture rate, organic carbon and texture of the soil, and various climatic conditions (1).

In this study, the isolated Acanthamoeba strains belonged to the T4 genotype with the highest distribution compared to other FLAs. The results of the current study showed that most Acanthamoeba isolates are in soil samples. This finding may be due to their high resistance to harsh environmental conditions (2). There are very few reports about Acanthamoeba genotypes in soil sources in Iran and around the world. In a previous Iranian study, the prevalence rate of Acanthamoeba spp. (all belonging to T4 genotype) was 26.9% in soil sources of recreational parks in Tehran, Iran (22). Other research in Ahvaz city (southern Iran) also showed the low prevalence of Acanthamoeba (26%) from soil samples and the isolated genotypes belonged to T4 and T5 (23).

Besides, the results of the present research agreed with another findings, that presented T4 is the most frequent genotype isolated from environmental sources and patients with AK and granulomatous amoebic encephalitis in the USA (24). The Acanthamoeba belonging to genotype T4 was isolated from 8 clinical samples collected from 35 patients referred to the eye clinic (25). The researchers reported an increased prevalence of keratitis due to the Acanthamoeba T4 genotype (6, 26). To date, 90% of the infection-causing Acanthamoeba isolates belonged to the T4 genotype. The frequency of this genotype in human infections is probably due to their high virulence and properties that increase their transmissibility as well as decreased their susceptibility to drugs (27). Later, Iovieno et al. showed bacterial endosymbionts (Legionella spp. and Neochlamydia spp.) in the T4 genotype considering as a pathogenic genotype (28).

In the present study, non-pathogenic Naegleria, N. americana, was detected for the first time in soil samples of Iran. In the examined soil samples, pathogenic species of the Naegleria, N. fowleri, were not detected. However, other researchers reported Naegleria spp. in Iran. The presence of Naegleria spp. such as N. americana, N. australiensis, N. dobsoni, N. pagei, N. fultoni, and N. polaris were reported in therapeutic hot springs in Mazandaran, northern Iran (9).

Unfortunately, the isolation of B. mandrillaris from soil was not successful in this study. It may be because of favorable growth conditions needed for the cultivation of Balamuthia in non-nutrient agar. To date in Iran, three environmental studies have reported Balamuthia in environmental sources, including one from a soil sample from north-western (7), one from hot springs in Mazandaran (8), and one case from urban dust in Tehran (6).

Conclusion

The present study indicates the occurrence of FLA in soil sources of the recreational and public places in Mazandaran province that could be a severe risk for native people and tourists. Therefore, posting warning signs in recreational sites may be an option for decreasing the hazard of FLA-related diseases.