The investigation of Acanthamoeba spp., Naegleria fowleri and Balamuthia mandrillaris in environmental samples collected from Izmir province
Project no. 174 · Status: Kapanmış · 2018
Summary
In this study, it was aimed to investigate the presence of Acanthamoeba spp., N. fowleri and B. mandrillaris, which are pathogenic for humans and animals, using non-nutrient agar (NNA) culture and quantitative real-time PCR (qPCR) assay in water, soil and swab samples collected from different regions of Izmir province. In addition, it was aimed to determine the pathogen genotypes by performing sequence analysis in Acanthamoeba positive samples. In İzmir province, water (n=148), soil (n=35), and swab samples (n=20) (air-conditioner, eyewash unit, sink, window) were collected from 23 various districts. Membrane filtration was applied to all collected water, and also soil samples, swab samples and half of the filter were cultured in the NNA plate. Also, direct DNA isolation from the other half of the filter water and soil samples was performed. Standard curves were established by plasmid controls with the qPCR method and their concentrations were determined. A phylogenetic tree was created using the obtained sequences to determine the pathogen genotypes of positive Acanthamoeba spp.. Of the examined samples, water 71/148 (47.97%), soil 24/35 (68.57%), and swap 4/20 (20%) samples were detected positive by NNA culture method for free-living amoebae (FLA). Acanthamoeba spp. were found positive in water 12.16% (n=18), soil 25.71% (n=9), and swab 5% (n=1) samples by NNA culture. Osmotolerance and thermotolerance tests in water of 55.5% (n=10) and soil samples 77.7% (n=7), a growth of 1 M mannitol osmolarity and at 42°C were observed, respectively. Besides, one swab sample was showed growth at 0.5-1 M mannitol osmolarity and 37-42 °C. With the qPCR assay; Acanthamoeba spp. and N. fowleri were found to be 18.24% (n=27), 2.7% (n=4), respectively, and B. mandrillaris could not be detected in any sample. Acanthamoeba spp., N. fowleri, and B. mandrillaris were found positive in soil samples of 45.71% (n=16), 17.4% (n=6) and 20% (n=7), respectively. The quantitative amount of Acanthamoeba spp. and N. fowleri was calculated and in water sources were determined 1.4x102-3.2x105 and 11x102-8x103 plasmid copies/liter and Acanthamoeba spp., N. fowleri and B. mandrillaris in soil samples were determined 6x102-18x105, 9x103-8x102, and 39x103-47x104 plasmid copies/gr, respectively. However, B. mandrillaris could not be detected in water samples by qPCR method. Genotypes T4 of Acanthamoeba spp. were found to be 51.85% (n=14), 56.25% (n=9), and 100% (n=1) in water, soil and swab samples, respectively. Genotype T5 22.22% (n=6), genotype T2 14.81% (n=4), and genotype T15 11.11% (n=3) in water samples and genotype T5 25% (n=4), genotype T2 18.75% (n=3) in soil samples were identified. In conclusion, the thesis study carried out is important in terms of being the first comprehensive study and detecting Acanthamoeba spp., N. fowleri and B. mandrillaris by qPCR assay in environmental samples collected from the places where the most human contact in İzmir province. Present study is the first report of both N. fowleri and B. mandrillaris in Turkey by qPCR method in soil samples. The risks of possible health the Acanthamoeba T4 genotype, which is the most common pathogen in humans and animals, has to be taken into consideration. Although the presence of the genotype T5 was reported for the second time in water samples, the first time in soil samples were reported ın Turkey. Therefore, people should be informed about the existence of hidden danger in their contact with water and soil. In addition, awareness should be increased about this risk among clinicians and public health professionals in this region.