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Molecular Identification of Free-living Amoebae Isolated from Artificial Water Bodies Located in Poland

Publication date: 31.10.2014

Acta Protozoologica, 2015, Volume 54, Issue 1, pp. 77 - 84

https://doi.org/10.4467/16890027AP.15.006.2193

Authors

,
Agata Leońska-Duniec
Department of Genetics, Szczecin University, Szczecin, Poland
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,
Bogumiła Skotarczak
Department of Genetics, Szczecin University, Szczecin, Poland
All publications →
Małgorzata Adamska
Department of Genetics, Szczecin University, Szczecin, Poland
All publications →

Titles

Molecular Identification of Free-living Amoebae Isolated from Artificial Water Bodies Located in Poland

Abstract

Free living amoebae (FLA) are amphizoic protozoa that are widely found in various environmental sources. They are known to cause serious human infections, including a fatal encephalitis, a blinding keratitis, and pneumonia. The main aim of the study was detection and molecular identification of Acanthamoeba spp., Naegleria fowleriBalamuthia mandrillarisSappinia pedata, and Vermoamoeba vermiformis (formerly Hartmannella vermiformis) in artificial water bodies in North-Western Poland. We examined 86 water samples collected during 2-year period from 43 water bodies, including outdoor and indoor swimming pools, firefighting reservoirs, fountains, as well as water network. The samples were filtrated using Filta-Max® membrane filters (IDEXX Laboratories, USA) and, in order to select potentially pathogenic, thermophilic strains and to limit the number of PCR examined samples, the thermal tolerance test was carried out. Obtained filtrates were transferred to non-nutrient agar plates with E. coli. The agar plates were incubated at 37°C and then proliferated amoebae were passaged at 42°C. DNA was extracted from the thermophilic trophozoites and then polymerase chain reactions and sequence analysis were performed for molecular identification of FLA. From the 86 collected water samples 57 strains of FLA were able to proliferate at 37°C and 7 of them showed ability to proliferate at 42°C. For molecular identification ofAcanthamoeba spp. and V. vermiformis, regions of 18S rDNA were amplified. In order to detect B. mandrillaris DNA, we used mitochondrial 16S rDNA as a marker, and for detection of N. fowleri andS. pedata – ITS regions. Based on molecular analysis, isolates were classified to the genusAcanthamoeba (T4 and T11 genotypes, as well as the new genotypes detected earlier in clinical samples and named T16) and V. vermiformis species. Detected strains were highly similar or identical to pathogenic strains detected earlier in patients. Our results show a wide distribution of potential pathogenic FLA, as Acanthamoeba T4, T11, T16 genotypes, and V. vermiformis species in various artificial water bodies located in North-Western Poland and suggest a potential threat to health of humans in this part of the country.

References

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Adamska M., Leonska-Duniec A., Lanocha N., Skotarczak B. (2014) Thermophilic potentially pathogenic amoebae isolated from natural water bodies in Poland and their molecular characterization. Acta Parasitol59: DOI: 10.2478/s11686-014-0266-7

Alsam S., Kim K. S., Stins M., Rivas A. O., Sissons J., Khan N. A. (2003) Acanthamoeba interactions with human brain microvascular endothelial cells. Microb. Patholog. 35: 235–241

Booton G. C., Rogerson G. C., Bonilla T.D., Seal D.V., Kelly D. J., Beattie T. K., Tomlinson A., Lares-Villa F., Fuerst P. A., Byers T. J. (2004) Molecular and physiological evaluation of subtropical environmental isolates of Acanthamoeba spp., casual agent of Acanthamoeba keratitis. J. Eukaryot. Microbiol. 51: 192–200

Booton G. C., Kelly D. J., Chu Y. W., Seal D. V., Houang E., Lam D. S., Byers T. J., Fuerst P. A. (2002) 18S ribosomal DNA typing and tracking of Acanthamoeba species isolates from corneal scrape specimens, contact lenses, lens cases, and home water supplies of Acanthamoeba keratitis patients in Hong Kong. J. Clin. Microbiol. 40: 1621–1625

Booton G. C., Visvesvara G. S., Byers T. J., Kelly D. J., Fuerst P. A. (2005) Identification and distribution of Acanthamoeba species genotypes associated with nonkeratitis infections. J. Clin. Microbiol. 43: 1689–1693

Brown M. W., Spiegel F. W., Silberman J. D. (2007) Amoeba at attention: phylogenetic affinity of Sappinia pedataJ. Eukaryot. Microbiol. 54: 511–519

Carlesso A. M., Artuso G. L., Caumo K., Rott M. B. (2010) Potentially pathogenic Acanthamoeba isolated from a hospital in Brazil. Curr. Microbiol. 60: 185–190

Carter R. F. (1970) Description of a Naegleria sp. isolated from two cases of primary amoebic meningoencephalitis, and of the experimental pathological change induced by it. J. Pathol. 100: 212–244

Centeno M. F., Rivera F., Cerva L., Tsutaumi V., Gallegos E., Calderon A., Ortiz R., Bonilla P., Ramirez E., Suarez G. (1996) Hartmannella vermiformis isolated from the cerebrospinal fluid of a young male patient with meningoencephalitis and bronchopneumonia. Arch. Med. Res. 27: 579–586

Corsaro D., Venditti D. (2011) More Acanthamoeba genotypes: limits to the use of rDNA fragments to describe new genotypes. Acta Protozool. 50: 49–54

Culbertson C. G., Smith J. W., Miner J. R. (1958) Acanthamoeba observation on animal pathogenicity. Science 127: 1506

Culbertson C. G., Smith J. W., Cohen H. K., Miner J. R. (1959) Experimental infection of mice and monkeys by AcanthamoebaAm. J. Pathol. 35: 185–197

De Jonckheere J. F. (1979) Pathogenic free-living amoebae in swimming pools: survey in Belgium. Ann. Microbiol. (Paris) 130B: 205–212

De Jonckheere J. F. (1980) Growth characteristics, cytopathic effect in cell culture, and virulence in mice of 36 type strains belonging to 19 different Acanthamoeba spp. Appl. Environ. Microbiol. 39: 681–685

De Jonckheere J. F., Brown S. (1998) There is no evidence that the free-living amoeba Hartmannella is a human parasite. Clin. Infect. Dis. 26: 773

Dyková I., Pindová Z., Fiala I., Dvoráková H., Machácková B. (2005) Fish-isolated strains of Hartmannella vermiformis page, 1967: morphology, phylogeny and molecular diagnosis of the species in tissue lesions. Folia Parasitol. (Praha) 52: 295–303

Fowler M., Carter R. F. (1965) Acute pyogenic meningitis probably due to Acanthamoeba sp.: a preliminary report. Br. Med. J. 2(5464): 740–742

Gast R. J. (2001) Development of an Acanthamoeba – specific reverse dot – blot and the discovery of a new ribotype. J. Eukaryot. Microbiol. 48: 609–615

Gelman B. B., Rauf S. J., Nader R., Popov V., Bokowski J., Chaljub G., Nauta H. W. (2001) Amoebic encephalitis due to Sappinia diploideaJ. Am. Med. Assoc. 285: 2450–2451

Gelman B. B., Popov V., Chaljub G., Nader R., Rauf S. J., Nauta H. W., Visvesvara G. S. (2003) Neuopathological and ultrastructural features of amoebic encephalitis caused by Sappinia diploideaJ. Neuropathol. Exp. Neurol. 62: 990–998

Gianinazzi C., Schild M., Zumkehr B., Wüthrich F., Nüesch I., Ryter R., Schürch N., Gottstein B., Müller N. (2010) Screening of Swiss hot spring resorts for potentially pathogenic free-living amoebae. Exp. Parasitol. 126: 45–53

Górnik K., Kuźna-Grygiel W. (2004) Presence of virulent strains of amphizoic amoebae in swimming pools of the city of Szczecin. Ann. Agric. Environ. Med. 11: 233–236

Greub G., Raoult D. (2004) Microorganisms resistant to free-living amoebae. Clin. Microbiol. Rev. 17: 413–433

Hsu B. M., Lin C. L., Shih F. C. (2009) Survey of pathogenic free-living amoebae and Legionella spp. in mud spring recreation area. Water Res. 43: 2817–2828

Jager B. V., Stamm W. P. (1972) Brain abscesses caused by free-living amoeba probably of the genus Hartmannella in a patient with Hodgkin’s disease. Lancet 2: 1343–1345

Kasprzak W., Mazur T., Červa L. (1982) Naegleria fowleri in thermally polluted waters. Folia Parasitol. (Praha) 29: 211–218

Kennedy S. M., Devine P., Hurley C., Ooi Y. S., Collum L. M. T. (1995) Corneal infection with Hartmannella vermiformis in contact-lens wearer. Lancet 346: 637–638

Khan N. A. (2006) Acanthamoeba: biology and increasing importance in human health. FEMS Microbiol. Rev. 30: 564–595

Khan N. A., Jarroll E. L., Paget T. A. (2002). Molecular and physiological differentiation between pathogenic and nonpathogenic AcanthamoebaCurr. Microbiol. 45: 197–202

Lares-Villa F., Hernández-Peña C. (2010) Concentration of Naegleria fowleri in natural waters used for recreational purposes in Sonora, Mexico (November 2007–October 2008). Exp. Parasitol. 126: 33–36

Lorenzo-Morales J., Monteverde-Miranda C. A., Jiménez C., Tejedor M. L., Valladares B., Ortega-Rivas A. (2005) Evaluation of Acanthamoeba isolates from environmental sources in Tenerife, Canary Islands, Spain. Ann. Agric. Environ. Med. 12: 233–236

Lorenzo-Morales J., Martínez-Carretero E., Batista N., Álvarez-Martín J., Bahaya Y., Walochnik J., Valladares B. (2007) Early diagnosis of amoebic keratitis due to a mixed infectrion with Acanthamoeba and HartmannellaParasitol. Res. 102: 167–169

Łanocha N., Kosik-Bogacka D., Maciejewska A., Sawczuk M., Wilk A., Kuźna-Grygiel W. (2009) The occurrence Acanthamoeba (free living ameba) in environmental and respiratory samples in Poland. Acta Protozool. 48: 271–279

Naginton J., Watson P. G., Playfair T. J., McGill J., Jones B. R., Steele A. D. (1974) Amoebic infection of the eye. Lancet 2: 1537–1540

Nagyová V., Nagy A., Janeček Š., Timko J. (2010) Morphological, physiological, molecular and phylogenetic characterization of new environmental isolates of Acanthamoeba spp. from the region of Bratislava, Slovakia. Biologia 65: 81–91

Nuprasert W., Putaporntip C., Pariyakanok L., Jongwutiwes S. (2010) Identification of a novel T17 genotype of Acanthamoeba from environmental isolates and T10 genotype causing keratitis in Thailand. J. Clin. Microbiol. 48: 4636–4640

Qvarnstrom Y., da Silva A. J., Schuster F. L., Gelman B. B., Visvesvara G. S. (2009) Molecular confirmation of Sappinia pedata as a causative agent of amoebic encephalitis. J. Infect. Dis. 199: 1139–1142

Qvarnstrom Y., Nerad T. A., Visvesvara G. S. (2013) Characterisation of a new pathogenic Acanthamoeba species, A. byersi n. sp., isolated from a human with fatal amoebic encephalitis. J. Euk. Microbiol. 

Schroeder J. M., Booton G. C., Hay J., Niszl I. A., Seal D. V., Markus M. B., Fuerst P. A., Byers T. J. (2001) Use of subgenic 18S ribosomal DNA PCR and sequencing for genus and genotype identification of Acanthamoebae from humans with keratitis and from sewage sludge. J. Clin. Microbiol. 39: 1903–1911

Smirnov A. V., Chao E., Nassonova E. S., Cavalier-Smith T. (2011) A revised classification of naked lobose amoebae (Amoebozoa: Lobosa). Protist 162: 545–570

Spanakos G., Tzanetou K., Miltsakakis D., Patsoula E., Malamou-Lada E., Vakalis N. C. (2006) Genotyping of pathogenic Acanthamoebae isolated from clinical samples in Greece-report of a clinical isolate presenting T5 genotype. Parasitol. Int. 55: 147–149

Stothard D. R., Schroeder-Diedrich J. M., Awwad M. H., Gast R. J., Ledee D. R., Rodriguez-Zaragosa S., Dean C. L., Fuerst P. A., Byers T. J. (1998) The evolutionary history of the genus Acanthamoeba and the identification of eight new 18S rRNA gene sequence types. J. Eukaryot. Microbiol. 45: 45–54

Stratford M. P., Griffiths A. J. (1978) Variations in the properties and morphology of cysts of Acanthamoeba castellaniiJ. Gen. Microbiol. 108: 33

Thomas V., Herrera-Rimann K., Blanc D. S., Greub G. (2006) Biodiversity of amoebae and amoeba-resisting bacteria in a hospital water network. Appl. Environ. Microbiol. 72: 2428–2438

Tsvetkova N., Schild M., Panaiotov S., Kurdova-Mintcheva R., Gottstein B., Walochnik J., Aspöck H., Lucas M. S., Müller N. (2004) The identification of free-living environmental isolates of amoebae from Bulgaria. Parasitol. Res. 92: 405–413

Visvesvara G. S., Martinez A. J., Schuster F. L., Leitch G. J., Wallace S. V., Sawyer T. K., Anderson M. (1990) Leptomyxid ameba, a new agent of amebic meningoencephalitis in humans and animals. J. Clin. Microbiol. 28: 2750–2756

Visvesvara G. S., Schuster F. L., Martinez A. J. (1993) Balamuthia mandrillaris, n. g., n. sp., agent of amoebic meningoencephalitis in humans and other animals. J. Eukaryot. Microbiol. 40: 504–514

Visvesvara G. S., Moura H., Schuster F. L. (2007) Pathogenic and opportunistic free-living amoebae: Acanthamoeba spp., Balamuthia mandrillarisNaegleria fowleri, and Sappinia diploideaFEMS Immunol. Med. Microbiol. 50: 1–26

Walochnik J., Haller-Schober E., Kölli H., Picher O., Obwaller A., Aspöck H. (2000) Discrimination between clinically relevant and nonrelevant Acanthamoeba strains isolated from contact lens-wearing keratitis patients in Austria. J. Clin. Microbiol. 38: 3932–3936

Yagi S., Schuster F. L., Visvesvara G. S. (2008) Demonstration of Balamuthia and Acanthamoeba mitochondrial DNA in sectioned archival brain and other tissues by the polymerase chain reaction. Parasitol. Res. 102: 491–497

Information

Information: Acta Protozoologica, 2015, Volume 54, Issue 1, pp. 77 - 84

Article type: Original article

Authors

Department of Genetics, Szczecin University, Szczecin, Poland

Department of Genetics, Szczecin University, Szczecin, Poland

Department of Genetics, Szczecin University, Szczecin, Poland

Published at: 31.10.2014

Received at: 13.11.2013

Corrected at: 06.03.2014

Article status: Open

Licence: None

Percentage share of authors:

Agata Leońska-Duniec (Author) - 33%
Bogumiła Skotarczak (Author) - 33%
Małgorzata Adamska (Author) - 34%

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