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Licensed Unlicensed Requires Authentication Published by De Gruyter October 18, 2018

Molecular characterization and phylogenetic analysis of Acanthamoeba isolates in tap water of Beni-Suef, Egypt

Wegdan M. Abd El Wahab, Ayman A. El-Badry and Doaa A. Hamdy
From the journal Acta Parasitologica

Abstract

The genus Acanthamoeba is a free-living amoeba widely distributed in various aquatic environments. It is an etiologic cause of amoebic encephalitis and keratitis particularly for immunocompromised individuals. The purpose of the present study was to investigate Acanthamoeba species prevalence in household and hospital potable water in Beni-Suef governorate, Egypt, and to employ sequencing methods to identify positive Acanthamoeba species isolates and their potential health risks. Sixty tap water samples (30 household and 30 governmental and private hospital settings) collected from Beni-Suef governorate, Egypt were filtered, cultured on non-nutrient agar, identified by morphotyping keys after staining with Giemsa stain and then confirmed by PCR using Acanthamoeba specific primers. Twenty positive samples were successfully genetically characterized and phylogenetically analyzed to identify Acanthamoeba species. The total detection rate for Acanthamoeba was 48/60 (80%); Acanthamoeba contamination in water collected from domestic houses was higher than in hospitals; 27/30 (90%) versus 21/30 (70%) with statistical significant value (P value = 0.05). Sequencing of 20 positive isolates revealed Acanthamoeba T4 in 65% and T2 in 35%. To our knowledge, this is the first research that documents the occurrence and phylogeny of Acanthamoeba species in Beni-Suef, Egypt. The presence of a higher percentage of Acanthamoeba species in tap water, in particular T4, highlights the potential health hazards for immunocompromised individuals and emphasizes the urgent need for the implementation of effective filtration and disinfection measures.

  1. Funding. This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors

  2. Competing Interest. The authors have declared that no competing interest exists.

  3. Ethical approval. this study was approved by ethical committee of Faculty of Medicine, Beni-Suef University.

Acknowledgment

The authors are kindly grateful to Dr. Eman S. El-wakil, Theodor Bilharz Research Institute for her consultation in the culturing techniques.

References

Al-Herrawy A.Z., Gad M.A. 2017. Assessment of Two Different Drinking Water Treatment Plants for the Removal of Free-living Amoebae, Egypt. Iranian Journal of Parasitology, 12, 413–422Search in Google Scholar

Al-Herrawy A., Bahgat M., Mohammed A., Ashour A., Hikal W. 2013. Morpho-physiological and biochemical criteria of Acanthamoeba spp. isolated from the Egyptian aquatic environment. Iranian Journal of Parasitology, 8, 302– 312Search in Google Scholar

Al-Herrawy A.Z., Mohamed S.H., Mohamed A.H., Zaghloul N.M. 2015. Surveillance of potentially pathogenic free-living amoebae through drinking water treatment processes in Fayoum Governorate, Egypt. International Environment, 4, 98–107Search in Google Scholar

Alsam S., Kim K.S., Stins M., Rivas A.O., Sissons J., Khan N.A. 2003. Acanthamoeba interactions with human brain microvascular endothelial cells. Microbial Pathogenesis, 35, 235–41. 10.1016/j.micpath.2003.07.001Search in Google Scholar PubMed

Astorga B., Lorenzo-Morales J., Martín-Navarro C.M., Alarcón V., Moreno J., González A.C., Navarrete E., Piñero J.E., Valladares B. 2011. Acanthamoeba belonging to T3, T4, and T11: genotypes isolated from air-conditioning units in Santiago, Chile. Journal of Eukaryotic Microbiology, 58, 542–4. 10.1111/j.1550-7408.2011.00584.xSearch in Google Scholar PubMed

Bagheri H.R., Shafiei R., Shafiei F., Sajjadi S.A. 2010. Isolation of Acanthamoeba Spp. from drinking waters in several hospitals of Iran. Iranian Journal of Parasitology, 5, 19–25Search in Google Scholar

Baquero R.A., Reyes-Batlle M., Nicola G.G., Martı´n-Navarro C.M., Lo´pez-Arencibia A., Esteban J.G., et al. 2014. Presence of potentially pathogenic free-living amoebae strains from well water samples in Guinea-Bissau. Pathogens and Global Health, 108, 206–211. 10.1179/2047773214Y.0000000143Search in Google Scholar PubMed PubMed Central

Berger P., Papazian L., Drancourt M., La Scola B., Auffray J.P., Raoult D. 2006. Ameba-associated microorganisms and diagnosis of nosocomial pneumonia. Emerging Infectious Diseases, 12, 248–255. 10.3201/eid1202.050434Search in Google Scholar PubMed PubMed Central

Boost M., Cho P., Lai S., Sun W.M.2008. Detection of Acanthamoeba in tap water and contact lens cases using polymerase chain reaction. Optometry and Vision Science, 85, 526–30. 10.1097/OPX.0b013e31817dba7cSearch in Google Scholar PubMed

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 non-keratitis infections. Journal of Clinical Microbiology, 43, 1689–1693. 10.1128/JCM.43.4.1689-1693.2005Search in Google Scholar PubMed PubMed Central

Bradbury R.S., French L.P., Blizzard L. 2014. Prevalence of Acanthamoeba spp. in Tasmanian intensive care clinical specimens. Journal of Hospital Infection, 86, 178–81. 10.1016/j.jhin.2013.12.009Search in Google Scholar PubMed

Carlesso A.M., Artuso G.L., Caumo K., Rott M.B. 2010. Potentially pathogenic Acanthamoeba isolated from a hospital in Brazil. Current Microbiology, 60, 185–190. 10.1007/s00284-009-9523-7Search in Google Scholar PubMed

Corsaro D., Walochnik J., Kohsler M., Rott M.B. 2015. Acanthamoeba misidentification and multiple labels: redefining genotypes T16, T19, and T20 and proposal for Acanthamoeba micheli sp. nov. (genotype T19). Parasitology Research, 114, 2481–90. 10.1007/s00436-015-4445-8Search in Google Scholar PubMed

Coşkun K.A., Özçelik S., Tutar L., Elaldı N., Tutar Y. 2013. Isolation and identifiation of free-living amoebae from tap water in Sivas, Turkey. Biomed Research International, 1–8. 10.1155/2013/675145Search in Google Scholar PubMed PubMed Central

Crary M.J. 2012. Genetic variability and its relationship to Acanthamoeba pathogenesis. PhD thesis, The Ohio State University, Columbus, Ohio, USASearch in Google Scholar

Dendana F., Sellami H., Jarraya F., Sellami A., Makni F., Cheikhrouhou F., et al. 2008. Free-living amoebae (FLA): Detection, morphological and molecular identification of Acanthamoeba genus in the hydraulic system of an haemodialysis unit in Tunisia. Parasite. 15, 137–42. 10.1051/para-site/2008152137Search in Google Scholar

Edagawa A., Kimura A., Kawabuchi-Kurata T., Kusuhara Y., Karanis P. 2009. Isolation and genotyping of potentially pathogenic Acanthamoeba and Naegleria species from tap-water sources in Osaka, Japan. Parasitology Research, 105, 1109–17. 10.1007/s00436-009-1528-4Search in Google Scholar PubMed

Ertabaklar H., Turk M., Dayanir V., Ertu S., Walochnik J. 2007. Acanthamoeba keratitis due to Acanthamoeba genotype T4 in a non-contact-lens wearer in Turkey. Parasitology Research, 100, 241–246. 10.1007/s00436-006-0274-0Search in Google Scholar PubMed

Ghadar-ghadr S.H., Solhjoo K., Norouz-nejad M.J., Rohi R., ZiaJahromi S. 2012. Isolation and identification of free livingamoeba (Naegleriaand Acanthamoeba) in Shiraz water resources by morphological criteria. Journal of Jahrom University of Medical Sciences, 10, 33–42. 10.29252/jmj. 10.3.33Search in Google Scholar

Hall T.A. 1999. BioEdit: a user-friendly biological sequence alignment editor and analysis program for Windows 95/98/NT. Nucleic Acids Symposium Series, 41, 95–98Search in Google Scholar

Hammersmith K.M. 2006. Diagnosis and management of Acanthamoeba Keratitis. Current Opinion in Ophthalmology, 17, 327–31. 10.1097/01.icu. 0000233949.56229.7dSearch in Google Scholar

Hassan A., Farouk H., Hassanein F., Abdul-Ghani R., Abdelhady A.H. 2012. Acanthamoeba contamination of hemodialysis and dental units in Alexandria, Egypt: A neglected potential source of infection. Journal of Infection and Public Health, 5, 304– 10. 10.1016/j.jiph.2012.06.001Search in Google Scholar PubMed

Jeong H.J., Yu H.S. 2005. The role of domestic tap water in Acanthamoeba contamination in contact lens storage cases in Korea. Korean Journal of Parasitology 43, 47–5010.3347/kjp.2005.43.2.47Search in Google Scholar PubMed PubMed Central

Kao P.M., Chou M.Y., Tao C.W., Huang W.C., Hsu B.M., Shen S.M. et al. 2013. Diversity and seasonal impact of Acanthamoeba species in a subtropical river shed. Biomed Research International, 7, 1–810.1155/2013/405794Search in Google Scholar PubMed PubMed Central

Khan N.A., Jarroll E.L., Paget T.A. 2002. Molecular and physiological differentiation between pathogenic and nonpathogenic Acanthamoeba. Current Microbiology, 45, 197–202. 10.1007/s00284-001-0108-3Search in Google Scholar PubMed

Khurana S., Biswal M., Kaur H., Malhotra P., Arora P., Megha K., Taneja N., Sehgal R. 2015. Free living amoebae in water sources of critical units in a tertiary care hospital in India. Indian Journal of Medical Microbiology, 33, 343–8. 10.4103/0255-0857.158543Search in Google Scholar PubMed

Kilic A., Tanyuksel M., Sissons J., Jayasekera S., Khan N.A. 2004. Isolation of Acanthamoeba isolates belonging to T2, T3, T4 and T7 genotypes from environmental samples in Ankara, Turkey. Acta Parasitologica, 49, 246–252Search in Google Scholar

Kilvington S., Price J. 1990. Survival of Legionella pneumophila within cysts of Acanthamoeba polyphaga following chlorine exposure. Journal of Applied Bacteriology, 68, 519–2510.1111/j.1365-2672.1990.tb02904.xSearch in Google Scholar PubMed

Kilvington S., Beeching J.R., White D.G. 1991. Differentiation of Acanthamoeba strains from infected corneas and the environment using restriction endonuclease digestion of wholecell DNA. Journal of Clinical Microbiology, 29, 310–410.1128/jcm.29.2.310-314.1991Search in Google Scholar PubMed PubMed Central

Kilvington S., Gray T., Dart J., Morlet N., Beeching J.R., Frazer D.G., et al. 2004. Acanthamoeba keratitis: the role of domestic tap water contamination in the United Kingdom. Invest. Optometry and Vision Science, 45, 165–16910.1167/iovs.03-0559Search in Google Scholar PubMed

Kumar S., Stecher G., Tamura K. 2016. MEGA7: Molecular Evolutionary Genetics Analysis version 7.0 for bigger datasets. Molecular Biology and Evolution, 33, 1870–1874. 10.1093/molbev/msw054Search in Google Scholar PubMed PubMed Central

Lasjerdi Z., Niyyati M., Haghighi A., Shahabi S., Biderouni F.T., Taghipour N., et al. 2011. Potentially pathogenic free-living amoebae isolated from hospital wards with immunodeficient patients in Tehran, Iran. Parasitology Research, 109, 575–80. 10.1007/s00436-011-2288-5Search in Google Scholar PubMed

Leiva B., Clasdotter E., Linder E., Winiecka-Krusnell J. 2007. Free-living Acanthamoeba and Negleria spp. Amebae in water sources of León, Nicaragua. Revista de Biología Tropical, 56, 439–4610.15517/rbt.v56i2.5598Search in Google Scholar PubMed

Lorenzo-Morales J., Khan N.A., Walochnik J. 2015. An update on Acanthamoeba keratitis: diagnosis, pathogenesis and treatment. Parasite, 22, 1–20. 10.1051/parasite/2015010Search in Google Scholar PubMed PubMed Central

Lorenzo-Morales J., Martı´n-Navarro C.M., Lo´pez-Arencibia A., Arnalich-Montiel F., Pin˜ero J.E., Valladares B. 2013. Acanthamoeba keratitis: an emerging disease gathering importance worldwide? Trends in Parasitology, 29, 181–7. 10.1016/j.pt.2013.01.006Search in Google Scholar PubMed

Lorenzo-Morales J., Ortega-Rivas A., Martı´nez E., Khoubbane M., Artigas P., Periago M.V. et al. 2006. Acanthamoeba isolates belonging to T1, T2, T3, T4 and T7 genotypes from environmental freshwater samples in the Nile Delta region, Egypt. Acta Tropica, 100, 63–9. 10.1016/j.actatropica.2006.09.008Search in Google Scholar PubMed

Lorenzo-Morales J., Ortega-Rivas A., Martinez E., Foronda P., Valladares B. 2005. Isolation and identification of pathogenic Acanthamoeba strains in Tenerife, Canary Island, Spain from water sources. Parasitology Research, 95, 273–277. 10.1007/s00436-005-1301-2Search in Google Scholar PubMed

Maciver S.K., Asif M., Simmen M.W., Lorenzo-Morales J.A. 2013. Systematic analysis of Acanthamoeba genotype frequency correlated with source and pathogenicity: T4 is confirmed as a pathogen-rich genotype. European Journal of Protistology, 49, 217–21. 10.1016/j.ejop.2012.11.004Search in Google Scholar PubMed

Maghsood A.H., Sissons J., Rezaian M., Nolder D., Warhurst D., Khan N.A. 2005. Acanthamoeba genotype T4 from the UK and Iran and isolation of the T2 genotype from clinical isolates. Journal of Medical Microbiology, 54, 755–9. 10.1099/jmm.0.45970-0Search in Google Scholar PubMed

Magliano A.C., da Silva F.M., Teixeira M.M., Alfieri S.C. 2009. Genotyping, physiological features and proteolytic activities of a potentially pathogenic Acanthamoeba sp. isolated from tap water in Brazil. Experimental Parasitology, 123, 231–5. 10.1016/j.exppara.2009.07.006Search in Google Scholar PubMed

Magnet A., Henriques-Gil N., Galván-Diaz A.L., Izquiedo F., Fenoy S., del Aguila C. 2014. Novel Acanthamoeba 18S rRNA gene sequence type from an environmental isolate. Parasitology Research, 113, 2845–2850. 10.1007/s00436-014-3945-2Search in Google Scholar PubMed

Mella C., Medina G., Flores-Martin S., Toledo Z., Simaluiza R.J., Pérez-Pérez G., Fernández H. 2016. Interaction between zoonotic bacteria and free living amoebas. A new angle of an epidemiological polyhedron of public health importance? Archivos de Medicina Veterinaria, 48, 1–10. 10.4067/S0301-732X2016000100001Search in Google Scholar

Morsy G.H., Al-Herrawy A.Z., Elsenousy W.M., Marouf M.A. 2016. Prevalence of free-living amoebae in tap water and biofilm, Egypt. Research Journal of Pharmaceutical, Biological and Chemical Sciences, 7, 752–759Search in Google Scholar

Niyyati M., Lasgerdi Z., Lorenzo-Morales J. 2015. Detection and Molecular Characterization of Potentially Pathogenic Freeliving Amoebae from Water Sources in Kish Island, Southern Iran. Microbiology Insights, 8 (Suppl 1), 1–6. 10.4137/MBI.S24099Search in Google Scholar PubMed PubMed Central

Onichandran S., Kumar T., Salibay C.C., Dungca J.Z., Tabo H.A., Tabo N., et al. 2014. Waterborne parasites: a current status from the Philippines. Parasites and Vectors. 7, 244. 10.1186/1756-3305-7-244Search in Google Scholar PubMed PubMed Central

Ovrutsky A.R., Chan E.D., Kartalija M., Bai X., Jackson M., Gibbs S., et al. 2013. Cooccurrence of free-living amoebae and nontuberculous Mycobacteria in hospital water networks, and preferential growth of Mycobacterium avium in Acanthamoeba lenticulata. Applied and Environmental Microbiology, 79, 3185–92. 10.1128/AEM.03823-12Search in Google Scholar PubMed PubMed Central

Özçelik S., Coşkun K.A., Yünlü Ö., Alim A., Malatyalı E. 2012. The Prevalence, Isolation and Morphotyping of Potentially Pathogenic Free-Living Amoebae from Tap Water and Environmental Water Sources in Sivas. Türkiye Parazitoloji Dergisi, 36, 198–203. 10.5152/tpd.2012.48Search in Google Scholar PubMed

Page F.C. 1976. An illustrated key to freshwater and soil amoebae. Freshwater Biological Association, Ferry House, Far Sawrey, Ambleside, England. 62–66.Search in Google Scholar

Rozej A., Cydzik-Kwiatkowska A., Kowalska B., Kowalski D. 2015. Structure and microbial diversity of biofims on different pipe materials of a model drinking water distribution systems. World Journal of Microbiology and Biotechnology, 31, 37– 47. 10.1007/s11274-014-1761-6Search in Google Scholar PubMed PubMed Central

Sadaka H.A., el-Nassery S.F., Abou Samra L.M., Awadalla H.N. 1994. Isolation and identification of free-living amoebae from some water sources in Alexandria. Journal of Egyptian Society of Parasitology, 24, 247–57Search in Google Scholar

Saitou N., Nei M. 1987. The neighbor-joining method: a new method for reconstructing phylogenetic trees. Molecular Biology and Evolution, 4, 406 – 425. 10.1093/oxfordjournals.mol-bev.a040454Search in Google Scholar

Sakran T.F., El-Shahawy A.G., Shalaby M.A., Sabry H.Y., Matooq P.M., Elmallah A.M. 2017. Detection rates of waterborne protozoa in water sources from Fayoum Governorate. Parasitology United Journal, 10, 30–38. 10.21608/PUJ.2017.4734Search in Google Scholar

Sarıca F.B., Tufan K., Çekinmez M., Erdoğan B., Altınors M.N. 2009. A rare but fatal case of Granulomatous Amebic Encephalitis with Brain abscess. The first case reported from Turkey. Turkish Neurosurgery, 19, 256–9Search in Google Scholar

Schroeder J.M., Booton G.C., Hay J., Niszl I.A., Seal D.V., Markus M.B., et al. 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. Journal of Clinical Microbiology, 39, 1903–11. 10.1128/JCM.39.5.1903-1911.2001Search in Google Scholar PubMed PubMed Central

Schuster F.L. 2002. Cultivation of pathogenic and opportunistic freeliving amebas. Clinical Microbiology Reviews, 15, 342–354. 10.1128/CMR.15.3.342-354.2002Search in Google Scholar PubMed PubMed Central

Stockman L.J., Wright C.J., Visvesvara G.S., Fields B.S., Beach M.J. 2011. The prevalence of Acanthamoeba spp. and other freeliving amoebae in household water, Ohio, USA 1990-1992. Parasitology Research, 108, 621–7. 10.1007/s00436- 010-2120-7Search in Google Scholar

Storey M.V., Winiecka-Krusnell J., Ashbolt N.J., Stenström T.A. 2004. The efficacy of heat and chlorine treatment against thermotolerant Acanthamoebae and Legionella. Scandinavian Journal of Infectious Diseases, 36, 656–62. 10.1080/00365540410020785Search in Google Scholar PubMed

Taher E.E., Méabed EMH, Abdallah I., Abdel Wahed W.Y. 2017. Acanthamoeba keratitis in noncompliant soft contact lenses users: Genotyping and risk factors, a study from Cairo, Egypt. Journal of Infection and Public Health, 28. pii: S1876–0341, 30247–2. 10.1016/j.jiph.2017.09.013Search in Google Scholar PubMed

Tawfeek G.M., Bishara S.A., Sarhan R.M., Taher E.E., Khayyal A.E. 2016. Genotypic, physiological, and biochemical characterization of potentially pathogenic Acanthamoeba isolated from the environment in Cairo, Egypt. Parasitology Research, 115, 1871–1881. 10.1007/s00436-016-4927-3Search in Google Scholar PubMed

Teixeira L.H., Rocha S., Pinto R.M.F., Caseiro M.M., da Costa S.O.P. 2009. Prevalence of Potentially Pathogenic Free-Living Amoebae from Acanthamoeba and Naegleria Genera in Non-Hospital, Public, Internal Environments from the City of Santos, Brazil. Brazilian Journal of Infectious Diseases, 13, 395–397. 10.1590/S1413-86702009000600001Search in Google Scholar PubMed

Thomas V., McDonnell G., Denyer S.P., Maillard J.Y. 2010. Free living amoebae and their intracellular pathogenic microorganisms: risks for water quality. FEMS Microbiology Reviews, 34, 231–59. 10.1111/j.1574-6976.2009.00190.xSearch in Google Scholar PubMed

Thomas V., Loret J.F., Jousset M., Greub G. 2008. Biodiversity of amoebae and amoebae-resisting bacteria in a drinking water treatment plant. Environmental Microbiology, 10, 2728–45. 10.1111/j.1462-2920.2008.01693.xSearch in Google Scholar PubMed

Trabelsi H., Dendana F., Sellami A., Cheikhrouhou F., Neji S., Makni F., et al. 2012. Pathogenic free living amoebae: epidemiology and clinical review. Pathologie Biologie, 60, 399–405. 10.1016/j.patbio.2012.03.002Search in Google Scholar PubMed

Trabelsi H., Sellami A., Dendena F., Sellami H., Cheikh-Rouhou F., Makni F., Ayadi A. 2010. Free-living Amoebae (FLA): Morphological and molecular identification of Acanthamoeba in dental unit water. Parasite. 17, 67–70 10.1051/para-site/2010171067Search in Google Scholar

Tsvetkova N., Schild M., Panaiotov S., Kurdova-Mintcheva R., Gottstein B., Walochnik J., et al. 2004. The identification of free-living environmental isolates of amoebae from Bulgaria. Parasitology Research, 92, 405–13. 10.1007/s00436-003- 1052-xSearch in Google Scholar

Wang W., Wei F., Li J., Li N., Liu Q. 2014. Isolation and Identification of Naegleria Species from Environmental Water in Changchun, Northeastern China. Iranian Journal of Parasitology, 9, 254–9Search in Google Scholar

Xuan Y., Shen Y., Ge Y., Yan G., Zheng S. 2017. Isolation and identification of Acanthamoeba strains from soil and tap water in Yanji, China. Environmental Health and Preventive Medicine, 22, 1–6. 10.1186/s12199-017-0655-2Search in Google Scholar PubMed PubMed Central

Yousuf F.A., Siddiqui R., Khan N.A. 2013. Acanthamoeba castellanii of the T4 genotype is a potential environmental host for Enterobacter aerogenes and Aeromonas hydrophila. Parasites and Vectors, 6, 169. 10.1186/1756-3305-6-169Search in Google Scholar PubMed PubMed Central

Received: 2018-08-09
Revised: 2018-08-24
Accepted: 2018-08-29
Published Online: 2018-10-18
Published in Print: 2018-12-19

© 2018 W. Stefański Institute of Parasitology, PAS