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      Isolation and Molecular Characterization of Free-Living Amoebae from Different Water Sources in Italy

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          Abstract

          Free-living amoebae (FLA) are protozoa ubiquitous in Nature, isolated from a variety of environments worldwide. In addition to their natural distribution, some species have been found to be pathogenic to humans. In the present study a survey was conducted in order to evaluate the presence and to characterize at molecular level the isolates of amoebic organisms collected from different water sources in Italy. A total of 160 water samples were analyzed by culture and microscopic examination. FLA were found in 46 (28.7%) of the investigated water samples. Groundwater, well waters, and ornamental fountain waters were the sources with higher prevalence rates (85.7%, 50.0%, and 45.9%, respectively). Identification of FLA species/genotypes, based on the 18S rDNA regions, allowed to identify 18 (39.1%) Acanthamoeba isolates (genotypes T4 and T15) and 21 (45.6%) Vermamoeba vermiformis isolates. Other FLA species, including Vahlkampfia sp. and Naegleria spp., previously reported in Italy, were not recovered. The occurrence of potentially pathogenic free-living amoebae in habitats related to human population, as reported in the present study, supports the relevance of FLA as a potential health threat to humans.

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          Use of subgenic 18S ribosomal DNA PCR and sequencing for genus and genotype identification of acanthamoebae from humans with keratitis and from sewage sludge.

          Michael J. M. Hay, Susan J. Schroeder, I Niszl … (2001)
          This study identified subgenic PCR amplimers from 18S rDNA that were (i) highly specific for the genus Acanthamoeba, (ii) obtainable from all known genotypes, and (iii) useful for identification of individual genotypes. A 423- to 551-bp Acanthamoeba-specific amplimer ASA.S1 obtained with primers JDP1 and JDP2 was the most reliable for purposes i and ii. A variable region within this amplimer also identified genotype clusters, but purpose iii was best achieved with sequencing of the genotype-specific amplimer GTSA.B1. Because this amplimer could be obtained from any eukaryote, axenic Acanthamoeba cultures were required for its study. GTSA.B1, produced with primers CRN5 and 1137, extended between reference bp 1 and 1475. Genotypic identification relied on three segments: bp 178 to 355, 705 to 926, and 1175 to 1379. ASA.S1 was obtained from single amoeba, from cultures of all known 18S rDNA genotypes, and from corneal scrapings of Scottish patients with suspected Acanthamoeba keratitis (AK). The AK PCR findings were consistent with culture results for 11 of 15 culture-positive specimens and detected Acanthamoeba in one of nine culture-negative specimens. ASA.S1 sequences were examined for 6 of the 11 culture-positive isolates and were most closely associated with genotypic cluster T3-T4-T11. A similar distance analysis using GTSA.B1 sequences identified nine South African AK-associated isolates as genotype T4 and three isolates from sewage sludge as genotype T5. Our results demonstrate the usefulness of 18S ribosomal DNA PCR amplimers ASA.S1 and GTSA.B1 for Acanthamoeba-specific detection and reliable genotyping, respectively, and provide further evidence that T4 is the predominant genotype in AK.
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            Pathogenic free-living amoebae: epidemiology and clinical review.

            H. Trabelsi, F Dendana, A Sellami … (2012)
            Free-living amoebae are widely distributed in soil and water. Small number of them was implicated in human disease: Acanthamoeba spp., Naegleria fowleri, Balamuthia mandrillaris and Sappinia diploidea. Some of the infections were opportunistic, occurring mainly in immunocompromised hosts (Acanthamoeba and Balamuthia encephalitis) while others are non opportunistic (Acanthamoeba keratitis, Naegleria meningoencephalitis and some cases of Balamuthia encephalitis). Although, the number of infections caused by these amoebae is low, their diagnosis was still difficult to confirm and so there was a higher mortality, particularly, associated with encephalitis. In this review, we present some information about epidemiology, ecology and the types of diseases caused by these pathogens amoebae. Copyright © 2012 Elsevier Masson SAS. All rights reserved.
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              Selection of models of DNA evolution with jModelTest.

              David Posada (2008)
              jModelTest is a bioinformatic tool for choosing among different models of nucleotide substitution. The program implements five different model selection strategies, including hierarchical and dynamical likelihood ratio tests (hLRT and dLRT), Akaike and Bayesian information criteria (AIC and BIC), and a performance-based decision theory method (DT). The output includes estimates of model selection uncertainty, parameter importance, and model-averaged parameter estimates, including model-averaged phylogenies. jModelTest is a Java program that runs under Mac OSX, Windows, and Unix systems with a Java Run Environment installed, and it can be freely downloaded from (http://darwin.uvigo.es).
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                Author and article information

                Contributors
                Roberto Spurio: Role: Academic Editor
                Duarte Tito: Role: Academic Editor
                Letizia Brandi: Role: Academic Editor
                Laura Mancini: Role: Academic Editor
                Journal
                Journal ID (nlm-ta): Int J Environ Res Public Health
                Journal ID (iso-abbrev): Int J Environ Res Public Health
                Journal ID (publisher-id): ijerph
                Title: International Journal of Environmental Research and Public Health
                Publisher: MDPI
                ISSN (Print): 1661-7827
                ISSN (Electronic): 1660-4601
                Publication date (Electronic): 24 March 2015
                Publication date (Print): April 2015
                Volume: 12
                Issue: 4
                Pages: 3417-3427
                Affiliations
                [1 ]Department of Experimental Medicine and Surgery, University of Rome Tor Vergata, Via Montpellier 1, 00133 Rome, Italy; E-Mails: montalbano.margherita89@ 123456gmail.com (M.M.F.); maristella5384@ 123456gmail.com (M.S.); dicave@ 123456uniroma2.it (D.C.)
                [2 ]Interdisciplinary Department of Medicine, University of Bari, Piazza G. Cesare 11, 70124 Bari, Italy; E-Mail: piero.lovreglio@ 123456uniba.it
                [3 ]Department of Basic Medical Science, Neuroscience and Sense Organ, University of Bari, Piazza G. Cesare 11, 70124 Bari, Italy; E-Mails: rosa.monno@ 123456uniba.it (R.M.); carmen.capolongo84@ 123456gmail.com (C.C.); carla.calia@ 123456virgilio.it (C.Cal.); luciana.fumarola@ 123456uniba.it (L.F.)
                [4 ]Department of Systems Medicine, University of Rome Tor Vergata, Via Montpellier 1, 00133 Rome, Italy; E-Mail: dalfonso@ 123456uniroma2.it
                [5 ]Laboratory of Parasitology, Foundation Polyclinic Tor Vergata, Viale Oxford 81, 00133 Rome, Italy
                Author notes
                [* ]Author to whom correspondence should be addressed; E-Mail: berrilli@ 123456uniroma2.it ; Tel.: +39-06-7259-6009.
                Article
                Publisher ID: ijerph-12-03417
                DOI: 10.3390/ijerph120403417
                PMC ID: 4410193
                PubMed ID: 25811766
                SO-VID: 7613ec5c-c1ba-48a0-816b-ee91f02ae5b7
                Copyright © © 2015 by the authors; licensee MDPI, Basel, Switzerland.
                License:

                This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution license ( http://creativecommons.org/licenses/by/4.0/).

                History
                Date received : 19 January 2015
                Date accepted : 16 March 2015
                Categories
                Subject: Article

                ScienceOpen disciplines: Public health
                Keywords: free-living amoebae,molecular characterization,water sources,italy
                Data availability:
                ScienceOpen disciplines: Public health
                Keywords: free-living amoebae, molecular characterization, water sources, italy

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