Presence of potentially pathogenic free-living amoebae strains from well water samples in Guinea-Bissau

Abstract

Free-living amoebae (FLA) include opportunistic pathogens such as Naegleria fowleri, Balamuthia mandrillaris, and the genera Sappinia and Acanthamoeba. In this study, a survey was conducted in order to evaluate the presence of potentially pathogenic amoebic strains in water samples collected from wells located in the western part of Guinea-Bissau. The samples were left to precipitate for 48 hours and then the sediments were seeded on non-nutrient agar plates containing Escherichia coli spread and cultures were checked daily for the presence of FLA. Identification of FLA strains was based on the morphological and polymerase chain reaction (PCR) using the 18S rDNA or 16S mitochondrial rDNA genes in the case of Naegleria and Balamuthia genera, respectively. In the case of positive samples of Acanthamoeba, strains were further classified at the genotype level by sequencing the diagnostic fragment 3 (DF3) region located in the 18S rDNA gene as previously described. Sappinia sp. was not isolated during the study and thus, no molecular analysis was performed for this genus. The obtained results revealed the presence of Acanthamoeba (genotypes T3 and T4), Naegleria fowleri, and Balamuthia mandrillaris. To the best of our knowledge, this is the first report demonstrating the presence of FLA in water bodies from Guinea-Bissau and the first report on the isolation of Balamuthia mandrillaris from environmental sources in Africa.

Keywords: Acanthamoeba,; Balamuthia mandrillaris,; Genotype,; Guinea-Bissau; Naegleria,; Well water,.

Figure 1

Sampling points surveyed during 2011 in three regions of Guinea-Bissau. Oio Region: 1, Culcunhe; 2, Watine; 3, Ufle; 4, Braia; 5, Clack N’djassé; 6, Bera; 7, Palche Iala; 8, Wedequeia; 9, Saw; 10, Djugudul Com; 11, Thugue; 12, Oco Grande. Quínara Region: 13, Ganjetra; 14, Fulacunda; 15, Brandao; 16, Madina Hatche; 17, Nova Sintra; 18, Empada. Tombalí Region: 19, Madina Contabane; 20, Afia Bunhio; 21, Botchi Minde; 22, Mato Forroba.

Figure 2

18S rDNA DF3 linearized neighbour-joining tree obtained by using the Kimura two-parameter distance algorithm, produced in MEGA 5.0. The isolates obtained in the present study are identified in the tree (boxes). The type sequences were taken from GenBank and are presented under the following numbers: A. astronyxis strain CCAP 1534/1 #AF239293, A. castellanii strain CDC:0981:V006 Accession #U07400, A. castellanii ATCC 50373 Accession # U07416, A. griffini S-7 ATCC 30731 Accession #U07412, A. griffini B-18 FB2 Acession #GU597017, A. griffini B-18 Acession #GU553135, A. griffini strain D4 Acession #HQ00740, A. lenticulata isolate 12-SO #KC694184, A. lenticulata isolate 33195463 Accession #KC438381, Acanthamoeba sp. isolate OSU09-002 Accession # JQ669657, Acanthamoeba sp. genotype T2 Isolate OSU09-006 #JQ669661, Acanthamoeba palestinensis isolate TW-2 Accession #KC694193, A. palestinensis strain CCAP 1547-1 #AF239296, Acanthamoeba sp. isolate BRO2-T16 Accession #JX683392, Acanthamoeba sp. UWC9 Accession #AF132134, Acanthamoeba sp. PM5 Accession #JX494395, Acanthamoeba jacobsi AC304 Accession #AY262364.