Molecular identification of bacterial endosymbionts of Sappinia strains

The genus Sappinia comprises free-living amoebae occurring worldwide in a variety of habitats such as soils, plant matter and freshwater ponds, but also animal faeces, and includes at present three species, S. pedata, S. diploidea and S. platani. The genus is potentially pathogenic, as indicated by the identification of S. pedata in a case of human amoebic encephalitis. Electron microscopy studies on some strains already revealed intracellular bacteria in Sappinia. In the current study, we performed 16S ribosomal RNA gene (rDNA) analysis of these bacterial endosymbionts. We first inferred relationships among Sappinia strains on the basis of 18S rDNA, demonstrating that S. pedata emerged as sister to a larger clade including S. diploidea, S. platani and a few 'S. diploidea-like' strains. Thus, bacterial 16S rDNA was searched for in representative strains of each Sappinia species/subgroup. We found that Sappinia strains were associated to distinct species of Flavobacterium or Pedobacter (phylum Bacteroidetes). These appear to be distributed following the amoebal host subgroups, and are not directly related to other Bacteroidetes species known as interacting with free-living amoebae. While all the endosymbionts' close relatives are known to grow on agar, bacteriological media inoculated with amoebal extracts remained negative. Overall, results indicate that the recovered bacteria are likely specific obligate endosymbionts of Sappinia species. Further studies, including additional amoebal strains and deep morphological and molecular analyses, will be necessary to confirm this hypothesis.