Members of the Cytophaga-Flavobacterium-Bacteroides phylum as intracellular bacteria of acanthamoebae: proposal of 'Candidatus Amoebophilus asiaticus'

Three Gram-negative, rod-shaped bacteria that were found intracellularly in two environmental and one clinical Acanthamoeba sp. isolates were analysed . Two endocytobionts showing a parasitic behaviour were propagated successf ully outside their amoebal host cells and were identified subsequently by c omparative 16S rRNA sequence analysis as being most closely affiliated with Flavobacterium succinicans (99% 16S rRNA sequence similarity) or Flavobact erium johnsoniae (98% 16S rRNA sequence similarity). One endocytobiont coul d neither be cultivated outside its original Acanthamoeba host (Acanthamoeb a sp. TUMSJ-321) nor transferred into other amoebae. Electron microscopy re vealed that the amoebal trophozoites and cysts were almost completely fille d with cells of this endosymbiont which are surrounded by a host-derived me mbrane. According to 16S rRNA sequence analysis, this endosymbiont could al so be assigned to the Cytophaga-Flavobacterium-Bacteroides (CFB) phylum, bu t was not closely affiliated to any recognized species within this phylogen etic group (less than 82% 16S rRNA sequence similarity). Identity and intra cellular localization of this endosymbiont were confirmed by application of a specific fluorescently labelled 16S rRNA-targeted probe. Based on these findings, we propose classification of this obligate Acanthamoeba endosymbi ont as 'Candidatus Amoebophilus asiaticus'. Comparative 18S rRNA sequence a nalysis of the host of 'Candidatus Amoebophilus asiaticus' revealed its mem bership with Acanthamoeba 18S rDNA sequence type T4 that comprises the majo rity of all Acanthamoeba isolates.