Environmental Microbiology (2001) 3(7), 440±449 Members of the Cytophaga±Flavobacterium± Bacteroides phylum as intracellular bacteria of acanthamoebae: proposal of `Candidatus Amoebophilus asiaticus' Matthias Horn, 1 * Marit D. Harzenetter, 1 Tanja Linner, 1 Ernst N. Schmid, 2 Karl-Dieter Mu È ller, 2 Rolf Michel 3 and Michael Wagner 1 1 Lehrstuhl fu È r Mikrobiologie, Technische Universita Èt Mu È nchen, D-83530 Freising, Germany. 2 Institut fu È r Medizinische Mikrobiologie der Universita Èt GH-Essen, D-45147 Essen, Germany. 3 Central Institute of the Federal Armed Forces Medical Service, D-56065 Koblenz, Germany. Summary 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 successfully outside their amoebal host cells and were identified subsequently by comparative 16S rRNA sequence analysis as being most closely affiliated with Flavobacterium succini- cans (99% 16S rRNA sequence similarity) or Flavo- bacterium johnsoniae (98% 16S rRNA sequence similarity). One endocytobiont could neither be cultivated outside its original Acanthamoeba host (Acanthamoeba sp. TUMSJ-321) nor transferred into other amoebae. Electron microscopy revealed that the amoebal trophozoites and cysts were almost completely filled with cells of this endosymbiont which are surrounded by a host-derived membrane. According to 16S rRNA sequence analysis, this endosymbiont could also be assigned to the Cyto- phaga ± Flavobacterium±Bacteroides (CFB) phylum, but was not closely affiliated to any recognized species within this phylogenetic group (less than 82% 16S rRNA sequence similarity). Identity and intra- cellular localization of this endosymbiont were con- firmed by application of a specific fluorescently labelled 16S rRNA-targeted probe. Based on these findings, we propose classification of this obligate Acantha- moeba endosymbiont as `Candidatus Amoebophilus asiaticus'. Comparative 18S rRNA sequence analysis of the host of `Candidatus Amoebophilus asiaticus' revealed its membership with Acanthamoeba 18S rDNA sequence type T4 that comprises the majority of all Acanthamoeba isolates. Introduction Acanthamoebae are ubiquitous, free-living amoebae that act as important predators controlling microbial commu- nities (Rodriguez-Zaragoza, 1994) and, in addition, have been recognized as opportunistic human pathogens causing keratitis or granulomatous amoebic encephalitis (GAE) (Visvesvara, 1995). Several types of interactions between acanthamoebae and bacteria leading to transi- ent, as well as long-term, associations have been reported (e.g. Fritsche et al., 1993; Barker and Brown, 1994). Besides being a food source for amoebae, some bacteria survive phagocytosis and are able to multiply as parasites within free-living amoebae. This capability, which may protect the bacteria from unfavourable environmental conditions, has, for example, been demon- strated for Legionella pneumophila (Fields, 1996), Chla- mydia pneumoniae (Essig et al., 1997), Listeria monocytogenes (Ly and Mu È ller, 1990) and Mycobacter- ium avium (Steinert et al., 1998). Even obligate anaerobic microorganisms like Mobiluncus curtisii can exploit amoebal hosts as transportation and protection vehicles (Tomov et al., 1999). Although long-term symbiotic associations between acanthamoebae and bacteria have already been observed for the first time in the 1970's (Proca-Ciobanu et al., 1975), the characterization of diversity and phylogeny of these obligate intracellular microorganisms had to await the recent development of culture-independent identification techniques. Compara- tive analysis of 16S rRNA sequences led to the descrip- tion of four major evolutionary lineages of Acanthamoeba endosymbionts. Two of these lineages are within the a- subclass of Proteobacteria, being most closely related with either the Rickettsiales (Fritsche et al., 1999) or the paramecium symbiont Caedibacter caryophilus (Horn et al., 1999; Birtles et al., 2000). In addition, several acanthamoebae endosymbionts form a monophyletic assemblage within the b-subclass of Proteobacteria Q 2001 Blackwell Science Ltd Received 18 April, 2001; accepted 5 June, 2001. *For correspon- dence. E-mail horn@mikro.biologie.tu-muenchen.de; Tel. (1 49) 8161 715459; Fax (1 49) 8161 715475.