In situ detection of novel bacterial endosymbionts of Acanthamoeba spp. phylogenetically related to members of the order Rickettsiales

Acanthamoebae are ubiquitous soil and water bactivores which may serve as a mplification vehicles for a variety of pathogenic facultative bacteria and as hosts to other, presently uncultured bacterial endosymbionts. The spectr um of uncultured endosymbionts includes gram-negative rods and gram-variabl e cocci, the latter recently shown to be members of the Chlamydiales. We re port here the isolation from corneal scrapings of two Acanthamocba strains that harbor gram-negative rod endosymbionts that could not be cultured by s tandard techniques. These bacteria were phylogenetically characterized foll owing amplification and sequencing of the near-full-length 16S rRNA gene. W e used two fluorescently labelled oligonucleotide probes targeting signatur e regions within the retrieved sequences to detect these organisms in situ, Phylogenetic analyses demonstrated that they displayed 99.6% sequence simi larity and formed an independent and well-separated lineage within the Rick ettsiales branch of the alpha subdivision of the Proteobacteria, Nearest re latives included members of the genus Rickettsia, with sequence similaritie s of approximately 85 to 86%, suggesting that these symbionts are represent atives of a new genus and, perhaps, family. Distance matrix, parsimony, and maximum-likelihood tree-generating methods all consistently supported deep branching of the 16S rDNA sequences within the Rickettsiales. The oligonuc leotide probes displayed at least three mismatches to all other available 1 6S rDNA sequences, and they both readily permitted the unambiguous detectio n of rod-shaped bacteria within intact acanthamoebae by confocal laser-scan ning microscopy. Considering the long-standing relationship of most Rickett siales with arthropods, the finding of a related lineage of endosymbionts i n protozoan hosts was unexpected and may have implications for the preadapt ation and/or recruitment of rickettsia-like bacteria to metazoan hosts.