Phylogenetic diversity among geographically dispersed Chlamydiales endosymbionts recovered from clinical and environmental isolates of Acanthamoeba spp.

The recently proposed reorganization of the order Chlamydiales and descript ion of new taxa are broadening our perception of this once narrowly defined taxon. We have recovered four strains of gram-negative cocci endosymbiotic in Acanthamoeba spp,, representing 5% of the Acanthamoeba sp. isolates exa mined, which displayed developmental life cycles typical of members of the Chlamydiales, One of these endosymbiont strains was found stably infecting an amoebic isolate recovered from a case of amoebic keratitis in North Amer ica, with three others found in acanthamoebae recovered from environmental sources in North America (two isolates) and Europe (one isolate). Analyses of nearly full-length 16S rRNA gene sequences of these isolates by neighbor joining, parsimony, and distance matrix methods revealed their clustering with other members of the Chlamydiales but in a lineage separate from those of the genera Chlamydia, Chlamydophila, Simkania, and Waddlia (sequence si milarities, <88%) and including the recently described species Parachlamydi a acanthamoebae (sequence similarities, 91.2 to 93.1%), With sequence simil arities to each other of 91.4 to 99.4%, these four isolates of intra-amoeba l endosymbionts may represent three distinct species and, perhaps, new gene ra within the recently proposed family Parachlamydiaceae, Fluorescently lab eled oligonucleotide probes targeted to 16S rRNA signature regions were abl e to readily differentiate two groups of intra-amoebal endosymbionts which corresponded to two phylogenetic lineages, These results reveal significant phylogenetic diversity occurring among the Chlamydiales in nontraditional host species and supports the existence of a large environmental reservoir of related species. Considering that all described species of Chlamydiales are known to be pathogenic, further investigation of intra-amoebal parachla mydiae as disease-producing agents is warranted.