The recently proposed reorganization of the order Chlamydiales and description 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 examined, 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 America, 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 similarities, <88%) and including the recently described species Parachlamydia acanthamoebae (sequence similarities, 91.2 to 93.1%). With sequence similarities to each other of 91.4 to 99.4%, these four isolates of intra-amoebal endosymbionts may represent three distinct species and, perhaps, new genera within the recently proposed family Parachlamydiaceae. Fluorescently labeled oligonucleotide probes targeted to 16S rRNA signature regions were able 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 parachlamydiae as disease-producing agents is warranted.All members of the order Chlamydiales are recognized pathogens of mammals, marsupials, or birds. The ability to produce respiratory disease, among other clinical presentations, is a feature of most species within the order and is especially characteristic of infections produced by Chlamydophila pneumoniae, Chlamydophila psittaci, certain serovars of Chlamydia trachomatis, and the recently described species Simkania negevensis (20,21,23). The recent finding of evidence for seroconversion to antigen of Parachlamydia acanthamoebae, an intra-amoebal Chlamydia-like bacterium, in a small number of humans experiencing community-acquired pneumonia, along with the findings of novel Parachlamydia-related 16S rRNA sequences in respiratory specimens, peripheral blood, and aortic tissue, suggests that this and related species of protozoal endosymbionts may also be of clinical significance, warranting further investigation (2, 5, 30).Previously, we have reported on the common occurrence of uncultured bacterial endosymbionts in protozoa of the genus Acanthamoeba (13). While 20% of axenically growing Acanthamoeba isolates recovered from clini...
