<i>Criblamydia sequanensis</i> , a new intracellular <i>Chlamydiales</i> isolated from Seine river water using amoebal co‐culture

<jats:title>Summary</jats:title> <jats:p> Accumulating evidence supports a role for <jats:italic>Chlamydia</jats:italic> ‐related organisms as emerging pathogens for human and animals. Assessment of their pathogenicity requires strain availability, at least for animal models and serological studies. As these obligate intracellular species are able to grow inside amoebae, we used co‐culture with <jats:italic>Acanthamoeba castellanii</jats:italic> in an attempt to recover new <jats:italic>Chlamydia</jats:italic> ‐related species from river water. We isolated two strains from eight water samples. The first strain is a new <jats:italic>Parachlamydia acanthamoebae</jats:italic> strain that differs from previously described isolates by only two bases in the complete 16S rRNA gene sequence. The second isolate is the first representative of a new <jats:italic>Chlamydiales</jats:italic> family, as demonstrated by genetic and phylogenetic analyses of the 16S rRNA, 23S rRNA, ADP/ATP translocase and RnpB encoding genes. Using fluorescent <jats:italic>in situ</jats:italic> hybridization and electron microscopy, we demonstrated that it grows in high numbers in amoebae, where it exhibits a <jats:italic>Chlamydia</jats:italic> ‐like developmental cycle with reticulate bodies and star‐like elementary bodies. Based on these results, we propose to name this new species ‘ <jats:italic>Criblamydia sequanensis</jats:italic> ’. This work confirmed that amoebal co‐culture is a relevant method to isolate new chlamydiae, and that it can be successfully applied to ecosystems colonized with a complex microbial community. </jats:p>