Molecular genotyping schemes

MLST genotyping of gene fragments of seven housekeeping genes (enoA, fumC, gatA, gidA, hemN, hlfX and oppA) of C. psittaci strain 84/2334 and representative strains from Chlamydiaceae species, was performed as previously described [28, 32]. Genes were concatenated, aligned using MAFFT and a phylogenetic tree estimated in TOPALi as detailed in the following section (Phylogenetic and network analysis). Cluster analysis based on defined allelic profiles or sequence types (ST) for each individual isolate, as well as STs from other representative isolates for all Chlamydiaceae species (except C. trachomatis) in the online database [36, 81] was conducted to generate minimum spanning trees that were visualised using GrapeTree [82]. Ribosomal MLST analysis to determine species classification was conducted using the rps gene database [34, 83]. Digital DNA-DNA hybridization to determine species was conducted using the Genome-to-Genome Distance Calculator 2.1 [33, 84].

A classification scheme, based on five discriminant proteins (Adk, FtsK, HemL, PepF and RpoN) for species designation, was applied to strain 84/2334, as previously described [35]. Protein sequences for all strains were identified by BLAST analysis. Sequence distances (% ID) for each of the five proteins from strain 84/2334 with each of the equivalent proteins from the C. psittaci and C. abortus strains and genotypes in Table ​Table22 were calculated by aligning sequences in MegAlign 15 (Lasergene software, DNASTAR Inc., Madison, WI, USA) Clustal Omega [85]. Proteins with a %ID ≥95% for Adk and HemL, ≥ 96% for PepF and RpoN, and ≥ 98% for FtsK, indicate they are classified as the same species.