Genetic diversity of the obligate intracellular bacterium Chlamydophila pneumoniae by genome-wide analysis of single nucleotide polymorphisms: evidence for highly clonal population structure

Abstract

Background: Chlamydophila pneumoniae is an obligate intracellular bacterium that replicates in a biphasic life cycle within eukaryotic host cells. Four published genomes revealed an identity of > 99 %. This remarkable finding raised questions about the existence of distinguishable genotypes in correlation with geographical and anatomical origin.

Results: We studied the genetic diversity of C. pneumoniae by analysing synonymous single nucleotide polymorphisms (sSNPs) that are under reduced selection pressure. We conducted an in silico analysis of the four sequenced genomes, chose 232 representative sSNPs and analysed the loci in 38 C. pneumoniae isolates. We identified 15 different genotypes that were separated in four major clusters. Clusters were not associated with anatomical or geographical origin. However, animal lineages are basal on the C. pneumomiae phylogeny, suggesting a recent transmission to humans through successive bottlenecks some 150,000 years ago. A lack of detectable variation in 17 isolates emphasizes the extraordinary genetic conservation of this species and the high clonality of the population. Moreover, the largest cluster, which encompasses 80% of all analysed strains, is an extremely young clade, that went through an important population expansion some 3,300 years ago.

Conclusion: sSNPs have proven useful as a sensitive marker to gain new insights into genetic diversity, population structure and evolutionary history of C. pneumoniae.

Figure 1

Distribution of SNPs within the genome. SNPs are equally distributed around the genome; no mutational hot-spot could be identified.

Figure 2

Phylogeny of C. pneumoniae reference isolates based on different SNP types (see table 2). Distance calculated as number of SNPs. Analysis revealed a phylogenetic proximity between CWL-029 and TW-183, and AR-39 and J-138, respectively. The topology of the tree was not significantly altered when sSNP, nsSNP or SNP of intergenic regions were analysed separately. Only sSNPs were not able to resolve the common branch of CWL-029 and TW-183.

Figure 3

C. pneumoniae consensus tree topology based on multiple and independent phylogenetic reconstructions (Neighbor-joining, Minimum Evolution and Parsimony) with C. abortus as an outgoup. At least one representative of the clusters of figure 4 are analysed. Animal strains are basal and the human strains are derived, suggesting a zoonosis scenario for C. pneumoniae.

Figure 4

Phylogeny of 38 C. pneumoniae isolates based on 232 sSNPs using the neighbor-joining method with 1000 bootstrap replicates and distances calculated using the number of different SNPs. The marked section of panel A is enlarged in a separate subtree in panel B (* CWL-029-Group: CWL-029; CV-15 to -18; PB-1 to -3; MUL-250, -2076, -2090, -2216; Wien-2, -3; Al-1, W-5, U-1360). Four separate cluster can be distinguished.

Figure 5

Mismatch distribution of cluster IV strains for the concatenated sequences (sSNPs). These curves represent the frequency distribution of pairwise differences. The dashed plot correspond to the observed data; the smoth curve corresponds to the sudden expansion model and the punctuated lines represent the upper and lower bounds of the 95% confidence interval on this model. P value represents the probability that the raggedness of the simulated data set is equal to or greater than the observed data set. The major demographic extension occurred 2 mutations ago for the entire concatenated genes (70 kb). The latter translates into 0.003% sequence divergence, which suggests that the maximum population expansion occurred about 3,300 years (700 to 7,200 years) ago.