Tracing the Spread of Clostridium difficile Ribotype 027 in Germany Based on Bacterial Genome Sequences

Abstract

We applied whole-genome sequencing to reconstruct the spatial and temporal dynamics underpinning the expansion of Clostridium difficile ribotype 027 in Germany. Based on re-sequencing of genomes from 57 clinical C. difficile isolates, which had been collected from hospitalized patients at 36 locations throughout Germany between 1990 and 2012, we demonstrate that C. difficile genomes have accumulated sequence variation sufficiently fast to document the pathogen's spread at a regional scale. We detected both previously described lineages of fluoroquinolone-resistant C. difficile ribotype 027, FQR1 and FQR2. Using Bayesian phylogeographic analyses, we show that fluoroquinolone-resistant C. difficile 027 was imported into Germany at least four times, that it had been widely disseminated across multiple federal states even before the first outbreak was noted in 2007, and that it has continued to spread since.

Fig 1. Maximum likelihood phylogenetic tree.

The phylogeny of C. difficile ribotype 027 was reconstructed based on 255 core-genome SNPs. Previously published genome sequences (indicated by shaded isolate names; [14]) were included to enable identification of clades FQR1 and FQR2. The tree was rooted by using the distantly related BI 3 genome [14] as an outgroup.

Fig 2. Maximum clade credibility tree based on BEAST analysis of C. difficile genome sequences.

Tips of the tree were constrained by sampling dates, the time scale is shown at the bottom. Blue bars indicate 95% Bayesian credibility intervals of bacterial divergence dates (node heights).

Fig 3. Bayesian reconstruction of the spread of C. difficile 027 in Germany.

Squares indicate the centroids of 11 regions and lines indicate the inferred spread of FQR1 (blue) and FQR2 (red). Note that, in this discrete analysis, arrival at a region centroid indicates arrival at that region, but trajectories do not represent precise transport routes.