A timescale for evolution, population expansion, and spatial spread of an emerging clone of methicillin-resistant Staphylococcus aureus

Abstract

Due to the lack of fossil evidence, the timescales of bacterial evolution are largely unknown. The speed with which genetic change accumulates in populations of pathogenic bacteria, however, is a key parameter that is crucial for understanding the emergence of traits such as increased virulence or antibiotic resistance, together with the forces driving pathogen spread. Methicillin-resistant Staphylococcus aureus (MRSA) is a common cause of hospital-acquired infections. We have investigated an MRSA strain (ST225) that is highly prevalent in hospitals in Central Europe. By using mutation discovery at 269 genetic loci (118,804 basepairs) within an international isolate collection, we ascertained extremely low diversity among European ST225 isolates, indicating that a recent population bottleneck had preceded the expansion of this clone. In contrast, US isolates were more divergent, suggesting they represent the ancestral population. While diversity was low, however, our results demonstrate that the short-term evolutionary rate in this natural population of MRSA resulted in the accumulation of measurable DNA sequence variation within two decades, which we could exploit to reconstruct its recent demographic history and the spatiotemporal dynamics of spread. By applying Bayesian coalescent methods on DNA sequences serially sampled through time, we estimated that ST225 had diverged since approximately 1990 (1987 to 1994), and that expansion of the European clade began in 1995 (1991 to 1999), several years before the new clone was recognized. Demographic analysis based on DNA sequence variation indicated a sharp increase of bacterial population size from 2001 to 2004, which is concordant with the reported prevalence of this strain in several European countries. A detailed ancestry-based reconstruction of the spatiotemporal dispersal dynamics suggested a pattern of frequent transmission of the ST225 clone among hospitals within Central Europe. In addition, comparative genomics indicated complex bacteriophage dynamics.

Figure 1. Radiation of ST225.

Minimum spanning trees; the tree in Figure 1a is based on 269 loci investigated in 73 ST225 isolates, and the position of the JH strain was resolved based on published genome sequences for isolates JH1 and JH9 . The ancestral node (‘root’) was determined by comparison to genome sequences from more distantly related isolates, including N315 (GenBank accession number BA000018), COL (CP000046), and MW2 (BA000033). Colours indicate the isolates' countries of origin. The tree in Figure 1b is based on 108 loci to show the relationship of ST225 to the previously reported ST5 radiation . Haplotype H5-22 represents two ST5 isolates and the JH strain. Red colour labels isolates harboring prophage ΦSaST5K.

Figure 2. Increase of DNA sequence variation over the sampling time interval.

Maximum-likelihood phylogenetic tree based on sequence variation among haplotypes (a). In this tree, each haplotype has a particular distance to the root. In (b), these root-to-tip genetic distances are plotted against sampling dates. The figure illustrates a positive correlation of divergence with sampling date, and, hence, a significant increase of DNA sequence variation over the sampling time interval.

Figure 3. Effect of date permutation on the estimate of divergence time.

Age of the ST225 clade (tmrca, time since the most recent common ancestor) and 95% confidence intervals on a log scale, determined by Bayesian phylogenetics analysis with the tips constrained by the correct dates and with the dates switched across isolates (permutations 1 to 5).

Figure 4. Effective population size through time in comparison to surveillance data.

Bayesian skyline plot (Figure 4a), showing the effective population size of ST225 through time (black line), estimated from the concatenated dataset. The shaded area represents 95% confidence intervals. Proportional abundance of ST225 among MRSA in Germany and the Czech Republic (Figure 4b). Data for Germany are based on 2,000 MRSA isolates on average typed per year at the national reference centre for staphylococci. These isolates were received from all over the country and were associated with approximately 10% of all MRSA infections in Germany . Data for the Czech Republic are based on 142 MRSA isolates recovered from blood samples in 13 different hospitals throughout the country.

Figure 5. Ancestry-based scenario for the spatial spread of ST225.

This figure shows the spatial spread of ST225, inferred for successive time windows by applying the SeqTrack algorithm. Each arrow represents an inferred ancestry, pointing from the ancestor to its descendent. Local ancestries are represented by colored dots for single isolates and dots with additional segments representing multiple ancestry events (one segment per isolate). Cumulative numbers of isolates are indicated with blue bubbles.