High-depth whole genome sequencing of blood culture plates reveals evolutionary dynamics in cases of persistent bacteremia due to methicillin-resistant Staphylococcus aureus

Within a single bacterial strain, DNA sequence variation is expected between individual clones. Whole genome sequencing (WGS) can be applied to clinical cultures to detect this polyclonal variation, enabling tracking of within-host evolution and transmission. Culture isolates from infected patients are often sequenced as individual colonies (c-seq). To increase the sensitivity of variant detection, cultures can also be sequenced to a high depth of coverage as a pool (p-seq), but the utility of this approach is not clear for most clinical specimens. To understand the performance of high-depth WGS in bacteremia, we applied p-seq to blood culture plates for 10 patients with persistent bacteremia due to methicillin-resistant Staphylococcus aureus. As a comparison, for six patients, we also applied c-seq to five colonies (c5-seq) from the same plates. p-seq was more sensitive than c5-seq for detecting low frequency variant alleles; however, the most important factor for new variant detection was the number of culture plates analyzed rather than the sequencing method used. We also used these data to construct Muller plots for three patients with especially diverse infecting populations, which enabled visualization of rapid evolutionary dynamics in response to antibiotic exposures. We identified 204 unique variant alleles, and our analysis provides additional evidence for parallel evolution of several different genes during S. aureus bacteremia. Overall, these data provide a detailed view of evolutionary dynamics during clinical cases of MRSA bacteremia and describe the merits and limitations of a c-seq versus p-seq strategy for analyzing blood culture plates using WGS. ImportanceAs bacterial whole genome sequencing (WGS) is increasingly used as a research tool for clinical samples, it is important to understand the pros and cons of different culture sampling methodologies. Here, we analyzed cases of persistent bacteremia due to methicillin-resistant Staphylococcus aureus by applying WGS to either each of five individual colonies isolated on blood culture plates (c5-seq) or the pooled bacterial population on each plate (p-seq). We found that c5-seq was a more practical and informative method to understand evolutionary dynamics.