Long-term intrahost evolution of methicillin resistant Staphylococcus aureus among cystic fibrosis patients with respiratory carriage

Staphylococcus aureus is the most commonly identified airway colonizer of cystic fibrosis (CF) patients, and infections with methicillin-resistant S. aureus (MRSA) are associated with poor outcomes. Yet, little is known about the intrahost evolution of S. aureus among CF patients. We investigated convergent evolution and adaptation of MRSA among four CF patients with long-term respiratory carriage. For each patient, we performed whole-genome sequencing on an average of 21 isolates (range: 19-23) carried for a mean of 1,403 days (range: 903-1,679), including 25 pairs of isolates collected on the same day. We then assessed intrahost diversity, population structure, evolutionary history, and signatures of adaptation in the context of patient age, antibiotic treatment, and co-colonizing microbes. We conducted tests of gene-wide diversifying selection and identified instances of switched intergenic regions (IGRs), which may be associated with gene expression. Phylogenetic analysis delineated distinct multilocus sequence type ST5 (n=3) and ST72 (n=1) clonal populations in addition to sporadic, non-clonal isolates, and uncovered a putative transmission event. Variation in antibiotic resistance was observed within clonal populations, even among isolates collected on the same day. Coalescent analysis revealed rates of molecular evolution ranging from 2.21 to 8.64 nucleotide polymorphisms per genome per year. Estimated lineage ages were consistent with acquisition of colonization in early childhood and subsequent persistence of multiple sub-populations. Selection analysis focused on 1,622 core genes shared by all four clonal populations (n=79). Eleven genes were variable in three subjects - most notable, ATP-dependent protease clpX, 2-oxoglutarate dehydrogenase odhA, fmtC, and transcription-repair coupling factor mfd. Only one gene, staphylococcal protein A (spa), was found to have evidence of gene-wide diversifying selection. We identified three instances of intrahost IGR switching events, two of which flanked genes related to quorum sensing. The ecology of microbes in the airways of CF patients is undeniably complex, posing challenges for management. We illustrate appreciable intrahost diversity as well as persistence of a dominant lineage. We also show that intrahost adaptation is a continual process, despite purifying selective pressure, and provide targets that should be investigated further for their function in CF adaptation.