Adaptive laboratory evolution and independent component analysis disentangle complex vancomycin adaptation trajectories.

Human infections with methicillin-resistant (MRSA) are commonly treated with vancomycin, and strains with decreased susceptibility, designated as vancomycin-intermediate (VISA), are associated with treatment failure. Here, we profiled the phenotypic, mutational, and transcriptional landscape of 10 VISA strains adapted by laboratory evolution from one common MRSA ancestor, the USA300 strain JE2. Using functional and independent component analysis, we found that: 1) despite the common genetic background and environmental conditions, the mutational landscape diverged between evolved strains and included mutations previously associated with vancomycin resistance (in , , , , and ) as well as novel adaptive mutations (SAUSA300_RS04225, , , and ); 2) the first wave of mutations affected transcriptional regulators and the second affected genes involved in membrane biosynthesis; 3) expression profiles were predominantly strain-specific except for and , which were the only two genes significantly differentially expressed in all clones; 4) three independent virulence systems (φSa3, SaeR, and T7SS) featured as the most transcriptionally perturbed gene sets across clones; 5) there was a striking variation in oxacillin susceptibility across the evolved lineages (from a 10-fold increase to a 63-fold decrease) that also arose in clinical MRSA isolates exposed to vancomycin and correlated with susceptibility to teichoic acid inhibitors; and 6) constitutive expression of the VraR regulon explained cross-susceptibility, while mutations in were associated with cross-resistance. Our results show that adaptation to vancomycin involves a surprising breadth of mutational and transcriptional pathways that affect antibiotic susceptibility and possibly the clinical outcome of infections.