Extended Data Fig. 1: Specific RpoB substitutions are associated with DAP resistance in VREfm.

a. Maximum-likelihood core-SNP-based phylogeny of clinical VREfm (n = 1000) inferred from 6574 SNPs, demonstrating the interspersing of daptomycin resistance. Overlaid are the results of in silico MLST and daptomycin phenotypic testing. In the first heat map, ST is not shown for uncommon STs (n ≤ 5). The scale bar indicates number of nucleotide substitutions per site (top), with an approximation of SNP distance (in parentheses). ST=sequence type. SNP=single nucleotide polymorphism; MLST=multi-locus sequence type. b. Competition assays for the WT and RpoB S491F (dark purple) or WT and RpoB S491F/RpoC T634K (light purple) mutant pairs, with the percentage of rifampicin resistant (RIF^R) to rifampicin susceptible (RIF^S) isolates determined by plate count, shown on the y-axis. The x-axis denotes time in hours (either 0 or 24). Differences were assessed using two-way analysis of variance (ANOVA). All data points for independent biological replicates (n = 6) are displayed. Horizontal lines depict mean and error bars show the standard deviation. Exact P values are provided when the P value is above P < 0.0001. c. Maximum-likelihood core-SNP-based phylogeny of ST203, RpoB S491F VREfm (n = 80) inferred from 1960 SNPs, demonstrating the spread of isolates across different hospital networks. Overlaid are the year of isolation and hospital network for each isolate, represented as hospital 1 through 10 (H1-H10). The scale bar indicates number of nucleotide substitutions per site. d. Rifampicin susceptibility data for the clinical strains containing a mutation in the RpoB RRDR region (n = 169 isolates) versus a random selection of clinical strains containing a wild-type RRDR (n = 169 isolates). Bars represent the count of isolates containing each MIC. Three independent replicates were performed for each isolate. RRDR=rifampicin resistance determining region; MIC=minimum inhibitory concentration.

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