Associated metadata of the study isolates and their respective phenotype, MLST, study grouping, NCBI accession, and isolate number.

Candidate daptomycin resistance genes as reported in Diaz et al

(38) with mutations occurring across multiple MLSTs removed (35).

Experimental Design

Isolates were obtained from patients with one of two types of VRE bacteremia: de novo resistant patients that converted from having daptomycin sensitive to daptomycin resistant VRE bacteremia or putatively transmitted resistance patients with daptomycin resistant on their initial culture. Three independent populations were evolved from the initial daptomycin sensitive strains, the converted daptomycin resistant strain, and the daptomycin strains from patients resistant on arrival for 32 days in BHI media. Figure was created using Biorender.com.

© 2024, Tracy et al. Parts of this image created with BioRender are made available under a Creative Commons Attribution License, which permits unrestricted use and redistribution provided that the original author and source are credited.

Population level of resistance during experimental evolution

Resistance is measured as the ratio of colonies growing on plates supplemented with daptomycin at 8 µg/mL to colonies on plates without antibiotic for each of three independent experimental populations evolved from each patient-derived isolate. Populations were tested on days 0, 8, 10, 17, 18, 25, and 32.

Maximum likelihood fit of three mixed models, as described in the text, to estimate the effect of group (i.e. being founded with a PT or DN clone), and the MIC of that founding clone, on the reduction in MIC after 32 days of evolution in antibiotic-free conditions. difference (individual) as output, does NOT include DNS group

MIC and fitness relationship

The relationship between the mathematically modelled fitness of a clone (see methods) and its MIC. For each PTR, DNR, and DNS isolate, the fitness and MIC of the initial clone and three clones from three experimental evolved replicate populations were estimated. For DN patients, the change from the initial DNS to the DNR clone or the difference between the DNS and the DNR clones was also estimated. Each color represents a group, with the red line representing in vivo changes between initial DNS and DNR isolates. The remaining lines represent the average changes from the initial clone to the average of the three clones from each replicate population. Colored shapes show the individual measurements of each clone from each replicate population.

Maximum likelihood fit of mixed models to estimate the effect of group (i.e. being founded with a PT or DN clone), fitness gain during adaptation, and starting MIC on the fitness of strains after 32 days of evolution in antibiotic-free conditions.

Mutations in laboratory evolved populations from patients with de novo resistance. Lollipop plot showing mutations occurring during in vivo and in vitro evolution. The line represents the chromosome of the sensitive isolate and each lollipop represents a mutation. Differences between the resistant and sensitive isolate (in vivo) evolution are above the chromosome, while mutations seen during experimental evolution are below. The color indicated the experimental replicate and the shape indicated the mutation class. Genes or operons with mutations in multiple experimental populations or when the gene has been associated with daptomycin resistance are labeled. Mutations labeled with one asterisk are mutations that have been previously associated with daptomycin resistance. Mutations labeled with three asterisks in DN1 appeared in the initial resistant isolate and only one of the evolved resistant populations. See text for further explanation.

Mutations in laboratory evolved populations from transmitted resistance patients

Lollipop plots showing genetic changes occurring in isolates from PT patients. Lollipops on the top half of the plot show mutations in genes associated with daptomycin resistance mapped against the daptomycin sensitive reference genome DO. Gene names with asterisks have multiple mutations within the gene. Lollipops on the bottom half of the plot show genetic differences in one clone selected from each of the three experimentally evolved populations.

Phylogenetic tree of the 18 initial, unevolved isolates

A neighbor-joining tree of the initial 18 founding isolates from the SNP distance matrix. The SNP distance matrix was derived from a core genome alignment of all 18 isolates.

Growth curves of de novo resistant strains

Each point represents one of 2 technical replicates from one of the three different biological replicates. The best fit from the growth model is superimposed. Each plot is labeled by Strain ID, Day, Experimental Population, and Clone

Growth curves of transmitted resistant strains

Each point represents one of 2 technical replicates from one of the three biological replicates. The best fit from the model is superimposed. Each plot is labeled by Strain ID, Day, Experimental Population, and Clone

Daptomycin resistance levels before and after evolution in an antibiotic-free environment

Minimum inhibitory concentration of daptomycin from clinical isolates (initial) and three isolates from each of three replicate populations following 320 generations of experimental evolution.