Fig. 1: Schematic representation of the main methods used in this study.
From: Bacteria can compensate the fitness costs of amplified resistance genes via a bypass mechanism
A Selection of resistant bacteria with increased copy number and fitness cost by serially streaking 4 heteroresistant strains at progressively higher concentrations of their respective antibiotic in 4 parallel replicates. The isolated mutants were characterized by measuring the fitness, MIC, and resistance gene copy number. Mutants isolated at 24X MIC exhibited the highest fitness cost and highest levels of gene amplifications and were whole genome sequenced and used for further studies. B Compensatory evolution was performed at 24X MIC antibiotic concentration with the mutants isolated at 24X MIC in 3 replicates for each of the 16 mutants isolated previously. After serial passaging for 100 generations, the mutants were characterized by measuring fitness, MIC, resistance gene copy number and whole genome sequencing. C The stability of the gene amplifications was measured in the compensated mutants and was compared to its respective parent isolated at 24X MIC. This was done by serially passaging these strains in antibiotic-free media for 150 generations and then measuring the changes in MIC and resistance gene copy number.
