Using Salmonella as a model organism, and a previously characterized Rif R mutation (rpoB R529C) as a starting point, independent lineages were evolved with selection for improved growth in the presence and absence of rifampicin. Compensatory mutations were identified in every lineage and were distributed between rpoA, rpoB and rpoC. Resistance was maintained in all strains showing that increased fitness by compensatory mutation was more likely than reversion. Genetic reconstructions demonstrated that the secondary mutations were responsible for increasing growth rate. Many of the compensatory mutations in rpoA and rpoC individually caused small but significant reductions in susceptibility to rifampicin, and some compensatory mutations in rpoB individually caused high-level resistance. These findings show that mutations in different components of RNA polymerase are responsible for fitness compensation of a Rif R mutant.

Ciprofloxacin is an important antibacterial drug targeting Type II topoisomerases, highly active against Gram-negatives including Escherichia coli. The evolution of resistance to ciprofloxacin in E. coli always requires multiple genetic changes, usually including mutations affecting two different drug target genes, gyrA and parC. Resistant mutants selected in vitro or in vivo can have many different mutations in target genes and efflux regulator genes that contribute to resistance. Among resistant clinical isolates the genotype, gyrA S83L D87N, parC S80I is significantly overrepresented suggesting that it has a selective advantage. However, the evolutionary or functional significance of this high frequency resistance genotype is not fully understood. By combining experimental data and mathematical modeling, we addressed the reasons for the predominance of this specific genotype. The experimental data were used to model trajectories of mutational resistance evolution under different conditions of drug exposure and population bottlenecks. We identified the order in which specific mutations are selected in the clinical genotype, showed that the high frequency genotype could be selected over a range of drug selective pressures, and was strongly influenced by the relative fitness of alternative mutations and factors affecting mutation supply. Our data map for the first time the fitness landscape that constrains the evolutionary trajectories taken during the development of clinical resistance to ciprofloxacin and explain the predominance of the most frequently selected genotype. This study provides strong support for the use of in vitro competition assays as a tool to trace evolutionary trajectories, not only in the antibiotic resistance field.

The compensatory mutations identified in Salmonella cluster in similar locations to the additional mutations found in M. tuberculosis isolates. These new data strongly support the idea that many of the previously identified rpoA, rpoB and rpoC mutations in rifampicin-resistant M. tuberculosis (rpoB S531L) are indeed fitness-compensatory mutations.

Experimental evolution is a powerful tool to study genetic trajectories to antibiotic resistance under selection. A confounding factor is that outcomes may be heavily influenced by the choice of experimental parameters. For practical purposes (minimizing culture volumes), most experimental evolution studies with bacteria use transmission bottleneck sizes of 5 × 106 cfu. We currently have a poor understanding of how the choice of transmission bottleneck size affects the accumulation of deleterious versus high-fitness mutations when resistance requires multiple mutations, and how this relates outcome to clinical resistance. We addressed this using experimental evolution of resistance to ciprofloxacin in Escherichia coli. Populations were passaged with three different transmission bottlenecks, including single cell (to maximize genetic drift) and bottlenecks spanning the reciprocal of the frequency of drug target mutations (108 and 1010). The 1010 bottlenecks selected overwhelmingly mutations in drug target genes, and the resulting genotypes corresponded closely to those found in resistant clinical isolates. In contrast, both the 108 and single-cell bottlenecks selected mutations in three different gene classes: 1) drug targets, 2) efflux pump repressors, and 3) transcription-translation genes, including many mutations with low fitness. Accordingly, bottlenecks smaller than the average nucleotide substitution rate significantly altered the experimental outcome away from genotypes observed in resistant clinical isolates. These data could be applied in designing experimental evolution studies to increase their predictive power and to explore the interplay between different environmental conditions, where transmission bottlenecks might vary, and resulting evolutionary trajectories.

These data identify mutations in RNA polymerase as novel contributors to the evolution of resistance to ciprofloxacin and show that the phenotype is mediated by increased MdtK-dependent drug efflux.