Colistin is an antimicrobial peptide that has become the only remaining alternative for the treatment of multidrug-resistant Gram-negative bacterial infections, but little is known of how clinical levels of colistin resistance evolve. We use in vitro experimental evolution and whole-genome sequencing of colistin-resistant Pseudomonas aeruginosa isolates from cystic fibrosis patients to reconstruct the molecular evolutionary pathways open for high-level colistin resistance. We show that the evolution of resistance is a complex, multistep process that requires mutation in at least five independent loci that synergistically create the phenotype. Strong intergenic epistasis limits the number of possible evolutionary pathways to resistance. Mutations in transcriptional regulators are essential for resistance evolution and function as nodes that potentiate further evolution towards higher resistance by functionalizing and increasing the effect of the other mutations. These results add to our understanding of clinical antimicrobial peptide resistance and the prediction of resistance evolution.

c Methicillin-resistant Staphylococcus aureus (MRSA) has acquired the mecA gene encoding a peptidoglycan transpeptidase, penicillin binding protein 2a (PBP2a), which has decreased affinity for ␤-lactams. Quickly spreading and highly virulent communityacquired (CA) MRSA strains recently emerged as a frequent cause of infection in individuals without exposure to the health care system. In this study, we found that the inactivation of the components of the ClpXP protease substantially increased the ␤-lactam resistance level of a CA-MRSA USA300 strain, suggesting that the proteolytic activity of ClpXP controls one or more pathways modulating ␤-lactam resistance. These pathways do not involve the control of mecA expression, as the cellular levels of PBP2a were unaltered in the clp mutants. An analysis of the cell envelope properties of the clpX and clpP mutants revealed a number of distinct phenotypes that may contribute to the enhanced ␤-lactam tolerance. Both mutants displayed significantly thicker cell walls, increased peptidoglycan cross-linking, and altered composition of monomeric muropeptide species compared to those of the wild types. Moreover, changes in Sle1-mediated peptidoglycan hydrolysis and altered processing of the major autolysin Atl were observed in the clp mutants. In conclusion, the results presented here point to an important role for the ClpXP protease in controlling cell wall metabolism and add novel insights into the molecular factors that determine strain-dependent ␤-lactam resistance.

Mutations that cause antibiotic resistance often produce associated fitness costs. These costs have a detrimental effect on the fate of resistant organisms in natural populations and could be exploited in designing drugs, therapeutic regimes, and intervention strategies. The streptomycin resistance (Str R ) mutations K42N and P90S in ribosomal protein S12 impair growth on rich medium. Surprisingly, in media with poorer carbon sources, the same Str R mutants grow faster than wild type. This improvement reflects a failure of these Str R mutants to induce the stress-inducible sigma factor RpoS (s S ), a key regulator of many stationary-phase and stress-inducible genes. On poorer carbon sources, wild-type cells induce s S , which retards growth. By not inducing s S , Str R mutants escape this self-imposed inhibition. Consistent with this interpretation, the Str R mutant loses its advantage over wild type when both strains lack an RpoS (s S ) gene. Failure to induce s S produced the following side effects: (1) impaired induction of several stress-inducible genes, (2) reduced tolerance to thermal stress, and (3) reduced translational fidelity. These results suggest that RpoS may contribute to long-term cell survival, while actually limiting short-term growth rate under restrictive growth conditions. Accordingly, the Str R mutant avoids short-term growth limitation but is sensitized to other stresses. These results highlight the importance of measuring fitness costs under multiple experimental conditions not only to acquire a more relevant estimate of fitness, but also to reveal novel physiological weaknesses exploitable for drug development.

SummaryWe examined how the fitness costs of mupirocin resistance caused by mutations in the chromosomal isoleucyl-tRNA synthetase gene (ileS) can be ameliorated. Mupirocin-resistant mutants were isolated and four different, resistance-conferring point mutations in the chromosomal ileS gene were identified. Fifty independent lineages of the lowfitness, resistant mutants were serially passaged to evolve compensated mutants with increased fitness. In 34/50 of the evolved lineages, the increase in fitness resulted from additional point mutations in isoleucine tRNA synthetase (IleRS). Measurements in vitro of the kinetics of aminoacylation of wild-type and mutant enzymes showed that resistant IleRS had a reduced rate of aminoacylation due to altered interactions with both tRNA Ile and ATP. The intragenic compensatory mutations improved IleRS kinetics towards the wild-type enzyme, thereby restoring bacterial fitness. Seven of the 16 lineages that lacked second-site compensatory mutations in ileS, showed an increase in ileS gene dosage, suggesting that an increased level of defective IleRS compensate for the decrease in aminoacylation activity. Our findings show that the fitness costs of ileS mutations conferring mupirocin resistance can be reduced by several types of mechanisms that may contribute to the stability of mupirocin resistance in clinical settings.