The evolution of antimicrobial peptide resistance in Pseudomonas aeruginosa is shaped by strong epistatic interactions

Abstract

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.

Rapid and Consistent Evolution of Colistin Resistance in Extensively Drug-Resistant Pseudomonas aeruginosa during Morbidostat Culture

“…Our results underscore the potential importance of mutator phenotypes in bacterial resistance evolution, as also reported by Jochumsen et al (18). Once the mutation rate is high, drug resistance mutations are much more rapidly discovered in the mutator lineage, in particular when multiple mutations are necessary to convey full resistance (35, 36, 51).…”

“…They also found parallel mutations in genes pmrB and lpxC. However, other loci that frequently mutated in experiments by Jochumsen et al (18), such as PA5194 (8 out of 9) and PA5005 (5 out of 9), rarely mutated in our experiments. We found two mutations in homologs of PA5194 (a nonsense mutation in v02 of PA77 at locus PSA77_04096 and G77R in v03 of PA83 at locus PSMA83_05923) and no mutation in opr86 or at locus PA5005.…”

“…In contrast to transposon knockout screens for polymyxin resistance (39), direct selection for resistance by the morbidostat and whole-genome deep sequencing allows the unbiased detection of loss-of-function as well as gain-of-function mutations associated with resistance (40). Furthermore, compared to classical experimental setups to investigate antibiotic resistance evolution, such as serial dilution protocols or chemostats (18,41), the morbidostat approach allows a higher degree of replication and control. Multifold replication is essential to quantify convergent evolution and prevalence of different evolutionary pathways and to map the mutations associated with resistance.…”

“…Many more mutations were observed in the experiment with isolate PA83 (Table 2). PA83 repeatedly mutated lpxC, which codes for a UDP-3-O- [3-hydroxymyristoyl]-Nacetylglucosamine deacetylase, which participates in the biosynthesis of lipid A and could, therefore, be involved in the progression to colistin resistance as shown in P. aeruginosa (18) and Acinetobacter baumannii (31). The gene was mutated in every culture of PA83 and suffered from multiple mutations in 5 out of 10 vials.…”

“…Despite the characterization of the role of these loci in colistin resistance, the evolutionary dynamics of colistin resistance remain poorly understood and reported adaptive mutations are likely to be incomplete. Since progression of colistin-susceptible to -resistant strains under antibiotic exposure is increasingly reported in clinical settings (17)(18)(19)(20), it is important to understand how fast and via which mutations colistin resistance emerges under antibiotic stress.…”