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

Abstract

Colistin is a last-resort antibiotic commonly used against multidrug-resistant strains of Pseudomonas aeruginosa. To investigate the potential for in situ evolution of resistance against colistin and to map the molecular targets of colistin resistance, we exposed two P. aeruginosa isolates to colistin using a continuous-culture device known as a morbidostat. As a result, colistin resistance reproducibly increased 10-fold within 10 days and 100-fold within 20 days, along with highly stereotypic yet strain-specific mutation patterns. The majority of mutations hit the pmrAB two-component signaling system and genes involved in lipopolysaccharide (LPS) synthesis, including lpxC, pmrE, and migA. We tracked the frequencies of all arising mutations by whole-genome deep sequencing every 3 to 4 days to obtain a detailed picture of the dynamics of resistance evolution, including competition and displacement among multiple resistant subpopulations. In 7 out of 18 cultures, we observed mutations in mutS along with a mutator phenotype that seemed to facilitate resistance evolution.

The Essential Role of Hypermutation in Rapid Adaptation to Antibiotic Stress

“…Previous studies have been conducted to identify genetic changes leading to polymyxin resistance in P. aeruginosa (11-13, 27, 28, 32-34). Since resistance has often been associated with hypermutation (12,13), which leads to the accumulation of a large number of nonadaptive hitchhiker mutations, not all mutations can be implicated in resistance. However, if the same gene is mutated during adaptation to polymyxin in different studies that use different experimental conditions, it is more likely an adaptative allele than a hitchhiker.…”

“…We identified such genes that were found to be mutated in our study as well as previous studies and arranged them in the following functional groups: a two-component system, pmrAB, lipopolysaccharide modification and biosynthesis genes (pagL and PA5194), long-chain fatty acid coenzyme A (CoA) ligase (fadD2), outer membrane protein (opr86), probable short-chain dehydrogenase (PA4089), and multidrug efflux transporter (mexB). While the role of some of these genes in polymyxin resistance has been validated (for example, in pmrAB, opr86, PA5194, pagL, and mexB), others have not been previously associated with resistance (12,13,28,32,35,36). While migA has been identified in colistin resistance-evolved P. aeruginosa samples in other studies (12,13) and in this work, the presence of migA mutations in the control population suggests that it most likely plays a role in adaptation to the medium or growth conditions rather than the antibiotic.…”

“…While the role of some of these genes in polymyxin resistance has been validated (for example, in pmrAB, opr86, PA5194, pagL, and mexB), others have not been previously associated with resistance (12,13,28,32,35,36). While migA has been identified in colistin resistance-evolved P. aeruginosa samples in other studies (12,13) and in this work, the presence of migA mutations in the control population suggests that it most likely plays a role in adaptation to the medium or growth conditions rather than the antibiotic. The other targets were mapped on the FIG 4 Genes mutated more frequently than expected identified using Fisher's exact test.…”

“…We used a combination of phylogenetic and statistical approaches to sort through these complex data, discern truly adaptive alleles from hitchhikers, and infer not just the major genetic changes associated with colistin resistance but also those alleles that in combination with the major players were essential to produce the high MICs observed in many clinical isolates (12,27). Our work has benefited strongly from a series of clinical and in vitro experimental evolution studies that have identified many of the major contributors to colistin resistance in P. aeruginosa (10,12,13,27,28).…”

Chromosomally Encoded mcr-5 in Colistin Non-susceptible Pseudomonas aeruginosa

“…Colistin resistance in P. aeruginosa has primarily been attributed to mutations in up to five different two-component regulatory systems (PhoPQ, PmrAB, ParR/S, ColR/S, and CprR/S) (Reviewed in (6)) but to the best of our knowledge, Mcr-mediated colistin resistance has not been described in P. aeruginosa to date. As mutations in the two-component regulatory systems could potentially contribute to colistin resistance in P. aeruginosa MRSN 12280, we examined the amino acid sequences of PmrA, PmrB, PmrE, PhoQ, ParR, ParS, ColR, ColS, MigA, LpxC, and CprS for non-synonymous mutations (6, 11-13). When compared to P. aeruginosa PA01, P. aeruginosa MRSN 12280 had non-synonymous mutations in PhoQ (Y85F), PmrA (L71R), PmrB (S2P, A4T, G68S, Y345H, G362S), ParR (L153R, S170N), and ParS (H398R).…”