Evolution of Pseudomonas aeruginosa Antimicrobial Resistance and Fitness under Low and High Mutation Rates

Abstract

Pseudomonas aeruginosa, a major cause of nosocomial and chronic infections, is considered a paradigm of antimicrobial resistance development. However, the evolutionary trajectories of antimicrobial resistance and the impact of mutator phenotypes remain mostly unexplored. Therefore, whole-genome sequencing (WGS) was performed in lineages of wild-type and mutator (ΔmutS) strains exposed to increasing concentrations of relevant antipseudomonal agents. WGS provided a privileged perspective of the dramatic effect of mutator phenotypes on the accumulation of random mutations, most of which were transitions, as expected. Moreover, a frameshift mutagenic signature, consistent with error-prone DNA polymerase activity as a consequence of SOS system induction, was also seen. This effect was evidenced for all antibiotics tested, but it was higher for fluoroquinolones than for cephalosporins or carbapenems. Analysis of genotype versus phenotype confirmed expected resistance evolution trajectories but also revealed new pathways. Classical mechanisms included multiple mutations leading to AmpC overexpression (ceftazidime), quinolone resistance-determining region (QRDR) mutations (ciprofloxacin), oprD inactivation (meropenem), and efflux pump overexpression (ciprofloxacin and meropenem). Groundbreaking findings included gain-of-function mutations leading to the structural modification of AmpC (ceftazidime), novel DNA gyrase (GyrA) modification (ciprofloxacin), and the alteration of the β-lactam binding site of penicillin-binding protein 3 (PBP3) (meropenem). A further striking finding was seen in the evolution of meropenem resistance, selecting for specific extremely large (>250 kb) genomic deletions providing a growth advantage in the presence of the antibiotic. Finally, fitness and virulence varied within and across evolved antibiotic-resistant populations, but mutator lineages showed a lower biological cost for some antibiotics.

FIG 1

Number of nonsynonymous mutations by gene function category documented in PAOMS derivatives exposed for 7 days to increasing concentrations of ceftazidime (CAZ), ciprofloxacin (CIP), meropenem (MER), or control without antibiotics. The mean ± SD values obtained for three independent lineages are shown. Statistically significant differences (P < 0.05, Student's t test) for one condition respect to each of the other 3 are indicated with an asterisk.

FIG 2

MiSeq Reporter run plots and PCR confirmation of deleted regions. Upper panel, MiSeq Reporter run plots of P. aeruginosa reference strain PAO1 and the 3 derivative lineages evolved in the presence of meropenem for 7 days. Lower panel, agarose gel electrophoresis results of PCR products obtained to confirm the deleted regions in PAO1.1-MER and PAO1.3-MER. PCRs for genes within right and left margins of the deletions were positive for PAO1 only. On the other hand, PCR amplifications of DNA regions flanking the deletion yielded the expected size in PAO1.1-MER and PAO1.3-MER and were negative for PAO1. In all cases, PCR products were confirmed by sequencing. MW, molecular weight.

FIG 3

Competition index (CI) values for 10 selected mutants (2 mutant CFU from each of 5 independent experiments) for each antibiotic series. The mean ± SD of 3 replicates are represented. The median CI values for the 10 mutants in each series are indicated by the red dashed line (with the value shown to the right).

FIG 4

Virulence on C. elegans of PAO1 and PAOMS lineages evolved in the presence of different antibiotics. The mean ± SD numbers of surviving nematodes at 0, 24, 72, and 168 h are shown. CAZ^r, ceftazidime resistant; CIP^r, ciprofloxacin resistant; MER^r, meropenem resistant.

FIG 5

Grow rates (OD_600nm) of wild-type PAO1 and the 3 derivative lineages evolved in the presence of meropenem. Growth was monitored in the presence of 0, 0.25, and 32 μg/ml meropenem.