Genomic Analysis Identifies Novel Pseudomonas aeruginosa Resistance Genes under Selection during Inhaled Aztreonam Therapy In Vivo

Abstract

Inhaled aztreonam is increasingly used for chronic Pseudomonas aeruginosa suppression in patients with cystic fibrosis (CF), but the potential for that organism to evolve aztreonam resistance remains incompletely explored. Here, we performed genomic analysis of clonally related pre- and posttreatment CF clinical isolate pairs to identify genes that are under positive selection during aztreonam therapy in vivo We identified 16 frequently mutated genes associated with aztreonam resistance, the most prevalent being ftsI and ampC, and 13 of which increased aztreonam resistance when introduced as single gene transposon mutants. Several previously implicated aztreonam resistance genes were found to be under positive selection in clinical isolates even in the absence of inhaled aztreonam exposure, indicating that other selective pressures in the cystic fibrosis airway can promote aztreonam resistance. Given its potential to confer plasmid-mediated resistance, we further characterized mutant ampC alleles and performed artificial evolution of ampC for maximal activity against aztreonam. We found that naturally occurring ampC mutants conferred variably increased resistance to aztreonam (2- to 64-fold) and other β-lactam agents but that its maximal evolutionary capacity for hydrolyzing aztreonam was considerably higher (512- to 1,024-fold increases) and was achieved while maintaining or increasing resistance to other drugs. These studies implicate novel chromosomal aztreonam resistance determinants while highlighting that different mutations are favored during selection in vivo and in vitro, show that ampC has a high maximal potential to hydrolyze aztreonam, and provide an approach to disambiguate mutations promoting specific resistance phenotypes from those more generally increasing bacterial fitness in vivo.

Keywords: Pseudomonas aeruginosa; ampC; antibiotic resistance; aztreonam; cystic fibrosis; genome analysis; selection; β-lactamase.

FIG 1

De novo mutations in proteins associated with aztreonam resistance. Mutations are displayed as lolliplots for PBP3 (A) and AmpC (B). Amino acid position is indicated along the x axis, and protein domains are shown as labeled, colored regions. Subregions in AmpC are indicated by dark gray shading, with labels shown at the bottom. The location and abundance of specific amino acid mutations are indicated by colored circles.