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

Inhaled aztreonam is increasingly used for chronic 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 We identified 16 frequently mutated genes associated with aztreonam resistance, the most prevalent being and , 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 alleles and performed artificial evolution of for maximal activity against aztreonam. We found that naturally occurring 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 and , show that 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 .