Evolved Aztreonam Resistance Is Multifactorial and Can Produce Hypervirulence in

While much attention has been focused on acquired antibiotic resistance genes, chromosomal mutations may be most important in chronic infections where isolated, persistently infecting lineages experience repeated antibiotic exposure. Here, we used experimental evolution and whole-genome sequencing to investigate chromosomally encoded mutations causing aztreonam resistance in and characterized the secondary consequences of resistance development. We identified 19 recurrently mutated genes associated with aztreonam resistance. The most frequently observed mutations affected negative transcriptional regulators of the efflux system and the target of aztreonam, While individual mutations conferred modest resistance gains, high-level resistance (1,024 µg/ml) was achieved through the accumulation of multiple variants. Despite being largely stable when strains were passaged in the absence of antibiotics, aztreonam resistance was associated with decreased growth rates, indicating an associated fitness cost. In some instances, evolved aztreonam-resistant strains exhibited increased resistance to structurally unrelated antipseudomonal antibiotics. Surprisingly, strains carrying evolved mutations which affected negative regulators of ( and ) demonstrated enhanced virulence in a murine pneumonia infection model. Mutations in these genes, and other genes that we associated with aztreonam resistance, were common in isolates from chronically infected patients with cystic fibrosis. These findings illuminate mechanisms of aztreonam resistance and raise the possibility that antibiotic treatment could inadvertently select for hypervirulence phenotypes. Inhaled aztreonam is a relatively new antibiotic which is being increasingly used to treat cystic fibrosis patients with airway infections. As for all antimicrobial agents, bacteria can evolve resistance that decreases the effectiveness of the drug; however, the mechanisms and consequences of aztreonam resistance are incompletely understood. Here, using experimental evolution, we have cataloged spontaneous mutations conferring aztreonam resistance and have explored their effects. We found that a diverse collection of genes contributes to aztreonam resistance, each with a small but cumulative effect. Surprisingly, we found that selection for aztreonam resistance mutations could confer increased resistance to other antibiotics and promote hypervirulence in a mouse infection model. Our study reveals inherent mechanisms of aztreonam resistance and indicates that aztreonam exposure can have unintended secondary effects.