Mutation-Driven Evolution of Pseudomonas aeruginosa in the Presence of either Ceftazidime or Ceftazidime-Avibactam

Ceftazidime-avibactam is a combination of β-lactam/β-lactamase inhibitor, the use of which is restricted to some clinical cases, including cystic fibrosis patients infected with multidrug-resistant , in which mutation is the main driver of resistance. This study aims to predict the mechanisms of mutation-driven resistance that are selected for when is challenged with either ceftazidime or ceftazidime-avibactam. For this purpose, PA14 was submitted to experimental evolution in the absence of antibiotics and in the presence of increasing concentrations of ceftazidime or ceftazidime-avibactam for 30 consecutive days. Final populations were analyzed by whole-genome sequencing. All evolved populations reached similar levels of ceftazidime resistance. In addition, they were more susceptible to amikacin and produced pyomelanin. A first event in this evolution was the selection of large chromosomal deletions containing (involved in pyomelanin production), (involved in β-lactams resistance), and (involved in aminoglycoside resistance). Besides mutations in and that regulate β-lactamase expression, mutations related to MexAB-OprM overexpression were prevalent. Ceftazidime-avibactam challenge selected mutants in the putative efflux pump and and in a two-component system ( and ), likely regulating its expression. All populations produced pyomelanin and were more susceptible to aminoglycosides, likely due to the selection of large chromosomal deletions. Since pyomelanin-producing mutants presenting similar deletions are regularly isolated from infections, the potential aminoglycoside hypersusceptiblity and reduced β-lactam susceptibility of pyomelanin-producing should be taken into consideration for treating infections caused by these isolates.