Contributions and Evolutionary Potential of ampC Mutations to confer Aztreonam Resistance in Pseudomonas aeruginosa

Abstract

Aztreonam is a synthetic monobactam antibiotic that is increasingly used for chronic suppressive therapy in Cystic Fibrosis (CF) patients with Pseudomonas aeruginosa airway infection. P. aeruginosa can accumulate mutations in the chromosomal ampC β-lactamase which promote aztreonam resistance, but the extent to which spontaneous mutations can increase ampC activity against aztreonam remains incompletely explored. To address this question, here we both examined ampC mutations that occur in P. aeruginosa clinical isolates derived from CF patients treated with aztreonam and additionally performed in vitro evolution of ampC for enhanced aztreonam hydrolysis. First, to catalog spontaneous ampC mutations that confer increased aztreonam resistance in P. aeruginosa, we sequenced a collection of 60 pre- and post-treatment clonal isolate pairs showing elevated resistance levels and identified 14 isolates with treatment-associated mutations in that gene. The 12 alleles, containing 13 unique mutations, had a variable effect on the ability of ampC to confer elevated aztreonam resistance relative to the wild-type allele (range 8-fold decrease to 64-fold increase) when expressed either in Escherichia coli or a P. aeruginosa laboratory strain deficient for native ampC. The alleles conferred variable resistance to ampicillin, ceftazidime, and cefpirome, but meropenem resistance was consistently unaffected. To more fully evaluate the potential of point mutations to increase ampC activity against aztreonam, we subsequently performed artificial evolution and aztreonam selection of plasmid-borne ampC expressed in E. coli. We recovered mutants showing up to 4,096-fold increases in aztreonam resistance (128 ug/mL) relative to wild-type ampC, with mainained or enhanced resistance provided to other β-lactam drugs. Collectively, these findings indicate that mutations in ampC which result in elevated aztreonam resistance are relatively frequent in P. aeruginosa isolates from CF patient lungs, and that the maximum potential for ampC to evolve activity against this synthetic antibiotic are greater than previously appreciated.