Deciphering β-lactamase-independent p-lactam resistance evolution trajectories in Pseudomonas aeruginosa

Background: While resistance related to the expression of beta-Eactamases, such as AmpC from Pseudomonas aeruginosa, has been deeply studied, this work addresses the gap in the knowledge of other potential bacterial strategies to overcome the activity of beta-lactams when beta-lactamases are not expressed. Methods: We analysed 13-lactam resistance evolution trajectories in a WT strain and in isogenic mutants either lacking AmpC (AmpC mutant) or unable to express it (AmpG mutant), exposed to increasing concentrations of ceftazidime for 7 days in quintuplicate experiments, Characterization of evolved lineages included susceptibility profiles, whole-genome sequences, resistance mechanisms, fitness (competitive growth assays) and virulence (Caenorhabditis elegans model). Results: Development of resistance was faster for the WT strain but, after 7 days, all strains reached clinical ceftazidime resistance levels, The main resistance mechanism in the WT strain was ampC overexpression, due to mutations in clacB and ampD or mpl, In contrast, ampC overexpression did not evolve in any of the AmpG lineages. Moreover, sequencing of the Delta AmpC and Delta AmpG evolved lineages revealed alternative resistance mutations (not seen in WT lineages) that included, in all cases, large (50-600 kb) deletions of specific chromosomal regions together with mutations leading to p-lactam target [ftsI (PBP3)] modification and/or the overexpression or structural modification of the efflux pump MexAB-OprM. Finally, evolved lineages from the AmpC and, especially, AmpG mutants showed a reduced fitness and virulence. Conclusions: In addition to providing new insights intop-lactam resistance mechanisms and evolution, our findings should be helpful for guiding future strategies to combat P. aeruginosa nfections.