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.

The present work examines the evolutionary trajectories of replicate Pseudomonas aeruginosa cultures in presence of the ribosome-targeting antibiotics tobramycin and tigecycline. It is known that large number of mutations across different genes – and therefore a large number of potential pathways – may be involved in resistance to any single antibiotic. Thus, evolution toward resistance might, to a large degree, rely on stochasticity, which might preclude the use of predictive strategies for fighting antibiotic resistance. However, the present results show that P. aeruginosa populations evolving in parallel in the presence of antibiotics (either tobramycin or tigecycline) follow a set of trajectories that present common elements. In addition, the pattern of resistance mutations involved include common elements for these two ribosome-targeting antimicrobials. This indicates that mutational evolution toward resistance (and perhaps other properties) is to a certain degree deterministic and, consequently, predictable. These findings are of interest, not just for P. aeruginosa, but in understanding the general rules involved in the evolution of antibiotic resistance also. In addition, the results indicate that bacteria can evolve toward higher levels of resistance to antibiotics against which they are considered to be intrinsically resistant, as tigecycline in the case of P. aeruginosa and that this may confer cross-resistance to other antibiotics of therapeutic value. Our results are particularly relevant in the case of patients under empiric treatment with tigecycline, which frequently suffer P. aeruginosa superinfections.

The analysis of trade-offs, as collateral sensitivity, associated with the acquisition of antibiotic resistance, is mainly based on the use of model strains. However, the possibility of exploiting these trade-offs for fighting already resistant isolates has not been addressed in depth, despite the fact that bacterial pathogens are frequently antibiotic-resistant, forming either homogeneous or heterogeneous populations. Using a set of Pseudomonas aeruginosa-resistant mutants, we found that ceftazidime selects pyomelanogenic tobramycin-hypersusceptible mutants presenting chromosomal deletions in the analyzed genetic backgrounds. Since pyomelanogenic resistant mutants frequently coexist with other morphotypes in patients with cystic fibrosis, we analyzed the exploitation of this trade-off to drive extinction of heterogeneous resistant populations by using tobramycin/ceftazidime alternation. Our work shows that this approach is feasible because phenotypic trade-offs associated with the use of ceftazidime are robust. The identification of conserved collateral sensitivity networks may guide the rational design of evolution-based antibiotic therapies in P. aeruginosa infections.

Acquisition of antibiotic resistance is a relevant problem for human health. The selection and spread of antibiotic-resistant organisms not only compromise the treatment of infectious diseases, but also the implementation of different therapeutic procedures as organ transplantation, advanced surgery or chemotherapy, all of which require proficient methods for avoiding infections. It has been generally accepted that the acquisition of antibiotic resistance will produce a general metabolic burden: in the absence of selection, the resistant organisms would be outcompeted by the susceptible ones. If that was always true, discontinuation of antibiotic use would render the disappearance of resistant microorganisms. However, several studies have shown that, once resistance emerges, the recovery of a fully susceptible population even in the absence of antibiotics is not easy. In the present study, we review updated information on the effect of the acquisition of antibiotic resistance in bacterial physiology as well as on the mechanisms that allow the compensation of the fitness costs associated with the acquisition of resistance.