Metabolic Compensation of Fitness Costs Is a General Outcome for Antibiotic- Resistant Pseudomonas aeruginosa Mutants Overexpressing Efflux Pumps

Abstract

ABSTRACT It is generally assumed that the acquisition of antibiotic resistance is as- sociated with a fitness cost. We have shown that overexpression of the MexEF-OprN efflux pump does not decrease the fitness of a resistant Pseudomonas aeruginosa strain compared to its wild-type counterpart. This lack of fitness cost was associated with a metabolic rewiring that includes increased expression of the anaerobic nitrate respiratory chain when cells are growing under fully aerobic conditions. It was not clear whether this metabolic compensation was exclusive to strains overexpressing MexEF-OprN or if it extended to other resistant strains that overexpress similar sys- tems. To answer this question, we studied a set of P. aeruginosa mutants that inde- pendently overexpress the MexAB-OprM, MexCD-OprJ, or MexXY efflux pumps. We observed increased expression of the anaerobic nitrate respiratory chain in all cases, with a concomitant increase in NO3 consumption and NO production. These efflux pumps are proton/substrate antiporters, and their overexpression may lead to intra- cellular H accumulation, which may in turn offset the pH homeostasis. Indeed, all studied mutants showed a decrease in intracellular pH under anaerobic conditions. The fastest way to eliminate the excess of protons is by increasing oxygen consump- tion, a feature also displayed by all analyzed mutants. Taken together, our results support metabolic rewiring as a general mechanism to avoid the fitness costs de- rived from overexpression of P. aeruginosa multidrug efflux pumps. The develop- ment of drugs that block this metabolic “reaccommodation” might help in reducing the persistence and spread of antibiotic resistance elements among bacterial popu- lations. IMPORTANCE It is widely accepted that the acquisition of resistance confers a fit- ness cost in such a way that in the absence of antibiotics, resistant populations will be outcompeted by susceptible ones. Based on this assumption, antibiotic cycling regimes have been proposed in the belief that they will reduce the persistence and spread of resistance among bacterial pathogens. Unfortunately, trials testing this possibility have frequently failed, indicating that resistant microorganisms are not al- ways outcompeted by susceptible ones. Indeed, some mutations do not result in a fitness cost, and in case they do, the cost may be compensated for by a secondary mutation. Here we describe an alternative nonmutational mechanism for compensat- ing for fitness costs, which consists of the metabolic rewiring of resistant mutants. Deciphering the mechanisms involved in the compensation of fitness costs of antibiotic-resistant mutants may help in the develop