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

It is generally assumed that the acquisition of antibiotic resistance is associated 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 systems. To answer this question, we studied a set of P. aeruginosa mutants that independently 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 intracellular 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 consumption, a feature also displayed by all analyzed mutants. Taken together, our results support metabolic rewiring as a general mechanism to avoid the fitness costs derived from overexpression of P. aeruginosa multidrug efflux pumps. The development of drugs that block this metabolic "reaccommodation" might help in reducing the persistence and spread of antibiotic resistance elements among bacterial populations.IMPORTANCE It is widely accepted that the acquisition of resistance confers a fitness 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 always 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 compensating 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 development of drugs that will reduce the persistence of resistance by increasing said costs.

Metabolic compensation of fitness costs associated with overexpression of the multidrug efflux pump MexEF-OprN in Pseudomonas aeruginosa.

The acquisition of antibiotic resistance has been associated with a possible nonspecific, metabolic burden that is reflected in decreased fitness among resistant bacteria. We have recently demonstrated that overexpression of the MexEF-OprN multidrug efflux pump does not produce a metabolic burden when measured by classical competitions tests but rather leads to a number of changes in the organism's physiology. One of these changes is the untimely activation of the nitrate respiratory chain under aerobic conditions. MexEF-OprN is a proton/substrate antiporter. Overexpression of this element should result in a constant influx of protons, which may lead to cytoplasmic acidification. Acidification was not observed in aerobiosis, a situation in which the MexEF-overproducing mutant increases oxygen consumption. This enhanced oxygen uptake serves to eliminate intracellular proton accumulation, preventing the cytoplasmic acidification that was observed exclusively under anaerobic conditions, a situation in which the fitness of the MexEF-OprN-overproducing mutant decreases. Finally, we determined that the early activation of the nitrate respiratory chain under aerobic conditions plays a role in preventing a deleterious effect associated with the overexpression of MexEF-OprN. Our results show that metabolic rewiring may assist in overcoming the potential fitness cost associated with the acquisition of antibiotic resistance. Furthermore, the capability to metabolically compensate for this effect is habitat dependent, as demonstrated by our results under anaerobic conditions. The development of drugs that prevent metabolic compensation of fitness costs may help to reduce the persistence and dissemination of antibiotic resistance.