MexXY-OprM Efflux Pump Is Necessary for Adaptive Resistance of Pseudomonas aeruginosa to Aminoglycosides

Abstract

Exposure of Pseudomonas aeruginosa to aminoglycosides frequently selects for recalcitrant subpopulations exhibiting an unstable, "adaptive" resistance to these antibiotics. In this study, we investigated the implication in the phenomenon of MexXY-OprM, an active efflux system known to export aminoglycosides in P. aeruginosa. Immunoblotting experiments demonstrated that the transporter MexY, but not the outer membrane pore OprM, was overproduced during the post-drug exposure adaptation period in wild-type strain PAO1. Furthermore, MexY production was dependent upon the degree of bacterial exposure to gentamicin (drug concentration). In contrast to parental strain PAO1, mutants defective in MexXY or in OprM were unable to develop adaptive resistance. Altogether, these results indicate that the resistance process requires the rapid production of MexXY and the interaction of these proteins with the constitutively produced component OprM.Aminoglycosides remain invaluable antibiotics in the treatment of severe infections caused by Pseudomonas aeruginosa. However, their in vivo efficacy may be compromised by the development of transiently resistant subpopulations (7,8,15,34). Exposure of susceptible P. aeruginosa to an aminoglycoside classically results in an early and rapid drug concentrationdependent killing followed by a phase of bacterial refractoriness characterized by a slow drug concentration-independent killing (27). This so-called adaptive resistance, which is distinct from the postantibiotic effect and which disappears when the organism is no longer in contact with the aminoglycoside, has been observed in vitro (5,11,19), in animal models of infection (12,40), and in patients with cystic fibrosis (6). Because of its ephemeral and reproducible nature, adaptive resistance is not believed to result from mutational events. Ribosomal alterations and drug inactivation are not considered plausible mechanisms, either, as they would result in specific patterns of susceptibility to aminoglycosides and not cross-resistance to these antibiotics (11). Reduced intracellular accumulation of aminoglycosides, which is concomitant to adaptive resistance, was first interpreted as the consequence of lower drug uptake across the bacterial envelopes (11,19). Supporting this assumption, pleiotropic changes in the protein profiles of the cytoplasmic membrane were detected in drug-exposed bacteria by some investigators (19). However, clear evidence for a substantial decrease in aminoglycoside transport across the inner membrane could not be obtained. The membrane potential ⌬ (the driving force for drug entry) appears to be marginally diminished in adaptively resistant bacteria (19), a finding which agrees well with the observation that surviving bacteria grow normally during the postexposure refractory phase (11,19). These characteristics are opposite of those of another drugrecalcitrant subpopulation also selectable by aminoglycosides, composed of energy-deficient variants (also called small-colony variants) (7, 34).Recently, severa...

Resistance suppression by high-intensity, short-duration aminoglycoside exposure against hypermutable and non-hypermutablePseudomonas aeruginosa

“…The static concentrations with complete removal of tobramycin at the end of each exposure period would have prevented the development of resistance mechanisms that require sub-MIC concentrations to occur. Common mechanisms involved in the resistance observed in PAO1 could be adaptive resistance via the upregulation of the MexXY-OprM efflux pump, 41,50 which is frequently caused by overexpression of the MexY component, or de novo mutations during treatment. 41,51,52 Previous studies have found that a resistance mechanism unrelated to this efflux pump has played a role in aminoglycoside resistance for PAODmutS.…”

“…Adaptive resistance against aminoglycosides is often caused by the overexpression of the MexXY-OprM efflux pump 41 or inhibition of energy-dependent uptake in P. aeruginosa, 42 both of which would decrease C intra , as described above. It was described by a twocompartment model for ADAPT 1 (Equation 9) and ADAPT 2 (Equation 10)…”

Breakpoints for Predicting Pseudomonas aeruginosa Susceptibility to Inhaled Tobramycin in Cystic Fibrosis Patients: Use of High-Range Etest Strips

“…Conversely, low to moderate resistance has been associated with impermeability or so-called adaptive resistance (2), which has mostly been observed in CF isolates. An inducible multidrug efflux system, MexXY-OprM, appears to be responsible for this adaptive mechanism of resistance (13). Such induction may explain the increase in transient low to moderate tobramycin resistance upon exposure of P. aeruginosa to antimicrobial concentrations near those of the MICs of aminoglycosides, a situation that may occur even under inhaled therapy (33).…”

Adaptive and Mutational Resistance: Role of Porins and Efflux Pumps in Drug Resistance

“…Indeed, this knowledge has been essential for the design of improved therapeutic regimens, which now involve the administration of a higher dose with a lower frequency within the range permitted by toxicity risks (16,45). To a large extent, this adaptive phenotype is due to the aminoglycoside-induced expression of the genes encoding the MexXY efflux pump, which varies according to the aminoglycoside concentration (94). This pump has also been shown to be involved in the antagonism between divalent cations and aminoglycosides (147).…”