MexXY is an inducible efflux system that contributes to the natural resistance of Pseudomonas aeruginosa to antibiotics. Experiments involving real-time PCR after reverse transcription in reference strain PAO1 showed concentration-dependent induction of gene mexY by various ribosome inhibitors (e.g., chloramphenicol, tetracycline, macrolides, and aminoglycosides) but not by antibiotics acting on other cellular targets (e.g., ␤-lactams, fluoroquinolones). Confirming a functional link between the efflux system and the translational machinery, ribosome protection by plasmid-encoded proteins TetO and ErmBP increased the resistance of a ⌬mexAB-oprM mutant of PAO1 to tetracycline and erythromycin, respectively, as well as the concentrations of both drugs required to induce mexY. Furthermore, spontaneous mutations resulting in specific resistance to dihydrostreptomycin or spectinomycin also raised the minimal drug concentration for mexXY induction in strain PAO1. While strongly upregulated in a PAO1 mutant defective in gene mexZ (which codes for a putative repressor of operon mexXY), gene mexY remained inducible by agents such as tetracycline, chloramphenicol, and spectinomycin, suggesting additional regulatory loci for mexXY. Altogether, these data demonstrate physiological interplays between MexXY and the ribosome and are suggestive of an alternative function for MexXY beyond antibiotic efflux.

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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...

Aminoglycosides are among the most potent antimicrobials to eradicate Pseudomonas aeruginosa. However, the emergence of resistance has clearly led to a shortage of treatment options, especially for critically ill patients. In the search for new antibiotics, we have synthesized derivatives of the small aminoglycoside, neamine. The amphiphilic aminoglycoside 3',4',6-tri-2-naphtylmethylene neamine (3',4',6-tri-2NM neamine) has appeared to be active against sensitive and resistant P. aeruginosa strains as well as Staphylococcus aureus strains (Baussanne et al., 2010). To understand the molecular mechanism involved, we determined the ability of 3',4',6-tri-2NM neamine to alter the protein synthesis and to interact with the bacterial membranes of P. aeruginosa or models mimicking these membranes. Using atomic force microscopy, we observed a decrease of P. aeruginosa cell thickness. In models of bacterial lipid membranes, we showed a lipid membrane permeabilization in agreement with the deep insertion of 3',4',6-tri-2NM neamine within lipid bilayer as predicted by modeling. This new amphiphilic aminoglycoside bound to lipopolysaccharides and induced P. aeruginosa membrane depolarization. All these effects were compared to those obtained with neamine, the disubstituted neamine derivative (3',6-di-2NM neamine), conventional aminoglycosides (neomycin B and gentamicin) as well as to compounds acting on lipid bilayers like colistin and chlorhexidine. All together, the data showed that naphthylmethyl neamine derivatives target the membrane of P. aeruginosa. This should offer promising prospects in the search for new antibacterials against drug- or biocide-resistant strains.

Cefepime (FEP) and ceftazidime (CAZ) are potent ␤-lactam antibiotics with similar MICs (1 to 2 g/ml) for wild-type strains of Pseudomonas aeruginosa. However, recent epidemiological studies have highlighted the occurrence of isolates more resistant to FEP than to CAZ (FEP r /CAZ s profile). We thus investigated the mechanisms conferring such a phenotype in 38 clonally unrelated strains collected in two French teaching hospitals. Most of the bacteria (n ‫؍‬ 32; 84%) appeared to stably overexpress the mexY gene, which codes for the RND transporter of the multidrug efflux system MexXY-OprM. MexXY up-regulation was the sole FEP resistance mechanism identified (n ‫؍‬ 12) or was associated with increased levels of pump MexAB-OprM (n ‫؍‬ 5) or MexJK (n ‫؍‬ 2), synthesis of secondary ␤-lactamase PSE-1 (n ‫؍‬ 10), derepression of cephalosporinase AmpC (n ‫؍‬ 1), coexpression of both OXA-35 and MexJK (n ‫؍‬ 1), or production of both PSE-1 and MexAB-OprM (n ‫؍‬ 1). Down-regulation of the mexXY operon in seven selected strains by the plasmid-borne repressor gene mexZ decreased FEP resistance from two-to eightfold, thereby demonstrating the significant contribution of MexXY-OprM to the FEP r /CAZ s phenotype. The six isolates of this series that exhibited wild-type levels of the mexY gene were found to produce ␤-lactamase PSE-1 (n ‫؍‬ 1), OXA-35 (n ‫؍‬ 4), or both PSE-1 and OXA-35 (n ‫؍‬ 1). Altogether, these data provide evidence that MexXY-OprM plays a major role in the development of FEP resistance among clinical strains of P. aeruginosa.Cefepime (FEP) and ceftazidime (CAZ) are broad-spectrum cephalosporins that display similar MICs (1 to 2 g/ml) for wild-type Pseudomonas aeruginosa. Both antibiotics have been approved for antipseudomonal chemotherapy (8) and are widely used to treat severely ill patients in hematology-oncology and intensive care units (13,23,38). Although MIC distribution patterns of both ␤-lactams (e.g., the MIC at which 50% of strains are inhibited [MIC 50 ]) appear to be identical for North American isolates of P. aeruginosa (14), several European studies have recently pointed out higher MIC 50 values for cefepime (4 to 8 g/ml) than for ceftazidime (2 to 4 g/ml) (4-6, 39). These epidemiological data suggested the occurrence of isolates less susceptible to cefepime than to ceftazidime (FEP r /CAZ s phenotype). Resistance of clinical strains of P. aeruginosa to antipseudomonal cephalosporins is mainly due to overexpression of the chromosomally encoded ␤-lactamase AmpC (with MICs of cefepime usually lower than those of ceftazidime) and occasionally to acquisition of extended-spectrum ␤-lactamases (41). However, while an increasing number of extended-spectrum ␤-lactamases have been characterized in ceftazidime-resistant isolates over the last decade (41), little information is available about ␤-lactamases that preferentially hydrolyze cefepime compared with ceftazidime. For instance, several FEP r /CAZ s strains were recently found to produce class D ␤-lactamases, such as OXA-1, OXA-10, OXA-31, and OXA-3...