Antibiotic-resistant mutants of Pseudomonas aeruginosa were generated using chloramphenicol and ciprofloxacin as selective agents. These mutants displayed a multidrug phenotype and overexpressed an outer membrane protein of 50 kDa, which was shown by Western blot analysis to correspond to OprN. A cosmid clone harbouring the oprN gene was isolated by partial complementation of a mutant deficient in OprM, the outer membrane component of the mexAB-oprM efflux operon. Antibiotic-accumulation studies indicated that OprN was part of an energy-dependent antibiotic-efflux system. Sequencing of a 6180bp fragment from the complementing cosmid revealed the presence of three open reading frames (ORFs), which exhibited amino acid similarity to the components of the mexAB-oprM and mexCD-oprJ efflux operons of P. aeruginosa. The ORFs were designated MexE, MexF and OprN. Mutation of the mexE gene eliminated the multidrug-resistance phenotype in an OprN-overexpressing strain, but did not affect the susceptibility profile of the wild-type strain. Expression of the mexEF-oprN operon was shown to be positively regulated by a protein encoded on a 1.5 kb DNA fragment located upstream of mexE and belonging to the LysR family of transcriptional activators. The presence of a plasmid containing this DNA fragment was sufficient to confer a multidrug phenotype onto the wild-type strain but not onto the mexE mutant. Evidence is provided to show that the mexEF-oprN operon may be involved in the excretion of intermediates for the biosynthesis of pyocyanin, a typical secondary metabolite of P. aeruginosa.

While meropenem MICs were strongly influenced by the presence or absence of the MexAB-OprM efflux pump in both OprD-proficient and -deficient strain backgrounds, MICs of imipenem and of ER-35786 remained unchanged, demonstrating that meropenem is a substrate of MexAB-OprM but not imipenem and ER-35786. In vitro, all three carbapenems selected loss of OprD as a first mechanism of resistance. However, in an OprD-deficient background, meropenem was able to select MexAB-OprM overproducers as a secondary resistance mechanism, while ER-35786 selected a mutant cross-resistant to sparfloxacin and cefpirome.

Resistance emerging after fluoroquinolone therapy was investigated in a murine model of Pseudomonas aeruginosa infection. Mice were infected intraperitoneally by one of six strains and treated with pefloxacin or ciprofloxacin. In mice challenged with a low inoculum (1.6 x 105 CFU), no resistance occurred. With a higher inoculum (1.5 x 108 CFU) and after a single dose of antibiotic, posttherapy (PT1) strains with decreased susceptibility to quinolones (4-to 32-fold less) were isolated at a variable rate. The presence of talcum (125 mg) in the peritoneal cavity increased the risk of resistance after therapy. Pefloxacin (25 or 200 mg/kg) and ciprofloxacin (25 mg/kg) yielded similar resistance rates (61 to 77%), but ciprofloxacin (10 mg/kg) produced more resistance (83%) than did ciprofloxacin (50 mg/kg) (44%) (P < 0.02). Combined with a quinolone, ceftazidime (P < 0.001) or amikacin (P < 0.01), but not piperacillin, reduced the emergence of resistance. After several doses of ciprofloxacin, it was found that 25-mg/kg doses every 12 h produced more resistance than did 25-mg/kg doses every 8 h or 50-mg/kg doses every 12 h. Compared with the preceding experiments using parent strains, ciprofloxacin and pefloxacin were less efficient in killing bacteria in mice infected with PT, strains.Moreover, in one of these mice, a highly resistant PT2 strain (64-fold MIC increase for the quinolones) emerged. Besides increased MICs of the quinolones, there was a two-to eightfold increase in imipenem MIC for all PT1 and PT2 strains without alteration of other ,-lactam and aminoglycoside susceptibility. Some PT, strains also showed a decreased susceptibility to trimethoprim and chloramphenicol. During therapy with a quinolone, resistance can emerge rapidly, especially when there is a large number of bacteria or a foreign body present. This risk may depend on the dosing schedule and may be reduced by combined therapy.During the past decade, the emergence of resistance after therapy with newer ,-lactam compounds has been an increasing concern (12). In our laboratories, we have reproduced this phenomenon by using an experimental model of Enterobacter cloacae infection (6a). We showed that the emergence of resistance varied with the P-lactam compound given to the animal. When the newer fluoroquinolones appeared, it was hoped that the development of resistance during therapy would be avoided (6). As a result of the improved antibacterial activity of drugs such as pefloxacin and ciprofloxacin, the resistant variants found in the bacterial populations, although less susceptible than the parent strains, might still be regarded as susceptible to these drugs (6). Unfortunately, this did not hold true, and several preliminary therapeutic trials have shown that resistant strains emerging after therapy could generate therapeutic difficulties, especially in Pseudomonas infections (11,16). In this paper, we compared the abilities of pefloxacin and ciprofloxacin to produce resistance in our murine model, with six strains of Pseudomonas aeruginosa ...

Pseudomonas aeruginosa possesses at least two multiple drug efflux systems which are defined by the outer membrane proteins OprM and OprJ. We have found that mutants overexpressing OprM were two- and eightfold more resistant than their wild-type parent to sulfamethoxazole (SMX) and trimethoprim (TMP), respectively. For OprJ-overproducing strains, MICs of TMP increased fourfold but those of SMX were unchanged. Strains overexpressing OprM, but not those overexpressing OprJ, became hypersusceptible to TMP and SMX when oprM was inactivated. The wild-type antibiotic profile could be restored in an oprM mutant by transcomplementation with the cloned oprM gene. These results demonstrate that the mexABoprM multidrug efflux system is mainly responsible for the intrinsic resistance of P. aeruginosa to TMP and SMX.

We investigated the unusual susceptibility to meropenem observed for seven imipenem-resistant clinical isolates of Pseudomonas aeruginosa. These strains were genetically closely related, expressed OprD, as determined by Western blot analyses, and were resistant to imipenem (>5 g/ml) but susceptible to meropenem (<1 g/ml). The oprD genes from two isolates were entirely sequenced, and their deduced protein sequences showed 93% identity with that of OprD of strain PAO1. The major alteration consisted of the replacement of a stretch of 12 amino acids, located in putative external loop L7 of OprD, by a divergent sequence of 10 amino acid residues. The oprD gene variants and the wild-type oprD gene were cloned and expressed in a defined oprD mutant. The meropenem MICs for strains carrying the oprD genes from clinical isolates were four times lower than that for the strain carrying the wild-type oprD gene. Imipenem activities, however, were comparable for all strains. Furthermore, meropenem hypersusceptibility was obtained with a hybrid OprD porin that consisted of the PAO1 oprD gene containing loop L7 from a clinical isolate. These results show that the C-terminal portion of OprD, in particular, loop L7, was responsible for the unusual meropenem hypersusceptibility. Competition experiments suggested that the observed OprD modifications in the clinical isolates did not affect antagonism between imipenem and the basic amino acid L-lysine. We further propose that shortening of putative loop L7 of the OprD porin by 2 amino acid residues sufficiently opens the porin channel to allow optimal penetration of meropenem and increase its activity. In contrast, this alteration would not affect susceptibility to a smaller carbapenem molecule, such as imipenem.Pseudomonas aeruginosa is an opportunistic organism causing difficult-to-treat infections. The high intrinsic resistance of this bacterium to antibiotics results from the complex interaction of several mechanisms, among which the rather impermeable porin pathway plays a key role (40). Substrate-specific transport systems in the outer membrane allow the diffusion of essential nutrients present at low concentrations in the vicinity of the cells and compensate for this low nonspecific permeability. The outer membrane porin OprD facilitates the uptake of basic amino acids (36), small peptides, and carbapenem antibiotics, such as imipenem and meropenem (35). All of these molecules share common binding sites inside the OprD channel (6).Carbapenems are potent inhibitors of P. aeruginosa because of their high stability to the chromosomally encoded AmpC ␤-lactamase produced by this species (15). However, resistance of clinical strains to both imipenem and meropenem is increasingly observed as the result of the loss of protein OprD (18, 29), alone or associated with the stable overexpression of the AmpC ␤-lactamase (16). Overproduction of the MexABOprM active efflux system in nalB mutants may also increase the resistance to meropenem but has no effect on the susceptibility of P. aerugi...