Earlier studies that used model membrane reconstitution methods have come to different conclusions regarding the exclusion limit of the outer membrane of Pseudomonas aeruginosa and whether OprF is the major channel-forming protein in the outer membrane. In this study, a 6.2-kbp SalI fragment, encoding only two cytoplasmic enzymes, alpha-galactosidase and sucrose hydrolase, and the inner membrane raffinose permease, was cloned behind the m-toluate-inducible tol promoter of vector pNM185 to create plasmid pFB71. P. aeruginosa strains harboring pFB71, when grown with inducer, produced both enzymes encoded by the insert and had acquired the ability to grow on the disaccharide melibiose and the trisaccharide raffinose. The rate of growth was dependent on the concentration and size of the saccharide and was decreased three- to fivefold by the absence of OprF, as examined by measuring the growth on melibiose and raffinose of an isogenic OprF-deficient omega insertion derivative, H636(pFB71). At high concentrations, di-, tri-, and tetrasaccharides could pass across the outer membrane to plasmolyze P. aeruginosa, as measured by light scattering and confirmed by electron microscopy. The initial rate kinetics of light-scattering changes were dependent on the size of the saccharide being used. Furthermore, the rates of change in light scattering due to raffinose and stachyose uptake across the outer membrane for strain H636 were fivefold or more lower than for its OprF-sufficient parent H103. These data are consistent with model membrane studies showing that OprF is the most predominant porin for compounds larger than disaccharides in P. aeruginosa and suggest that the exclusion limit for this porin and the outer membrane is greater than the size of a tetrasaccharide. In addition, these data confirmed the existence of other porins with a predominant function in monosaccharide uptake and a more minor function in the uptake of larger saccharides.

Mechanisms of ceftriaxone resistance were examined in Enterobacter cloacae. Clones were selected from four strains: susceptible (S), resistant (Rl), selected by plating on ceftriaxone-containing agar, and highly resistant (R2), selected in ceftriaxone-treated mice infected with S clones. According to 14C-labeled ,-lactam

The fourth generation cephalosporins, cefpirome and cefepime, demonstrate better activity against strains of Enterobacter cloacae with derepressed beta-lactamase than the third generation compounds cefotaxime and ceftriaxone. Several methodological refinements were used to measure the parameters, predicted by the Zimmermann-Rosselet equation to be important in the efficacy of beta-lactams. Outer membrane permeability was measured by a novel HPLC method. The kinetics of interaction of purified beta-lactamase with beta-lactams were estimated to calculate the inhibition and catalytic constants. The periplasmic concentration of beta-lactams leading to growth inhibition of cells was determined by substituting the above parameters into the Zimmermann-Rosselet equation. Consideration of these three factors allowed accurate prediction of MICs in isogenic E. cloacae strains with differing porin or beta-lactamase contents. The fourth generation cephalosporins had markedly reduced affinity for beta-lactamase and increased outer membrane permeability when compared to the third generation cephalosporins. Such advantages were only partly offset by a lower stability of complexes with beta-lactamase and reduced affinity for their targets.

An enoxacin-resistant Pseudomonas aeruginosa mutant (G49) isolated during patient therapy was characterized in detail. The G49 mutant was cross resistant to several classes of antibiotics including quinolones, I-lactams, chloramphenicol, and tetracycline, but not imipenem or aminoglycosides. Compared with its paired pretherapy isolate G48, this mutant had several alterations in outer membrane proteins including a complete loss of the major porin protein OprF and a substantially altered lipopolysaccharide profile. Revertants were selected at a frequency of approximately 1% after enrichment for OprF+ cells on low-salt proteose peptone no. 2 medium. Ninety-seven of these OprF+ revertants were as susceptible to carbenicillin and norfloxacin as the pretherapy isolate. One of these revertants was characterized in more detail and shown to be indistinguishable in all properties from the pretherapy isolate. It is proposed that the multiple-antibiotic-resistance (Mar) phenotype of this mutant resulted from a single pleiotropic mutation.Enoxacin is a difluorinated quinolone with strong activity for gram-negative bacteria (7,41). Enoxacin is now marketed in 11 countries, including South America, the United Kingdom, and other parts of Europe, for use in the therapy of urinary tract infections and in some countries for respiratory tract infections. As in other bacteria, the primary target for quinolones in Pseudomonas aeruginosa is DNA gyrase. nalA, nfxA, norA, and cipA are alleles ofgyrA and encode A subunits that are less susceptible to inhibition by quinolones (14,16,33,34). Other mutations affecting quinolone activity, but not DNA gyrase, in P. aeruginosa have also been described. Many laboratory mutants contain alleles of nalB and require nalidixic acid MICs of >500 ,ug/ml and are cross resistant to carbenicillin, ureidopenicillins, chloramphenicol, and novobiocin (33, 34). Resistant strains have arisen during experimental P. aeruginosa infections, with several classes of mutants isolated with mutations resembling naL4 and nfxC (10,22,23). Quinolone-resistant P. aeruginosa isolated from patients may also have the nal4 or nalB phenotype (43) or a nalB-like phenotype lacking OprF. Such strains have been isolated from patients with chronic obstructive airway disease (30), burn wound sepsis (17), cystic fibrosis (6), and empyema (20). While several strains with decreased susceptibilities to several agents have been isolated, which suggests a mutation in nalB, there are biochemical differences that are commensurate with different mutations encoding multiple resistance. Some quinolone-resistant P. aeruginosa strains contain a new 54-kDa outer membrane protein (14, 18) or lack a 31.5-kDa outer membrane protein (9, 17), possibly OprF, or have decreased levels of proteins D2 and Hi or Gl (5,20,23). Several workers have also reported changes in lipopolysaccharide (LPS) (6,18,34).An open study to evaluate the efficacy of enoxacin in respiratory tract infections with particular attention to those caused by P. aeruginosa was perform...