Nobuhisa Masuda

Substrate Specificities of MexAB-OprM, MexCD-OprJ, and MexXY-OprM Efflux Pumps in Pseudomonas aeruginosa

ABSTRACT To find the exact substrate specificities of three species of tripartite efflux systems of Pseudomonas aeruginosa, MexAB-OprM, MexCD-OprJ, and MexXY-OprM, we constructed a series of isogenic mutants, each of which constitutively overproduced one of the three efflux systems and lacked the other two, and their isogenic mutants, which lacked all these systems. Comparison of the susceptibilities of the constructed mutants to 52 antimicrobial agents belonging to various groups suggested the following substrate specificities. All of the efflux systems extrude a wide variety of antimicrobial agent groups, i.e., quinolones, macrolides, tetracyclines, lincomycin, chloramphenicol, most penicillins (all but carbenicillin and sulbenicillin), most cephems (all but cefsulodin and ceftazidime), meropenem, and S-4661, but none of them extrude polymyxin B or imipenem. Extrusion of aminoglycosides is specific to MexXY-OprM, and extrusion of a group of the β-lactams, i.e., carbenicillin, sulbenicillin, ceftazidime, moxalactam, and aztreonam, is specific to MexAB-OprM. Moreover, MexAB-OprM and MexCD-OprJ extrude novobiocin, cefsulodin, and flomoxef, while MexXY-OprM does not. These substrate specificities are distinct from those reported previously.

Contribution of the MexX-MexY-OprM Efflux System to Intrinsic Resistance in Pseudomonas aeruginosa

ABSTRACT To test the possibility that MexX-MexY, a new set of efflux system components, is associated with OprM and contributes to intrinsic resistance in Pseudomonas aeruginosa, we constructed a series of isogenic mutants lacking mexXY and/ormexAB and/or oprM from a laboratory strain PAO1, and examined their susceptibilities to ofloxacin, tetracycline, erythromycin, gentamicin, and streptomycin. Loss of either MexXY or OprM from the MexAB-deficient mutant increased susceptibility to all agents tested, whereas loss of MexXY from the MexAB-OprM-deficient mutant caused no change in susceptibility. Introduction of an OprM expression plasmid decreased the susceptibility of themexAB-oprM-deficient-/mexXY-maintaining mutant, yet caused no change in the susceptibility of amexAB-oprM- and mexXY-deficient double mutant. Immunoblot analysis using anti-MexX polyclonal rabbit serum generated against synthetic oligopeptides detected expression of MexX in the PAO1 cells grown in medium containing tetracycline, erythromycin, or gentamicin, although expression of MexX was undetectable in the cells incubated in medium without any agent. These results suggest that MexXY induced by these agents is functionally associated with spontaneously expressed OprM and contributes to the intrinsic resistance to these agents.

Outer membrane proteins responsible for multiple drug resistance in Pseudomonas aeruginosa.

Three types of multiple-drug-resistant mutants which were phenotypically similar to previously described nalB, nfxB, and nfxC mutants were isolated from Pseudomonas aeruginosa PAO1 and two clinical isolates. Type 1 (nalB-type) mutants showed cross-resistance to meropenem, cephems, and quinolones. They overproduced an outer membrane protein with an apparent molecular mass of 50 kDa (OprM). Type 2 (nfxB-type) mutants showed cross-resistance to quinolones and new cephems, i.e., cefpirome and cefozopran, concomitant with overproduction of an outer membrane protein with an apparent molecular mass of 54 kDa (OprJ). Type 3 (nfxC-type) mutants showed cross-resistance to carbapenems and quinolones. They produced decreased amounts of OprD and increased amounts of a 50-kDa protein (OprN), which was almost the same molecular weight as that of OprM, but it was distinguishable from OprM by its heat modifiability on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. In the presence of salicylate, the parent strains showed an increased level of resistance to carbapenems and quinolones and produced decreased amounts of OprD and increased amounts of OprN. Salicylate caused the repression of OprJ production and the loss of resistance to cefpirome and cefozopran in two of the three OprJ-overproducing mutants, although salicylate slightly increased the level of resistance in the parent strains. The changes in susceptibilities were transient in the presence of salicylate. These data suggest that at least three different outer membrane proteins, OprM, OprJ, and OprN, are associated with multiple drug resistance in P. aeruginosa.

In Vitro and In Vivo Antibacterial Activities of CS-023 (RO4908463), a Novel Parenteral Carbapenem

ABSTRACT CS-023 (RO4908463, formerly R-115685) is a novel 1β-methylcarbapenem with 5-substituted pyrrolidin-3-ylthio groups, including an amidine moiety at the C-2 position. Its antibacterial activity was tested against 1,214 clinical isolates of 32 species and was compared with those of imipenem, meropenem, ceftazidime, ceftriaxone, ampicillin, amikacin, and levofloxacin. CS-023 exhibited a broad spectrum of activity against gram-positive and -negative aerobes and anaerobes, including methicillin-resistant Staphylococcus aureus (MRSA), methicillin-resistant Staphylococcus epidermidis, penicillin-resistant Streptococcus pneumoniae (PRSP), β-lactamase-negative ampicillin-resistant Haemophilus influenzae, and Pseudomonas aeruginosa. CS-023 showed the most potent activity among the compounds tested against P. aeruginosa and MRSA, with MICs at which 90% of isolates tested were inhibited of 4 μg/ml and 8 μg/ml, respectively. CS-023 was stable against hydrolysis by the β-lactamases from Enterobacter cloacae and Proteus vulgaris. CS-023 also showed potent activity against extended-spectrum β-lactamase-producing Escherichia coli. The in vivo efficacy of CS-023 was evaluated with a murine systemic infection model induced by 13 strains of gram-positive and -negative pathogens and a lung infection model induced by 2 strains of PRSP (serotypes 6 and 19). Against the systemic infections with PRSP, MRSA, and P. aeruginosa and the lung infections, the efficacy of CS-023 was comparable to those of imipenem/cilastatin and vancomycin (tested against lung infections only) and superior to those of meropenem, ceftriaxone, and ceftazidime (tested against P. aeruginosa infections only). These results suggest that CS-023 has potential for the treatment of nosocomial bacterial infections by gram-positive and -negative pathogens, including MRSA and P. aeruginosa.

Increase in susceptibility of Pseudomonas aeruginosa to carbapenem antibiotics in low-amino-acid media.

The in vitro susceptibility of Pseudomonas aeruginosa PAO1 to carbapenem antibiotics, such as CS-533, was influenced by various concentrations of basic amino acids, i.e., L-lysine, L-histidine, and L-arginine, in agar media. P. aeruginosa PAO1 showed higher susceptibility to carbapenems in minimal medium than it did in rich media such as Mueller-Hinton II agar. The susceptibility was decreased by the addition of a basic amino acid to the minimal medium, whereas it was influenced less by other amino acids. The susceptibility of PAO1 to cephalosporins, piperacillin, quinolones, and gentamicin was not influenced by the addition of a basic amino acid to the minimal medium. A significant change in susceptibility to carbapenems by the addition of a basic amino acid was not observed with D2 protein-deficient mutants of PAO1. Clinical isolates of P. aeruginosa also showed an increase in susceptibility in minimal medium. L-Lysine in minimal medium did not have any influence on the production of D2 protein, beta-lactamases, or penicillin-binding proteins of PAO1 or on the chemical degradation of CS-533. These results strongly indicate that the increase in susceptibility of P. aeruginosa to carbapenems relates to less competition with basic amino acids for permeation through the D2 protein channel of P. aeruginosa. Images

Potent In Vitro Activity of Tomopenem (CS-023) against Methicillin-Resistant Staphylococcus aureus and Pseudomonas aeruginosa

ABSTRACT Tomopenem (formerly CS-023) is a novel 1β-methylcarbapenem with broad-spectrum coverage of gram-positive and gram-negative pathogens. Its antibacterial activity against European clinical isolates of methicillin-resistant Staphylococcus aureus (MRSA) and Pseudomonas aeruginosa was compared with those of imipenem and meropenem. The MICs of tomopenem against MRSA and P. aeruginosa at which 90% of the isolates tested were inhibited were 8 and 4 μg/ml, respectively, and were equal to or more than fourfold lower than those of imipenem and meropenem. The antibacterial activity of tomopenem against MRSA was correlated with a higher affinity for the penicillin-binding protein (PBP) 2a. Its activity against laboratory mutants of P. aeruginosa with (i) overproduction of chromosomally coded AmpC β-lactamase; (ii) overproduction of the multidrug efflux pumps MexAB-OprM, MexCD-OprJ, and MexEF-OprN; (iii) deficiency in OprD; and (iv) various combinations of AmpC overproduction, MexAB-OprM overproduction, and OprD deficiency were tested. The increases in the MIC of tomopenem against each single mutant compared with that against its parent strain were within a fourfold range. Tomopenem exhibited antibacterial activity against all mutants, with an observed MIC range of 0.5 to 8 μg/ml. These results suggest that the antibacterial activity of tomopenem against the clinical isolates of MRSA and P. aeruginosa should be ascribed to its high affinity for PBP 2a and its activity against the mutants of P. aeruginosa, respectively.

Hypersusceptibility of the Pseudomonas aeruginosa nfxB Mutant to β-Lactams Due to Reduced Expression of the AmpC β-Lactamase

ABSTRACT The Pseudomonas aeruginosa nfxB mutant lackingmexAB-oprM showed hypersusceptibility to 9 out of 24 β-lactams tested. This hypersusceptibility was found for thenfxB mutant lacking mexAB-oprM-mexXY(N108) but not for the nfxB mutant lacking bothmexAB-oprM-mexXY and ampC. The level of the AmpC β-lactamase induction was reduced in N108. Thus, the reduced AmpC induction must be the cause of the hypersusceptibility.

Affinity of Tomopenem (CS-023) for Penicillin-Binding Proteins in Staphylococcus aureus, Escherichia coli, and Pseudomonas aeruginosa

ABSTRACT Tomopenem (formerly CS-023), a novel 1β-methylcarbapenem, exhibited high affinity for penicillin-binding protein (PBP) 2 in Staphylococcus aureus, PBP 2 in Escherichia coli, and PBPs 2 and 3 in Pseudomonas aeruginosa, which are considered major lethal targets. Morphologically, tomopenem induced spherical forms in E. coli and short filamentation with bulges in P. aeruginosa, which correlated with the drugs PBP profiles. The potential of resistance of these bacteria to tomopenem was comparable to that to imipenem.

In vitro and in vivo activities of DS-2969b, a novel GyrB inhibitor, and its water-soluble prodrug, DS11960558, against methicillin-resistant Staphylococcus aureus

ABSTRACT DS-2969b is a novel GyrB inhibitor under clinical development. In this study, the in vitro activity of DS-2969b and the in vivo activities of DS-2969b and its water-soluble prodrug, DS11960558, against methicillin-resistant Staphylococcus aureus (MRSA) were evaluated. DS-2969b inhibited the supercoiling activity of S. aureus DNA gyrase and the decatenation activity of its topoisomerase IV. DS-2969b showed antibacterial activity against Gram-positive aerobes but not against Gram-negative aerobes, except for Moraxella catarrhalis and Haemophilus influenzae. DS-2969b was active against MRSA with an MIC90 of 0.25 μg/ml, which was 8-fold lower than that of linezolid. The presence of a pulmonary surfactant did not affect the MIC of DS-2969b. DS-2969b showed time-dependent slow killing against MRSA. The frequency of spontaneous resistance development was less than 6.2 × 10−10 in all four S. aureus isolates at 4× MIC of DS-2969b. In a neutropenic MRSA-induced murine muscle infection model, DS-2969b was more efficacious than linezolid by both the subcutaneous and oral routes. DS-2969b and DS11960558 showed efficacy in a neutropenic murine MRSA lung infection model. The pharmacokinetics and pharmacodynamics of DS-2969b and DS11960558 against MRSA were characterized in a neutropenic murine thigh infection model; the percentage of time during the dosing period in which the free drug concentration exceeded the MIC (fTMIC) correlated best with in vivo efficacy, and the static percent fTMIC was 43 to 49%. A sufficient fTMIC was observed in a phase 1 multiple-ascending-dose study of DS-2969b given orally at 400 mg once a day. These results suggest that DS11960558 and DS-2969b have potential for use as intravenous-to-oral step-down therapy for treating MRSA infections with a higher efficacy than linezolid.

A Phase 1 Study in Healthy Subjects to Assess the Safety, Tolerability, Pharmacokinetics, and Pharmacodynamics of Multiple Oral Doses of DS-2969b, a Novel GyrB Inhibitor

ABSTRACT DS-2969b is a novel GyrB inhibitor in development for the treatment of Clostridium difficile infection (CDI). The aim of this study was to assess the safety, tolerability, pharmacokinetics, and effects on the normal gastrointestinal microbiota of multiple daily oral ascending doses of DS-2969b in healthy subjects. The study enrolled three sequential ascending-dose cohorts (60 mg, 200 mg, and 400 mg). In each cohort, subjects received an oral dose of DS-2969b or placebo (six subjects received DS-2969b, and two received placebo) each morning for 14 days. DS-2969b was safe and well tolerated at all dose levels examined. All adverse events related to DS-2969b were mild and predominantly related to the gastrointestinal tract. DS-2969a (free form of DS-2969b) plasma concentrations increased with increasing doses; however, both the maximum concentration of drug in serum (Cmax) and the area under the concentration-time curve (AUC) increased less than dose proportionally. In all cohorts, sufficient fecal levels of DS-2969a were achieved within 24 h following the administration of the first dose and maintained for at least 17 days. Following treatment with DS-2969b, clear reductions in the populations of Clostridium coccoides and Bifidobacterium groups were observed. However, populations of three other bacterial groups examined (Bacteroides fragilis, Clostridium leptum, and Prevotella) were not affected. Data from this study support and encourage the further development of DS-2969b as a novel treatment for CDI.