Kazuki Hoshino

Identification and Characterization of Inhibitors of Multidrug Resistance Efflux Pumps in Pseudomonas aeruginosa: Novel Agents for Combination Therapy

ABSTRACT Whole-cell assays were implemented to search for efflux pump inhibitors (EPIs) of the three multidrug resistance efflux pumps (MexAB-OprM, MexCD-OprJ, MexEF-OprN) that contribute to fluoroquinolone resistance in clinical isolates of Pseudomonas aeruginosa. Secondary assays were developed to identify lead compounds with exquisite activities as inhibitors. A broad-spectrum EPI which is active against all three known Mex efflux pumps from P. aeruginosa and their close Escherichia coli efflux pump homolog (AcrAB-TolC) was discovered. When this compound, MC-207,110, was used, the intrinsic resistance of P. aeruginosa to fluoroquinolones was decreased significantly (eightfold for levofloxacin). Acquired resistance due to the overexpression of efflux pumps was also decreased (32- to 64-fold reduction in the MIC of levofloxacin). Similarly, 32- to 64-fold reductions in MICs in the presence of MC-207,110 were observed for strains with overexpressed efflux pumps and various target mutations that confer resistance to levofloxacin (e.g., gyrA andparC). We also compared the frequencies of emergence of levofloxacin-resistant variants in the wild-type strain at four times the MIC of levofloxacin (1 μg/ml) when it was used either alone or in combination with EPI. In the case of levofloxacin alone, the frequency was ∼10−7 CFU/ml. In contrast, with an EPI, the frequency was below the level of detection (<10−11). In summary, we have demonstrated that inhibition of efflux pumps (i) decreased the level of intrinsic resistance significantly, (ii) reversed acquired resistance, and (iii) resulted in a decreased frequency of emergence of P. aeruginosa strains that are highly resistant to fluoroquinolones.

Impact of Siderophore Production on Pseudomonas aeruginosa Infections in Immunosuppressed Mice

ABSTRACT Pseudomonas aeruginosa produces siderophores, pyoverdin and pyochelin, for high-affinity iron uptake. To investigate their contribution to P. aeruginosa infections, we constructed allelic exchange mutants from strain PAO1 which were deficient in producing one or both of the siderophores. When inoculated into the calf muscles of immunosuppressed mice, pyochelin-deficient and pyoverdin-deficient mutants grew and killed the animals as efficiently as PAO1. In contrast, the pyochelin- and pyoverdin-deficient (double) mutant did not show lethal virulence, although it did infect the muscles. On the other hand, when inoculated intranasally, all mutants grew in the lungs and killed immunosuppressed mice. Compared with PAO1, however, the pyoverdin-deficient mutant and the double mutant grew poorly in the lungs, and the latter was significantly attenuated for virulence. Irrespective of the inoculation route, the pyoverdin-deficient and doubly deficient mutants detected in the blood were significantly less numerous than PAO1. Additionally, in vitro examination demonstrated that the growth of the double mutant was extremely reduced under a free-iron-restricted condition with apotransferrin but that the growth reduction was completely canceled by supplementation with hemoglobin as a heme source. These results suggest that both pyoverdin and pyochelin are required for efficient bacterial growth and full expression of virulence in P. aeruginosainfection, although pyoverdin may be comparatively more important for bacterial growth and dissemination. However, the siderophores were not always required for infection. It is possible that non-siderophore-mediated iron acquisition, such as via heme uptake, might also play an important role in P. aeruginosainfections.

Interplay between Efflux Pumps May Provide Either Additive or Multiplicative Effects on Drug Resistance

The effects of simultaneous expression of several efflux pumps on antibiotic resistance were investigated in Escherichia coli and Pseudomonas aeruginosa. Several combinations of efflux pumps have been studied: (i) simultaneous expression of a single-component efflux pump, which exports antibiotics into the periplasm, in combination with a multicomponent efflux pump that accomplishes efflux directly into the external medium; (ii) simultaneous expression of two single-component pumps; and (iii) simultaneous expression of two multicomponent pumps. It was found that when efflux pumps of different structural types were combined in the same cell (the first case), the observed antibiotic resistance was much higher than that conferred by each of the pumps expressed singly. Simultaneous expression of pairs of single-component or multicomponent efflux pumps (the second and third cases) did not produce strong increases in antibiotic resistance.

Comparison of inhibition of Escherichia coli topoisomerase IV by quinolones with DNA gyrase inhibition.

In order to examine the inhibitory activities of quinolones against topoisomerase IV, both subunits of this enzyme, ParC and ParE, were purified from Escherichia coli. The specific activity of topoisomerase IV decatenation was found to be more than five times greater than that of topoisomerase IV relaxation. Thus, the decatenation activity of topoisomerase IV seems the most relevant activity for use in studies of drug inhibition of this enzyme. Although topoisomerase IV was less sensitive to quinolones than DNA gyrase, the 50% inhibitory concentrations for decatenation were significantly lower than those for type I topoisomerases. Moreover, there was a positive correlation between the inhibitory activity against topoisomerase IV decatenation and that for DNA gyrase supercoiling. These results imply that topoisomerase IV could be a target for the quinolones in intact bacteria and that quinolones could inhibit not only supercoiling of DNA gyrase but also decatenation of topoisomerase IV when high concentrations of drug exist in bacterial cells. Images

Structural basis for the inhibition of bacterial multidrug exporters

The multidrug efflux transporter AcrB and its homologues are important in the multidrug resistance of Gram-negative pathogens. However, despite efforts to develop efflux inhibitors, clinically useful inhibitors are not available at present. Pyridopyrimidine derivatives are AcrB- and MexB-specific inhibitors that do not inhibit MexY; MexB and MexY are principal multidrug exporters in Pseudomonas aeruginosa. We have previously determined the crystal structure of AcrB in the absence and presence of antibiotics. Drugs were shown to be exported by a functionally rotating mechanism through tandem proximal and distal multisite drug-binding pockets. Here we describe the first inhibitor-bound structures of AcrB and MexB, in which these proteins are bound by a pyridopyrimidine derivative. The pyridopyrimidine derivative binds tightly to a narrow pit composed of a phenylalanine cluster located in the distal pocket and sterically hinders the functional rotation. This pit is a hydrophobic trap that branches off from the substrate-translocation channel. Phe 178 is located at the edge of this trap in AcrB and MexB and contributes to the tight binding of the inhibitor molecule through a π–π interaction with the pyridopyrimidine ring. The voluminous side chain of Trp 177 located at the corresponding position in MexY prevents inhibitor binding. The structure of the hydrophobic trap described in this study will contribute to the development of universal inhibitors of MexB and MexY in P. aeruginosa.

Antimicrobial activity of DU-6859, a new potent fluoroquinolone, against clinical isolates.

DU-6859, (-)-7-[(7S)-amino-5-azaspiro(2,4)heptan-5-yl]-8-chloro-6- fluoro-1-[(1R,2R)-cis-2-fluoro-1-cyclopropyl]-1,4-dihydro-4-oxoquinol one-3- carboxylic acid, is a new fluoroquinolone with antibacterial activity which is significantly better than those of currently available quinolones. The MICs for 90% of methicillin-susceptible and -resistant Staphylococcus aureus and Staphylococcus epidermidis clinical isolates (MIC90s) were 0.1, 3.13, 0.1, and 0.39 microgram/ml, respectively. MIC50s of DU-6859 against quinolone-resistant, methicillin-resistant S. aureus were 8-, 32-, 64-, and 128-fold lower than those of tosufloxacin and sparfloxacin, ofloxacin and fleroxacin, ciprofloxacin, and lomefloxacin, respectively. DU-6859 inhibited the growth of all strains of Streptococcus pneumoniae and Streptococcus pyogenes at 0.1 and 0.2 microgram/ml, respectively, and was more active against enterococci than the other quinolones tested. Although the activity of DU-6859 against Pseudomonas aeruginosa was roughly comparable to that of ciprofloxacin at the MIC50 level, it was fourfold more active than ciprofloxacin at the MIC90 level. DU-6859 was also more active against other glucose-nonfermenting bacteria, Haemophilus influenzae, Moraxella catarrhalis, and Neisseria gonorrhoeae, than the other drugs tested. Strains of Bacteroides fragilis and Peptostreptococcus spp. were susceptible to DU-6859; MIC90s were 0.39 and 0.2 microgram/ml, respectively. DU-6859 generally showed activities twofold or greater than those of ciprofloxacin and the other drugs against almost all members of the family Enterobacteriaceae. The action of DU-6859 against the clinical isolates was bactericidal at concentrations near the MICs. DU-6859 activity was not affected by different media, pH, inoculum size, or human serum but was decreased in human urine.

Requirement of the Pseudomonas aeruginosa tonB Gene for High-Affinity Iron Acquisition and Infection

ABSTRACT To investigate the contribution of the TonB protein to high-affinity iron acquisition in Pseudomonas aeruginosa, we constructed tonB-inactivated mutants from strain PAO1 and its derivative deficient in producing the siderophores pyoverdin and pyochelin. The tonB mutants could not grow in a free-iron-restricted medium prepared by apotransferrin addition, even though the medium was supplemented with each purified siderophore or with a heme source (hemoglobin or hemin). The tonBinactivation was shown to make P. aeruginosa unable to acquire iron from the transferrin with either siderophore. Introduction of a plasmid carrying the intact tonB gene restored growth of the tonB mutant of PAO1 in the free-iron-restricted medium without any supplements and restored growth of thetonB mutant of the siderophore-deficient derivative in the medium supplemented with pyoverdin, pyochelin, hemoglobin, or hemin. In addition, animal experiments showed that, in contrast to PAO1, thetonB mutant of PAO1 could not grow in vivo, such as in the muscles and lungs of immunosuppressed mice, and could not kill any of the animals. The in vivo growth ability and lethal virulence were also restored by introduction of the tonB-carrying plasmid in the tonB mutant. These results indicate clearly that the intact tonB gene—and, therefore, the TonB protein encoded by it—is essential for iron acquisition mediated by pyoverdin and pyochelin and via heme uptake in P. aeruginosa and suggest that the TonB-dependent iron acquisition may be essential for P. aeruginosa to infect the animal host.

Inhibitory effects of quinolones on DNA gyrase of Escherichia coli and topoisomerase II of fetal calf thymus.

The in vitro inhibitory effects of quinolones on the bacterial DNA gyrase of Escherichia coli KL-16 and topoisomerase II of fetal calf thymus were compared. All the quinolones tested required higher concentrations to inhibit the topoisomerase II than to inhibit the DNA gyrase, and no correlation existed among their inhibitory activities against both enzymes. However, there was a large difference among the quinolones in their selectivities between the bacterial enzyme and its eucaryotic counterpart. The selectivity of ofloxacin was highest, and the selectivities of CI-934 and nalidixic acid were lowest.

Contribution of the C-8 substituent of DU-6859a, a new potent fluoroquinolone, to its activity against DNA gyrase mutants of Pseudomonas aeruginosa.

Inhibitory effects of five quinolones against DNA gyrases purified from four quinolone-resistant clinical isolates of Pseudomonas aeruginosa and the quinolone-susceptible strain PAO1 were examined. All of the quinolone-resistant strains tested were found to be DNA gyrase mutants. The 50% inhibitory concentrations (IC50s) of the quinolones for these DNA gyrases roughly correlated with their MICs. Interestingly, gyrase inhibition by DU-6859a was found to be significantly less affected by these mutations that inhibition by other currently available quinolones. To assess the enhanced activity shown by DU-6859a, the effects of quinolones with altered substituents at the N-1, C-7, and C-8 positions of the quinolone ring of DU-6859a were tested. Measurement of MICs for four DNA gyrase mutants and IC50s for their purified DNA gyrases showed that removal of the C-8 chlorine of DU-6859a significantly increased MICs and IC50s for DNA gyrase mutants. However, no deleterious effects were observed when either the fluorine on the cyclopropyl substituent at the N-1 position or the cyclopropyl ring at the C-7 substituent was removed. Moreover, removal of the C-8 chlorine also increased the MIC for 19 of 20 quinolone-resistant clinical isolates. Our results led to the conclusion that DU-6859a is much more active against quinolone-resistant clinical isolates of P. aeruginosa than other currently available quinolones, probably because of its strong inhibitory effects against mutant quinolone-resistant DNA gyrases, and that the C-8 chlorine is necessary for these potent effects.

In Vitro and In Vivo Antibacterial Activities of DC-159a, a New Fluoroquinolone

ABSTRACT DC-159a is a new 8-methoxy fluoroquinolone that possesses a broad spectrum of antibacterial activity, with extended activity against gram-positive pathogens, especially streptococci and staphylococci from patients with community-acquired infections. DC-159a showed activity against Streptococcus spp. (MIC90, 0.12 μg/ml) and inhibited the growth of 90% of levofloxacin-intermediate and -resistant strains at 1 μg/ml. The MIC90s of DC-159a against Staphylococcus spp. were 0.5 μg/ml or less. Against quinolone- and methicillin-resistant Staphylococcus aureus strains, however, the MIC90 of DC-159a was 8 μg/ml. DC-159a was the most active against Enterococcus spp. (MIC90, 4 to 8 μg/ml) and was more active than the marketed fluoroquinolones, such as levofloxacin, ciprofloxacin, and moxifloxacin. The MIC90s of DC-159a against Haemophilus influenzae, Moraxella catarrhalis, and Klebsiella pneumoniae were 0.015, 0.06, and 0.25 μg/ml, respectively. The activity of DC-159a against Mycoplasma pneumoniae was eightfold more potent than that of levofloxacin. The MICs of DC-159a against Chlamydophila pneumoniae were comparable to those of moxifloxacin, and DC-159a was more potent than levofloxacin. The MIC90s of DC-159a against Peptostreptococcus spp., Clostridium difficile, and Bacteroides fragilis were 0.5, 4, and 2 μg/ml, respectively; and among the quinolones tested it showed the highest level of activity against anaerobic organisms. DC-159a demonstrated rapid bactericidal activity against quinolone-resistant Streptococcus pneumoniae strains both in vitro and in vivo. In vitro, DC-159a showed faster killing than moxifloxacin and garenoxacin. The bactericidal activity of DC-159a in a murine muscle infection model was revealed to be superior to that of moxifloxacin. These activities carried over to the in vivo efficacy in the murine pneumonia model, in which treatment with DC-159a led to bactericidal activity superior to those of the other agents tested.