Masaki Hosaka

New norfloxacin resistance gene in Pseudomonas aeruginosa PAO

A new type of norfloxacin-resistant mutant of Pseudomonas aeruginosa PAO was isolated. This mutant showed cross resistance to imipenem and chloramphenicol and hypersusceptibility to beta-lactam and aminoglycoside antibiotics. The new norfloxacin resistance gene nfxC was mapped near catA (46 min) on the PAO chromosome. Norfloxacin accumulation was decreased in the nfxC mutant; furthermore, the rate of imipenem diffusion through the outer membrane of the nfxC mutant was lower than that of the parent strain. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of outer membrane proteins showed a decrease of a 46-kilodalton protein and an increase of a 50-kilodalton protein in the nfxC mutant. We conclude the nfxC is a new norfloxacin resistance gene that affects outer membrane permeability to quinolones and other antimicrobial agents. Images

In vitro and in vivo antibacterial activities of AM-1155, a new 6-fluoro-8-methoxy quinolone.

AM-1155 is a new quinolone with a wide spectrum of antibacterial activity against various bacteria including anaerobes and Mycoplasma pneumoniae. AM-1155 was 2- to 16-fold more active than ciprofloxacin and ofloxacin against Staphylococcus aureus including methicillin-resistant strains, Staphylococcus epidermidis, Streptococcus pneumoniae, and Enterococcus faecalis; its MICs for 90% of strains tested were 0.10 to 0.78 micrograms/ml. The activity of AM-1155 was comparable to that of ciprofloxacin against members of the family Enterobacteriaceae, Branhamella catarrhalis, Haemophilus influenzae, and Neisseria gonorrhoeae, but was fourfold less than that of ciprofloxacin against Pseudomonas aeruginosa. Against Xanthomonas maltophilia, Acinetobacter calcoaceticus, and Campylobacter jejuni, AM-1155 was two- to fourfold more active than ciprofloxacin. At a concentration of 1.56 micrograms/ml, AM-1155 inhibited 90% of Bacteroides fragilis strains tested; its activity was 8- to 10-fold higher than those of ofloxacin and ciprofloxacin. Development of resistance to AM-1155 in S. aureus and S. epidermidis occurred at a lower frequency than did that to ciprofloxacin after eight transfers in the presence of drug. In the oral treatment of mouse systemic infections, AM-1155 was four- to eightfold more effective than ciprofloxacin against gram-positive cocci and was as active as ciprofloxacin against gram-negative rods. The efficacy of an oral or a subcutaneous dose of AM-1155 was two- to fivefold greater than that of ofloxacin. Against experimental pneumonia with Klebsiella pneumoniae and P. aeruginosa, AM-1155 was two- to fourfold more active than ciprofloxacin and ofloxacin. AM-1155 also had good efficacy against mouse ascending urinary tract infections with Escherichia coli and P. aeruginosa. These results suggest that AM-1155 may be a potent antibacterial agent applicable to various infections.

Target Preference of 15 Quinolones against Staphylococcus aureus, Based on Antibacterial Activities and Target Inhibition

ABSTRACT The antibacterial activities and target inhibition of 15 quinolones against grlA and gyrA mutant strains were studied. The strains were obtained from wild-type Staphylococcus aureus MS5935 by selection with norfloxacin and nadifloxacin, respectively. The antibacterial activities of most quinolones against both mutant strains were lower than those against the wild-type strain. The ratios of MICs for the gyrA mutant strain to those for the grlA mutant strain (MIC ratio) varied from 0.125 to 4. The ratios of 50% inhibitory concentrations (IC50s) of quinolones against topoisomerase IV to those against DNA gyrase (IC50 ratios) also varied, from 0.177 to 5.52. A significant correlation between the MIC ratios and the IC50ratios was observed (r = 0.919; P < 0.001). These results suggest that the antibacterial activities of quinolones against the wild-type strain are involved not only in topoisomerase IV inhibition but also in DNA gyrase inhibition and that the target preference in the wild-type strain can be anticipated by the MIC ratios. Based on the MIC ratios, the quinolones were classified into three categories. Type I quinolones (norfloxacin, enoxacin, fleroxacin, ciprofloxacin, lomefloxacin, trovafloxacin, grepafloxacin, ofloxacin, and levofloxacin) had MIC ratios of <1, type II quinolones (sparfloxacin and nadifloxacin) had MIC ratios of >1, and type III quinolones (gatifloxacin, pazufloxacin, moxifloxacin, and clinafloxacin) had MIC ratios of 1. Type I and type II quinolones seem to prefer topoisomerase IV and DNA gyrase, respectively. Type III quinolones seem to target both enzymes at nearly the same level in bacterial cells (a phenomenon known as the dual-targeting property), and their IC50 ratios were approximately 2.

nfxC-type quinolone resistance in a clinical isolate of Pseudomonas aeruginosa.

Quinolone resistance gene nqr-T91 in a clinical isolate of Pseudomonas aeruginosa P1481 was cotransducible with catA1 in P. aeruginosa PAO. The nqr-T91 transductant, PKH-T91, was resistant to norfloxacin, imipenem, and chloramphenicol and showed less norfloxacin accumulation than the parent strain did. Loss of the 46-kDa outer membrane protein (D2) and an increase in the 50-kDa outer membrane protein in PKH-T91 were observed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Lipopolysaccharides in the transductant were also changed. These alterations were considered to be related to lower levels of norfloxacin accumulation in PKH-T91. These genetic and biochemical properties suggested that an nfxC type of quinolone-resistant mutation occurred in a clinical isolate of P. aeruginosa P1481.

Contributions of the 8-methoxy group of gatifloxacin to resistance selectivity, target preference, and antibacterial activity against Streptococcus pneumoniae

ABSTRACT Gatifloxacin (8-methoxy, 7-piperazinyl-3′-methyl) at the MIC selected mutant strains that possessed gyrA mutations at a low frequency (3.7 × 10−9) from wild-type strainStreptococcus pneumoniae IID553. AM-1147 (8-methoxy, 7-piperazinyl-3′-H) at the MIC or higher concentrations selected no mutant strains. On the other hand, the respective 8-H counterparts of these two compounds, AM-1121 (8-H, 7-piperazinyl-3′-methyl) and ciprofloxacin (8-H, 7-piperazinyl-3′-H), at one and two times the MIC selected mutant strains that possessed parC mutations at a high frequency (>2.4 × 10−6). The MIC of AM-1147 increased for the gyrA mutant strains but not for theparC mutant strains compared with that for the wild-type strain. These results suggest that fluoroquinolones that harbor 8-methoxy groups select mutant strains less frequently and prefer DNA gyrase, as distinct from their 8-H counterparts. The in vitro activities of gatifloxacin and AM-1147 are twofold higher against the wild-type strain, eight- and twofold higher against the first-stepparC and gyrA mutant strains, respectively, and two- to eightfold higher against the second-step gyrA andparC double mutant strains than those of their 8-H counterparts. These results indicate that the 8-methoxy group contributes to enhancement of antibacterial activity against target-altered mutant strains as well as the wild-type strain. It is hypothesized that the 8-methoxy group of gatifloxacin increases the level of target inhibition, especially against DNA gyrase, so that it is nearly the same as that for topoisomerase IV inhibition in the bacterial cell, leading to potent antibacterial activity and a low level of resistance selectivity.

Contribution of the 8-Methoxy Group to the Activity of Gatifloxacin against Type II Topoisomerases of Streptococcus pneumoniae

ABSTRACT The inhibitory activities (50% inhibitory concentrations [IC50s]) of gatifloxacin and other quinolones against both DNA gyrase and topoisomerase IV of the wild-type Streptococcus pneumoniae IID553 were determined. The IC50s of 10 compounds ranged from 4.28 to 582 μg/ml against DNA gyrase and from 1.90 to 35.2 μg/ml against topoisomerase IV. The inhibitory activity against DNA gyrase was more varied than that against topoisomerase IV among fluoroquinolones. The IC50s for DNA gyrase of the 8-methoxy quinolones gatifloxacin and AM-1147 were approximately seven times lower than those of their 8-H counterparts AM-1121 and ciprofloxacin, whereas the IC50s for topoisomerase IV were 1.5 times lower. Moreover, the IC50 ratios (IC50 for DNA gyrase/IC50 for topoisomerase IV) of gatifloxacin, AM-1147, and moxifloxacin, which possess 8-methoxy groups, were almost the same. The 8-methoxy quinolones showed higher antibacterial activity and less mutant selectivity against IID553 than their 8-H counterparts. These results suggest that the 8-methoxy group enhances both target inhibition, especially for DNA gyrase, leading to potent antipneumococcal activity and dual inhibition against both DNA gyrase and topoisomerase IV in the bacterial cell.

Contribution of the C-8-Methoxy Group of Gatifloxacin to Inhibition of Type II Topoisomerases of Staphylococcus aureus

Gatifloxacin is a recently marketed quinolone with an enhanced activity against gram-positive bacteria compared with that of ciprofloxacin (4, 12, 13, 18). The structural differences between gatifloxacin and ciprofloxacin are substituents at the C-8 position of the quinolone nucleus (gatifloxacin, C-8-methoxy; ciprofloxacin, C-8-H) and at the C-3′ position of the piperazinyl moiety of the C-7 position (gatifloxacin, C-3′-methyl; ciprofloxacin, C-3′-H). Several studies have directed attention to substituents at the C-8 position of the quinolone nucleus (1, 2, 5-10, 14, 15, 19, 20). Introduction of the methoxy group at the C-8 position was thought to ensure potent activity against gram-positive bacteria (2, 6, 9, 20). However, there have been few studies on the effect of C-8 substituents against type II topoisomerases. Thus, to investigate the effect of the C-8 and C-3′ substituents of gatifloxacin against type II topoisomerases in Staphylococcus aureus, the activities of the quinolones AM-1121 (C-8-H, C-3′-methyl) and AM-1147 (C-8-methoxy, C-3′-H), which are structurally related to gatifloxacin,​gatifloxacin, were compared with those of gatifloxacin and ciprofloxacin. TABLE 1. Target inhibition and antibacterial activity of gatifloxacin and its related compounds The quinolones tested were synthesized in-house. The bacterial strain used in this study was the quinolone-susceptible clinical isolate S. aureus MS5935 (3). MICs were determined by using agar dilution methodology according to NCCLS guidelines (11). The GyrA and GyrB subunits of DNA gyrase and the GrlA and GrlB subunits of topoisomerase IV of MS5935 were prepared by a method described previously (16, 17). The activities of each enzyme were determined by a method described previously (16, 17). The effects of the quinolones were evaluated based on the concentrations required to inhibit 50% of the enzyme reaction (IC50s). The IC50s of C-8-methoxy compounds (gatifloxacin and AM-1147) against topoisomerase IV were almost equal to those of their C-8-H counterparts (AM-1121 and ciprofloxacin), whereas the inhibitory activities of C-8-methoxy compounds against DNA gyrase were about six times higher than those of their C-8-H counterparts (Table ​(Table1;1; Fig. ​Fig.1).1). The C-3′-methyl moiety had little effect on the inhibitory activity against both DNA gyrase and topoisomerase IV. These results indicate that introduction of the C-8-methoxy group to the quinolone ring enhances the inhibitory activity against DNA gyrase without exerting influence on topoisomerase IV inhibition. FIG. 1. Inhibitory activities of gatifloxacin and its related compounds against topoisomerase IV (A) and DNA gyrase (B). The C-8-methoxy compounds showed two- to fourfold-lower MICs than their C-8-H counterparts. On the other hand, the antibacterial activities of C-3′-methyl compounds were similar or slightly superior to those of their C-3′-H counterparts. These results indicate that the C-8-methoxy group of these compounds has a greater effect on the increment of the activity against the wild-type strain, whereas the C-3′-methyl group is slightly involved in the increment of this activity. It was previously reported that the inhibition of both target enzymes contributes to the antibacterial activities of quinolones because the antibacterial activities of most quinolones were decreased by both the grlA and gyrA mutations (17). Therefore, it is suggested that an increase in the inhibitory activity of the C-8-methoxy compounds against DNA gyrase is the possible mechanism underlying their potent antibacterial activity. In summary, introduction of the methoxy group at the C-8position of the quinolone nucleus enhances inhibitory activity against DNA gyrase, leading to the potent activity of gatifloxacin against S. aureus.

Bactericidal activity of gatifloxacin (AM-1155) against Pseudomonas aeruginosa and Enterococcus faecalis in an in vitro bladder model simulating human urinary concentrations after oral administration

The bactericidal activity of gatifloxacin, a new 6-fluoro-8-methoxy quinolone, was determined in a dynamic in vitro model mimicking complicated lower urinary tract infection. Strains of Pseudomonas aeruginosa and Enterococcus faecalis with different susceptibility were exposed to changing gatifloxacin concentrations, simulating human urinary concentrations afer oral treatment with 200 mg twice daily for 3 consecutive days. Bacterial numbers of P. aeruginosa (minimal inhibitory concentrations, MIC: ≤ 32 μg/ml) and of E. faecalis (MIC: 16 μg/ml) were reduced to undetectable levels during exposure. For the strains with lower susceptibility, gatifloxacin showed bactericidal activity, but eradication was not complete. Thus, in a complicated urinary tract infection model, breakpoint MICs of gatifloxacin for uropathogenic organisms were presumed to range from 16 to 32 μg/ml. At least 86% of recent clinical isolates of P. aeruginosa and E. faecalis were inhibited at its breakpoint MIC. These results suggest that gatifloxacin may be useful in the treatment of urinary tract infections.

Uptake and Intracellular Activity of Fleroxacin in Phagocytic Cells

The uptake and intracellular activity of fleroxacin in murine J774.1 macrophages and human polymorphonuclear leukocytes were studied. The uptake of fleroxacin by J774.1 macrophages was rapid and reversible. The cellular to extracellular concentration ratios of fleroxacin in both types of phagocytes ranged from 5 to 6. These ratios were almost equal to those of ofloxacin and ciprofloxacin, and higher than those of the beta-lactam antibiotics, flomoxef and piperacillin. The intracellular activity of fleroxacin in J774.1 macrophages, examined with Staphylococcus aurenus as a test bacterium, showed that its bactericidal action was dependent on both the extracellular concentration and the exposure time. Fleroxacin reduced the number of viable cells of ingested S. aureus at an extracellular concentration that simulated the clinical serum levels, that is, killing more than 70% of the bacteria at 4 micrograms/ml. The bactericidal activity of fleroxacin in phagocytes was superior to that of erythromycin, flomoxef and piperacillin. These results indicate that fleroxacin is taken up well by phagocytes, reaching a concentration several fold higher than the extracellular concentration, and that it has potent activity against intracellular pathogens.