Many pathogenic bacteria utilize two-component systems consisting of a histidine protein kinase (HPK) and a response regulator (RR) for signal transduction. During the search for novel inhibitors, several chemical series, including benzoxazines, benzimidazoles, bis-phenols, cyclohexenes, trityls, and salicylanilides, were identified that inhibited the purified HPK-RR pairs KinA-Spo0F and NRII-NRI, with 50% inhibitory concentrations (IC50s) ranging from 1.9 to >500 μM and MICs ranging from 0.5 to >16 μg/ml for gram-positive bacteria. However, additional observations suggested that mechanisms other than HPK inhibition might contribute to antibacterial activity. In the present work, representative compounds from the six different series of inhibitors were analyzed for their effects on membrane integrity and macromolecular synthesis. At 4× MIC, 17 of 24 compounds compromised the integrity of the bacterial cell membrane within 10 min, as measured by uptake of propidium iodide. In this set, compounds with lower IC50s tended to cause greater membrane disruption. Eleven of 12 compounds inhibited cellular incorporation of radiolabeled thymidine and uridine >97% in 5 min and amino acids >80% in 15 min. The HPK inhibitor that allowed >25% precursor incorporation had no measurable MIC (>16 μg/ml). Fifteen of 24 compounds also caused hemolysis of equine erythrocytes. Thus, the antibacterial HPK inhibitors caused a rapid decrease in cellular incorporation of RNA, DNA, and protein precursors, possibly as a result of the concomitant disruption of the cytoplasmic membrane. Bacterial killing by these HPK inhibitors may therefore be due to multiple mechanisms, independent of HPK inhibition.

The bacterial enzyme MurA catalyzes the transfer of enolpyruvate from phosphoenolpyruvate (PEP) to uridine diphospho-N-acetylglucosamine (UNAG), which is the first committed step of bacterial cell wall biosynthesis. From high-throughput screening of a chemical library, three novel inhibitors of the Escherichia coli MurA enzyme were identified: the cyclic disulfide RWJ-3981, the purine analog RWJ-140998, and the pyrazolopyrimidine RWJ-110192. When MurA was preincubated with inhibitor, followed by addition of UNAG and PEP, the 50% inhibitory concentrations (IC 50 s) were 0.2 to 0.9 M, compared to 8.8 M for the known MurA inhibitor, fosfomycin. The three compounds exhibited MICs of 4 to 32 g/ml against Staphylococcus aureus; however, the inhibition of DNA, RNA, and protein synthesis in addition to peptidoglycan synthesis by all three inhibitors indicated that antibacterial activity was not due specifically to MurA inhibition. The presence of UNAG during the MurA and inhibitor preincubation lowered the IC 50 at least fivefold, suggesting that, like fosfomycin, the three compounds may interact with the enzyme in a specific fashion that is enhanced by UNAG. Ultrafiltration and mass spectrometry experiments suggested that the compounds were tightly, but not covalently, associated with MurA. Molecular modeling studies demonstrated that the compounds could fit into the site occupied by fosfomycin; exposure of MurA to each compound reduced the labeling of MurA by tritiated fosfomycin. Taken together, the evidence indicates that these inhibitors may bind noncovalently to the MurA enzyme, at or near the site where fosfomycin binds.

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The postantibiotic subminimum inhibitory concentration effect (PA SME) may simulate in vivo drug exposure more accurately than the postantibiotic effect (PAE) since subinhibitory concentrations of drug persist between antibiotic dosings. In this study, we compared the PAEs and PA SMEs of levofloxacin and ciprofloxacin for clinical isolates of fluoroquinolone-susceptible Staphylococcus aureus and Streptococcus pneumoniae. At two times the MIC, PAEs of levofloxacin were an average of 0.6 h longer than the PAEs obtained for ciprofloxacin for methicillin-susceptible and methicillin-resistant S. aureus strains. The PAEs of levofloxacin and ciprofloxacin ranged from 1.8 to 3.1 and 1.1 to 2.4 h, respectively. Continued exposure of the methicillin-resistant strain to 1/16, 1/8, and 1/4 the MIC resulted in PA SMEs of 6.5, 15.3, and >22.3 h, respectively, for levofloxacin and 3.8, 8.0, and 12.3 h, respectively, for ciprofloxacin. For isolates of S. pneumoniae, at two times the MIC of both fluoroquinolones, the average PAEs of levofloxacin and ciprofloxacin were equivalent: 1.3 h for the penicillin-susceptible isolate and 0.6 h for the penicillin-resistant isolate. Continued exposure of the penicillin-susceptible S. pneumoniae strain to 1/16, 1/8, and 1/4 the MIC resulted in average PA SMEs of 1.0, 1.4, and 2.8 h, respectively, for levofloxacin and 1.8, 2.0, and 2.5 h, respectively, for ciprofloxacin. Continued exposure of penicillin-resistant S. pneumoniae to 1/16, 1/8, and 1/4 the MIC of the same fluoroquinolones resulted in average PA SMEs of 0.6, 1.1, and 2.9 h, respectively, for levofloxacin and 0.6, 1.1, and 1.5 h, respectively, for ciprofloxacin. The PA SMEs observed demonstrate the superior activity of levofloxacin against methicillin-susceptible or methicillin-resistant S. aureus. Although PAEs were similar for the penicillin-susceptible and penicillin-resistant S. pneumoniae strains, the PA SME of levofloxacin at one-fourth the MIC was longer for penicillin-resistant S. pneumoniae.

Oral levofloxacin was compared to oral ciprofloxacin in aStaphylococcus aureus subcutaneous abscess model in rabbits. Rabbits were surgically prepared with subcutaneous wiffle balls (43 mm in diameter) and allowed to recover for 4 to 6 weeks. Rabbits were infected by direct injection into the capsule withS. aureus ATCC 29213 (5 × 105 CFU) and were allowed to remain infected for 8 days before the initiation of anti-infective treatment. Efficacy was determined by assessing the bacterial load within the capsule over a 10-day treatment period. In single-dose pharmacokinetic studies in infected rabbits, similar area under the concentration-time curve/MIC ratios were obtained in the plasma and abscess fluid for levofloxacin at 45 mg/kg of body weight and ciprofloxacin at 200 mg/kg of body weight. Similar efficacies were seen with levofloxacin at 45 mg/kg/day and ciprofloxacin 400 mg/kg/day by day 10. In this model, levofloxacin was significantly more efficacious than ciprofloxacin (P < 0.01).

Levofloxacin, the active L-isomer of ofloxacin, has demonstrated strong activity against Staphylococcus aureus both in vitro and in vivo. In a murine model of hematogenous pyelonephritis, the in vivo efficacies of levofloxacin and ciprofloxacin were evaluated against two methicillin-susceptible and two methicillin-resistant S. aureus strains. All four isolates had virtually identical susceptibilities to levofloxacin and ciprofloxacin. Pyelonephritis was induced in carrageenan-primed mice by an intravenous injection of 0.5 ml of 10(7) CFU of methicillin-susceptible S. aureus isolates per ml or 10(8) CFU of methicillin-resistant S. aureus isolates per ml. At 1 h postinfection, the mice were treated orally with levofloxacin or ciprofloxacin once a day or twice a day (total daily dose of 20 to 160 mg/kg of body weight) for 4 days. Mice were euthanized 24 h after the final treatment, and the kidneys were excised and weighed. The kidneys were prepared for histological examination or were homogenized to determine the numbers of CFU per gram of tissue quantitatively. The reduction in the mean log10 number of CFU per gram as a function of total daily dose was recorded. A dose-response analysis showed that levofloxacin was superior to ciprofloxacin for all four isolates at any dose or regimen tested, independent of the methicillin susceptibility of the isolates. By using an inverse prediction technique, the equivalent effective doses of levofloxacin (once a day) were less than those of ciprofloxacin (twice a day) by 5.2 and 3.2 times, respectively, for methicillin-susceptible S. aureus 9039 and 3087. For methicillin-resistant S. aureus 667 and 2878, the equivalent effective doses of levofloxacin (once a day) were less than those of ciprofloxacin (twice a day) by 4.1 and 6.4 times, respectively. In a separate study, histological examination of all infected, untreated mice showed moderate to marked hematogenous pyelonephritis. Levofloxacin-treated mice (40 mg/kg once a day) showed no evidence of pyelonephritis in the kidneys, whereas the kidneys of mice treated with the same dose of ciprofloxacin showed only a reduction in the severity of the lesions. Treatment with ciprofloxacin (80 mg/kg twice a day) demonstrated a histology comparable to that of treatment with levofloxacin (40 mg/kg once a day). Levofloxacin (40 mg/kg once a day) reduced the log10 numbers of CFU per gram by 5 log10; however, ciprofloxacin (80 mg/kg twice a day) reduced the numbers of CFU per gram by only 3 log10. In the present murine model of pyelonephritis, levofloxacin was superior to ciprofloxacin in preventing pyelonephritis and eradicating S. aureus.