The mechanism of action of the fungicidal peptide auristatin PHE was investigated in Cryptococcus neoformans. Since auristatin PHE causes budding arrest in C. neoformans (T. Woyke, G. R. Pettit, G. Winkelmann, and R. K. Pettit, Antimicrob. Agents Chemother. 45:3580-3584, 2001), microtubule integrity and nuclear localization in auristatin PHE-treated cells were examined. Iterative deconvolution in conjunction with an optimized C. neoformans microtubule immunolabeling procedure enabled detailed visualization of the microtubule cytoskeleton in auristatin PHE-treated C. neoformans. The effect of auristatin PHE on C. neoformans microtubule organization was compared with that of the tubulin-binding agent nocodazole. Both drugs produced complete disruption first of cytoplasmic and then of spindle microtubules in a time- and concentration-dependent manner. Sub-MICs of auristatin PHE caused complete microtubule disruption within 4.5 h, while 1.5 times the nocodazole MIC was required for the same effect. For both drugs, disruption of microtubules was accompanied by blockage of nuclear migration and of nuclear and cellular division, resulting in cells arrested in a uninucleate, large-budded stage. Nocodazole and the linear peptide auristatin PHE are remarkably different in structure and spectrum of activity, yet on the cellular level, they have similar effects.
In a previous study, zidovudine (ZDV) was shown to cause a concentration-dependent inhibition of antigen-specific cytotoxic T-lymphocyte (CTL) clonal expansion (S. Francke, C. G. Orosz, K. A. Hayes, and L. E. Mathes, Antimicrob. Agents Chemother. 44:1900-1905, 2000). However, this suppressive effect was lost if exposure to ZDV was delayed for 24 to 48 h during the antigen sensitization period, suggesting that antigen-primed CTL may be less susceptible than naive T lymphocytes to the suppressive effects of ZDV. The present study was undertaken to determine if naive T lymphocytes were more sensitive to the suppressive effects of ZDV than T lymphocytes previously exposed to antigen. The 50% inhibitory concentration (IC50) values of ZDV were determined on naive and antigen-primed T-cell responses in an alloantigen system. Lymphocyte cultures with continuous antigen exposure (double prime) were more resistant to ZDV suppression (IC50 = 316 μM) than were naive lymphocytes (IC50 = 87.5 μM). Interestingly, lymphocytes that were antigen primed but deprived of antigen during the final 7 days of culture (prime/hold) were exquisitely sensitive to ZDV suppression (IC50 = 29.3 μM). The addition of 80 μM ZDV during the initial priming of the single-prime (prime/hold) and double-prime cultures did not select for a more drug-resistant cell population. The differences in ZDV sensitivities are likely a reflection of the physiological properties of the lymphocytes related to their activation state.
The prevalence of antimicrobial resistance among 4,940 U.S. pneumococcal isolates collected during 1999 was as follows: penicillin, 16.2%; amoxicillin-clavulanate, 12.2%; cefuroxime, 28.1%; ceftriaxone, 3.6%; trimethoprim-sulfamethoxazole, 30.3%; azithromycin, 21.4%; levofloxacin, 0.6%; and moxifloxacin, 0.1%. Compared to the previous 1997-1998 study (Jones et al., Antimicrob. Agents Chemother. 44:2645-2652, 2000), increases were noted for resistance to penicillin (3.7%; P < 0.001), amoxicillin-clavulanate (3.9%; P < 0.001), cefuroxime (5.7%; P < 0.001), azithromycin (2.4%; P = 0.014), trimethoprim-sulfamethoxazole (15.4%; P < 0.001), and levofloxacin (0.3%; P = 0.017). Resistance to ceftriaxone (0.1%; P = 0.809) and moxifloxacin (0.03%; P = 0.570) decreased. Concurrently, multidrug resistance increased (P < 0.001) from 6.3% to 11.3%.
In previous studies, we demonstrated the leishmanicide effect of coronaridine, a natural indole alkaloid isolated from stem bark of Peschiera australis (Delorenzi et al., Antimicrob. Agents Chemother. 45:1349-1354, 2001). In this study we show the leishmanicidal effect of the synthetic coronaridine and its racemic 18-methoxylated analog, 18-methoxycoronaridine. Both alkaloids revealed a potent leishmanicide effect against Leishmania amazonensis, a causative agent of cutaneous and diffuse cutaneous leishmaniasis in the New World. Despite their potent leishmanicide effect, both alkaloids were neither toxic to murine macrophages nor did they modulate their oxidative or cytokine production responses.
Mechanisms of resistance were studied in 22 macrolide-resistant mutants selected in vitro from 5 parental strains of macrolide-susceptible Streptococcus pneumoniae by serial passage in various macrolides (T. A. Davies, B. E. Dewasse, M. R. Jacobs, and P. C. Appelbaum, Antimicrob. Agents Chemother., 44:414–417, 2000). Portions of genes encoding ribosomal proteins L22 and L4 and 23S rRNA (domains II and V) were amplified by PCR and analyzed by single-strand conformational polymorphism analysis to screen for mutations. The DNA sequences of amplicons from mutants that differed from those of parental strains by their electrophoretic migration profiles were determined. In six mutants, point mutations were detected in the L22 gene (G95D, P99Q, A93E, P91S, and G83E). The only mutant selected by telithromycin (for which the MIC increased from 0.008 to 0.25 μg/ml) contained a combination of three mutations in the L22 gene (A93E, P91S, and G83E). L22 mutations were combined with an L4 mutation (G71R) in one strain and with a 23S rRNA mutation (C2611A) in another strain. Nine other strains selected by various macrolides had A2058G (n = 1), A2058U (n = 2), A2059G (n = 1), C2610U (n = 1), and C2611U (n = 4) mutations (Escherichia coli numbering) in domain V of 23S rRNA. One mutant selected by clarithromycin and resistant to all macrolides tested (MIC, >32 μg/ml) and telithromycin (MIC, 4 μg/ml) had a single base deletion (A752) in domain II. In six remaining mutants, no mutations in L22, L4, or 23S rRNA could be detected.
The recent emergence of methicillin-resistant Staphylococcus aureus (MRSA) with decreased susceptibility to vancomycin has intensified the search for alternative therapies for the treatment of infections caused by this organism. One approach has been to identify a β-lactam with improved affinity for PBP 2a, the target enzyme responsible for methicillin resistance in staphylococci. BMS-247243 is such a candidate, with MICs that inhibit 90% of isolates tested (MIC90s) of 4, 2, and 8 μg/ml for methicillin-resistant strains of S. aureus, S. epidermidis, and S. haemolyticus, respectively, as determined on plates with Mueller-Hinton agar and 2% NaCl. The BMS-247243 MICs for MRSA were minimally affected by the susceptibility testing conditions (inoculum size, prolonged incubation, addition of salt to the test medium) or by staphylococcal β-lactamases. BMS-247243 MIC90s for methicillin-susceptible staphylococcal species ranged from ≤0.25 to 1 μg/ml. The BMS-247243 MIC90 for β-lactamase-producing S. aureus strains was fourfold higher than that for β-lactamase-nonproducing strains. BMS-247243 is hydrolyzed by staphylococccal β-lactamases at 4.5 to 26.2% of the rates measured for cephaloridine. The affinity of BMS-247243 for PBP 2a was >100-fold better than that of methicillin or cefotaxime. BMS-247243 is bactericidal for MRSA, killing the bacteria twice as fast as vancomycin. These in vitro activities of BMS-247243 correlated with its in vivo efficacy against infections in animals, including the neutropenic murine thigh and rabbit endocarditis models involving MRSA strains. In conclusion, BMS-247243 has in vitro and in vivo activities against methicillin-resistant staphylococci and thus may prove to be useful in the treatment of infections caused by these multidrug-resistant organisms.
In this study, the safety, tolerability, and pharmacokinetics of intravenous (i.v.)- to oral-dose regimens of voriconazole were evaluated with a group of 42 healthy men, 41 of whom completed the study. Two cohorts of subjects participated in the study. Cohort 1 (n = 28) took part in two study periods, each consisting of 14 days separated by a minimum 7-day washout. In one of the periods, 14 subjects received 6 mg/kg i.v. twice a day (b.i.d.) on day 1 followed by 3 mg/kg i.v. b.i.d. on days 2 to 7 and were then switched to 200 mg orally b.i.d. for days 8 to 14. In the other period, subjects received 6 mg/kg i.v. b.i.d. on day 1 followed by 5 mg/kg i.v. b.i.d. on days 2 to 7and were then switched to 400 mg orally b.i.d. for days 8 to 14. The remaining 14 subjects in cohort 1 received a matching placebo throughout the study. In cohort 2 (n = 14), 7 subjects received 6 mg/kg i.v. b.i.d. on day 1 followed by 4 mg/kg i.v. b.i.d. on days 2 to 7 and were then switched to 300 mg orally b.i.d. for days 8 to 14. The remaining seven subjects in cohort 2 received a matching placebo. Blood samples were taken prior to dosing on days 1 to 6 and on days 8 to 13. Blood samples were drawn prior to dosing and at frequent intervals up to 12 h following the morning dose on days 7 and 14 of each study period. The samples were assayed for voriconazole by a high-performance liquid chromatography method. The maximum concentration in plasma (Cmax) occurred at the end of the 1-h i.v. infusion and between 1.4 and 1.8 h after oral administration. Voriconazole exhibited nonlinear pharmacokinetics, possibly due to saturable metabolism. For cohort 1, both Cmax and the area under the concentration-time curve within a dosage interval (AUCτ) increased disproportionately with dose for both i.v. and oral dosing. For i.v. dosing, a 1.7-fold increase in dose resulted in 2.4- and 3.1-fold increases in Cmax and AUCτ, respectively. Similarly, a 2-fold increase in oral dosing resulted in 2.8- and 3.9-fold increases in Cmax and AUCτ, respectively. The mean values for Cmax observed following oral dosing were lower than those obtained after i.v. administration, ranging from 62.7 to 89.6% of the i.v. value. After the switch from i.v. to oral dosing, most subjects achieved steady state by day 4, and mean minimum concentrations in plasma remained above clinically important MICs. The pharmacokinetic profiles for saliva followed a pattern similar to those observed for plasma; there was a highly significant correlation between plasma and saliva voriconazole concentrations (P < 0.0001). Voriconazole was well tolerated; the most commonly reported adverse events in voriconazole-treated subjects were mild to moderate headache, rash, and abnormal vision. Visual function tests detected no further abnormalities during voriconazole treatment.
The activity of a new oxazolidinone antimicrobial, AZD2563, was assessed against >500 clinical isolates of enterococci representing six species. All isolates, including those resistant to other antibiotic classes, were inhibited by AZD2563 at concentrations ≤2 μg/ml, except for four strains resistant to linezolid. In most cases, AZD2563 was twofold more active than linezolid against enterococci.
Ertapenem (INVANZ) is a new once-a-day parenteral β-lactam antimicrobial shown to be effective as a single agent for treatment of various community-acquired and mixed infections. The single- and multiple-dose pharmacokinetics of ertapenem at doses up to 3 g were examined in healthy young men and women volunteers. Plasma and urine samples collected were analyzed using reversed-phase high-performance liquid chromatography with UV detection. Ertapenem is highly bound to plasma protein. The protein binding changes from ∼95% bound at concentrations of <50 μg/ml to ∼92% bound at concentrations of 150 μg/ml (concentration at the end of a 30-min infusion following the 1-g dose). The nonlinear protein binding of ertapenem resulted in a slightly less than dose proportional increase in the area under the curve from 0 h to infinity (AUC0-∞) of total ertapenem. The single-dose AUC0-∞ of unbound ertapenem was nearly dose proportional over the dose range of 0.5 to 2 g. The mean concentration of ertapenem in plasma ranged from ∼145 to 175 μg/ml at the end of a 30-min infusion, from ∼30 to 34 μg/ml at 6 h, and from ∼9 to 11 μg/ml at 12 h. The mean plasma t1/2 ranged from 3.8 to 4.4 h. About 45% of the plasma clearance (CLP) was via renal clearance. The remainder of the CLP was primarily via the formation of the β-lactam ring-opened metabolite that was excreted in urine. There were no clinically significant differences between the pharmacokinetics of ertapenem in men and women. Ertapenem does not accumulate after multiple once-daily dosing.
We did not observe signs of chondrotoxicity in immature rats treated orally with garenoxacin (BMS-284756) at doses up to five times 600 mg/kg of body weight or with ciprofloxacin, whereas ofloxacin induced typical cartilage lesions. The peak plasma garenoxacin concentration was 25.5 mg/liter after administration of a dose of 600 mg/kg once daily for 5 days. Assuming that this model is predictive of human risk, BMS-284756 and ciprofloxacin should be more suitable for pediatric use than ofloxacin.
Cethromycin (ABT-773), a new ketolide, possesses potent in vitro activity against Streptococcus pneumoniae. The objective of this study was to investigate the in vivo bactericidal activity of cethromycin against macrolide-susceptible and -resistant S. pneumoniae in a murine pneumonia model and to describe the pharmacodynamic (PD) profile of cethromycin. Eight (two macrolide susceptible, six macrolide resistant) clinical isolates of S. pneumoniae were investigated. Cyclophosphamide administration rendered ICR mice transiently neutropenic prior to intratracheal inoculation with 0.05 ml of an S. pneumoniae suspension containing 107 to 108 CFU/ml. Oral cethromycin was initiated 12 to 14 h postinoculation over a dosage range of 0.1 to 800 mg/kg of body weight/day. Lungs from seven to eight mice per treatment and control groups were collected at 0 and 24 h posttherapy to assess bacterial density. The cumulative mortality (n = 12 to 13) was assessed at 120 h (end of therapy) and at 192 h (3 days posttherapy). Recovery of pneumococci from the lungs of infected animals prior to the initiation of therapy ranged from 4.6 to 7.2 log10 CFU. Growth in untreated control animals over a 24-h study period increased 0.3 to 2.7 log10 CFU. Cethromycin demonstrated a substantial bactericidal effect, regardless of macrolide susceptibility. Correlation between changes in bacterial density (24 h) and survival over both 120 and 192 h were statistically significant. All three PD parameters demonstrated a significant correlation with changes in log10 CFU/lung (Spearman's correlation coefficient, P < 0.001); however, the goodness of fit as assessed with the maximum effect (Emax) model revealed that the maximum concentration of free drug in serum (Cmax free)/MIC and the area under the free drug concentration-time curve (AUCfree)/MIC best explained the relationship between drug exposure and reductions in viable bacterial counts. These data reveal that an approximate cethromycin AUCfree/MIC of 50 or Cmax free/MIC of 1 results in bacteriostatic effects, while higher values (twofold) maximize survival.
We compared the disk diffusion and broth microdilution methods for susceptibility testing of caspofungin against Aspergillus (n = 78) and Fusarium (n = 22) isolates. Microdilution testing followed the NCCLS M-38P guidelines but was performed in antibiotic medium 3 supplemented to 2% glucose (AM3). Disk diffusion assays were performed on AM3 agar plates with a 2-μg caspofungin disk. By both methods, caspofungin showed favorable activity against Aspergillus isolates and no activity against Fusarium isolates. In the disk-based format, intrazonal growth that was not influenced by the drug concentration gradient was consistently observed for all of the Aspergillus isolates tested.
Streptococcus pyogenes BM4478 and Staphylococcus aureus BM4479 were isolated from a patient undergoing rifampin therapy. High-level resistance to rifampin was due to the following mutations in the rpoB gene: Ser522Leu in strain BM4478 and His526Asn and Ser574Leu in strain BM4479.