Comparison of the bactericidal activities and post-antibiotic effects of the Des-F(6)-quinolone BMS-284756, levofloxacin, and ciprofloxacin against methicillin-susceptible and methicillin-resistant Staphylococcus aureus.

The bactericidal activities and post-antibiotic effects of BMS-284756 (T-3811ME), levofloxacin, and ciprofloxacin were evaluated against a methicillin-susceptible and a methicillin-resistant Staphylococcus aureus strain. Minimum inhibitory concentrations (MICs), minimum bactericidal concentrations, post-antibiotic effects, and post-antibiotic sub-MIC effects were determined and time-kill studies were performed for BMS-284756, levofloxacin, and ciprofloxacin. At 4-times and 10-times the MIC, time-kill kinetics over 3 h and over 24 h were similar for all three quinolones when effects were considered as multiples of the MIC. All three quinolones achieved a 3 log10 reduction in cfu/ml within 2 h. At 10-times the MIC, the post-antibiotic effects of BMS-284756, levofloxacin, and ciprofloxacin were 1.6-2.6 h for the methicillin-susceptible Staphylococcus aureus strain and 1.5-1.9 h for the methicillin-resistant Staphylococcus aureus strain. When actual concentrations were considered, BMS-284756 achieved results comparable to levofloxacin and ciprofloxacin at concentrations nearly 10-fold less. When relating the pharmacokinetic properties of the three quinolones to their in vitro activities, the resulting Cmax/MIC and AUC/MIC ratios were. respectively, 120-240.7 and 1,321.7-2,643 for BMS-284756, 22.8 and 190 for levofloxacin, and 5.9-11.9 and 54.8-109.6 for ciprofloxacin. The greater in vitro activity and favorable human pharmacokinetics of BMS-284756 may translate to improved clinical effectiveness of this agent compared to currently marketed quinolones.

Comparison of efficacies of oral levofloxacin and oral ciprofloxacin in a rabbit model of a staphylococcal abscess.

Oral levofloxacin was compared to oral ciprofloxacin in a Staphylococcus 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 with S. aureus ATCC 29213 (5 x 10(5) 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).

39th Interscience Conference on Antimicrobial Agents and Chemotherapy of the American Society for Microbiology.

The 39th ASM ICAAC Meeting, attended by over 16,000 delegates, highlighted numerous late-stage development antibacterials. The presentation of over 600 reports (posters, symposia and oral presentations) regarding resistance emergence underscores the evolving landscape of antibiotic susceptibility worldwide. Although bacterial genomic efforts are slowly expanding the potential sources of novel targets, the established antibacterial classes, including quinolones, beta-lactams, protein synthesis inhibitors and carbapenems, were most prevalent among the presentations. Quinolones dominated this conference, with many presentations of moxifloxacin (> 72 presentations), gatifloxacin (> 66 presentations) and gemifloxacin (> 59 presentations). The new class of antibacterials, the oxazolidinones, represented by Linezolid, was also highlighted.

Antibacterial agents that inhibit two-component signal transduction systems.

A class of antibacterials has been discovered that inhibits the growth of Gram-positive pathogenic bacteria. RWJ-49815, a representative of a family of hydrophobic tyramines, in addition to being a potent bactericidal Gram-positive antibacterial, inhibits the autophosphorylation of kinase A of the KinA::Spo0F two-component signal transduction system in vitro. Analogs of RWJ-49815 vary greatly in their ability to inhibit growth of bacteria and this ability correlates directly with their activity as kinase A inhibitors. Compared with the potent quinolone, ciprofloxacin, RWJ-49815 exhibits reduced resistance emergence in a laboratory passage experiment. Inhibition of the histidine protein kinase::response regulator two-component signal transduction pathways may present an opportunity to depress chromosomal resistance emergence by targeting multiple proteins with a single inhibitor in a single bacterium. Such inhibitors may represent a class of antibacterials that potentially may represent a breakthrough in antibacterial therapy.

Importance of antifungal drug-resistance: clinical significance and need for novel therapy.

The incidence of fungal infections has increased dramatically over the past few decades due to the increase in the members of the population susceptible to such infections. This population includes individuals undergoing chemotherapy for cancer, those enduring long-term treatment with antibacterial agents, those receiving immunosuppressive drugs following transplantations, or those immunosuppressed due to diseases, such as AIDS, or malignancies. Newer antifungal agents, namely the triazoles, have aided in both the treatment of fungal infections and in the prevention of disease in susceptible individuals. However, resistance to the azoles, as well as to the polyenes, has resulted in clinical failures. Only a few potential antifungal targets have been exploited to date and there is a critical need for the discovery and development of novel antifungal agents that will result in improved therapy in this ever-expanding patient population. An increased intensity in the study of fungal pathogens at the molecular level holds the key to such advances.

98th General Meeting of the American Society for Microbiology. May 17-21, 1998; Atlanta, GA.

The 98th General Meeting of the American Society for Microbiology was held from May 17-21, 1998, in Atlanta, Georgia and was attended by well over 10,000 scientists. The theme of antibiotic resistance dominated the meeting with numerous presentations on resistance mechanisms, new targets and potential antimicrobial agents. Many new insights into the understanding of microbial physiology were provided. Microbial genomics was shown to be revolutionizing the way in which scientists can probe and explore bacteria and fungi.

The 97th Annual Meeting of the American Society for Microbiology.

The Annual Meeting of the American Society for Microbiology took place in Miami Beach, Florida, from May 4-8, 1997. Over 9000 scientists attended this meeting, which covers all major aspects of prokaryotic research (basic, applied, medical, and diagnostic). Genomics discussions were a major part of the meeting agenda, with scientists detailing both basic and applied research effort using genomics and bioinformatics. New ideas for potential novel antimicrobials have also surfaced as the tools to pursue Drug Discovery have fallen into place and pharmaceutical companies have ;rediscovered' anti-infectives.

Comparison of the postantibiotic and postantibiotic sub-MIC effects of levofloxacin and ciprofloxacin on Staphylococcus aureus and Streptococcus pneumoniae.

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.

Assessment of the synergistic interactions of levofloxacin and ampicillin against Enterococcus faecium by the checkerboard agar dilution and time-kill methods.

Multidrug-resistant enterococci have become increasingly difficult to eradicate in a growing number of nosocomial infections. With the emergence of vancomycin-resistant enterococci, the use of synergistic antibiotic combinations has become one of the only remaining therapeutic options. Levofloxacin, the active l-isomer of ofloxacin, is a new oral and intravenous fluoroquinolone with a broad spectrum of activity against numerous Gram-positive, Gram-negative, and atypical organisms. The in vitro activity of levofloxacin, alone and in combination with ampicillin, against recent clinical isolates of Enterococcus faecium was assessed for synergistic interactions using the checkerboard agar dilution technique and time-kill methodology. Against all strains, the static technique of checkerboard agar dilution demonstrated indifferent or additive effects for the ampicillin + levofloxacin combination. With the dynamic time-kill technique, synergy was demonstrated for ampicillin (16 micrograms/ml) + levofloxacin (2 micrograms/ml) combination against three levofloxacin-sensitive, ampicillin-resistant isolates. At 24 h, the combination yielded a > or = 2-log10 decrease in CFU/ml compared to levofloxacin alone, while ampicillin had negligible effects. Against both a levofloxacin-intermediate, ampicillin-resistant isolate, and a highly levofloxacin-resistant, ampicillin-resistant isolate, none of the ampicillin+levofloxacin combinations tested demonstrated a synergistic interaction. The time-kill method suggested synergy for the ampicillin+levofloxacin combination against some strains of E. faecium.

Interscience Conference on Antimicrobial Agents and Chemotherapy (ICAAC).

Over 10,000 research scientists attended the ICAAC meeting in Toronto, Canada. The continued increase in the rate of resistance in bacteria and fungi and the underlying mechanisms of resistance were the focus of many presentations this year. So, too, were the attempts to combat this emerging resistance crisis. Many papers described new modifications to older antimicrobials, novel classes of antimicrobials and the potential use of new antibacterial and antifungal targets.

In vivo oral efficacy of levofloxacin for treatment of systemic Pseudomonas aeruginosa infections in a murine model of septicemia.

The in vivo efficacies of levofloxacin and ciprofloxacin in lethal, systemic Pseudomonas aeruginosa infections in mice were compared. MICs of levofloxacin and ciprofloxacin ranged from 0.5 to 2.0 micrograms/ml and from 0.12 to 1.0 microgram/ml respectively. Infecting doses ranged from 5.0 x 10(1) to 3.2 x 10(3) CFU per mouse, depending on the isolate. Test fluoroquinolones were administered orally at 1 h (single dose) or at 1 and 3 h (divided dose) postinfection, with 10 infected mice used for each of six concentrations of each fluoroquinolone tested (1 to 40 mg/kg of body weight) in each dosing regimen. Whether given in a single or a divided dose, the total daily dose was the same for each fluoroquinolone. For mice treated 1 h postinfection with levofloxacin and ciprofloxacin, the effective doses for 50% of the infected mice ranged from 2.09 to 13.80 mg/kg and from 2.34 to 11.22 mg/kg, respectively, and for those treated 1 and 3 h postinfection, the effective doses for 50% of the infected mice ranged from 3.71 to 16.98 mg/kg and from 2.95 to 13.18 mg/kg, respectively. Although the potency varied for both levofloxacin and ciprofloxacin among all strains of P. aeruginosa tested, there were small differences within the same strain for levofloxacin and ciprofloxacin when given in the same dosing regimen. Levofloxacin proved nearly as effective as ciprofloxacin against a systemic P. aeruginosa infection in mice.

In vivo efficacies of levofloxacin and ciprofloxacin in acute murine hematogenous pyelonephritis induced by methicillin-susceptible and-resistant Staphylococcus aureus strains.

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.

The effect of elastase-specific monoclonal and polyclonal antibodies on the virulence of Aspergillus fumigatus in immunocompromised mice.

Elastase has been implicated as a potential virulence factor involved in the invasion process of the opportunistic pathogen, Aspergillus fumigatus. Monoclonal and polyclonal antibodies, known to inhibit elastase in vitro, were employed in an immunocompromised mouse model of invasive aspergillosis to determine if the antibodies could protect mice from fatal infection. Individual monoclonal antibodies, known to inhibit elastase partially (13 to 23%), or combinations of monoclonal antibodies, known to inhibit elastase 70 to 100%, were tested in the mouse model. No individual nor combination of monoclonal antibodies protected immunosuppressed, infected mice in the doses tested. Similarly, elastase-specific polyclonal antibodies, raised in mice or rabbits, did not exhibit a protective effect, nor did immunization of mice with elastase prior to immunosuppression and infection. Histological examination of the lungs of immunosuppressed, infected mice showed no amelioration of fungal invasiveness by treatment with elastase-specific monoclonal or polyclonal antibodies. However, immunocompetent mice, instilled with a spore inoculum much higher than used in the preceding studies and treated with antibodies, survived, while control mice not treated with antibodies were overwhelmed by the massive spore dose and died. Nevertheless, overall evidence suggests that elastase may not be the primary virulence factor involved in invasive pulmonary aspergillosis.

Production and characterization of monoclonal antibodies to the carboxyl-terminal heptapeptide of endothelin-1.

Two cell lines, RR5.ET-1 and RR1.ET-1, that produce monoclonal antibodies specific for the carboxyl-terminal heptapeptide of endothelin-1 (ET-1) have been cloned and stabilized. An RIA was developed to facilitate the evaluation and characterization of these monoclonal antibodies. The affinity constant of each MAb for ET-1, as determined by Scatchard analysis, was 5.74 x 10(8) M-1 for RR5.ET-1 and 4.15 x 10(7) M-1 for RR1.ET-1. The antibodies reacted specifically with the carboxyl-terminus (ET15-21) and did not cross-react with the amino-terminal amino acids (ET1-16). As expected, the antibodies cross-reacted with endothelin-2 (ET-2) and endothelin-3 (ET-3), and to a lesser extent, with the closely related sarafotoxins. Both MAbs retained about 55% reactivity with the ET-1 terminal sequence of Asp-Ile-Ile-Trp (ET18-21) but had no reactivity with the ET sequence His-Leu-Asp-Ile-Ile-Trp-Val-Asn (ET16-23) nor with Big ET-1 (ET1-39). These data strongly suggest that the terminal four amino acids of ET-1 are included in the MAb binding site. More importantly, the terminal Trp21 must be free, not linked to Val22 to retain reactivity with either of the MAbs.

Purification and properties of the elastase from Aspergillus fumigatus.

Elastase, a potential virulence factor from the opportunistic pathogen Aspergillus fumigatus, was purified 220-fold from culture broth by fast-performance liquid chromatography employing anion exchange (Q Sepharose fast flow), cation exchange (S Sepharose fast flow), and gel filtration (Superose 12). Purified to near homogeneity, the elastase had an apparent molecular mass of 32 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (silver stain) but a mass of about 19.1 kDa as determined by gel filtration on Superdex 75. The elastase is not glycosylated and is positively charged at neutral pH, having a pI of 8.75. Inhibition by 0.2 mM phenylmethylsulfonyl fluoride (100%) and 0.21 mM leupeptin (60%) implies that the elastase is a serine protease. However, the enzyme is also inhibited by 5 mM EDTA (100%) and 10 mM 1,10-orthophenanthroline (30%), suggesting a requirement for divalent cations. The enzyme acts optimally at pH 7.4 and 45 degrees C in 50 mM sodium borate buffer, but in Tris HCl, the pH optimum shifts to 8.8.

Inhibition of Aspergillus fumigatus elastase with monoclonal antibodies produced by using denatured elastase as an immunogen.

In preparing monoclonal antibodies to the elastase from Aspergillus fumigatus, we found that the enzyme was weakly immunogenic in BALB/c mice. Antiserum titers were only 1:1,000 to 1:5,000, and hybridomas secreted nonspecific immunoglobulin M (IgM). Denaturing the elastase in 0.5% sodium dodecyl sulfate at 80 degrees C for 10 min prior to injection increased titers of antiserum against the nondenatured (native) enzyme 10-fold. Of eight hybridomas selected following immunization with the denatured enzyme, seven produced IgG reactive with the native enzyme and one produced nonspecific IgM. The nondenatured immunogen tested again yielded mainly IgM producers. Immunoblots and enzyme-linked immunosorbent assay showed that the IgG monoclonal antibodies were reactive with both the denatured and nondenatured fungal elastases; none cross-reacted with human neutrophil elastase, porcine pancreatic elastase, or Pseudomonas elastase. Elastase-specific polyclonal antibody produced in mice inhibited elastase activity beginning at a molar ratio (antibody to elastase) of 4:1, and activity was completely inhibited at 14.5:1. Some individual monoclonal antibodies partially inhibited elastase, but certain pairs, at a molar ratio of each antibody to elastase of 5.4:1, acted synergistically to inhibit the activity completely.