In vitro activity of levofloxacin, ofloxacin and other quinolones against coagulase-negative staphylococci.

The in vitro activity of levofloxacin against coagulase-negative staphylococci (CNS) was investigated. In vitro, on the basis of MIC90 values, levofloxacin was as active as ciprofloxacin, inhibiting both methicillin-sensitive and -resistant staphylococci at 0.5 microgram/ml. The frequency of one-step development of levofloxacin-resistant CNS was < 1 x 10(-9). After repeated transfer of CNS in the presence of increasing concentrations of levofloxacin or other quinolones, there were 4-fold increases in MIC50 values for both levofloxacin and ofloxacin, whereas there were 64- to 128-fold increases for ciprofloxacin, norfloxacin and enoxacin. Based on MIC50 values, the concentrations of 1 or 2 micrograms/ml are clinically relevant. The mutant strains induced by levofloxacin showed less virulence based on the LD50 value in acute systemic murine infection.

In vitro and in vivo antibacterial activities of FK037, a novel parenteral broad-spectrum cephalosporin.

FK037, a new parenteral cephalosporin, is an oxime-type cephem antibiotic with a 1-hydroxyethyl-5-amino-pyrazole moiety at the 3 position. FK037 was evaluated for antimicrobial activity in vitro and in vivo. In vitro, FK037 was twofold or more active than ceftazidime, cefoperazone, cefotaxime, and ceftriaxone against Pseudomonas aeruginosa (MIC for 90% of the strains [MIC90] = 32 micrograms/ml), members of the family Enterobacteriaceae (MIC90 < or = 2 micrograms/ml), group A streptococci (MIC90 = 0.015 microgram/ml), and methicillin-sensitive or -resistant coagulase-negative staphylococci (MIC90 = 2 and 16 micrograms/ml, respectively). In addition, the activity of FK037 was equal to or greater than that of ceftazidime, cefotaxime, or ceftriaxone against Streptococcus pneumoniae (MIC90 = 0.12 microgram/ml) and methicillin-sensitive or -resistant Staphylococcus aureus (MIC90 = 2 and 16 micrograms/ml, respectively). FK037 was more active in vitro than cefepime (two- to fourfold) and cefpirome (twofold) against S. aureus. In murine systemic infection models, FK037 showed potent activity against P. aeruginosa, Escherichia coli, and methicillin-sensitive and methicillin-resistant S. aureus. FK037 was also efficacious in a mouse model of pyelonephritis caused by S. aureus or Klebsiella pneumoniae and in a mouse model of pneumococcal pneumonia caused by S. pneumoniae. Additional studies on this compound to assess its potential clinical utility are warranted.

Induction of resistance of Streptococcus pneumoniae to quinolones in vitro.

Twelve strains of Streptococcus pneumoniae were serially exposed to increasing concentrations of levofloxacin, temafloxacin, ciprofloxacin and norfloxacin for five passages. Wild-type and passaged strains were tested for susceptibility to quinolones, macrolide and penicillin G. The MIC90 data showed a 2-fold increase for levofloxacin but a 32-fold increase for ciprofloxacin, a 16-fold increase for temafloxacin and an 8-fold increase for norfloxacin. Among 16 ciprofloxacin-induced resistant strains, 14 were susceptible to levofloxacin. No cross-resistance to other antibiotics was observed.

In vitro activity of l-ofloxacin against norfloxacin-resistant coagulase-negative staphylococci.

The in vitro activity of l-ofloxacin was determined against coagulase-negative staphylococci that were induced to norfloxacin resistance. l-Ofloxacin was the most active agent tested with an MIC90 of 4 micrograms/ml compared with greater than 128, 32, and 128 micrograms/ml for norfloxacin, ciprofloxacin, and enoxacin, respectively. Rifampin-resistant, coagulase-negative staphylococci were not cross-resistant to the quinolones tested. Among the rifampin-resistant organisms tested, l-ofloxacin was also the most active agent with an MIC90 of 0.25 micrograms/ml.

The postantibiotic suppressive effect of L-ofloxacin, an optically active isomer of ofloxacin.

The postantibiotic suppressive effect (PAE) of L-ofloxacin was studied and compared with those of ciprofloxacin and norfloxacin. The PAE of L-ofloxacin was observed against all Gram-positive organisms tested: Staphylococcus aureus, S. epidermidis, and Enterococcus faecalis. At the achievable serum concentrations L-ofloxacin showed a longer PAE than ciprofloxacin and norfloxacin. Exposure of organisms to 4 micrograms/ml of L-ofloxacin for 2 hr produced a 3.1 and 4.2 hr PAE for methicillin-sensitive and methicillin-resistant staphylococci, respectively. Against E. faecalis and S. epidermidis, the PAEs of L-ofloxacin were 1.9 and 1.6 hr, respectively.

In vitro and in vivo antibacterial activities of levofloxacin (l-ofloxacin), an optically active ofloxacin.

The antibacterial activity of levofloxacin was compared with those of ofloxacin, ciprofloxacin, and other antibiotics. In general, levofloxacin was equally active or up to fourfold more active than ofloxacin against all 801 organisms tested. Levofloxacin was twofold [corrected] more active than ciprofloxacin against Streptococcus pneumoniae and 2- to 4-fold more active than ciprofloxacin against Staphylococcus aureus, Xanthomonas maltophilia, and Bacteroides fragilis. Levofloxacin was two- to eightfold more active than ciprofloxacin against coagulase-negative staphylococci and Acinetobacter spp., although these improvements in potency may not be clinically relevant. Levofloxacin inhibited 90% of streptococci when it was used at concentrations of 1 to 2 micrograms/ml. Levofloxacin was two- to fourfold less active than ciprofloxacin against most members of the family Enterobacteriaceae, such as Escherichia coli; Klebsiella pneumoniae; Citrobacter, Proteus, Providencia, Salmonella, and Yersinia spp.; and Pseudomonas aeruginosa. Both compounds were equally active against Pseudomonas cepacia. The in vitro DNA gyrase inhibitory activity of levofloxacin was as potent as that of ciprofloxacin, with a 50% inhibitory concentration of 0.65 micrograms/ml against an E. coli enzyme. In vivo, oral treatment with levofloxacin was as efficacious or more efficacious than that with ciprofloxacin in systemic as well as pyelonephritis infections in mice. Levofloxacin achieved higher concentrations in the serum and tissue of mice than did ciprofloxacin. This study presents some potential advantages of the pure L isomer of ofloxacin over ciprofloxacin and other quinolones.

In vitro and in vivo antidermatophytic activity of saperconazole, a new fluorinated triazole.

The in vitro activity of saperconazole against selected isolates of dermatophytes and its in vivo efficacy in a guinea pig dermatophytic infection model using Trichophyton mentagrophytes were evaluated. Susceptibility testing was determined with an agar dilution method in three media: yeast nitrogen base agar (YNBA), brain heart infusion agar (BHIA) and Sabouraud dextrose agar (SDA). An inoculum of 1 x 10(5) CFU of T. mentagrophytes spores was placed onto the surface of these agars. Incubation was at 32 degrees C for 72 h. The MIC of saperconazole against all isolates was less than 1 microgram/ml, whereas the MIC ranged from 0.1 to > 128 micrograms/ml for fluconazole. The MIC range of saperconazole against Trichophyton species was < or = 0.002 to 0.25 micrograms/ml; against Microsporum species it was < 0.001 to 0.1 microgram/ml; and against Epidemophyton species was < or = 0.002 to 0.25 micrograms/ml. These data showed that saperconazole was the most active compound tested against these selected dermatophytes. The activities of saperconazole against T. mentagrophytes, T. rubrum and M. canis were not affected by the medium. The MICs against these organisms were < or = 0.008 micrograms/ml in SDA, YNBA or BHIA. There were 2- to 4-fold decreases in activity for fluconazole at the same conditions. In vivo, topical treatment with saperconazole at concentrations of 0.125% and 0.25% resulted in 50% and 75% microbiological cure rates, respectively, in the guinea pig topically infected with T. mentagrophytes.

Saperconazole: in vitro and in vivo anticandidal activity.

The in vitro activity of saperconazole against eight candidal species (81 strains) was determined and compared with fluconazole, Sch 39304 and amphotericin B. Using brain heart infusion broth with an inoculum of 10(4) CFU/ml, the MIC ranges (micrograms/ml) of saperconazole were: less than or equal to 0.015- greater than 32 for Candida albicans, less than or equal to 0.015-16 for C. tropicalis, less than or equal to 0.015-32 for C. glabrata, less than or equal to 0.015-32 for C. parapsilosis, less than or equal to 0.015-0.12 for C. guilliermondii and less than or equal to 0.015-0.06 for C. krusei. Saperconazole was the most active agent tested against Candida species. Saperconazole and 5-fluorocytosine combinations showed synergistic interactions against Candida species, and no antagonistic interaction was demonstrated. In a rat vaginal candidiasis infection model, saperconazole and fluconazole were equipotent producing 75-100% cures at levels of 0.016-0.25%, respectively, when dosed intravaginally. After single oral dosing, saperconazole was 5-fold more potent than fluconazole with an ED50 value of 0.53 mg/kg. These data demonstrate that saperconazole is effective in a rat vaginal candidiasis infection either with a single oral dose or by intravaginal treatment.

In-vivo evaluation of ofloxacin in Salmonella typhimurium infection in mice.

The effects of ofloxacin in Salmonella typhimurium infection in mice were compared with those of ciprofloxacin, ampicillin and chloramphenicol. Oral administration of ofloxacin at 10, 50 or 100 mg/kg once per day for seven days significantly (P less than 0.02) increased survival (20.1 days at 100 mg/kg) of infected mice relative to non-treated controls (4.1 days). In addition, after oral treatment with 100 mg/kg of each of the antibiotics, ofloxacin was significantly more effective than ampicillin (9.9 days), chloramphenicol (8.4 days) or ciprofloxacin (14.9 days) in prolonging the mean survival time of these mice. A comparison of oral potencies indicates that ofloxacin is five times more potent than ciprofloxacin (oral ED50 = 13.3 mg/kg vs ciprofloxacin = 69.9 mg/kg) and over eight times more potent than either of the other two antibiotics. When the number of bacteria from livers and spleens was quantitated, only ofloxacin (25 or 100 mg/kg,po) significantly (P less than 0.02) reduced the number of viable bacteria in both of these tissues in comparison with untreated controls, and, relative to the other antibiotics, ofloxacin (100 mg/kg) caused a significantly greater reduction. Single oral dosing of 20 mg/kg of either ofloxacin or ciprofloxacin showed that ofloxacin achieves approximately a four-fold higher peak serum or liver concentration than ciprofloxacin, which may contribute to its better efficacy in this infection model. These results taken together suggest that oral ofloxacin may be of value in treating systemic salmonella infections in humans.

Clearance of a novel recombinant tissue plasminogen activator in rabbits.

The pharmacokinetic properties of hPA(B), characterized by the insertion of a urokinase kringle coding region before the double kringle of tPA plus the complete tPA coding region, were investigated and compared to those of melanoma tPA (mtPA). Mean peak plasma concentrations at the end of infusion were 4.7 micrograms/ml for hPA(B) and 4.6 micrograms/ml for mtPA. The pharmacokinetics of both hPA(B) and mtPA showed a biexponential disappearance from plasma which is consistent with a two-compartment model of t 1/2 (lambda 1) = 2 minutes, t 1/2 (lambda 2) = 58 minutes for hPA(B), and t 1/2 (lambda 1) = 2.2 minutes, t 1/2 (lambda 2) = 61 minutes for mtPA. However, this very fast decaying lambda 1 phase of mtPA lasted five times longer than that of hPA(B) which resulted in very low concentrations of mtPA. Thus, hPA(B) exhibited larger AUC, slower clearance rate, and smaller volume of distribution (P less than 0.01) than those of mtPA. The fibrinolytic activity of hPA(B) in rabbit plasma as determined by zymography lasted up to 120 minutes after the end of infusion as compared to that of 2 minutes for mtPA. This indicates that mtPA, despite its t 1/2 (lambda 2) being similar to that of hPA(B), is no longer at physiologically meaningful concentrations at the start of the lambda 2 phase.

Disposition of a novel recombinant tissue plasminogen activator, delta 2-89 TPA, in mice.

The pharmacokinetic characteristics of delta 2-89 tPA, characterized by the deletion of the first 89 amino acids at the NH2-terminus of tPA, were evaluated and compared to those of recombinant tPA (rtPA). When they were administered intravenously to mice, a biexponential disposition curve was observed for both tPAs. The plasma half-lives of lambda 1 and lambda 2 phases of delta 2-89 tPA were 15 minutes and 180 minutes which are significantly higher than those of rtPA. A zymogram of mouse plasma taken at various time intervals showed that delta 2-89 tPA retained fibrinolytic activity up to 30 minutes, whereas rtPA could be detected only up to 5 minutes after injection. Autoradiography revealed that most of 125I-delta 2-89 tPA was associated with plasma protein complex.

Structure-function analysis with tissue-type plasminogen activator. Effect of deletion of NH2-terminal domains on its biochemical and biological properties.

Tissue-type plasminogen activator (t-PA) is a mosaic protein containing several distinct structural domains attached to the serine protease catalytic unit present at its COOH terminus. To investigate structure-function relationships in t-PA, we deleted the NH2-terminal domains, finger and epidermal growth factor, by genetic engineering. The genes for the parent and mutant t-PA were expressed in a bovine papilloma virus-dependent mammalian cell system. The secreted proteins were purified to homogeneity. The mutant protein was processed to the expected size of about 60 kDa compared to approximately 68 kDa for the parent t-PA, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and fibrin autography. While the mutant t-PA had amidolytic activity comparable to native t-PA, it did not bind appreciably to fibrin. Consequently, fibrin-dependent enzymic activity, i.e. plasminogen activation in the presence of soluble fibrin and fibrinolysis were lower than with native recombinant t-PA. The effect of deletion of NH2-terminal domains on the plasma half-life (t1/2) was investigated by injecting native and mutant t-PA into mice. While the majority of the t-PA disappeared initially with a t1/2 of about 2 min, mutant t-PA cleared at a much slower rate with t1/2 of about 50 min. These findings suggest that the NH2-terminal domains of t-PA not only determine its specificity for binding to fibrin but also mediate its clearance from plasma in vivo. Furthermore, the catalytic unit in t-PA seems to function autonomously.

Elimination of antibiotic-resistant plasmids by quinolone antibiotics.

Of 7 plasmids we tested, the plasmid pORF2 was eliminated in vitro with the most efficiency by treatment with subinhibitory concentrations of novobiocin, coumermycin and 10 quinolones. It showed a cure rate of 43% by enoxacin; 12% by novobiocin, pefloxacin, ciprofloxacin and CI-934; 7% by coumermycin and ofloxacin; 9% by amifloxacin; and 4% by AM-833. On the other hand, pSC194, pBR322 and pMH612 were poorly cured in vitro by quinolones, except pSC194 which was cured 33% by enoxacin. R1, pP1603, and pUB110 were unaffected by the treatment. Mice were challenged intraperitoneally with a 2XLD50 of Escherichia coli carrying the ORF2 plasmid and were treated per os with 1 X or 1/2 X ED50 of either enoxacin or CI-934. The frequency of loss of ampicillin resistance determined 3 h after treatment shows curing effects of 92% for CI-934, 89% for enoxacin and 20% for untreated control.

Therapeutic efficacy of cefpiramide-ciprofloxacin combination in experimental Pseudomonas infections in neutropenic mice.

The therapeutic efficacy of cefpiramide and ciprofloxacin alone and in combination was investigated and compared with that of ticarcillin plus tobramycin against pseudomonal infections in mice made neutropenic by administration of cyclosphosphamide. Therapy with cefpiramide plus ciprofloxacin was significantly more effective than that by either antibiotic alone. These results were consistent with in-vitro synergistic effects. At a higher dose of ciprofloxacin (4 mg/kg) plus cefpiramide (50 mg/kg), the combination therapy protected all neutropenic mice from fatal bacteraemia, and was more protective than ticarcillin (200 mg/kg) plus tobramycin (1 mg/kg). The peak serum concentration of cefpiramide in infected neutropenic mice was 51 mg/l when they were given 50 mg/kg subcutaneously. Ciprofloxacin attained a peak serum concentration of 1.2 mg/l and a serum half-life of 34 min.

Therapeutic efficacy and pharmacokinetic properties of ciprofloxacin in intra-abdominal abscesses caused by Bacteroides fragilis and Escherichia coli.

Experimental intra-abdominal abscesses were produced in mice by intraperitoneal injections of Bacteroides fragilis and Escherichia coli. The therapeutic efficacy of ciprofloxacin was investigated in this mixed intra-abdominal abscess model and was compared with that of rifampicin. Treatment with ciprofloxacin at 0.2 to 20 mg/kg or rifampicin at 20 mg/kg prevented all mice from death, as compared to the 60% mortality rate observed in the vehicle-treated controls. Rifampicin concentrations at 10 and 20 mg/kg were effective in preventing abscess formation and eradicated bacterial abscess. Ciprofloxacin at all the levels tested neither reduced the incidence of abscess nor eradicated Bact. fragilis from abscesses. However, ciprofloxacin at levels of 20, 10, 5, and 1 mg/kg reduced significantly the number of E. coli cells in the abscess. The peak serum level of ciprofloxacin at the oral dose of 20 mg/kg was 0.43 mg/l which was well above the MIC values for E. coli but not for Bact. fragilis.

Interaction of clavulanic acid, sulbactam and cephamycin antibiotics with beta-lactamases.

The inhibitory effects of clavulanic acid, sulbactam and cephamycin antibiotics on chromosomally-mediated or plasmid-mediated beta-lactamases were investigated. The inhibition constants were determined by a non-linear regression analysis. Clavulanic acid and sulbactam had high affinities for the purified plasmid-mediated beta-lactamases such as SHV-1, TEM-1 and PSE-4, and were potent inhibitors as shown by their low Ki values. Except for Bacteroides beta-lactamase, which is sensitive to inhibition by cephamycin antibiotics, clavulanic acid and sulbactam were found not to be as effective against chromosomally-mediated beta-lactamases. The cephamycin antibiotics were better inhibitors of chromosomally-mediated beta-lactamases than those that are plasmid mediated. Except for P99 beta-lactamase, against which sulbactam and clavulanic acid were inactive, the cephamycin antibiotics were less effective inhibitors than sulbactam and clavulanic acid.

Purification and properties of a beta-lactamase from Proteus penneri.

A cephalosporin-hydrolyzing enzyme from strains of Proteus penneri resistant to beta-lactam antibiotics was purified and characterized. The enzyme gave a single protein band on SDS-polyacrylamide gel electrophoresis with a molecular weight of 30,000. This cephalosporinase has an isoelectric point of 6.8, a pH optimum of 6.5 and a temperature optimum of 45 degrees C. The enzyme hydrolyzed cephaloridine, cephalothin, cefuroxime, and cefotaxime more rapidly than penicillins. The relative rate, with cephaloridine as 100, were: cephalothin, 50; cefuroxime, 93; cefotaxime, 48; ceftriaxone, 23; cefoperazone, 11; benzylpenicillin, 3; ampicillin, 9; and carbenicillin, less than 1. Cephamycins had low affinities for the enzyme. However, clavulanic acid and sulbactam, with high affinities for the enzyme, were inhibitors of this enzyme.

Synergistic activity of apalcillin and gentamicin in a combination therapy in experimental Pseudomonas bacteraemia of neutropenic mice.

The therapeutic activity of a combination of apalcillin and gentamicin was evaluated in experimental Pseudomonas aeruginosa infection in neutropenic mice. Mice made neutropenic by administration of cyclophosphamide were more susceptible to P. aeruginosa infection than normal mice. At both challenge levels of 2LD50 and 20LD50, therapy with gentamicin alone was more effective than that with apalcillin or piperacillin. However, therapy with apalcillin-gentamicin combinations was significantly more effective than that by either component alone, and was as active as that with piperacillin-gentamicin. These in-vivo findings correlated with those of in-vitro studies, thus establishing a synergistic effect when apalcillin and gentamicin were combined. The results show that apalcillin when combined with gentamicin is effective in treating serious P. aeruginosa bacteraemia in neutropenic mice.

Experimental efficacy of apalcillin and cefpiramide compared with that of six other antipseudomonal agents in Pseudomonas aeruginosa burn infections.

The therapeutic efficacy of cefpiramide and apalcillin was evaluated and compared with that of six other antipseudomonal beta-lactam antibiotics in an experimental mouse burn infection due to Pseudomonas aeruginosa. Both cefpiramide and apalcillin were as potent as cefsulodin and more potent than carbenicillin, cefotaxime, cefoperazone, piperacillin and gentamicin in protecting the infected mice from fatal bacteraemia and in eradicating Ps. aeruginosa from the infected site.

Therapeutic efficacy of cefpiramide and cefoperazone alone and in combination with gentamicin against pseudomonal infections in neutropenic mice.

Cefpiramide and cefoperazone alone and in combination with gentamicin were compared for therapeutic efficacy against pseudomonal infections in normal mice and in mice made neutropenic by administration of cyclophosphamide. Neutropenic mice were more susceptible to infection with Pseudomonas aeruginosa than normal mice. At all challenge doses, combination therapy with either cefpiramide-gentamicin or cefoperazone-gentamicin was more effective than that with a single agent. Therapy with the cefpiramide-gentamicin combination was significantly more active than that with the cefoperazone-gentamicin combination in protecting mice from fatal bacteremia. Pharmacokinetic studies in mice showed that cefpiramide attained a peak serum concentration of 12 micrograms/ml and a serum half-life of 40 min, which are higher than attained by cefoperazone with values of 4 micrograms/ml and 18 min. These factors may have caused the combined cefpiramide-gentamicin therapy to result in significantly improved survival rates in mice as well as in higher bactericidal titers than the cefoperazone-gentamicin combination. The results show that cefpiramide when combined with gentamicin is effective in treating serious infections with P. aeruginosa in neutropenic mice.