Akira Imada

Asparaginase and Glutaminase Activities of Micro-organisms

Summary: l-Asparaginase and l-glutaminase activities were detected in many microorganisms and the distribution of these activities was found to be related to the classification of micro-organisms. Among 464 bacteria, the activities occurred in many Gram-negative bacteria and in a few Gram-positive bacteria. Most members of the family Enterobacteri-aceae possessed l-asparaginase. l-Asparaginase and l-glutaminase occurred together in a large proportion of pseudomonads. Among Gram-positive bacteria many strains of Bacillus pumilus showed strong l-asparaginase activity. Amidase activities were also observed in several strains in other families. l-Asparaginase activity was not detected in culture filtrates of 261 strains of species of the genera Streptomyces and Nocardia, but l-asparaginase and l-glutaminase were detected when these organisms were sonicated. The amidase activities in culture filtrates of 4158 fungal strains were tested. All the strains of Fusarium species formed l-asparaginase. Organisms of the genera Hypomyces and Nectria, which are regarded as the perfect stage of the genus Fusarium, also formed l-asparaginase. Several Penicillium species formed l-asparaginase. Two organisms of the family Moniliaceae formed l-glutaminase together with l-asparaginase, and a fewascomycetous fungi formed l-asparaginase or l-glutaminase. Among 1326 yeasts, l-asparaginase or l-glutaminase occurred frequently in certain serological groups of yeasts: VI (Hansenula) group, Cryptococcus group and Rhodotorula group. Many strains of Sporobolomyces species also showed l-asparaginase activity. Several strains of Cryptococcus and Rhodotorula group possessed l-glutaminase and l-asparaginase. l-Glutaminase alone was formed in many strains of Candida scottii and Cryptococcus albidus, both of which are related to Basidiomycetes.

Therapeutic effect of cefozopran (SCE-2787), a new parenteral cephalosporin, against experimental infections in mice.

The therapeutic effect of cefozopran (SCE-2787), a new semisynthetic parenteral cephalosporin, against experimental infections in mice was examined. Cefozopran was more effective than cefpiramide and was as effective as ceftazidime and cefpirome against acute respiratory tract infections caused by Klebsiella pneumoniae DT-S. In the model of chronic respiratory tract infection caused by K. pneumoniae 27, cefozopran was as effective as ceftazidime. The therapeutic effect of cefozopran against urinary tract infections caused by Pseudomonas aeruginosa P9 was superior to that of cefpirome and was equal to those of ceftazidime and cefclidin. In addition, cefozopran was more effective than ceftazidime and was as effective as flomoxef in a thigh muscle infection caused by methicillin-sensitive Staphylococcus aureus 308A-1. Against thigh muscle infections caused by methicillin-resistant S. aureus N133, cefozopran was the most effective agent. The potent therapeutic effect of cefozopran in those experimental infections in mice suggests that it would be effective against respiratory tract, urinary tract, and soft tissue infections caused by a variety of gram-positive and gram-negative bacteria in humans.

In vitro and in vivo activities of SCE-2787, a new parenteral cephalosporin with a broad antibacterial spectrum.

SCE-2787, a new cephalosporin having a condensed azolium moiety in the 3 position and an aminothiadiazolyl group in the 7 beta side chain, was evaluated for its in vitro and in vivo activities in comparison with those of ceftazidime, flomoxef, cefpirome, and E1040. Against methicillin-susceptible strains of Staphylococcus aureus and Staphylococcus epidermidis, SCE-2787 was more active than ceftazidime and E1040 and was as active as flomoxef and cefpirome, with MICs for 90% of strains tested (MIC90s) being 1.56 micrograms/ml or less. SCE-2787 was also active against Pseudomonas aeruginosa, for which the MIC90 was 6.25 micrograms/ml, which was lower than that of cefpirome and comparable to that of ceftazidime. SCE-2787 was marginally active against methicillin-resistant strains of staphylococci and Enterococcus faecalis, although its MIC90s were the lowest among those of the antibiotics tested. The activities of SCE-2787 against Streptococcus species, most members of the family Enterobacteriaceae, and Haemophilus influenzae exceeded those of ceftazidime and flomoxef and were comparable to those of cefpirome. Furthermore, MIC90s of SCE-2787 were significantly lower than those of ceftazidime for ceftazidime-resistant isolates of Citrobacter freundii and Enterobacter cloacae. SCE-2787 was resistant to hydrolysis by various types of beta-lactamases, including the Bush group 1 beta-lactamases, and had low affinities for these enzymes, with Km or Ki values of greater than 100 microM. The in vitro activity of SCE-2787 was reflected in its efficacy in mouse protection tests. Thus, SCE-2787 appears to be a promising cephalosporin that should be further evaluated in clinical trials.

SCE-963, a New Potent Cephalosporin with High Affinity for Penicillin-Binding Proteins 1 and 3 of Escherichia coli

A few biochemical activities of SCE-963, a new cephalosporin with potent antibacterial activities against gram-negative bacteria, were compared with those of several currently available cephalosporins against strains of Escherichia coli K-12. The minimum inhibitory concentrations of SCE-963, cefazolin, cephaloridine, cephalothin, and cephalexin were 0.2, 1.56, 3.13, 12.5, and 25 μg/ml, respectively. Affinities of these cephalosporins for the penicillin-binding protein (PBP) 1B of E. coli correlated well with their antibacterial activities; among tested cephalosporins, SCE-963 showed the highest affinity for PBP 1B. SCE-963 inhibited cross-linking of peptidoglycan in a cell-free system the most strongly suggesting that this inhibition results from its high affinity for PBP 1B. SCE-963 also showed the highest affinity for PBP 3; it caused filamentation of cells over a wide range of relatively lower concentrations. Thus its superior antibacterial activity is believed to be manifested through its high affinity for the PBPs. Images

Comparative pharmacokinetics of SCE-2787 and related antibiotics in experimental animals.

The pharmacokinetic properties of SCE-2787 administered intravenously at a dose of 20 mg/kg of body weight were studied with mice, rats, rabbits, dogs, and monkeys and were compared with those of ceftazidime, cefpirome, and cefclidin in mice and dogs. The area under the concentration-time curve for plasma after intravenous administration was the largest in monkeys, followed by those in dogs, rabbits, rats, and mice, in that order. The elimination half-life ranged from 0.2 to 0.3 h in mice and rats to 0.7 to 1.3 h in rabbits, dogs, and monkeys. In young dogs, the concentrations of SCE-2787 in plasma were somewhat lower than those in the mature dogs. SCE-2787 was distributed well to the tissues, and the highest concentration was found in the kidneys in all species tested; the distribution to the lungs, liver, and spleen was also good, but the concentrations in these tissues were lower than those in the plasma. The pharmacokinetic parameters and urinary excretion of SCE-2787 in mice and dogs were similar to those of ceftazidime, cefpirome, and cefclidin. The maximum concentrations in the cerebrospinal fluid of rats and rabbits were 0.8 and 1.3 micrograms/ml, and the relative percentages of the area under the concentration-time curve of SCE-2787 in the cerebrospinal fluid to that in the plasma were 4.6 and 6.4%, respectively. SCE-2787 was excreted mainly in the urine; the recovery rate ranged from 74% (rats) to 90% (dogs) of the dose. The biliary excretion of SCE-2787, however, was low, amounting to about 1.4% for mice and rats and less than 0.5% for rabbits and dogs. In rats, there was no accumulation in the tissues and no delay in urinary excretion upon multiple intravenous administration of 20 mg of SCE-2787 per kg once daily for 7 days. No active metabolites were found in the plasma or urine of animals given SCE-2787. The binding of SCE-2787 to serum protein in mice, rats, dogs, monkeys, and humans was less than 11% and similar to that of cefclidin.

Regulation of Glucosamine Utilization in Staphylococcus aureus and Escherichia coli

Glucosamine- or N-acetylglucosamine-requiring mutants of Staphylococcus aureus 209P and Escherichia coli K12, which lack glucosamine-6-phosphate synthetase [2-amino-2-deoxy-D-glucose-6-phosphate ketol-isomerase (amino-transferring); EC 5.3.1.19], were isolated. Growth of these mutants on glucosamine was inhibited by glucose, but growth on N-acetylglucosamine was not. Addition of glucose to mutant cultures growing exponentially on glucosamine inhibited growth and caused death of bacteria, though chloramphenicol prevented death. Uptake of glucosamine by S. aureus and E. coli mutants was severely inhibited by glucose whereas uptake of N-acetylglucosamine was only slightly inhibited. Uptake of glucose was not inhibited by either glucosamine or N-acetylglucosamine. In glucosamine auxotrophs, glucose causes glucosamine deficiency which interrupts cell wall synthesis and results in some loss of viability in the presence of continued protein synthesis.

In vitro and in vivo antibacterial activities of carumonam (AMA-1080), a new N-sulfonated monocyclic beta-lactam antibiotic.

The in vitro and in vivo antibacterial activities of carumonam (AMA-1080), a synthetic sulfazecin derivative, were compared with those of aztreonam, cefoperazone, ceftazidime, and cefsulodin. Carumonam was highly active in vitro against members of the family Enterobacteriaceae, Pseudomonas aeruginosa, and Haemophilus influenzae and weakly active against Streptococcus pneumoniae, but it was not active against Staphylococcus aureus. The MICs of carumonam for 90% of 1,156 clinical Enterobacteriaceae isolates were between 0.013 and 25 micrograms/ml, which were the lowest MICs of the antibiotics tested. The MIC of carumonam for 90% of Klebsiella oxytoca was 0.2 micrograms/ml, whereas that of aztreonam was 50 micrograms/ml. The superiority of carumonam to aztreonam and the reference cephalosporins was also demonstrated by their activities against Klebsiella pneumoniae and Enterobacter cloacae. The MIC of carumonam for 90% of P. aeruginosa was 12.5 micrograms/ml, which was comparable to the MICs of aztreonam and ceftazidime. Carumonam showed a high affinity for the penicillin-binding protein 3 of gram-negative bacteria, but not for the penicillin-binding proteins of S. aureus and Bacteroides fragilis. Carumonam was resistant to hydrolysis by 12 plasmid-mediated beta-lactamases and 7 chromosomal beta-lactamases. It was more stable than aztreonam to hydrolysis by the beta-lactamase of K. oxytoca; this stability is related to the superiority of the in vitro and in vivo activities of carumonam to those of aztreonam against this species. In general, the protective activities (50% effective dose) of carumonam and reference antibiotics in mice with experimental intraperitoneal infections correlated with the in vitro activities (MIC); carumonam showed excellent protective activity against most aerobic gram-negative bacteria. Images

Formation of L-Asparaginase by Fusarium Species

SUMMARY: L-Asparaginase was formed in the culture filtrates of a number of Fusarium species, as well as in those of ascomycetous fungi having a Fusarium imperfect state, such as species of Hypomyces and Nectria. Species of Gibberella, though having a Fusarium state, formed little L-asparaginase. The distribution of the ability to form the enzyme was related to taxonomic position.

Comparative pharmacokinetics of carumonam and aztreonam in mice, rats, rabbits, dogs, and cynomolgus monkeys.

The pharmacokinetic properties of carumonam (AMA-1080, Ro 17-2301) were studied in mice, rats, rabbits, dogs, and cynomolgus monkeys and compared with those of aztreonam. Carumonam administered subcutaneously in mice or intramuscularly in rats, rabbits, dogs, and cynomolgus monkeys at a dose of 20 mg/kg was readily absorbed and distributed at high concentrations in the plasma, kidneys, liver, and lungs, as was aztreonam. The peak level of carumonam in plasma, ranging from 41 micrograms/ml in mice to 68 micrograms/ml in monkeys; the area under the plasma concentration-time curve, ranging from 20 micrograms X h/ml in mice to 80 micrograms X h/ml in monkeys; the plasma half-life, ranging from 0.24 h in mice to 1.10 h in dogs; and the plasma clearance, ranging from 4.5 ml/min per kg in monkeys to 16.7 ml/min per kg in mice, resembled respective values of aztreonam. In rats, carumonam was eliminated faster than aztreonam. The levels of both antibiotics in the kidneys and liver were usually higher than respective levels in plasma. The level of carumonam in the kidney was usually higher than that of aztreonam, whereas the level of aztreonam in the liver was usually higher than that of carumonam. Both antibiotics showed similar distribution in the lung and spleen; the levels in these tissues were less than the levels in plasma. Carumonam was excreted mainly in the urine; the recovery ranged from 52% (from dogs) to 73% (from rabbits). The urinary recovery of carumonam from mice, rats, and monkeys was higher, but the recovery of carumonam from rabbits and dogs was lower than that of aztreonam. The biliary excretion of carumonam, amounting to 4.1% from rats and less than 0.3% from rabbits and dogs, was smaller than that of aztreonam, amounting to 19.1% from rats and around 1% from rabbits and dogs. The extent of protein binding at 20 micrograms of carumonam per ml was lower than that of aztreonam. For all species except dogs, which have very low binding in their serum (11% for carumonam and 20% for aztreonam), the binding of carumonam ranged from 21% (in rabbits) to 36% (in rats), whereas that of aztreonam ranged from 55% (in rabbits) to 85% (in rats). Thus, the plasma pharmacokinetics of carumonam and aztreonam were generally similar for all animals tested except dogs, but the two antibiotics differed slightly in their distribution in tissue, excretion, and protein binding in serum.

Novel morphological changes in gram-negative bacteria caused by combination of bulgecin and cefmenoxime.

The mode of action of bulgecin was investigated by examining its bactericidal and bacteriolytic activities, its effect on bacterial morphology, and its interaction with penicillin-binding proteins (PBPs). Bulgecin alone did not show any antibacterial activity against Escherichia coli and Serratia marcescens, but in concert with cefmenoxime, it induced potent growth-inhibitory and bactericidal activities. Electron microscopic examination of E. coli cells exposed to bulgecin combined with cefmenoxime revealed that a bulge developed in the middle of the cell, and additional smaller bulges were formed halfway between the central bulge and the polar ends. At the site of bulge development, vesicular mesosomelike structures appeared in the cytoplasm, the peptidoglycan layer facing them became faint, and the outer membrane protruded to form blebs. These morphological changes were quite different from those caused by the mecillinam-cefmenoxime combination that produces big bulges in E. coli. When S. marcescens was exposed to the combination of bulgecin and cefmenoxime, not only bulge formation, but also branching of the cells was observed. Bulgecin neither showed affinity for any PBPs of E. coli nor affected the binding of cefmenoxime or mecillinam to the PBPs. Images