Biomedical subjects
Publications and source records attributed to S Mitsuhashi.
The purified beta-lactamase from Bacteroides fragilis hydrolyzed newly introduced cephalosporins including cefuroxime and HR 756, and was inhibited by 7 alpha-methoxylated cephalosporins such as 6059-S and YM09330.
A new chemotherapeutic agent, AB206, shows potent antibacterial activity against gram-negative bacteria, including most of the nalidixic acid-resistant strains tested. It strongly inhibits deoxyribonucleic acid (DNA) synthesis in Escherichia coli, but only slightly inhibits ribonucleic acid and protein synthesis. Its activity on DNA synthesis in vivo and in vitro is greater than that of nalidixic acid. AB206 also strongly inhibits in vivo DNA synthesis in nalidixic acid-susceptible and -resistant clinical isolates of Proteus and Serratia. AB206 shows high penetrability into E. coli cells, as demonstrated by antibacterial activity with or without ethylenediaminetetraacetic acid, inhibition of in vivo and in vitro DNA synthesis, and uptake of the drug into cells, as compared to nalidixic acid. It appears that the high antibacterial activity of AB206 may be explained both by its potent inhibitory action against DNA synthesis and also by its high penetrability into bacterial cells.
Strains of bacteria resistant to beta-lactam antibiotics have been increasing in number and are becoming troublesome in clinical medicine. The in vitro antibacterial activity of amoxicillin combined with clavulanic acid was determined on selected ampicillin-resistant clinical isolates. Synergistic effects were produced by amoxicillin with clavulanic acid against ampicillin-resistant strains of Staphylococcus aureus, Escherichia coli, Klebsiella pneumoniae, Proteus mirabilis, Proteus vulgaris, and Bacteroides fragilis. Inhibition of the beta-lactamases produced by the ampicillin-resistant strains was confirmed, especially against the penicillinases mediated by the R factor and the cephalosporinases produced by P.vulgaris and B. fragilis. The inhibitory effect of clavulanic acid against beta-lactamases was irreversible because of the high affinity of clavulanic acid to the enzymes.
Cefotaxime, a new semisynthetic cephalosporin derivative, showed a broad spectrum of antibacterial activity against clinically isolated strains of gram-positive and gram-negative bacteria. This cephalosporin was slightly less active than cefazolin against Staphylococcus aureus but 4 to 300 times as active as carbenicillin against gram-negative organisms, including Pseudomonas aeruginosa, Pseudomonas cepacia, Enterobacter cloacae, and Serratia marcescens. Cefotaxime was the most active compound against members of the Enterobacteriaceae and 20- to 100-fold more active than cefoxitin against the indole-positive Proteus group. The minimal bactericidal concentrations of the compound were identical to, or two times higher than, the minimal inhibitory concentrations against Escherichia coli and P. aeruginosa and four times higher against S. marcescens. A reduction of inoculum size decreased greatly the minimal inhibitory and bactericidal concentrations of cefotaxime against E. coli P. aeruginosa, and S. marcescens. The antibiotic was very stable to penicillinase and cephalosporinase produced by gram-negative bacteria, including Proteus vulgaris.
Cefroxadine (GCP-9000; CXD), 7 beta[D-2-amino-2-(1,4-cyclohexadienyl)-acetamido]-3-methoxy-ceph-3-em-carboxylic acid, is a new orally active cephalosporin derivative. The spectrum of antibacterial activity of CXD is identical with that of cephalexin (CEX), but CXD was twofold more effective than CEX against Escherichia coli and Klebsiella pneumoniae, CXD was as stable to penicillinase as CEX, but it was hydrolyzed by cephalosporinase, with a relative rate of hydrolysis similar to that of CEX. The affinities of CXD and CEX to penicillin-binding proteins of E. coli were estimated; the affinity of CXD to penicillin-binding protein 1Bs was higher than that of CEX. Consistent with this, CXD had more intensive lytic activity than CEX. In vivo antibacterial activities of CXD and CEX were compared using systemic infections of mice with E. coli and K. pneumoniae; CXD was consistently more active than CEX.
Cefoperazone (T-1551, CFP) a new semisynthetic cephalosporin, has a broad spectrum of antibacterial activity. We investigated the affinity of CFP to penicillin-binding proteins (PBPs) and the inhibition of peptidoglycan synthesis by CFP. CFP had high affinities for Escherichia coli PBP-3, -1Bs, -2, and -1A, in descending order, and low affinities for PBP-4, -5, and -6. Similarly, CFP showed high affinity for Pseudomonas aeruginosa PBP-3, -1A, -1B, -2, and -4, in descending order. It is known that E. coli PBP-3 and P. aeruginosa PBP-3 participate in cell division. These results are in good agreement with the formation of filamentous cells of E. coli and P. aeruginosa treated with CFP. CFP had lower inhibitory activities on D-alanine carboxypeptidase IA and IB of E. coli than that of penicillin G, but its inhibitory activities on the cross-link formation in peptidoglycan synthesis were the same as those of penicillin G and higher than those of ampicillin.
Cephalosporin beta-lactamase (cephalosporinase) was purified from a strain of Escherichia coli resistant to beta-lactam antibiotics. The purified enzyme preparation gave a single protein band on polyacrylamide gel electrophoresis, and its molecular weight was 39,000 from sodium dodecyl sulfate-acrylamide gel electrophoresis. Its isoelectric point was 8.7. The specific activity was 31.7 mumol/min per mg of protein of the purified enzyme for the hydrolysis of cephaloridine. The optimal pH was about 8.0, and the optimal temperature was 36 degrees C. The enzyme activity was inhibited by iodine, some divalent metallic ions, semisynthetic penicillins, cefuroxime-type cephalosporins, and cephamycin derivatives. The enzymological properties of the purified preparation have been compared with those of beta-lactamases from other gram-negative bacteria.
The induction of beta-lactamase in Enterobacter cloacae GN5797 was studied by using 23 beta-lactam antiobiotics, including newly introduced drugs, as inducers. the beta-lactam antibiotics can be classified into three groups on the basis of their inducer activity. Among the tested cephalosporins, cephamycin derivatives such as cefoxitin, cefmetazole, and YM09330 had high inducer activity even at low drug concentrations. On the other hand, cefoperazone, cefsulodin, piperacillin, and apalcillin showed low inducer activity when compared with the other cephalosporins.
beta-Lactamase produced by Proteus rettgeri was found to be a typical cephalosporin beta-lactamase on the basis of its substrate hydrolysis profile. The enzyme activity was enhanced by prior treatment with an inducer. The enzyme was purified 166-fold by carboxymethyl-Sephadex column chromatography which indicated that its molecular weight was 42,000 +/- 2,000 and its isoelectric point was 8.7. Cefoperazone, cefoxitin, cefusulodin, cefmetazole, cefotaxime, 6059-S, FK749, YM-09330, carbenicillin, and cloxacillin were stable to this enzyme and possessed the function of competitive inhibition, as shown by their affinity for the beta-lactamase. The enzyme activity was inhibited by iodine, p-chloromerburibenzoate, and HG2+ ion. Clavulanic acid and CP-45899 displayed poor inhibitory activity toward this enzyme. The optimal pH was 8.0, and the optimal temperature was 50 degrees C.
N-Formimidoyl thienamycin (MK0787) was active against beta-lactamase-producing strains and was an effective inhibitor of beta-lactamases extracted and purified from these strains except for plasmid-mediated penicillinase type IV.
A cephalosporin beta-lactamase (cephalosporinase) was extracted from Enterobacter cloacae GN7471 and purified by means of column chromatography. The resulting preparation gave a single protein band upon polyacrylamide gel electrophoresis. The enzyme's isoelectric point was 8.4, and its molecular weight was 44,000. The optimal pH was 8.5, and the optimal temperature was 40 degrees C. The enzyme hydrolyzed cephalosporins much more readily than penicillins. The enzyme activity was inhibited by iodine, semisynthetic penicillins, cefuroxime-type cephalosporins, and cephamycin derivatives. The enzymological properties of the purified enzyme were compared with those of beta-lactamases derived from other gram-negative enteric bacteria.
A mutant temperature-sensitive for R-plasmid replication, Rms201ts14, was isolated from composite plasmid Rms201 after mutagenesis of P1 transducing lysate with 100 mM hydroxylamine for 40 h at 37 degrees C. When Escherichia coli ML1410(Rms201ts14)(+) was grown at temperatures between 40 and 42 degrees C in L broth, antibiotic-sensitive cells were segregated. When the incubation temperature of ML1410(Rms201ts14)(+) in L-broth was shifted to 42 from 30 degrees C, the increase in the number of antibiotic-resistant cells ceased 90 min after the temperature shift. However, the total number of cells continuously increased, and only 3% of the cells retained the plasmid at 5 h after the temperature shift to 42 degrees C. At 30 degrees C the amounts of covalently closed circular deoxyribonucleic acid per chromosome of Rms201ts14 and Rms201 were 3.8 and 6.3%, respectively. Incorporation of radioactive thymidine into the covalently closed circular deoxyribonucleic acid of Rms201ts14 did not take place at 42 degrees C, whereas radioactive thymidine was incorporated into the covalently closed circular deoxyribonucleic acid of Rms201 at a rate of 4%/chromosome even at 42 degrees C. The synthesis of plasmid covalently closed circular deoxyribonucleic acid in a cell harboring Rms201ts14 was almost completely blocked at 42 degrees C. These results indicated that the gene(s) responsible for plasmid deoxyribonucleic acid replication was affected in the mutant Rms201ts14. Temperature-sensitive miniplasmid pMSts214, which has a molecular weight of 5.3 x 10(6) and encodes ampicillin resistance, was isolated from Rms201ts14. Similarly, miniplasmid pMS201, which encodes single ampicillin resistance, was isolated from its parent, Rms201, and its molecular weight was 4.7 x 10(6). These results indicate that the gene(s) causing temperature sensitivity for replication of Rms201 resides on the miniplasmid.
Plasmid Rms312, specifying resistance to tetracycline (Tc), chloramphenicol (Cm), streptomycin (Sm), sulfonamide (Su), and mercury chloride (Mer), deletes both Tc and Cm Sm Su Mer determinants at a high frequency in Salmonella typhimurium LT2. S. typhimurium mutants that were stable carriers of Rms312 were isolated by alternate culture of R-bearing cells in a medium containing either tetracycline or chloramphenicol. In one of these mutants the deletion frequency of drug resistance determinants was decreased by about 100-fold not only Rms312, but also in R100, R1, and R6-5. This mutation caused a slight reduction of ultraviolet resistance but did not affect generalized genetic recombination, indicating that the mutation is different from recA. The mutation, designated dor (deletion of r-determinants), was mapped to a position near 57 units in the new linkage map of S. typhimurijm LT2 (K. E. Sanderson and P. E. Hartman, Microbiol. Rev. 42:471-519, 1978). The dor mutation had no effect on IS1-mediated illegitimate deletion, indicating that the dor mutation is different from the del mutation described by Nevers and Saedler (P. Nevers and H. Saedler, Mol. Gen. Genet. 160:209-214, 1978).
Four lysozyme-sensitive mutants were isolated after nitrosoguanidine mutagenesis of a lysozyme-insensitive strain of Staphylococcus aureus. One mutant was sufficiently effective for the isolation of macromolecules, such as plasmid deoxyribonucleic acids, from a cell after lysozyme-induced cell lysis.
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Two types of mutants highly sensitive to beta-lactam antibiotics were obtained from Pseudomonas aeruginosa PAO 2142 by treatment with N-methyl-N'-nitro-N-nitrosoguanidine. One type of mutant showed over 30 times higher sensitivity to mecillinam, carbenicillin and sulbenicillin than did the parent, but not to most other beta-lactam antibiotics tested. In contrast, the other type mutant was about 30 times more sensitive to ampicillin, cephaloridine, cefoxitin and cefmetazole, but resistant to mecillinam, carbenicillin and sulbenicillin at the same level as the parent. Beta-lactamase activity of these mutants ws not different from that of the parent. Defect in either of penicillin-binding proteins 1A/1B or 5 was observed in some mutants of P. aeruginosa highly sensitive to beta-lactam antibiotics.
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