A constitutively produced beta-lactamase was purified from Flavobacterium odoratum GN14053. The purified enzyme gave a single protein band on polyacrylamide gel electrophoresis. The isoelectric point was 5.8, and the molecular weight was estimated to be about 26,000. The enzyme activity was inhibited by EDTA, iodine, p-chloromercuribenzoate, HgCl2, and CuSO4 but not by clavulanic acid, sulbactam, imipenem, and cephamycin derivatives. The enzyme showed a broad substrate profile, hydrolyzing oxyiminocephalosporins, cephamycins, imipenem, and some penicillins.

ME1207 (pivaloyloxymethyl ester of ME1206) is a new oral cephalosporin. ME1206 is (6R,7R)-7-[(Z)-2-(2-aminothiazol-4-yl)-2-(methoxyimino)- acetamido]-3-[(Z)-2-(4-methylthiazol-5-yl)-ethyl]-cephem-4-carboxy lic acid. The susceptibilities of about 1,600 clinical isolates to ME1206 were determined by the agar dilution method. ME1206 showed a broad spectrum of activity against gram-positive and gram-negative bacteria. ME1206 was more active than cefaclor, T-2525, and cefixime against Staphylococcus aureus and Staphylococcus epidermidis. Against gram-negative bacteria, the activity of ME1206 was comparable with that of T-2525, but ME1206 was less active than cefixime. Against Streptococcus pneumoniae, Haemophilus influenzae, and Neisseria gonorrhoeae, ME1206 had high activity (MIC, less than or equal to 0.05 microgram/ml). ME1206 was stable against various beta-lactamases, except beta-lactamases from Providencia rettgeri, Pseudomonas cepacia, and Escherichia coli W3630 (Rms213). The 50% effective doses of ME1207 after oral administration against systemic infections in mice were comparable with those of T-2588 against gram-negative bacteria and about one-fourth that of T-2588 against Staphylococcus aureus Smith.

omp mutants, MH1160 (MC4100 ompRI) and MH760 (MC4100 ompR2), by transformation. Effects of the combination of the omp mutations and these P-lactamases on the susceptibility of E. coli strains were studied with 15 ,B-lactam antibiotics including cephalosporins, cephamycins, penicillins, imipenem, and aztreonam. The ompRI mutant, MH1160, lacks OmpF and OmpC, and it showed reduced susceptibility to 11 of the 15 P-lactam agents. The reduction in susceptibility to cefoxitin, moxalactam, and flomoxef was much greater than reduction in susceptibility to the other agents. When the ompRI mutant produced the cephalosporinase of C. freundii, the susceptibility of the mutant to 12 of the 15 P-lactam antibiotics decreased. The reduction in susceptibility of MH1160 to 10 of the 12 agents affected by the enzyme was two-to fourfold greater than that observed in MC4100. Such a synergistic effect was also observed with the cephalosporinase of P. vulgaris and ompRI mutation against six cephalosporins, moxalactam, and aztreonam.P-Lactamases of gram-negative bacteria play an important part in bacterial resistance to P-lactam antibiotics. They are mediated by chromosomes or by plasmids. Both expression of P-lactamases, i.e., amount of the enzymes, and substrate profiles of the enzymes are major factors that determine resistance levels. Also, permeability of r-lactam antibiotics through the bacterial outer membrane affects susceptibility to r-lactam agents (21). Since OmpF and OmpC porins of Escherichia coli were found to act as pores for diffusion through the outer membrane, the role of the porin channels in permeation of r-lactams has been studied actively (24, 29). Some P-lactam antibiotics, e.g., cefoxitin, are known to penetrate much more rapidly through the OmpF porin than through the OmpC porin (11). Decreased permeability of the outer membrane and alterations in the outer membrane proteins also have been studied as factors in resistance to P-lactam antibiotics (1,6, 12). Recently, the contributions of 1-lactamases and outer membrane permeability to resistance have been studied simultaneously (15,20,27). Such an approach is more appropriate when both hydrolytic rate and permeation rate are slow (5). In our studies on bacterial resistance to newer cephalosporins by cephalosporinases, a decrease in permeability intensified the reduction in antimicrobial activity by cephalosporinase against cefotaxime and ceftazidime in E. coli (7,8

A /Mactamasewas purified from Klebsiella oxytoca strain GN10650. The enzymewas chromosomally-mediated and gave a single protein band on polyacrylamide gel electrophoresis. Its pi was 5.34 and its MWwas approximately 27,000. The optimal pH and temperature were about 7.0 and 50°C, respectively. The specific activity of the enzyme was 1,207 units per mgof protein for hydrolysis of penicillins and cephalosporins, including cefuroxime, cefotaxime, and aztreonam. The enzyme activity was inhibited by /?-chloromercuribenzoate, iodine, ferrous ion, and by clavulanic acid. Rabbit antibodies raised against the purified K. oxytoca enzymeshowedno cross-reactivity in neutralization tests with /Mactamases produced by other species of Gram-negative bacteria.