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The ampC and ampR genes of Enterobacter cloacae GN7471 were cloned into pMW218 to yield pKU403. Four mutant plasmids derived from pKU403 (pKU404, pKU405, pKU406, and pKU407) were isolated in an AmpD mutant of Escherichia coli ML4953 by selection with ceftazidime or aztreonam. The beta-lactamase activities expressed by pKU404, pKU405, pKU406, and pKU407 were about 450, 75, 160, and 160 times higher, respectively, than that expressed by the original plasmid, pKU403. These mutant plasmids all carried point mutations in the ampR gene. In pKU404 and pKU405, Asp-135 was changed to Asn and Val, respectively. In both pKU406 and pKU407, Arg-86 was changed to Cys. The ease of selection of AmpR mutations at a frequency of about 10(-6) in this study strongly suggests that derepressed strains, such as AmpD or AmpR mutants, could frequently emerge in the clinical setting.

T-3262, a new fluoroquinolone, showed a broad spectrum of activity against gram-positive and gram-negative bacteria. T-3262 had most potent activity against gram-positive cocci, such as Staphylococcus, Streptococcus, and Enterococcus spp. The MICs of T-3262 for 90% of strains tested were between 0.05 and 1.56 micrograms/ml. Against members of the family Enterobacteriaceae and Pseudomonas aeruginosa, the activities of T-3262 were almost equal to those of ciprofloxacin. Obligate anaerobes were also susceptible to T-3262. T-3262 was bactericidal for one strain each of Staphylococcus aureus, Escherichia coli, and P. aeruginosa at concentrations near its MIC; and fluoroquinolones, including T-3262, inhibited DNA gyrase activity at low concentrations. The 50% effective dose of T-3262 after oral administration against systemic infections with S. aureus in mice was about 6 times lower than that of ofloxacin and about 20 times lower than that of norfloxacin.

DNA gyrase is a bacterial enzyme which catalyzes the ATP-dependent negative supercoiling of DNA. It is the accepted target of quinolones. The enzyme from Citrobacter freundii IID976 was purified by affinity chromatography on novobiocin-Sepharose and heparin-Sepharose. It had two subunits, designated A and B, which closely resembled those of the enzyme from Escherichia coli and Micrococcus luteus in enzymatic requirements. The inhibitory effects of the quinolones on the supercoiling activities of the enzyme correlated with their antibacterial activities. New quinolones were better inhibitors of DNA gyrase than nalidixic acid and pipemidic acid. We also purified DNA gyrase from a spontaneous nalidixic acid-resistant mutant (M2-5). The gyrases from IID976 and M2-5 were defined as mixtures of subunits As+Bs (s, susceptible) and Ar+Br (r, resistant), respectively. The supercoiling activities of reconstituted Ar+Br and Ar+Bs were more resistant to quinolones than As+Bs and As+Br. These findings indicate that one mechanism of C. freundii resistance against quinolones is resistance modification of the A subunit protein.

T-2588, the pivaloyloxymethyl ester of T-2525, [6R, 7R]-7-[(z)-2-(2-aminothiazol-4-yl)-2-methoxyiminoacetoamido] -3- [(5-methyl-2H-tetrazol-2-yl)methyl]-3-cephem-4-carboxylic acid, is a new oral cephalosporin. T-2525 had a widely expanded antibacterial spectrum against gram-negative and gram-positive bacteria. T-2525 was more active in vitro than cefaclor, cephalexin, and amoxicillin against members of the family Enterobacteriaceae and Branhamella catarrhalis. Moreover, it exhibited superior in vitro activity against Streptococcus pyogenes, Streptococcus pneumoniae, Haemophilus influenzae, and Neisseria gonorrhoeae. T-2525 was highly stable to various beta-lactamases, which were classified as Richmond and Sykes types Ia, Ib, Ic, III, IV, and Vc. It had high affinities for the lethal (essential) penicillin-binding proteins of Escherichia coli, Clostridium perfringens, and Bacteroides fragilis. T-2588 had excellent therapeutic effect on systemic infections in mice with various species of gram-negative bacteria, including beta-lactamase-producing bacteria.

CS-807 is a new oral prodrug of R-3746, a cephalosporin derivative, with potent in vitro and in vivo antibacterial activity against both gram-positive and gram-negative bacteria. The susceptibility of about 1,200 clinical isolates to R-3746 was determined by the agar dilution method. Ninety percent or more of pathogens such as Staphylococcus aureus, streptococci, Escherichia coli, Klebsiella pneumoniae, Klebsiella oxytoca, indole-positive and indole-negative Proteus spp., Providencia rettgeri, and Haemophilus influenzae were inhibited at concentrations ranging less than or equal to 0.01 to 1.56 micrograms/ml. Furthermore, at a concentration of 3.13 micrograms/ml, 50% or more of Staphylococcus epidermidis, Morganella morganii, Citrobacter freundii, and Serratia marcescens strains were also inhibited. Pseudomonas aeruginosa and Xanthomonas maltophilia were resistant to R-3746. The activity of R-3746 was scarcely influenced by several growth conditions. R-3746 was highly resistant to hydrolysis by beta-lactamases derived from various species of bacteria. Killing-curve studies demonstrated bactericidal activity of R-3746 at concentrations above the MIC. R-3746 showed high affinity for penicillin-binding proteins 1, 3, and 4 of Staphylococcus aureus and 1A, 1Bs, and 3 of Escherichia coli. Systemic infections in mice caused by various pathogens, including beta-lactamase-producing strains, responded well to therapy with oral doses of CS-807.

NY-198 [1-ethyl-6,8-difluoro-1,4-dihydro-7-(3-methyl-1-piperazinyl)-4-oxo-3- quinolinecarboxylic acid hydrochloride] is a new difluorinated quinolone characterized by the presence of a C-methyl group at the 3 position of the piperazine moiety. It has a broad antibacterial spectrum. The in vitro antibacterial activity of NY-198 was almost the same as those of ofloxacin and norfloxacin, but far exceeded that of pipemidic acid. NY-198 was more active than norfloxacin against Pseudomonas maltophilia, Acinetobacter calcoaceticus, and anaerobic microorganisms. Cross resistance was not observed between NY-198 and various antibiotics including methicillin, gentamicin, and ampicillin. NY-198 had bactericidal activity at the MIC or slightly higher than the MIC. It showed excellent antibacterial activity against various systemic infections in mice. After oral administration, NY-198 was two times more active than or as active as ofloxacin and two to four times more active than norfloxacin.

An inducible beta-lactamase was purified from a beta-lactam antibiotic-resistant strain (GN11164) of Pseudomonas cepacia. The purified enzyme preparation gave a single protein band on polyacrylamide gel electrophoresis. The specific enzyme activity for the hydrolysis of cephalothin as a substrate was 314.5 U/mg of protein. The optimal pH was about 8.0, and the optimal temperature was 45 degrees C. The isoelectric point was 9.3, and the molecular weight was estimated to be about 22,000 to 24,000 from gel filtration on a Sephadex G-200 column and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The enzyme activity was inhibited by iodine, p-chloromercuribenzoate, clavulanic acid, CP 45899, and cloxacillin. The beta-lactamase showed a unique substrate profile by hydrolyzing most of the cephalosporins, including cefuroxime, cefotaxime (HR 756), ampicillin, and penicillin G, at a high rate.

The activity of AT-4140, a new fluoroquinolone, was evaluated against a wide range of clinical bacterial isolates and compared with those of existing analogs. AT-4140 had a broad spectrum and a potent activity against gram-positive and -negative bacteria, including Legionella spp. and Bacteroides fragilis. The activity of AT-4140 against gram-positive and -negative cocci, including Acinetobacter calcoaceticus, was higher than those of ciprofloxacin, ofloxacin, and norfloxacin. Its activity against gram-negative rods was generally comparable to that of ciprofloxacin. Some isolates of methicillin-resistant Staphylococcus aureus (MIC of methicillin, greater than or equal to 12.5 micrograms/ml) were resistant to existing quinolones, but many of them were still susceptible to AT-4140 at concentrations below 0.39 micrograms/ml. The MICs of AT-4140, ciprofloxacin, ofloxacin, and norfloxacin for 90% of clinical isolates of methicillin-resistant S. aureus were 0.2, 12.5, 6.25, and 100 micrograms/ml, respectively. AT-4140 was bactericidal for each of 20 clinical isolates of Staphylococcus aureus, Escherichia coli, Klebsiella pneumoniae, Serratia marcescens, and Pseudomonas aeruginosa at concentrations near the MICs. AT-4140 inhibited the supercoiling activity of DNA gyrase from E. coli.

Amifloxacin showed potent inhibitory activity against DNA gyrase of Escherichia coli. The difference in the susceptibilities of lipopolysaccharide-deficient Salmonella typhimurium mutants and their parent strain was less than twofold, and the difference in the susceptibilities of porin-deficient E. coli mutants and their parent strain was less than twofold. There was cross resistance among the quinolone group of agents; however, the decrease in MIC for norB mutants was slightly lower than that of other fluoroquinolones. Cell lysis was induced with combined treatment of amifloxacin and sodium dodecyl sulfate in E. coli. The frequency of mutants spontaneously resistant to amifloxacin was extremely low in all species tested.

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.

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.

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.

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.

Four types of beta-lactamases consisting of a penicillinase type I (TEM-1), a penicillinase type II (OXA-1), a cephalosporinase of Citrobacter freundii, and a cephalosporinase of Proteus vulgaris were introduced into Escherichia coli MC4100 and its omp mutants, MH1160 (MC4100 ompR1) and MH760 (MC4100 ompR2), by transformation. Effects of the combination of the omp mutations and these beta-lactamases on the susceptibility of E. coli strains were studied with 15 beta-lactam antibiotics including cephalosporins, cephamycins, penicillins, imipenem, and aztreonam. The ompR1 mutant, MH1160, lacks OmpF and OmpC, and it showed reduced susceptibility to 11 of the 15 beta-lactam agents. The reduction in susceptibility to cefoxitin, moxalactam, and flomoxef was much greater than reduction in susceptibility to the other agents. When the ompR1 mutant produced the cephalosporinase of C. freundii, the susceptibility of the mutant to 12 of the 15 beta-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 ompR1 mutation against six cephalosporins, moxalactam, and aztreonam.

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.

The susceptibilities of 52 clinical isolates of Bacteroides fragilis to five monoanionic cephalosporins were examined. Cefoperazone showed the highest antibacterial activity, followed by ceftezole, cefazolin, cefamandole, and cephalothin. There were two groups of resistant strains: one group (ca. 15%), of which B. fragilis G-232 was a typical sample, was resistant to ceftezole (MIC, 100 micrograms/ml), cefazolin (MIC, 100 micrograms/ml), and cephalothin (MIC, 200 micrograms/ml) but not cefoperazone (MIC, 6.25 micrograms/ml) or cefamandole (MIC, 25 micrograms/ml). On the basis of studies of stability to beta-lactamase, outer membrane permeation, and affinity for penicillin-binding proteins (PBPs), we conclude that decreased affinity for PBP 3 may play an important role in the resistance to ceftezole, cefazolin, and cephalothin in B. fragilis G-232. Another group (also ca. 15%), of which B. fragilis G-242 was a representative, was resistant to all five cephalosporins (MIC, 100 to 400 micrograms/ml) and produced a high amount of beta-lactamase. Similar broad-spectrum resistance was seen in a mutant of strain G-232 that had a greater-than-30-fold increase in beta-lactamase production.

Subunit A and B proteins of DNA gyrase were separately purified from fluoroquinolone-resistant Escherichia coli GN14181 (MIC of ofloxacin, 100 micrograms/ml) and susceptible strain KL-16. The supercoiling activities of reconstituted Ar+Br (r, resistant) and Ar+Bs (s, susceptible) were 250-fold more resistant to new fluoroquinolones than those of As+Bs and As+Br.

For clarification of the nature of the side effects of macrolide antibiotics on the gastrointestinal tract, the motor-stimulating activity of these agents was studied in unanesthetized dogs. The results showed that erythromycin and oleandomycin, the 14-membered macrolides with two side chain sugars combined at C3 and C5 in a glycosidic linkage in parallel, strongly stimulate gastrointestinal motor activity, an action accompanied by vomiting at large doses. On the other hand, leucomycin, acetylspiramycin, and tylosin, belonging to a 16-membered macrolide with two side chain sugars in series combined at C5 of the lactone ring, did not induce contractions of the gastrointestinal tract. Motor-stimulating activity by erythromycin and oleandomycin was greatly inhibited by atropine sulfate. These results point to structure-physiological activity relationships.

An inducible cephalosporinase was purified from Pseudomonas maltophilia GN12873. The pI was 8.4, and the molecular weight was ca. 56,000 by gel filtration or 27,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, suggesting that this enzyme had two subunits. The optimal pH and optimal temperature were 7.5 and 45 degrees C, respectively. Enzyme activity was inhibited by clavulanic acid, sulbactam, cephamycin derivatives, carbapenem antibiotics, iodine, HgCl2, and p-chloromercuribenzoate. The enzyme showed a broad substrate profile, hydrolyzing cephaloridine, cefazolin, cefsulodin, penicillin G, ceftizoxime, and ampicillin at a high rate.

E-0702 is an antipseudomonal cephalosporin derivative which has a broad spectrum of antibacterial activity against clinical isolates of gram-positive and gram-negative bacteria. Its in vitro antibacterial activities were less than those of cefoperazone and cefotaxime against Staphylococcus aureus and Staphylococcus epidermidis, but were significantly high against gram-negative bacteria including the Pseudomonas group. It was most characteristic that E-0702 showed high antibacterial activity against Pseudomonas aeruginosa. E-0702 was relatively stable to inactivation by plasmid-mediated penicillinases and cephalosporinases produced by gram-negative bacteria.

The susceptibility of 53 clinical isolates of Bacteroides fragilis to cephamycins was examined. Judging from the MICs for 50% of the strains tested, moxalactam was the most active, however, judging from the MICs for 90% of the strains tested, cefbuperazone was more effective than moxalactam. A correlation was observed between in vitro activity of benzylpenicillin and cephaloridine and beta-lactamase production. Inactivation due to enzymatic hydrolysis of cephamycins over a short time was not observed; however, inactivation was detected by a double disk diffusion test, and moxalactam was most easily inactivated. We conclude that inactivation due to enzymatic hydrolysis of cephamycins over a long time may play an important role in resistance to some cephamycins in strains of B. fragilis.

The in vitro antibacterial activity of Sch 34343 against 1,328 strains of clinical isolates was compared with those of imipenem and ceftazidime. Sch 34343 had a broad spectrum of antibacterial activity against both gram-positive and gram-negative bacteria but was inactive against Pseudomonas aeruginosa and Pseudomonas maltophilia. Sch 34343 was quite stable to hydrolysis by beta-lactamases, including both penicillinases and cephalosporinases. However, Sch 34343 was slightly hydrolyzed by a new type of beta-lactamase (oxyiminocephalosporin beta-lactamase), as was imipenem. Sch 34343 was slightly hydrolyzed by renal dehydropeptidase 1 but was somewhat more stable than other carbapenems.

An inducible penicillin beta-lactamase was purified from a strain of Alcaligenes faecalis resistant to beta-lactam antibiotics. The purified enzyme preparation gave a single protein band on polyacrylamide gel electrophoresis, and its molecular weight was 29,000 based on sodium dodecyl sulfate-acrylamide gel electrophoresis. Its isoelectric point was 5.9. The enzyme more rapidly hydrolyzed penicillins, such as penicillin G, ampicillin, carbenicillin, piperacillin, and cloxacillin, than it hydrolyzed cephalosporins. For the hydrolysis of penicillin G, the optimal pH was 5.5, and the optimal temperature was 35 degrees C. The enzyme activity was inhibited by iodine, Cu2+, Hg2+, and EDTA but was not inhibited by clavulanic acid and sulbactam.

Bacteroides fragilis strains were isolated from clinical specimens. B. fragilis G-237 was highly resistant to beta-lactam antibiotics due to beta-lactamase production. The purified enzyme from this strain gave a single protein band on polyacrylamide gel electrophoresis. The isoelectric point was 4.8, and the molecular weight was estimated to be 26,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The enzyme activity was inhibited by p-chloromercuribenzoate and iodine but not by clavulanic acid or sulbactam. The purified enzyme showed a unique substrate profile by hydrolyzing at a high rate most of the cephalosporins, including cephamycin derivatives, penicillins, and imipenem (formerly imipemide, N-formimidoyl thienamycin, or MK 0787).

AT-2266 [1-ethyl-6-fluoro-1,4-dihydro-4-oxo-7-(1-piperazinyl)-1,8-naphthyridine-3-carbo xylic acid] showed a broad spectrum of antibacterial activity against gram-positive and gram-negative microorganisms, including Pseudomonas aeruginosa. The in vitro antibacterial activity of AT-2266 was in general comparable to that of norfloxacin, but much higher than that of pipemidic or nalidixic acid. The 90% minimal inhibitory concentrations (MIC90s) of AT-2266 for P. aeruginosa resistant to gentamicin (MIC range, 25 to greater than 200 microgram/ml) and Enterobacteriaceae resistant to nalidixic acid (25 to greater than 1,600 micrograms/ml) were 3.13 and 12.5 micrograms/ml, respectively. The to nalidixic acid (25 to 1,600 micrograms/ml) were 3.13 and 12.5 micrograms/ml, respectively. The MICs of AT-2266 were only slightly affected by the addition of horse serum or sodium cholate, by the pH of the medium, and by inoculum size. AT-2266 was sodium cholate, by the pH of the medium, and by inoculum size. AT-2266 was bactericidal at concentrations near its MIC value. The 50% effective doses of AT-2266 after oral administration against systemic infections in mice were about 1/2 those of norfloxacin, about 1/10 those of pipemidic acid, and between 1/20 and 1/40 those of nalidixic acid.

The in vitro and in vivo activities of Sm-1652 were compared with those of other cephalosporins. SM-1652 possessed a wide antibacterial spectrum which included activity against Pseudomonas aeruginosa. It also exhibited potent antibacterial activities against gram-positive cocci and clinical isolates of glucose nonfermentative bacteria. Most notably, its activity against glucose nonfermentative bacteria was the highest of all of the drugs tested. The bactericidal activity of SM-1652 was compared with that of cefoperazone. The difference between the minimum bactericidal concentration and the minimum inhibitory concentration of SM-1652 was actually smaller than that of cefoperazone for Escherichia coli and clinical isolates of indole-positive Proteus spp. SM-1652 was stable for most cephalosporinases but was hydrolyzed to some extent by penicillinases. The in vivo therapeutic effect of SM-1652 against infections in mice was better than those of cefazolin and cefoxitin. The in vivo antipseudomonal activity of SM-1652 was second to that of cefsulodin.

A highly penicillin-resistant strain of Bacteroides fragilis, strain GN11499, was found among 80 clinical isolates of the B.fragilis group and appears to produce a new type of penicillinase. Penicillinase activity was detected in crude extracts and had a specific activity of 0.25 U/mg of protein. About 20% of the enzyme was released into the surrounding medium during growth. The enzyme hydrolyzed ampicillin and cloxacillin more rapidly than it did penicillin G, carbenicillin, and cephaloridine. Relative rates in a crude extract with penicillin G as 100 were ampicillin, 357; carbenicillin, 57; cloxacillin, 271; and cephaloridine, 71. Enzyme activity was inhibited by clavulanic acid, CP-45, 899, N-formimidoyl thienamycin, cefoxitin, moxalactam, and p-chloromercuribenzoate. The enzyme had a molecular weight of approximately 41,500 and an isoelectric point of 6.9. Penicillinase production and tetracycline resistance were transferred from B.fragilis GN11499 to two susceptible strains of B.fragilis and Bacteroides vulgatus by filter mating.

DL-8280, 9-fluoro-3-methyl-10-(4-methyl-1-piperazinyl)-7-oxo-2,3-dihydro-7H- pyrido-(1,2,3-de)1,4-benzoxazine-6-carboxylic acid, is a new nalidixic acid analog with a broad spectrum of antibacterial activity against gram-negative and gram-positive bacteria, including obligate anaerobes. The activity of DL-8280 against Enterobacteriaceae, Pseudomonas aeruginosa, Haemophilus influenzae, Neisseria gonorrhoeae, and Clostridium perfringens was roughly comparable to that of norfloxacin and far exceeded that of pipemidic acid and nalidixic acid. DL-8280 had greater activity against Staphylococcus spp., Streptococcus spp., Pseudomonas maltophilia, Acinetobacter spp., and Bacteroides fragilis than did norfloxacin, pipemidic acid, and nalidixic acid. Nalidixic acid-resistant Enterobacteriaceae, ampicillin-resistant gonococci, and clindamycin-resistant obligate anaerobes were also susceptible to DL-8280. The activity of DL-8280 was affected very little by inoculum size, and its action was bactericidal at two times the minimal inhibitory concentrations at most. Administered orally to mice experimentally infected with Staphylococcus aureus, Streptococcus pyogenes, Escherichia coli, Proteus mirabilis, Serratia marcescens, or P. aeruginosa, DL-8280 was 2 to 7 times more effective than norfloxacin and 7 to more than 50 times more active than pipemidic acid.

Two types of beta-lactamase were found in the cell-free extract from Pseudomonas maltophilia GN12873. One was an inducible penicillin beta-lactamase, and the other was an inducible cephalosporin beta-lactamase. The purified penicillin beta-lactamase gave a single protein band on polyacrylamide gel electrophoresis. The isoelectric point was 6.9, and the approximate molecular weight was 118,000 by gel filtration and 26,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, suggesting that this enzyme consisted of four subunits. For the hydrolysis of penicillin G, the optimal pH was 8.0 and the optimal temperature was 35 degrees C. The enzyme activity was inhibited by cephamycin derivatives, carpetimycins A and B, iodine, and HgCl2, but not by clavulanic acid. Furthermore, beta-lactamase activity was almost completely inhibited by EDTA but was recovered by the addition of zinc ion. The enzyme showed a unique substrate profile, hydrolyzing N-formimidoyl thienamycin at a significant rate.