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Eight new dimeric bromopyrrole alkaloids, nagelamides A−H (1−8) and a monomeric one, 9,10-dihydrokeramadine (9), have been isolated from the Okinawan marine sponge Agelas sp., and the structures were elucidated from spectroscopic data. Nagelamides A−H (1−8) exhibited antibacterial activity against Gram-positive bacteria. Nagelamide G (7) inhibited protein phosphatase 2A activity.

The carbonic anhydrases (CAs) fall into three evolutionarily distinct families designated alpha-, beta-, and gamma-CAs based on their primary structure. beta-CAs are present in higher plants, algae, and prokaryotes, and are involved in inorganic carbon utilization. Here, we describe the novel x-ray structure of beta-CA from the red alga, Porphyridium purpureum, at 2.2-A resolution using intrinsic zinc multiwavelength anomalous diffraction phasing. The CA monomer is composed of two internally repeating structures, being folded as a pair of fundamentally equivalent motifs of an alpha/beta domain and three projecting alpha-helices. The motif is obviously distinct from that of either alpha- or gamma-CAs. This homodimeric CA appears like a tetramer with a pseudo 222 symmetry. The active site zinc is coordinated by a Cys-Asp-His-Cys tetrad that is strictly conserved among the beta-CAs. No water molecule is found in a zinc-liganding radius, indicating that the zinc-hydroxide mechanism in alpha-CAs, and possibly in gamma-CAs, is not directly applicable to the case in beta-CAs. Zinc coordination environments of the CAs provide an interesting example of the convergent evolution of distinct catalytic sites required for the same CO(2) hydration reaction.

Genes for two group 1 beta-lactamases, SRT-1 and SST-1, were sequenced. These beta-lactamases were produced by clinical isolates of Serratia marcescens, isolates GN16694 and GN19450, respectively. The resulting enzymes were 96% identical. SRT-1 hydrolyzed oxyimino cephalosporins, but SST-1 hardly hydrolyzed them. At residue 213 in the third motif, which is conserved among group 1 beta-lactamases, SRT-1 and SST-1 had Lys and Glu, respectively. By site-directed mutagenesis, the substitution of Glu by Lys at residue 213 in SST-1 resulted in an enzyme that hydrolyzed oxyimino cephalosporins.

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.

Copy number mutants Rms201ts15 and Rms201ts16 were isolated at 30 degrees C from a temperature-sensitive replication mutant (Rms201ts14) of the conjugative plasmid Rms201. The numbers of plasmids per chromosome of ML1410(Rms201ts14), ML1410(Rms201ts15), and ML1410(Rms201ts16) grown at 30 degrees C were 2.2, 7.4, and 20, respectively. The synthesis of covalently closed circular plasmid deoxyribonucleic acid stopped in Rms201ts14, Rms201ts15, and Rms201ts16 immediately after a "shift-up" in temperature (42 degrees C). At 42 degrees C, antibiotic-sensitive derivatives appeared after a certain lag time: the lag times of ML1410(Rms201ts14), ML1410(Rms201ts15), and ML1410(Rms201ts16) were 2.5, 5, and 6.8 generations, respectively. After these times, plasmid-positive cells in the populations decreased at a rate of about 50% per generation in all of the mutants. From these results we conclude that plasmid segregation (partition) of Rms201 occurs by regular segregation (partition).

Esamining 12 plasmids in Pseudomonas aeruginosa, we found two types of interaction in their transfer (inhibition and facilitation), using donor cells carrying two compatible plasmids. (i) Ten plasmids representing incompatibility groups P-1, P-2, P-5, P-6, and P-7 were all transmissible at a high frequency, 10-2 to 10-1, except for one with a lower frequency of about 10-3. The transfer of P-5 plasmids was inhibited by P-2 plasmids reciprocally or unilaterally, and the unilateral transfer inhibition was observed in other combinations between plasmids belonging to groups P-1, P-2, P-6, and P-7. It was characteristic of Pseudomonas plasmids that most plasmids with high transferability inhibited the transfer of other coexisting plasmids without distinct inhibition of their own transfer. (ii) Two plasmids, Rms149 of P-8 group and Rlb679, which was not classified, were transmissible at an exceptionally low frequency of 10-7 to 10-6, but their transfer was facilitated by plasmids with high transferability.

A cyclophilin type peptidyl-prolyl cis-transisomerase was purified from a halophilic archaeum, Halobacterium cutirubrumDSM 669. The activity increased with an increase in KCl concentration up to 4 M. Sensitivity to cyclosporin A was comparable to that of eukaryotic cyclophilin and was also affected by KCl. IC50for Cyclosporin A was 1.5 × 10−8M in 2.9 M KCl but 1.4 × 10−7M in 1.4 M KCl. The apparent molecular weight was 31 K by SDS-PAGE and 22 K in 2.9 M KCl and 150 K in 0 M KCl by gel filtration chromatography. The N-terminal amino acid sequence (33 residues) sheared 4 completely and 5 highly conserved amino acid residues with other reported cyclophilin family PPIases.

We isolated a new transposon Tn2101, from plasmid Rms433 in Enterobacter cloacae. Tn2101 encoded the formation of type IV (carbenicillin-hydrolyzing) beta-lactamase and multiple resistance to streptomycin, sulfanilamide, spectinomycin, and mercury in addition to ampicillin. Tn2101 was transposable between conjugative (or nonconjugative) plasmids and the host chromosome. Transposition occurred independently of the general recombination ability of the host cell. Tn2101 had a molecular size of 9.5 x 10(6) and contained short inverted repeat terminal sequences.

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.

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.

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.

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.

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.

AB206 (5,8-dihydro-5-methoxy-8-oxo-2H-1,3-dioxolo-4,5-g quinoline 7-carboxylic acid) is a novel antibacterial agent structurally related to nalidixic acid. AB206 has potent antibacterial activity against clinical isolates of gram-negative bacteria and is 4 to 18 times more active than nalidixic acid. AB206 inhibited the growth of Staphylococcus aureus and Pseudomonas aeruginosa, which were resistnat to nalidixic acid. Cross-resistance was not observed between AB206 and various antibiotics, and most bacterial strains resistant to nalidixic acid were susceptible to AB206. AB206 showed bactericidal activity against Escherichia coli, Proteus, Klebsiella pneumoniae, and P. aeruginosa. Chemotherapeutic effects after oral administration of AB206 in mice experimentally infected with E. coli, K. pneumoniae, and Proteus morganii showed that AB206 was two to four times more potent than nalidixic acid.

KW1070, a new aminoglycoside antibiotic with the novel aminocyclitol, fortamine, has a broad spectrum of activity in vitro and in vivo against gram-positive and gram-negative bacteria. The minimum inhibitory concentrations of KW1070 are similar to those of kanamycin against aminoglycoside-susceptible strains, slightly less than those of gentamicin or 3',4'-dideoxykanamycin B. Minimal bactericidal concentrations were found to be near minimal inhibitory concentrations. KW1070 was active in vitro against many aminoglycoside-resistant bacterial strains that possess aminoglycoside-inactivating enzymes, particularly AAC(6'), AAC(2'), AAD(2''), and APH(3'). The activities of KW1070 in mice infected with Staphylococcus aureus, Escherichia coli, Proteus sp., and Serratia marcescens compared favorably with the activities of amikacin and kanamycin; KW1070 was also singificantly active in mice infected with resistant strains bearing the aminoglycoside-inactivating enzymes listed above.

T-1220, sodium 6-[d-(-)-α-(4-ethyl-2,3-dioxo-1-piperazinylcarbonyl-amino)- α-phenylacetamido] penicillanate, is a new semisynthetic penicillin derivative that possesses a broad spectrum of in vitro antibacterial activity against gram-positive and gram-negative bacteria. T-1220 is more effective than carbenicillin (CB-PC) against Pseudomonas aeruginosa, Klebsiella pneumoniae, Proteusspecies, and Serratia marcescens. Addition of human serum to culture media did not significantly alter the antibacterial activity of T-1220. Greater bactericidal activity toward various strains of gram-negative bacteria was demonstrated with T-1220 than with CB-PC. T-1220, like penicillin G, was hydrolyzed by penicillinase, but was sable to a type IV penicillinase produced by P. aeruginosastrains. In vivo antibacterial activities of T-1220, ampicillin (AB-PC), and CB-PC were compared, using systemic infections of mice with P. aeruginosa, K. pneumoniae, and Escherichia coli. The 50% effective doses (milligrams per kilogram) of T-1220 were consistently lower than those of AB-PC and CB-PC.

R plasmids identified in Pseudomonas aeruginosastrains isolated in our laboratories were classified by their incompatibility characters by using intraspecies conjugation in P. aeruginosa. The fertility factor, FP2, was compatible with R plasmids belonging to all groups, and was classified in a new group. From these data, the plasmids examined have been placed into seven or eight incompatibility groups.

Cefoperazone, a new semisynthetic cephalosporin, has a broad spectrum of antibacterial activity. It is as active as cefazolin and cefamandole against gram-positive bacteria and is more active than cefazolin and cefamandole against such gram-negative bacilli as Escherichia coli, Klebsiella pneumoniae, Proteus species, Pseudomonas aeruginosa, Citrobacter freundii, Enterobacter cloacae, and Serratia marcescens. The superiority of cefoperazone over cefazolin and cefamandole with respect to activity against P. aeruginosa by more than 200-fold was especially remarkable. As with other beta-lactam antibiotics, there was only a small spread between the minimum inhibitory concentrations and the minimum bactericidal concentrations of cefoperazone and a significant decrease in activity with an increase in inoculum size. Activity was not altered significantly by the addition of human serum to the test medium. Cefoperazone is relatively stable to hydrolysis to beta-lactamases produced by gram-negative bacteria. Relative rates of hydrolysis of cefoperazone by cephalosporinases are 7.0 to 0.01, with reference to cephaloridine hydrolysis (base, 100). Cefoperazone is also more stable than penicillin G and cephaloridine to various types of penicillinases.

Carbonic anhydrase (CA) from Porphyridium purpureum, a unicellular red alga, was purified >209-fold to a specific activity of 1,147 units/mg protein. cDNA clones for this CA were isolated. The longest clone, comprising 1,960 base pairs, contained an open reading frame which encoded a 571-amino acid polypeptide with a calculated molecular mass of 62,094 Da. The N- and C-terminal halves of the putative mature Porphyridium CA have amino acid sequence homology to each other (>70%) and to other prokaryotic-type CAs. Both regions contain, at equivalent positions, one set of three possible zinc-liganding amino acid residues conserved among prokaryotic-type CAs. CA purified from Porphyridium contained two atoms of zinc per molecule. We propose that the Porphyridium CA has evolved by duplication of an ancestral CA gene followed by the fusion of the duplicated CA gene. The CA truncated into the putative mature form was overexpressed in Escherichia coli, and the expressed protein was active. Clones expressing separately the N- and C-terminal halves of the CA were constructed. CA activity was present in extracts of E. coli cells expressing the N-terminal half, while no detectable activity was found in cells expressing the C-terminal half.

Subunits A and B of DNA gyrase were purified from Pseudomonas aeruginosa PAO1 and its mutant, which was resistant to nalidixic acid. Inhibition tests of DNA gyrases reconstituted with a combination of subunits from the two strains showed that an alteration of subunit A but not subunit B caused bacteria to resist fluoroquinolones.

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.

The in vitro and in vivo antibacterial activities of KB-5246, a tetracyclic quinolone, were compared with those of ciprofloxacin, ofloxacin, and norfloxacin. KB-5246 demonstrated a broad antibacterial spectrum. The in vitro activity of KB-5246 against gram-negative bacteria was higher than that of ofloxacin or norfloxacin and was comparable to that of ciprofloxacin. KB-5246 demonstrated the greatest activity against gram-positive bacteria of the four agents tested. Among Streptococcus pyogenes strains resistant to 1.56 micrograms of norfloxacin per ml, there were 26 strains susceptible to 0.2 micrograms of KB-5246 per ml. Similarly, among the Staphylococcus aureus and Staphylococcus epidermidis strains resistant to 3.13 micrograms of norfloxacin per ml, there were 23 S. aureus and 11 S. epidermidis strains susceptible to 0.39 micrograms of KB-5246 per ml. Among the Streptococcus pneumoniae and Enterococcus faecalis strains resistant to 12.5 micrograms of norfloxacin per ml, there were 5 S. pneumoniae and 10 E. faecalis strains susceptible to 0.39 micrograms of KB-5246 per ml. KB-5246 had bactericidal activity at the MIC. KB-5246 demonstrated excellent antibacterial activity against various systemic infections in mice. After oral administration, KB-5246 was as active as ofloxacin and about two times more active than norfloxacin.

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.

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.

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.

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.

Two genetically distinct classes of norfloxacin-resistant Pseudomonas aeruginosa PAO4009 mutants were isolated spontaneously. Two norfloxacin resistance genes, nfxA and nfxB, were mapped hex-9001 and leu-9005 and between pro-9031 and ilv-9023, respectively, on the P. aeruginosa PAO chromosome. The nfxA gene was shown to be an allele of nalA by transductional analysis with bacteriophage F116L. The nfxB mutant showed a 16-fold increase in resistance to norfloxacin and a slight increase in resistance to nalidixic acid. The nfxB mutant was unique in that it showed hypersusceptibility to beta-lactam and aminoglycoside antibiotics. This mutant had about a threefold-lower rate of norfloxacin uptake than that of the wild-type strain or nfxA mutant. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of outer membrane proteins demonstrated the appearance of a 54,000-dalton protein in the nfxB mutant. These findings suggested that the norfloxacin resistance mechanism in the nfxB mutant might be an alteration in outer membrane permeability to norfloxacin.

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.

We isolated spontaneous mutants from Escherichia coli K-12 with low-level resistance to norfloxacin. These mutants were classified into the following three types on the basis of their properties: (i) NorA appeared to result for mutation in the gyrA locus for the A subunit of DNA gyrase; (ii) NorB showed low-level resistance to quinolones and other antimicrobial agents (e.g., cefoxitin, chloramphenicol, and tetracycline), and the norB gene was considered to map at about 34 min on the E. coli K-12 chromosome; (iii) NorC was less susceptible to norfloxacin and ciprofloxacin but was hypersusceptible to hydrophobic quinolones such as nalidixic acid and rosoxacin, hydrophobic antibiotics, dyes, and detergents. Susceptibility to bacteriophages and the hydrophobicity of the NorC cell surface also differed from that of the parent strain. The norC gene was located near the lac locus at 8 min on the E. coli K-12 chromosome. Both NorB and NorC mutants had a lower rate of norfloxacin uptake, and it was found that the NorB mutant was altered in OmpF porin and that the NorC mutant was altered in both OmpF porin and apparently in the lipopolysaccharide structure of the outer membrane.

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.