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Clavulanic acid (CA) is a potent β-lactamase inhibitor produced by Streptomyces clavuligerus and has been successfully used in combination with β-lactam antibiotics (for example, Augmentin) to treat infections caused by β-lactamase-producing pathogens. Since the discovery of CA in the late 1970s, significant information has accumulated on its biosynthesis, and regarding molecular mechanisms involved in the regulation of its production. Notably, the genes directing CA biosynthesis are clustered along with the genes responsible for the biosynthesis of the β-lactam antibiotic, cephamycin C, and co-regulated, which makes this organism unique in that the production of an antibiotic and production of a small molecule to protect the antibiotic from its enzymatic degradation are controlled by shared mechanisms. Traditionally, the industrial strain improvement programs have relied significantly on random mutagenesis and selection approach. However, the recent availability of the genome sequence of S. clavuligerus along with the capability to build metabolic models, and ability to engineer the organism by directed approaches, has created exciting opportunities to improve strain productivity more efficiently. This review will include focus mainly on the gene organization of the CA biosynthetic genes, regulatory mechanisms that affect its production, and will include perspectives on improving strain productivity.

Cephalosporin beat-lactamase (cephalosporinase, CSase) was purified from a strain of Pseudomonas aeruginosa resistant to beta-lactam antibiotics. The purified enzyme preparation gave a single protein band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and its molecular weight was about 34,000. The specific activity was 49.7 mumoles/minute/mg of protein of the purified enzyme for the hydrolysis of cephaloridine. The optimal pH and optimal temperature were about 8.0 and 40 degrees C, respectively. Its isoelectric point was 8.7. The enzyme activity was inhibited by iodine, some divalent ions, and some semisynthetic beta-lactam antibiotics, including cephamycin derivatives such as moxalactam and YM09330. Mouse antiserum obtained against the purified enzyme showed no cross-reaction with other types of beta-lactamase in neutralization test.

The affinities of cefoxitin, a cephamycin antibiotic, to penicillin-binding proteins of Escherichia coli were reexamined using a recently developed method for separating penicillin-binding proteins. The inhibitions by this antibiotic of four measurable penicillin-sensitive enzymatic reactions, the reactions of D-alanine carboxypeptidases IA and IB, cross-bridge formation and concomitant release of D-alanine, were also measured. An approximate correlation was found between the affinities of cefoxitin to the penicillin-binding proteins responsible for these reactions and its rates of inhibition of the respective penicillin-sensitive reactions.

The susceptibility of 80 Bacteroides fragilis group strains isolated from clinical specimens to beta-lactam antibiotics was investigated by agar dilution method. Twenty strains showed high resistance to the antibiotics. The resistance level of the isolates to cephaloridine was related to the amount of beta-lactamase activity (cephalosporinase; CSase) produced. B. fragilis GN-11477, B. thetaiotaomicron GN11478 and B. vulgatus GN11479 were selected from among the CSase producing strains, and the enzymes were purified about 300-fold by affinity chromatography employed ampicillin as ligand bound to activated CH Sepharose 4B. The enzyme preparations gave a single protein band on polyacrylamide gel electrophoresis. The molecular weights of the three enzymes were estimated to be approximately 32,000 and their isoelectric points were 5.2, 4.9 and 4.5, respectively. The optimal pH and the optimal temperature of the enzymes were 7.2 and 37 degrees C, respectively. The enzyme activities were inhibited by iodine, some divalent ions, p-chloromercuribenzoate, clavulanic acid, cephamycin derivatives and cloxacillin. The enzymes showed hydrolytic activity against cephaloridine, cephalothin, cefazolin, cefuroxime and also newly introduced cephalosporins such as cefotaxime, cefoperazone and cefmenoxime. Each mouse antisera obtained against the purified enzymes showed cross-reactions with its each enzyme and others in neutralization test.

Cefoxitin, a semi-synthetic derivative of cephamycin C, is an analogue of cephalo-thin, and the two compounds were found to have many properties in common. A total of 232 strains of bacteria, representing 13 genera, isolated recently from clinical material, have been tested for sensitivity to both antibiotics. Against Gram-positive organisms, cephalothin was at least 8 times more active than cefoxitin, but against sensitive Gram-negative strains the activities of the two compounds were similar. Cefoxitin was active against a higher proportion of Bacteroides fragilis, Enterobacter spp. and Klebsiella spp. than was cephalothin, and was effective against 11 out of 12 indole-producing Proteus strains tested, all but one of which were resistant to cephalothin. Cefoxitin was remarkably stable in the presence of organisms which produce β-lactamase. Thus, on microbiological grounds, cefoxitin represents a significant advance over presently avail-able conventional cephalosporins.

Two new species of Streptomyces, S. heteromorphus and heteromorphus and S. panayensis, were found to produce a new atibiotic named C-2801X together with cephamycins A and B. The antibiotics were separated from each other by column chromatography on Amberlite XAD-2 and isolated in pure form as mono-sodium salts. C-2801X mono-sodium salt has a molecular formula C25H28N3O12SNa, and exhibits antibacterial activity against Gram-positive and Gram-negative bacteria including those insensitive to cephamycins A and B. From its physicochemical and biological properties, C2801X was considered to be a new cephamycin-type antibiotic.

Two hundred anaerobic isolates from clinical specimens were examined for beta-lactamase production by Nitrocefin methodology. Altogether, 77 strains were beta-lactamase producers. Organisms belonging to Bacteroides melaninogenicus group, i.e., B. intermedius and B. bivius, were found to have a significant frequency of beta-lactamase production. Substrate profile and sensitivity to the beta-lactamase inhibitors, sulbactam, clavulanic acid, cloxacillin, and cefmetazole, a cephamycin derivative, were determined for these enzymes. All enzymes hydrolyzed cephalosporins more rapidly than penicillins. Cefmetazole was not hydrolyzed at all. Strains of B. fragilis, B. thetaiotaomicron, B. uniformis, B. ovatus and B. oralis produced beta-lactamases sensitive to all four inhibitors. Strains of B. intermedius, B. bivius and B. disiens produced enzymes of different nature which were inhibited only by cefmetazole. B. vulgatus enzyme was inhibited by three of the four inhibitors. These results suggest that the beta-lactamases of the genus Bacteroides may be classified by substrate profile and inhibitor pattern.

D-Amino acid oxidase (EC 1.4.3.3) systems from Trigonopsis variabilis SANK 59963 were found to catalyze cephamycin C. The reaction products were assigned to 7 beta-(5-carboxy-5-oxovalerylamido)-7 alpha-methoxy-3-carbamoyl-3-cephem-4-carboxylic acid, and 7 beta-(4-carboxy-butyrylamido)-7 alpha-methoxy-3-carbamoyl-3-cephem-4-carboxylic acid, respectively. D-Amino acid oxidase from hog kidney was not able to catalyze cephamycin C.

A specific acylase designated A933 acylase was isolated and purified to 90% protein homogeneity from Streptomyces fulvoviridis A933 17M9 which produces PS-5, epithienamycins A and C and MM 17880 together with minor carbapenem analogs, penicillin N and cephamycin C. This enzyme was found to catalyze the depantothenylation of OA-6129 carbapenems; the acyl exchange of OA-6129 carbapenems with acyl CoA's; the deacetylation of N-acetyl-L-amino acids; and the acylation of NS-5 and 6-aminopenicillanate with acyl CoA's, whereas the deacetylation of PS-5 and N-acetyl-D-amino acids; and the deacylation of benzylpenicillin and cephalosporin C were not observed. Similar enzyme activities were also detected in Streptomyces cattleya, Streptomyces cremeus subsp. auratilis and Streptomyces argenteolus which are all carbapenem producers.

The effect of chemical modification of the D-amino acid function, which represents the C-7 beta substituent of cephamycin MT-141 on in vitro antibacterial activity was examined. MT-141 was more active on Gram-negative organisms than Gram-positive ones. It showed strong bacteriolytic activity on Gram-negative organisms. Lysis of Escherichia coli K-12 strain JE1011 treated with a low concentration of this antibiotic was preceded by frequent formation of multiple bulges from the cells. Amidation or decarboxylation, removing the acidic function from the D-amino acid of MT-141, resulted in an increase in activity against Gram-positive bacteria, and a decrease against Gram-negative ones. Cells treated with the amide or the decarboxylate did not form multiple bulges but formed single bulges. N-Acetylation of the D-amino acid moiety removing the basic function, caused a marked drop in activity against both Gram-positive and Gram-negative bacteria. The bacteriolytic activity on E. coli was reduced, and cells treated with the N-acetate became filamentous. Conversion of the D-amino acid function of MT-141 to the L configuration caused a moderate drop in activity against both Gram-positive and Gram-negative organisms. Both bacteriolytic and bactericidal activities against E. coli were reduced in the L-congener. Cefoxitin, cefmetazole and latamoxef used as reference antibiotics were less active than MT-141 in the bactericidal activity against E. coli, and induced single bulge formation or filamentation of the cells around MIC levels. Cell-surface permeability, stability to beta-lactamases, and binding affinity to PBPs of E. coli did not differ between MT-141 and its derivatives.

SQ 14,359 is a new cephamycin-type (7alpha-OCH3) antibiotic belonging to a series containing a 7alpha-ureidoacetyl substituent. The compound is the most potent extended spectrum derivative of this type yet reported, surpassing CS-1170 and cefoxitin by a wide margin. This activity in vitro which extends throughout the Enterobacteriaceae is particularly prominent against Gram-negative organisms that are producers of "cephalosporinase-type" beta-lactamases such as Enterobacter, Serratia, Citrobacter and indole-positive Proteus species. Superior activity also is demonstrated in vitro against streptococci, beta-lactamase-producing staphylococci, Haemophilus influenzae, Neisseria gonorrhoeae, and many Gram-negative pathogens resistant to aminoglycoside antibiotics. Experimental chemotherapeutic studies have confirmed these observations in wound and selected systemic infections in mice as well as acute pyelonephritis and meningitis in rats. The pharmacokinetics for each drug including antibiotic bound to serum was similar in both mice and rats. The pharmacokinetic profile in blood and cerebrospinal fluid favored SQ 14,359.

The antibacterial activity of MT-141 against Escherichia coli and Proteus morganii in compromised mice was investigated and compared with that of latamoxef, cefmetazole and cefoxitin. The bactericidal activity of MT-141 in short-term contact with E. coli and P. morganii was markedly enhanced when combined with mouse serum, and the activity of MT-141 was greater than the activities of the three reference drugs. The antibacterial activities of MT-141 in the liver, spleen and kidney of neutropenic and Sarcoma 180 tumor-bearing mice infected with E. coli and P. morganii were superior to the activities of the reference drugs. MT-141 was more potent than cefmetazole and cefoxitin, and similar to latamoxef in potency against systemic P. morganii infection in Sarcoma 180 tumor-bearing mice.