Clavulanic Acid: a Beta-Lactamase-Inhibiting Beta-Lactam from Streptomyces clavuligerus

Abstract

A novel ,8-lactamase inhibitor has been isolated from Streptomyces clavuligerus ATCC 27064 and given the name clavulanic acid. Conditions for the cultivation of the organism and detection and isolation of clavulanic acid are described. This compound resembles the nucleus of a penicillin but differs in having no acylamino side chain, having oxygen instead of sulfur, and containing a (3-hydroxyethylidine substituent in the oxazolidine ring. Clavulanic acid is a potent inhibitor of many 8-lactamases, including those found inEscherichia coli (plasmid mediated), Klebsiella aerogenes, Proteus mirabilis, and Staphylococcus aureus, the inhibition being ofa progressive type. The cephalosporinase type of /8-lactamase found in Pseudomonas aeruginosa and Enterobacter cloacae P99 and the chromosomally mediated (8-lactamase ofE. coli are less well inhibited. The minimum inhibitory concentrations of ampicillin and cephaloridine against ,8-lactamase-producing, penicillin-resistant strains of S. aureus, K. aerogenes, P. mirabilis, and E. coli have been shown to be considerably reduced by the addition of low concentrations of clavulanic acid.Streptomyces clavuligerus ATCC 27064 (NRRL 3585) has been described as producing several antibiotics structurally related to cephalosporin C, namely, the 3-carbamoyloxymethyl analogue, the 7-methoxy derivative of the latter compound (cephamycin C), and deacetoxy cephalosporin C, as well as penicillin N (8, 9, 12, 16; M. Gorman, M. M. Hoehn, R. Nagarajan, L. D. Boeck, E. A. Presti, J. G. Whitney, and R. L. Hamill, Prog. Abstr. Intersci. Conf. Antimicrob. Agents Chemother., 11th, Atlantic City, N.J., abstr. 14, p. 7, 1971). During an investigation of the metabolites produced by this culture, a pronounced /8-lactamase inhibitory activity was detected in the culture filtrate using a special bioassay procedure based on /8-lactamase inhibition (4). The substance responsible for the /3-lactamase inhibitory activity was named clavulanic acid (4) and has been shown to have the structure given in Fig. 1 (10). We describe below the detection, isolation, and preliminary information on the /3-lactamase-inhibitory properties of clavulanic acid.MATERIALS AND METHODSCultural conditions for S. clavuligerus. S. clavuligerus ATCC 27064 (NRRL 3585) was grown at 26°C on agar slopes containing 1% Yeatex yeast extract, 1% glucose, and 2% Oxoid agar no. 3, pH 6.8. Mycelium and spores from the slope were used to inoculate flasks containing a seed stage medium consisting of (wt/vol) 1% malt extract (Oxoid), 1% bacteriological peptone (Oxoid), and 2% glycerol. The medium was made up using tap water and adjusted to pH 7.0 with sodium hydroxide solution. Inoculated flasks were shaken for 3 days at 26°C.Production stage flasks containing the DAS medium were inoculated with 5% vegetative inoculum from the seed flasks. The DAS medium consisted of 2% dextrin, 1% Arkasoy 50 soyabean flour (British Arkady Co., Manchester, U.K.), 0.1% Scotasol dried distillers solubles (Thomas Borthwick Ltd, Glasgow, U.K.), and 0.01% FeSO4 7H2O...

Targeting Antibiotic Resistance

“…The first β‐lactamase inhibitor to be used was clavulanic acid ( 51 , discovered at Beecham Pharmaceuticals) in the mid‐1970s,166 followed over the next decade by sulbactam ( 52 )167 and tazobactam ( 53 ) 168. As seen in Figure 35, these β‐lactamase inhibitors all possess a β‐lactam core, but they have been shown to have only limited antibiotic activity.…”

Beta-Lactamase Stability of HR 756, a Novel Cephalosporin, Compared to That of Cefuroxime and Cefoxitin

“…There is great interest in antibiotics that are resistant to fl-lactamase hydrolysis and in compounds that are inhibitors of 8B-lactamases (2,5,11 (11). Recently, naturally occurring inhibitors of ,B-lactamases produced by streptomyces have been discovered (1,14). Cefoxitin and cefuroxime, two new fi-lactam compounds, have been reported to be resistant to B8-lactamases of gram-negative bacilli (3,12).…”

MOLECULAR AND MORPHOLOGICAL IDENTIFICATION OF STREPTOMYCES SP. NRC-88 NOVA SPECIES AS Β-Lactamase INHIBITOR FOR PHARMACEUTICAL APPLICATION

“…The isolates were primarily tested for their ability of β-lactamase inhibitor by the specific synergistic bioassay [20] using a resistant test strain of E. coli. CA was detected by thin-layer chromatography (TLC) [21]. It was determined by spectrophotometric method at 312 nm after derivatization with imidazole according to Bird et al [22], and confirmed the structure of the CA by high performance liquid chromatography (HPLC) assay [23].…”