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 betalactamases was irreversible because of the high affinity of clavulanic acid to the enzymes.Bacterial strains resistant to beta-lactam antibiotics have been increasing in number. The resistance of these bacteria is due mainly to the production of beta-lactamase (1), and newer semisynthetic beta-lactam antibiotics, i.e., cefuroxime (8) and cefoxitin (12), resistant to betalactamase have been developed. Clavulanic acid (CVA) (2, 10) and CP-45899 (4) were found to be beta-lactamase inhibitors, although they have poor antibacterial activity. Combination of these products with other beta-lactam antibiotics possessing high antibacterial activity but less stability to beta-lactamase will be a new approach to chemotherapy against resistant strains capable of producing beta-lactamase. This paper deals with the combined antibacterial activity of amoxicillin (AMC) with CVA against ampicillin (APC)-resistant strains ofbacteria. MATERIALS AND METHODSTest strains. Stock strains of clinical isolated, maintained in the Reference Laboratory of Bacterial Resistance, Gunma University, were used in this study.Conjugation methods. R factors were conjugally transfered to Escherichia coli strains by a method previously described (5). E. coli ML1410 Nalr (resistant to nalidixic acid) and E. coli x1037 Rifr (resistant to rifampin) were used as the recipients for the conjugal transfer of R plasmid.Antibiotics. CVA, APC, AMC, and cloxacillin were provided by Beecham Yakuhin Co. Ltd., Tokyo, Japan. Cefoxitin was provided by Daiichi Pharmaceutical Co., Ltd., Tokyo, Japan. Other antibiotics, penicillin G and cephalothin, were commercially available.was used for the determination of antibacterial activity. Peptone water was used for liquid culture and consisted of 1% peptone and 0.5% NaCl. GAM (Nissui, Tokyo, Japan) broth and agar were used for the anaerobic bacteria. Antibiotic medium 3 (Difco) was used for the study of bactericidal activity.
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.
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