A ,-lactamase from Branhamella catarrhalis was purified by column chromatography. The purified enzyme hydrolyzed penicillins, such as ampicillin, carbenicillin, and piperacillin, more rapidly than cephalosporins. Furthermore, the enzyme hydrolyzed cefotaxime and cefmenoxime. The molecular weight of the enzyme was 33,000. The pl was 5.4.Branhamella catarrhalis, a gram-negative diplococcus, has recently been isolated with increasing frequency from clinical specimens (1,10,13,14,16). P-Lactamase has been considered to be one of the important biochemical mechanisms of resistance to P-lactam antibiotics in bacteria. Several investigations have already described the P-lactamase from B. catarrhalis (3-5, 9, 18-20). However, there have been no reports on the biochemical properties of the purified P-lactamase. This paper deals with the enzymological and physicochemical properties of the purified P-lactamase produced by B. catarrhalis NNBR-8303.Sensitivity disk agar-N and sensitivity test broth (Nissui Pharmaceutical Co., Ltd.) were used. Penicillin G (PCG), ampicillin, carbenicillin, cloxacillin, piperacillin, ticarcillin, cephaloridine, cephalothin, and cefazolin were commercially available materials. We received the following compounds as gifts from their manufacturers: cefoperazone, cefpiramide, cefsulodin, cefuroxime, cefotaxime, ceftizoxime, cefmenoxime, cefoxitin, cefotetan, moxalactam, clavulanic acid, sulbactam, imipenem, and aztreonam. The MICs were determined by a standard agar dilution method with sensitivity disk agar-N. Sensitivity disk agar-N plates containing serial twofold dilutions of a drug were inoculated with one loopful (ca. 5 Rd; ca. 108 CFU/ml) of an overnight culture in sensitivity test broth. The MICs were scored after 18 h of incubation at 37°C.For extraction and purification of the enzyme, bacteria were grown overnight in 1,000 ml of sensitivity test broth with shaking at 37°C. The culture was diluted 10-fold with fresh sensitivity test broth and then grown at the same temperature with shaking until the optical density at 570 nm reached about 0.8. The bacterial cells were harvested by centrifugation at 4°C, washed twice with 50 mM phosphate buffer (pH 7.0), and suspended in the same buffer at 5% of the original volume. The cell suspension was sonicated at 75 W for 3 min on an ice bath. The disrupted cell suspension was centrifuged at 100,000 x g for 1 h at 4°C. Streptomycin sulfate was added to the resulting supernatant fluid at a final concentration of 2% (wt/vol) for the removal of nucleic acids. The mixture was allowed to stand for 5 h, the precipitate was removed by centrifugation, and the superna-* Corresponding author. tant was dialyzed against distilled water for 10 h. The precipitate that formed after dialysis was removed by centrifugation, and the resulting supernatant was dialyzed against 5 mM phosphate buffer (pH 7.0). The dialyzed solution was used as the crude enzyme preparation.The crude enzyme solution was applied to a DEAESepharose column (3.0 by 30 cm) equilibrated with 5 mM ph...
The in-vitro synergic activities of BRL42715, a new beta-lactamase inhibitor, clavulanic acid, sulbactam, and tazobactam combined with ampicillin, piperacillin, cephalothin, or cefoperazone were tested against various bacteria producing known types of beta-lactamase. BRL42715 showed the best synergistic activity among the inhibitors tested against strains producing penicillinases of type I, II, III, V, and that from Klebsiella pneumoniae, cephalosporinases, and oxyiminocephalosporinases (except that from Klebsiella oxytoca). Clavulanic acid combined with the beta-lactams tested showed the best synergic activity of the inhibitors against strains producing type IV penicillinase and oxyiminocephalosporinase from K. oxytoca. The 50% inhibitory doses of BRL42715 were superior to those of clavulanic acid against various types of beta-lactamases except for type IV penicillinase and the oxyiminocephalosporinase from K. oxytoca. The inhibitory activity of BRL42715 against cephalosporinases from various bacteria was 10(4) to 10(6)-fold greater than that of clavulanic acid. The synergic effects of BRL42715 and clavulanic acid on the activity of piperacillin were compared against six clinical isolates of bacteria resistant to piperacillin. The synergic activity of BRL42715 was greater than that of clavulanic acid in all six isolates.
Rabbit antiserum was prepared to the penicillinase of Alcaligenes denitrificans subsp. xylosoxydans GN14062. The antibody gave a single precipitin line in double immunodiffusion assays with the penicillinases from strain GN14062 and from two other A. denitrificans subsp. xylosoxydans strains. The precipitin line given by the GN14062 enzyme gave a reaction of complete identity with the precipitin lines of the penicillinases from the other A. denitrificans subsp. xylosoxydans strains. Furthermore, activity of the penicillinases from all three A. dentrificans subsp. xylosoxydans strains was neutralized by the antiserum to the GN14062 enzyme. Thus it was proved that the penicillinases produced by the three A. denitrificans subsp. xylosoxydans strains were immunologically identical. Penicillinases types I, II, III, IV, V, SHV-1, TEM-1, TEM-2, and the chromosomal penicillinases of Klebsiella pneumoniae GN69 and A. faecalis GN14061 formed no immunoprecipitate with, nor were neutralized by, the antiserum to the GN14062 enzyme. From these results, it was concluded that the penicillinase produced by A. denitrificans subsp. xylosoxydans was immunologically distinct from the penicillinases produced by these other bacteria.
A broad substrate-spectrum /3-lactamase was purified from Flavobacterium meningosepticum GN14059.The purified enzyme gave a single protein band on SDS-polyacrylamide gel electrophoresis. The molecular weight was estimated to be about 30,000 and the isoelectric point was 5.1. The enzyme hydrolyzed various /5-lactam antibiotics including oxyiminocephalosporins and aztreonam. Relative rates, with cephaloridine as 100, were cephalothin 200, cefazolin 48, cefuroxime 153, cefotaxime 51, ceftazidime 20, ampicillin 26, carbenicillin 19, and aztreonam 20. The enzymeactivity was inhibited by clavulanic acid, sulbactam, imipenemand cephamycins.
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