Immunological properties of a novel beta-lactamase produced by Bacteroides fragilis.

In the previous paper1), we reported the comparative study of the enzymatic and physicochemical properties of a novel 3-lactamase produced by Bacteroides fragilis G-237 with those of a typical B. fragilis /3-lactamase produced by B. fragilis G-242. The enzyme produced by B. fragilis G-237 showed a unique substrate profile in hydrolyzing cephalosporins including 7amethoxycephalosporins, penicillins and carbapenems. The enzyme activity was inhibited bypchloromercuribenzoate and iodine, but not by clavulanic acid and sulbactam. The isoelectric point was 4.8, and molecular weight was estimated to be 26,000. This paper deals with the immunological properties of purified /3-lactamase from B. fragilis G-237. Bacterial strains used in this study were: B. fragilis G-237 and G-242, Bacteroides vulgatus G-101, Bacteroides thetaiotaomicron G-282, Bacteroides ovatus G-53, Bacteroides distasonis G-50, Bacteroides uniformis G-294, Proteus vulgaris GN79712', Pseudomonas cepacia GN111643', Rtk3+/Escherichia coil TK-3 (penicillinase type I) (Richmond class III)4,5,6), Rms2l3+/E. coli W3630 (PCase type II) (Richmond class V type a)°', Rte16+/E. coli W3630 (PCase type III) (Richmond class V type b)8', Rms219+/Pseudomonas aeruginosa M1 (PCase type IV) (Richmond class V type d), Staphylococcus aureus F-186 (PCase type V), E. coli GN54829' and Enterobacter cloacae H-27. For the preparation of crude enzymes, we followed the method described previously"). For the production of specific anti-enzyme serum, the enzymes from B. fragilis G-237 and G-242 were purified in a previously-described manner1). Other enzymes were purified by absorption and elution on a DEAE-Sephadex A-50 column or CM-Sephadex C-50 column11). Antisera against purified B. fragilis G-237 and G-242 enzymes were obtained from rabbits. One-tenth milligram of enzyme protein was dissolved in 0.5 ml of saline, emulsified with 0.5 ml of Freund complete adjuvant (Difco), and used for the first injection. An injection of 0.5 ml was given under the skin of the footpad, and the remaining dose was given in the thigh. Three weeks after the first injection, a booster injection containing 0.1 mg of enzyme protein in 0.5 nil of saline was administered intravenously. Antisera were collected 2 weeks after the last injection. The immunological identification of a 3-lactamase was demonstrated by a neutralization test using spectrophotometric method10). The enzyme solution was mixed with the antiserum at 37"C for 1 hour and then kept at 0"C for 18 hours. The mixture was centrifuged for 10 minutes at 3,000 rpm, and the remaining enzyme activity in the supernatant was assayed12). The lactamase activity was inhibited by specific anti-enzyme serum. The standard neutralization curve was obtained by adding increasing quantities of antiserum to a fixed quantity of antigen (Fig. 1). The enzyme activities were al-