The induction of f,-lactamase in Enterobacter cloacae GN5797 was studied by using 23 .-lactam antibiotics, including newly introduced drugs, as inducers. The ,B-lactam antibiotics can be classified into three groups on the basis of their inducer activity. Among the tested cephalosporins, cephamycin derivatives such as cefoxitin, cefmetazole, and YMO9330 had high inducer activity even at low drug concentrations. On the other hand, cefoperazone, cefsulodin, piperacillin, and apalcillin showed low inducer activity when compared with the other cephalosporins. ,B-Lactamases specified by drug-resistant plasmids have been found to be of the penicillinase type (3). Most of the gram-negative bacteria isolated from clinical materials, however, produce cephalosporinases which are species specific in their substrate profiles (4). The inducible formation of ,B-lactamase is of special importance in clinical medicine and in the development of new ,B-lactam antibiotics and has received relatively little attention (1,5,6,11). This paper deals with the inducer activity of a number of,B-lactam antibiotics, including some of the newly developed drugs, for the production of /8-lactamase by Enterobacter cloacae. MATERIALS AND METHODSBacterial strain. A clinical isolate E. cloacae GN5797, a stock culture in this laboratory, was used in this study.Media. Brain heart infusion broth (Difco Laboratories, Detroit, Mich.) was used for both the growth of E. cloacae and the induction of fi-lactamase. Heart infusion agar (Eiken, Tokyo, Japan) was used for the determination of the minimal inhibitory concentration of the antibiotics by the agar dilution method.Antibiotics. Cephaloridine, cefazolin, cephalothin, cephalexin, penicillin G, ampicilhin, carbenicillin, cloxacillin, and methicillin were commercially available materials. Cefsulodin and cefotiam (Takeda Pharmaceutical, Osaka, Japan), cefamandole and moxalactam (6059S) (Shionogi Pharmaceutical, Osaka), cefoperazone and piperacillin (Toyama Chemical, Tokyo), cefuroxime (Shinnihon Jitsugyo, Tokyo), ceftizoxime (FK-749) (Fujisawa Yukuhin, Osaka) (12), cefoxitin (Daiichi Pharmaceutical, Tokyo), cefmetazole (Sankyo, Tokyo), YM09330 (Yamanouchi Pharmaceutical, Tokyo) (13), apalcillin (Sumitomo Chemical, Osaka), and clavulanic acid (Beecham Yakuhin, Tokyo) were the newly introduced drugs.Determination of the minimal inhibitory concentration. The minimal inhibitory concentration was determined by a serial dilution technique. Overnight cultures of test strains in brain heart infusion broth were diluted to a final concentratiua of 106 cells per ml, and one loopful (0.005 ml) of culture was inoculated onto heart infusion agar plates by use of a Microplanter (Sakuma, Tokyo) inoculator. The minimal inhibitory concentration was determined after overnight incubation at 37°C.Enzyme assay. ,B-Lactamase activity was determined either by a spectrophotometric method (10, 14) measuring the decrease in absorbance at an appropriate wavelength of the substrate (100 IM) in a temperature-controlled spectro...

Bacteroidesfragilis strains were isolated from clinical specimens. B. fragilis G-237 was highly resistant to P-lactam antibiotics due to P-lactamase production.The purified enzyme from this strain gave a single protein band on polyacrylamide gel electrophoresis. The isoelectric point was 4.8, and the molecular weight was estimated to be 26,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The enzyme activity was inhibited by p-chloromercuribenzoate and iodine but not by clavulanic acid or sulbactam. The purified enzyme showed a unique substrate profile by hydrolyzing at a high rate most of the cephalosporins, including cephamycin derivatives, penicillins, and imipenem (formerly imipemide, N-formimidoyl thienamycin, or MK 0787).P-Lactamase production plays a significant role in resistance to P-lactam antibiotics (8, 14, 20). The enzymes from gram-positive bacteria are inducible, are extracellular, and have predominantly penicillinase activity (5, 10). The enzymes from gram-negative bacteria have been classified into several groups by substrate profile, inhibitory profile, and physicochemical and immunological properties (8,14,20).Bacteroidesfragilis strains have been isolated from clinical specimens with increasing frequency and are known to be moderately or highly resistant to penicillins and cephalosporins (4,19). Resistance has been ascribed to P-lactamase production by many investigators (1,9,(11)(12)(13)(16)(17)(18). Two kinds of P-lactamase from B. fragilis are known: cephalosporinases (1,11,12,16,18) and penicillinases (9,13,17).We selected two strains from B. fragilis that had been isolated due to their resistance levels against various P-lactam antibiotics and purified their ,-lactamases. This paper compares the properties of a novel P-lactamase produced by B. fragilis G-237 with those of a typical B. fragilis P-lactamase produced by strain G-242. Media. GAM agar and GAM broth (18) were the products of Nissui Pharmaceutical Co., Ltd.Antibiotic susceptibility testing. Drug resistance was determined by the agar dilution method described previously (17).Purification of ,B-lactamase. For the preparation of crude enzymes, we followed the method described previously (17). The P-lactamases were purified by absorption and elution on a DEAE-cellulose DE-52 column, gel filtration on a Sephadex G-150 column, and gel filtration on a Sephadex G-75 superfine column.Assay of P-lactamase and inhibition study. For the assay of P-lactamase and the inhibition study, we followed methods described previously (17). The millimolar absorbancy differences (Ac-) (7)

The susceptibilities of 52 clinical isolates of Bacteroides fragilis to five monoanionic cephalosporins were examined. Cefoperazone showed the highest antibacterial activity, followed by ceftezole, cefazolin, cefamandole, and cephalothin. There were two groups of resistant strains: one group (ca. 15%), of which B. fragilis G-232 was a typical sample, was resistant to ceftezole (MIC, 100 micrograms/ml), cefazolin (MIC, 100 micrograms/ml), and cephalothin (MIC, 200 micrograms/ml) but not cefoperazone (MIC, 6.25 micrograms/ml) or cefamandole (MIC, 25 micrograms/ml). On the basis of studies of stability to beta-lactamase, outer membrane permeation, and affinity for penicillin-binding proteins (PBPs), we conclude that decreased affinity for PBP 3 may play an important role in the resistance to ceftezole, cefazolin, and cephalothin in B. fragilis G-232. Another group (also ca. 15%), of which B. fragilis G-242 was a representative, was resistant to all five cephalosporins (MIC, 100 to 400 micrograms/ml) and produced a high amount of beta-lactamase. Similar broad-spectrum resistance was seen in a mutant of strain G-232 that had a greater-than-30-fold increase in beta-lactamase production.

The purified cephalosporinase from Proteus vulgaris hydrolyzed a variety of cephalosporins, including cefuroxime, at a high level; its activity was inhibited by clavulanic acid.

The ~-lactam antibiotics can be classified into 3 groups on the basis of their inducer activity for r3-lactamase in Proteus rettgeri and Proteus vulgaris.In our previous paper1), we reported that the S-lactam antibiotics could be classified into several groups on the basis of their inducer activity for (3-lactamase in Enterobacter cloacae. There are a few reports concerned with the inducible formation of (3-lactamase2-6). Proteus rettgeri and Proteus vulgaris also produce inducible cephalosporinases (CSase) which are species-specific in their substrate profiles7-9). This paper deals with the inducer activity of a number of (3-lactam antibiotics for the production of ,3-lactamases by P. rettgeri and by P. vulgaris. P. rertgeri GN4430 and P. vulgaris GN76 are clinical isolates maintained as stock cultures in this laboratory. For determination of inducer activity, various concentrations of each drug were added to a mid log phase culture and shaking of the culture continued. After a further 2 hours of incubation, the cells were harvested and washed once with 0.1 M phosphate buffer (pH 7.0). The drug concentrations added to the culture ranged from 10 x MIC to 0.01 x MIC. MIC (Minimal inhibitory concentration) was determined by the agar dilution method. A loopful (5 /l) of 106/ml cells was inoculated on a heart infusion (HI) agar (Difco) plate containing a series of serial twofold dilutions of a drug. The MIC was scored after 18 hours of incubation at 37°C. Under these conditions most of the drugs showed little or no effect on the growth of P. rettgeri GN4430 when their concentrations were less than 100 jig/ml. Antibacterial activity and inducer activity of various 13-lactam antibiotics against P. rettgeri GN4430 are shown in Table 1. P. rettgeri GN4430 was resistant to many 13-lactam antibiotics except for the newly-introduced drugs. Both the cefuroxime type cephalosporins such as cefotaxime and ceftizoxime, and cephamycin derivatives such as latamoxef and cefotetan were highly active against P. rettgeri GN4430. Based on inducer activity, the j3-lactam antibiotics can be classified into three groups, i.e., with high (A), intermediate (B), and low (C) inducibility.Cephaloridine, cephalothin, cephalexin and cefazolin which are easily hydrolyzed by the cephalosporinase, and the cephamycin derivatives such as cefoxitin, cefinetazole and cefotetan which are not hydrolyzed by the cephalosporinase, showed very high inducer activities for enzyme induction. Cefamandole, cefotiam, cefsulodin, cefuroxime, benzylpenicillin and ampicillin are also in the same group (A). Methicillin and cloxacillin10) and clavulanic acid'l~ also showed high inducer activity. However, latamoxef, carbenicillin and apalcillin showed intermediate inducer activity (group (B)). Cefoperazone, cefotaxime and piperacillin showed low inducer activity (group (C)).