Activity of cefoperazone and two beta-lactamase inhibitors, sulbactam and clavulanic acid, against Bacteroides spp. correlated with beta-lactamase production.

A total of 102 isolates of Bacteroides spp. were studied for beta-lactamase production and susceptibility to cefoperazone alone or in combination with either of the beta-lactamase inhibitors sulbactam and clavulanic acid. The geometric mean minimal inhibitory concentration of cefoperazone alone was 31.5 micrograms/ml and when combined with 10 micrograms of sulbactam per ml or 2 micrograms of clavulanic acid per ml was reduced to 5.4 and 9.2 micrograms/ml, respectively. When bacterial suspensions were tested for beta-lactamase production with nitrocefin, 91 (89.2%) of these isolates produced the enzyme. The geometric mean minimal inhibitory concentrations of cefoperazone rose only slightly for isolates with low or intermediate enzyme activity but rose significantly for those with high activity. The addition of EDTA to cefoperazone significantly more frequently enhanced the activity of cefoperazone against beta-lactamase-negative as opposed to beta-lactamase-positive isolates. Furthermore, EDTA resulted in synergistic activity of the cefoperazone-sulbactam combination on beta-lactamase-positive isolates for which the combination had previously not shown a synergistic effect. This study demonstrates the relationship between beta-lactamase production and the resistance of Bacteroides spp. to cefoperazone and shows that inhibition of these enzymes can reverse this resistance.     

Activity of beta-lactamase produced by Bacteroides fragilis against newly introduced cephalosporins

The purified beta-lactamase from Bacteroides fragilis hydrolyzed newly introduced cephalosporins including cefuroxime and HR 756, and was inhibited by 7 alpha-methoxylated cephalosporins such as 6059-S and YM09330.     

Augmentation effect of clavulanic acid with penicillin, cephalothin and ticarcillin against Bacteroides fragilis

The effect of clavulanic acid on the in vitro activity of beta-lactam antibiotics against Bacteroides fragilis (154 strains) was tested. The MIC90 on 154 strains of B. fragilis tested was greater than 64 micrograms/ml for penicillin and cephalothin, and greater than 128 for ticarcillin alone. 32 strains of B. fragilis relatively resistant to the test beta-lactam antibiotics (most of them beta-lactamase producers) were retested with the addition of clavulanic acid. 90% of the strains were then inhibited by less than or equal to 8 micrograms/ml of penicillin and cephalothin, and by 32 micrograms/ml of ticarcillin. The strongest beta-lactamase producers were the most susceptible, and this was not influenced by change in pH of the diluent for clavulanic acid from 6.0 to 7.4. Both penicillin and cephalothin when combined with clavulanic acid were highly effective against B. fragilis but the therapeutic relevance of these combinations remains to be evaluated.     

Synergistic activity of mecillinam in combination with the beta-lactamase inhibitors clavulanic acid and sulbactam

The beta-lactamase inhibitors clavulanic acid and sulbactam were combined with mecillinam. beta-Lactamase-containing Escherichia coli resistant to mecillinam was synergistically inhibited by both clavulanic acid and sulbactam. beta-Lactamase-containing Enterobacter was synergistically inhibited, but strains lacking beta-lactamases were not synergistically inhibited. Synergistic inhibition was noted for beta-lactamase-containing, mecillinam-resistant Klebsiella, Citrobacter, Serratia, and Salmonella isolates, but only 18% of beta-lactamase-containing Proteus mirabilis, Providencia rettgeri, Providencia stuartii, and Morganella morganii were synergistically inhibited by the combinations.     

The effects of clavulanic acid and sulbactam on beta-lactamase biosynthesis

Clavulanic acid and sulbactam were tested as inducers of beta-lactamase in 21 strains of Gram-negative bacteria. beta-Lactamase synthesis was inducible in all these strains, as demonstrated by the increased enzyme activities obtained when cells were grown in the presence of 10 mg/l of cefoxitin, the increase varying from 11- to 734-fold However, at the same concentration, neither inhibitor induced significant amounts of beta-lactamase, except in one strain of Providencia rettgeri, where both were potent inducers. When steps were taken to overcome the inhibitory effects of clavulanic acid and sulbactam, they were still found to be ineffective inducers. We conclude that, at therapeutic concentrations, clavulanic acid and sulbactam are generally poor inducers of beta-lactamase. Amoxycillin and ampicillin induced more beta-lactamase than the inhibitors, but were much less effective than cefoxitin, except for the strain of Prov. rettgeri.     

Activity of trospectomycin against Bacteroides fragilis and other Bacteroides species.

The in vitro activity of trospectomycin (U-63366; 6'-n-propyl spectinomycin pentahydrate sulfate) was evaluated against 189 clinical isolates of the Bacteroides fragilis group and 65 Bacteroides species isolates. At less than or equal to 8 micrograms/ml, the activity of trospectomycin compared favorably with those of clindamycin and cefoxitin against B. fragilis, Bacteroides distasonis, and Bacteroides vulgatus, and there was no cross resistance to these three drugs among the strains of the B. fragilis group. All the Bacteroides species were susceptible to trospectomycin. The results of this in vitro study indicate that trospectomycin possesses excellent activity against Bacteroides species.     

Augmentation of the in vitro activity of azlocillin against Bacteroides fragilis by clavulanic acid

Azlocillin was active against 90% of 154 strains of Bacteroides fragilis at a concentration of 64 micrograms/ml. Twenty-eight strains of B. fragilis with an azlocillin MIC of greater than or equal to 8 micrograms/ml were retested with a combination of azlocillin plus clavulanic acid. Of these strains, 71% showed a 4- to 32-fold decrease in the MIC of azlocillin plus clavulanic acid.     

Comparative in vitro and in vivo activities of piperacillin combined with the beta-lactamase inhibitors tazobactam, clavulanic acid, and sulbactam.

Tazobactam (YTR-830H), a novel beta-lactamase inhibitor, was compared with clavulanic acid and sulbactam for enhancement of the activity of piperacillin against beta-lactamase-producing, piperacillin-resistant clinical isolates. Piperacillin MICs were determined in media containing a fixed concentration of 2 or 4 micrograms of the inhibitors per ml. The higher concentration was generally more effective. Tazobactam was superior to sulbactam in enhancing the spectrum and potency of piperacillin. Although the calvulanic acid combination was more potent, tazobactam was effective for a similar spectrum of resistant gram-negative clinical isolates containing beta-lactamase. MICs were reduced to the susceptible range for Escherichia coli, Klebsiella pneumoniae, Proteus spp., Salmonella spp., and Shigella spp. Combinations with tazobactam and sulbactam, but not clavulanic acid, were effective against Morganella spp. Some antagonism of the activity of piperacillin was observed with clavulanic acid but not with tazobactam or sulbactam. The inhibitors were similarly effective with piperacillin against beta-lactamase-positive Staphylococcus spp. and the Bacteroides fragilis group. Piperacillin-tazobactam was more effective against a broader spectrum of gram-negative enteric bacteria than ticarcillin plus clavulanic acid was. Combinations with tazobactam or clavulanic acid had a broader spectrum of activity than combinations with sulbactam against bacteria that produce characterized plasmid-mediated enzymes of clinical significance. In particular, piperacillin with tazobactam or clavulanic acid, but not with sulbactam, inhibited TEM-1, TEM-2, and SHV-1 enzymes. In vitro activity was reflected in vivo. Tazobactam and clavulanic acid were superior to sulbactam in enhancing the therapeutic efficacy of piperacillin in mice infected with beta-lactamase-positive E. coli, K. pneumoniae, Proteus mirabilis, and Staphylococcus aureus. Only combinations with tazobactam and sulbactam were effective against the Morganella infection. Tazobactam has a good potential for enhancing the clinical efficacy of piperacillin.     

Purification and properties of beta-lactamase from Bacteroides fragilis.

Beta-Lactamase activity was detected either biologically or using the chromogenic cephalosporin 87/312 in 20 clinical isolates of Bacteroides fragilis with penicillin G minimal inhibitory concentrations of 10 to 100 micrograms/ml. Strain AM78 (minimal inhibitory concentration, greater than 1,000 micrograms/ml) was used to optimize the conditions for production, assay, and storage of the enzyme. The enzymes are cell associated, with less than 1% of activity being found in culture fluids during growth, and can be released from the cell surface by modified osmotic shock procedure. This procedure causes concomitant release of cyclic phosphodiesterase activity. Substrate profiles and the effects of inhibitors were determined for enzymes partially purified by osmotic shock release and gel filtration. The enzymes are cephalosporinases with some penicillinase activity and are inhibited by p-chloromercuribenzoate, cloxacillin, and carbenicillin. The molecular weight, as determined by gel filtration, is 29,000 to 31,000. A method for the purification of the beta-lactamase from strain AM78 is described: the specific activity of the purified enzyme was 3,424 U/mg, about 3,000-fold that of the crude, cell-associated enzyme.     

Cephalosporin-resistance in the Bacteroides fragilis group and the effect of clavulanic acid

Thirty-five beta-lactam-resistant isolates of the Bacteroides fragilis group were examined for the susceptibility to eight cephalosporins, alone, and in combination with a subinhibitory concentration of the beta-lactamase inhibitor, clavulanic acid. The majority of strains tested became fully susceptible to cephaloridine, cefotaxime and ceftriaxone in the presence of clavulinic acid whereas the effect of the inhibitor on the susceptibility to compounds such as cefsulodin and ceftazidime and to a lesser extent cefoperazone varied according to species. Bact. fragilis strains almost invariably showed substantial reductions in MICs whereas those of other species, especially Bact. thetaiotaomicron, were influenced to a lesser extent. Susceptibility to cefoxitin was unaffected by the inhibitor but a few strains with high level resistance to latamoxef (moxalactam) became susceptible in the presence of clavulanic acid. All strains were found to produce beta-lactamase as determined by the nitrocefin test and these were characterized by isoelectric focusing.     

Increase in the activity of third-generation cephalosporins in combination with clavulanic acid and Sulbactam against Bacteroides fragilis

We studied the sensitivity of 160 strains of Bacteroides fragilis (74 beta-lactamase-positive and 86 beta-lactamase-negative) to four third-generation cephalosporins, alone as well as in combination with clavulanic acid and Sulbactam. For susceptibility testing we used a dilution method in agar. Detection of beta-lactamase production by this micro-organism was performed using chromogenic cephalosporin (Nitrocefin). There was a substantial improvement in cephalosporin activity with both positive and negative strains when they were combined with the inhibitors, although this was more significant in beta-lactamase-producing organisms. Generally, the results achieved in combination with clavulanic acid were better than those with Sulbactam, the highest increase in activity being obtained with Ceftizoxime in combination with any one of the inhibitors.     

Comparative activities of clavulanic acid, sulbactam, and tazobactam against clinically important beta-lactamases.

Clavulanic acid, sulbactam, and tazobactam are inhibitors of a variety of plasmid-mediated beta-lactamases. However, inhibition data for these three inhibitors with a wide range of different plasmid-mediated beta-lactamases have not yet been compared under the same experimental conditions. A number of groups have inferred that clavulanic acid inhibits extended-spectrum TEM and SHV beta-lactamases, but inhibition data have rarely been published. In this study, the 50% inhibitory concentrations of these three beta-lactamase inhibitors for 35 plasmid-mediated beta-lactamases have been determined. Of these 35 beta-lactamases, 20 were extended-spectrum TEM- or SHV-derived beta-lactamases. The other 15 enzymes were conventional-spectrum beta-lactamases such as TEM-1 and SHV-1. Clavulanic acid was a more potent inhibitor than sulbactam for 32 of the 35 plasmid-mediated beta-lactamases tested. In particular, clavulanic acid was 60 and 580 times more potent than sulbactam against TEM-1 and SHV-1, respectively, currently the two most clinically prevalent gram-negative plasmid-mediated beta-lactamases. Statistical analysis of the data of the 50% inhibitory concentrations showed that clavulanic acid was 20 times more active overall than sulbactam against the conventional-spectrum enzymes. In addition, clavulanic acid was 14 times more potent than sulbactam at inhibiting the extended-spectrum enzymes. Tazobactam also showed significantly greater activity than sulbactam against the two groups of beta-lactamases. There were no significant differences between the overall activities of tazobactam and clavulanic acid against the extended-spectrum TEM and SHV enzymes and conventional-spectrum enzymes, although differences in their inhibition profiles were observed.     

Effect of clavulanic acid on the activities of ten beta-lactam agents against members of the Bacteroides fragilis group.

Clavulanic acid reduced the MICs of amoxicillin, carbencillin , cefamandole, cefotaxime, ceftazidime, ceftizoxime, cephalothin, and penicillin G, but not of cefoxitin or moxalactam, against 77 isolates of the Bacteroides fragilis group, all rapidly beta-lactamase positive by the nitrocefin slide test. It had no effect on the susceptibilities of eight Bacteroides distasonis strains that were slowly beta-lactamase positive (18 h of incubation).     

Properties of novel beta-lactamase produced by Bacteroides fragilis.

Bacteroides fragilis strains were isolated from clinical specimens. B. fragilis G-237 was highly resistant to beta-lactam antibiotics due to beta-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).     

In vitro activities of the beta-lactamase inhibitors clavulanic acid, sulbactam, and tazobactam alone or in combination with beta-lactams against epidemiologically characterized multidrug-resistant Acinetobacter baumannii strains

Acinetobacter baumannii is an important nosocomial pathogen usually in the context of serious underlying disease. Multidrug resistance in these organisms is frequent. The beta-lactamase inhibitors clavulanic acid, sulbactam, and tazobactam have intrinsic activity against Acinetobacter strains. To evaluate their potential therapeutic usefulness, we determined the in vitro activity of ampicillin, sulbactam, ampicillin-sulbactam, cefoperazone, cefoperazone-sulbactam, piperacillin, piperacillin-sulbactam, tazobactam, piperacillin-tazobactam, amoxicillin, clavulanic acid, amoxicillin-clavulanic acid, ticarcillin, and ticarcillin-clavulanic acid against multidrug-resistant A. baumannii. All isolates were epidemiologically characterized by RAPD [random(ly) amplified polymorphic DNA] analysis and/or pulsed-field gel electrophoresis and represented different strain types, including sporadic strains, as well as outbreak-related strains. The MICs were determined by agar dilution on Mueller-Hinton agar (using fixed concentrations, as well as fixed ratios for beta-lactamase inhibitors) and the E-test. The majority of E-test results were within two dilutions of those recorded by agar dilution, with the exception of piperacillin-tazobactam. Sulbactam was superior to clavulanic acid and tazobactam and may represent an alternative treatment option for infections due to multiresistant A. baumannii strains. beta-Lactamase inhibitors have intrinsic activity but do not enhance activity of beta-lactams against A. baumannii. Testing with the inhibitor added at a fixed concentration as recommended for piperacillin-tazobactam and ticarcillin-clavulanic acid by the National Committee for Clinical Laboratory Standards may falsely suggest high activity or gives uninterpretable results due to trailing. If combinations are used for testing, fixed ratios may give more useful results.     

Antibacterial characteristics of YTR 830, a sulfone beta-lactamase inhibitor, compared with those of clavulanic acid and sulbactam.

The antibacterial activity, binding to penicillin-binding proteins, and morphological changes effected by YTR 830, a sulfone beta-lactamase inhibitor, were studied in comparison with those of other beta-lactamase inhibitors. YTR 830 had very poor antibacterial activity, bound to PBP 2 of gram-negative organisms, and at the MIC caused rapid lysis of spheroplasts formed.     

Hydrolysis of Cefotaxime by a beta-lactamase from Bacteroides fragilis.

A beta-lactamase isolated from a strain of Bacteroides fragilis subsp. fragilis possessed hydrolytic activity toward cefotaxime. This antibiotic was degraded to a lower extent than was cephalothin, cephaloridine, and cefamandole, whereas cefoxitin remained unaffected by the enzyme. Kinetic parameters Vmax and Km for cefotaxime were calculated at 0.172 mumol/min and 1.1 X 10(-2) mM, respectively.     

Activity of newer beta-lactam agents against clinical isolates of Bacteroides fragilis and other Bacteroides species.

The in vitro activities of beta-lactam antibiotics against Bacteroides fragilis and B. fragilis group isolates are presented. Clinical isolates from 1986 were compared with strains from 1979 to 1982. Imipenem, ticarcillin-clavulanic acid, and ceftizoxime were the most active agents. Cefotetan was equivalent to cefoxitin against B. fragilis but less active against B. fragilis group isolates. Enhancement of cefotaxime by its desacetyl metabolite was minimal.