Structural analysis indicates that affinity for the penta-coordinated zinc can be modulated by neighboring residues, perhaps explaining the absence of the second zinc in the B. cereus structure. Models of bound substrates suggest that the active-site channel can accommodate a wide variety of beta-lactams. We propose that the zinc cluster prepares an hydroxide, probably the hydroxide that ligates both zincs, for nucleophilic attack on the carbonyl carbon atom of the beta-lactam. The resulting negatively charged tetrahedral intermediate implicated in catalysis is stabilized by an oxyanion hole formed by the side chain of the invariant Asn 193 and the tetrahedral zinc.

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Tazobactam was shown to be a potent inhibitor of group 1, 2a, 2b, and 2b' 13-lactamases. Extended kinetic studies with class A and C serine 13-lactamases showed that the PC1, TEM-2, and P99 enzymes all were reversibly inhibited prior to inactivation of the enzymes. The CcrA metallo-1-lactamase was less well inhibited, with a 50%o inhibitory concentration at least 3 orders of magnitude less favorable than those for most serine 13-lactamases. The numbers of hydrolytic turnovers of tazobactam before inactivation were 2 for PC1, 125 for TEM-2, 50 for P99, and 4,000 for the CcrA enzyme. In spectral studies, transient intermediates were formed after reaction of tazobactam with the PC1, TEM-2, and CcrA 1-lactamases, corresponding to enzymeassociated intermediates responsible for hydrolysis of tazobactam. Chromophores absorbing at 270 nm (CcrA) and 288 nm (TEM-2 and PC1) were observed for these reaction intermediates. The P99 cephalosporinase formed a stable complex with a UV maximum at 295 nm. Incubation of tazobactam with all of the enzymes resulted in accumulation of a tazobactam reaction product with a short-wavelength absorbance. This product has characteristics similar to those of the major eucaryotic metabolite of tazobactam. Possible reaction mechanisms are presented to explain the findings. In conclusion, both serine-based and metallo-13-lactamases were irreversibly inactivated by tazobactam following an initial transient inhibition phase.Tazobactam, a triazolyl-substituted penicillanic acid sulfone (3), is a ,B-lactamase inhibitor that has been successfully combined with piperacillin to protect this broad-spectrum penicillin from ,B-lactamase-mediated hydrolysis (17, 31). Although extensive microbiological studies have been described for the piperacillin-tazobactam combination (24, 29), little has been reported concerning the mechanism of inhibition of standard ,3-lactamases. In contrast to the elaborate biochemical studies describing clavulanic acid and sulbactam inactivation of the TEM-2 (7, 15, 18, 21) and PC1 ,-lactamases (13), no comparative enzymological studies have been performed with tazobactam.In this set of studies, a metallo-p-lactamase and three serine ,-lactamases from different functional groups (8,9) were examined in detail to determine the kinetics of inhibition by tazobactam. These enzymes include the P99 cephalosporinase not inhibited by clavulanic acid (Bush group 1); the PC1 penicillinase (group 2a) and the broad-spectrum TEM-2 P-lactamase (group 2b), both well inhibited by clavulanic acid; and the CcrA metallo-,-lactamase from Bacteroides fragilis (group 3), an enzyme not inhibited by the classical 1-lactamase inhibitors. The PC1 and TEM-2 ,-lactamases are members of structural class A, with serine as the active-site acylating residue (2). The CcrA enzyme is a zinc-dependent enzyme of structural class B (2, 32). P99 is a class C enzyme with a higher molecular weight, also with an active-site serine (20). Evidence is presented to show that each enzyme undergoes reversible reactions with...

Bacteroides fragilis TAL3636 produces a class B, Zn2+-requiring 13-lactamase. The gene, ccrA, was cloned and expressed in Escherichia coli. The gene was sequenced and shown to share greater than 33% identity with the metalloenzyme from Bacillus cereus 569/H.The unique class B 1-lactamases require a metal cofactor,

Ceftazidime-resistant KkebsieUla pneumoniae strains began to appear when ceftazidime usage was increased in two unrelated Chicago hospitals. These strains produced a P-lactamase with an isoelectric point of 5.6 (RP-5.6) and strong hydrolyzing activity against ceftazidime. Two different restriction digest profiles were associated with the ceftazidime resistance plasmids. A second P-lactamase with a pl of 5.2 (RP-5.2) was coproduced in two representative strains. The second ,-lactamase hydrolyzed ceftazidime, cefotaxime, and aztreonam with relative hydrolysis rates of <8% of that observed for benzylpenicillin. Both enzymes were inhibited by clavulanic acid and tazobactam. Nucleotide sequencing of the genes coding for RP-5.2 and RP-5.6 revealed sequences identical to those of the TEM-12 and TEM-10 I8-lactamase genes, respectively. Both genes were derived from a TEM-1 sequence related to that of the gene encoded on the Tn2 transposon. Single point mutations are required to progress from TEM-1 to TEM-12 and from TEM-12 to TEM-10. Extracts from broths grown from single cell isolates of the strain producing TEM-12 and TEM-10 were shown to contain both enzymes. Transconjugants producing either the TEM-12 or the TEM-10 j-lactamase were obtained. A significant finding was that both enzymes were encoded by plasmids with identical restriction digest patterns.These studies show that mutations leading to extended-spectrum ,-lactamases can occur sequentially in the same organism, with the genes encoding both enzymes maintained stably.

In 1984, a year prior to the U.S. approval of imipenem for clinical use, a wound isolate and a bile isolate of Enterobacter cloacae were obtained from two patients in a California hospital. These isolates were resistant to imipenem, penicillins, and inhibitor combinations; early cephalosporins such as cephalothin, cefamandole, and cefoxitin; and cefoperazone. However, they were susceptible (MICs, < 4 micrograms/ml) to cefotaxime, ceftriaxone, ceftazidime, and moxalactam. Both strains produced an apparent TEM-1 beta-lactamase; an inducible NmcA-type imipenem-hydrolyzing beta-lactamase, IMI-1, with a pl of 7.05; and an inducible beta-lactamase with a pI of 8.1, typical of an E. cloacae AmpC beta-lactamase. Purified IMI-1 hydrolyzed imipenem and benzylpenicillin at modest rates, but more slowly than cephaloridine. The enzyme was inhibited by clavulanic acid and tazobactam. EDTA did not inhibit the cephaloridine-hydrolyzing activity. The beta-lactamase gene encoding IMI-1, imiA1, was cloned from E. cloacae 1413B. Sequence analysis identified the imiA1 gene as encoding a class A serine beta-lactamase. Both the imiA1 DNA and encoded amino acid sequences shared greater than 95% identity with the NmcA gene and its encoded protein. DNA sequence analysis also identified a gene upstream of imiA1 that shares > 95% identity with nmcR and that may encode a regulatory protein. In conclusion, IMI-1, a carbapenem-hydrolyzing beta-lactamase inhibited by clavulanic acid, was identified as a group 2f, class A, carbapenem-hydrolyzing cephalosporinase.

The development of antibiotic resistance in anaerobic bacteria has a tremendous impact on the selection of antimicrobial agents for empirical therapy. Susceptibility studies have documented the emergence of antimicrobial resistance and indicate distinct differences in resistance patterns related to individual hospitals, geographic regions, and antibiotic-prescribing regimens. Resistance to beta-lactam drugs, clindamycin, tetracyclines, and 5-nitroimidazoles (metronidazole) has been observed. The prime mechanism for resistance to beta-lactam agents is the production of beta-lactamases. Resistance to clindamycin is mediated by modification of the ribosome. Tetracycline resistance is mediated by both tetracycline efflux and ribosomal protection. 5-Nitroimidazole resistance appears to be caused by a combination of decreased antibiotic uptake and decreased nitroreductase activity. The level of chloramphenicol susceptibility remains quite high, whereas uniform resistance to aminoglycosides and quinolones is observed. Understanding the mechanisms of resistance is critical for both informed selection of antimicrobial therapy and the design of new antimicrobial agents.

Four ceftazidime-resistant Escherichia coli strains were isolated from elderly nursing home patients in a New York hospital during 1993. Strains MCQ-2, MCQ-3, and MCQ-4 were determined to be identical by pulsed-field gel electrophoresis and plasmid profiles, whereas strain MCQ-1 was unique. Strain MCQ-1 was determined to produce a TEM-10 beta-lactamase. Strains MCQ-2, MCQ-3, and MCQ-4 were also noted to be resistant to cefotaxime. These three strains produced two beta-lactamases with pIs of 5.4 (TEM-1) and 7.6. beta-Lactamase assays revealed that the pI 7.6 enzyme hydrolyzed cefotaxime faster (at a relative hydrolysis rate of 30% compared with that of benzylpenicillin) than either ceftazidime or aztreonam (relative hydrolysis rates of 13 and 3.3%, respectively). Nucleotide sequencing of the gene encoding the pI 7.6 beta-lactamase from strain MCQ-3 revealed a blaSHV-type gene differing from the gene encoding SHV-1 at four nucleotides which resulted in amino acid substitutions: phenylalanine for isoleucine at position 8, serine for arginine at position 43, serine for glycine at position 238, and lysine for glutamate at position 240. This novel SHV-type extended-spectrum beta-lactamase is designated SHV-7.