Ceftriaxone attenuates acute cocaine-evoked dopaminergic neurotransmission in the nucleus accumbens of the rat.

BACKGROUND AND PURPOSE: Ceftriaxone is a ß-lactam antibiotic and glutamate transporter activator that reduces the reinforcing effects of psychostimulants. Ceftriaxone also reduces locomotor activation following acute psychostimulant exposure, suggesting that alterations in dopamine transmission in the nucleus accumbens contribute to its mechanism of action. In the present studies we tested the hypothesis that pretreatment with ceftriaxone disrupts acute cocaine-evoked dopaminergic neurotransmission in the nucleus accumbens. EXPERIMENTAL APPROACH: Adult male Sprague-Dawley rats were pretreated with saline or ceftriaxone (200 mg kg(-1) , i.p. × 10 days) and then challenged with cocaine (15 mg kg(-1) , i.p.). Motor activity, dopamine efflux (via in vivo microdialysis) and protein levels of tyrosine hydroxylase (TH), the dopamine transporter and organic cation transporter as well as α-synuclein, Akt and GSK3ß were analysed in the nucleus accumbens. KEY RESULTS: Ceftriaxone-pretreated rats challenged with cocaine displayed reduced locomotor activity and accumbal dopamine efflux compared with saline-pretreated controls challenged with cocaine. The reduction in cocaine-evoked dopamine levels was not counteracted by excitatory amino acid transporter 2 blockade in the nucleus accumbens. Pretreatment with ceftriaxone increased Akt/GSK3ß signalling in the nucleus accumbens and reduced levels of dopamine transporter, TH and phosphorylated α-synuclein, indicating that ceftriaxone affects numerous proteins involved in dopaminergic transmission. CONCLUSIONS AND IMPLICATIONS: These results are the first evidence that ceftriaxone affects cocaine-evoked dopaminergic transmission, in addition to its well-described effects on glutamate, and suggest that its ability to attenuate cocaine-induced behaviours, such as psychomotor activity, is due in part to reduced dopaminergic neurotransmission in the nucleus accumbens.

Mechanism of inhibition of the class A beta -lactamases PC1 and TEM-1 by tazobactam. Observation of reaction products by electrospray ionization mass spectrometry.

The reactions of class A beta-lactamases PC1 and TEM-1 with tazobactam (TZB), a potent penicillanic sulfone inhibitor for class A beta-lactamases, were studied using electrospray ionization mass spectrometry (ESI/MS). Following inactivation of the beta-lactamases by TZB, new abundant high mass components were observed including three with molecular masses of 52, 70, and 88 Da greater than PC1 and TEM-1, respectively, and a component with a molecular mass of 300 Da greater than PC1. In addition, three TZB reaction products with molecular masses of 248, 264, and 280 Da were observed. High performance liquid chromatography (HPLC)/ESI/MS analysis of the TZB-PC1 adduct digested with Glu-C revealed three new components with masses 52, 70, and 88 Da greater than that of the peptide composed of amino acid residues 58-82 and one new component with a mass 70 Da greater than that of the peptide composed of amino acid residues 125-141. HPLC/ESI/MS/MS analysis of the two digested peptides whose masses increased by 70 Da indicated that Ser-70 and Ser-130 were the most likely TZB-modified amino acid residues. Based on these data, a mechanism for the inactivation of the class A beta-lactamases by TZB is proposed. In this scheme, initial acylation of Ser-70 by TZB and opening of the lactam ring are followed by one of several different events: (1) the rapid decomposition of TZB with loss of the enamine moiety to form the propiolylated enzyme, (2) an intramolecular nucleophilic displacement of the imine or enamine moiety by Ser-130 to form a cross-linked vinyl ether, and (3) hydrolysis of the imine or enamines to form a Ser-70-linked aldehyde.

Biochemical characterization of novel tetrahydrofuranyl 1beta-methylcarbapenems: stability to hydrolysis by renal dehydropeptidases and bacterial beta-lactamases, binding to penicillin binding proteins, and permeability properties.

The biochemical properties of tetrahydrofuranyl (THF) carbapenems, carbapenems with THF substituents, were evaluated with respect to enzyme stability, binding to penicillin-binding proteins (PBPs), and penetration into gram-negative organisms. THF carbapenems showed increased stability to hog renal dehydropeptidases (DHPs) compared to that of imipenem or meropenem and were more stable to human DHP than imipenem (<10% hydrolysis compared to that for imipenem). THF carbapenems were stable to hydrolysis by all serine beta-lactamases tested. CL 191,121, a prototype THF carbapenem, was more stable to hydrolysis by carbapenem-hydrolyzing serine beta-lactamases such as IMI-1 and Sme-1 than imipenem, with a relative k(cat) value of <20% for imipenem. Similar to imipenem and meropenem, THF carbapenems were not stable to the metallo beta-lactamases CcrA and L1. However, CL 191,121 bound to all Staphylococcus aureus PBPs at concentrations that were less than or equal to the MICs. The THF carbapenems bound to PBPs from Escherichia coli and Pseudomonas aeruginosa, with the highest affinities being for PBPs 2 and 4, as noted with imipenem. The affinities for PBPs 1a and 1b in E. coli were reduced for the THF carbapenems compared to that for imipenem, even though the MICs of the THF carbapenems for E. coli strains were lower than those of imipenem. The penetrability of the THF carbapenems into Serratia marcescens S6, which produces the Sme-1 carbapenem-hydrolyzing beta-lactamase, was 2.4 to 7.8 times less than that of imipenem. Compounds CL 190,294 and CL 188,624 showed good penetrability, with permeability coefficient values comparable to those of the rapidly penetrating agents cephaloridine, imipenem, meropenem, and biapenem. Decreased penetration into wild-type P. aeruginosa was suggested by the high MICs of the THF carbapenems (MICs, 16 to 32 microg/ml), despite equivalent or better binding to P. aeruginosa PBPs than that of imipenem. However, the MICs of the THF carbapenems for wild-type P. aeruginosa compared to that for an OprD2 mutant generally varied no more than 2-fold, but those of imipenem and other carbapenems differed 16-fold. These data indicated that THF carbapenems do not appear to enter through protein OprD2. In conclusion, the THF carbapenems exhibited stability to hydrolysis by renal DHPs and serine beta-lactamases, exhibited strong binding to essential PBPs from E. coli and S. aureus, and penetrated gram-negative enteric bacteria at rates comparable to those for meropenem and biapenem.

Class A beta-lactamases--enzyme-inhibitor interactions and resistance.

Beta-Lactamases of Ambler's Class A are the most commonly encountered mechanism of bacterial resistance to beta-lactam antibiotics. In the face of selective pressure arising from use of either newer cephalosporins or beta-lactam/beta-lactamase inhibitor combinations, mutations arose among Class A beta-lactamase genes, leading to resistance. Clavulanic acid, a naturally occurring clavam, and the penicillanic acid sulfones sulbactam and tazobactam are the inhibitors in clinical use. This review focuses on the mechanism of inhibition by these currently marketed beta-lactamase inhibitors and on the mechanism by which specific amino acid substitutions might lead to resistance. The key amino acid positions important for inhibitor-resistance include Met69, Ser130, Arg244, Arg275, and Asn276. Ser130 is vital to the chemical mechanism of inhibition. Arg244 appears to be coordinated to Arg275 and Asp276 by hydrogen bonds. Arg244 is involved in positioning beta-lactams, especially penicillins and beta-lactamase inhibitors, via their carboxyl groups. Site-directed mutagenesis studies confirm the role of Arg244 and its coordinating partners in beta-lactam turnover and in the reactions leading to enzyme inactivation. This mechanism is dependent on the donation of a proton via a water coordinated to Arg244 and Val216 to clavulanic acid to allow formation of a favorable leaving group. This proton donation is probably not required for formation of a favorable leaving group for the sulfone inhibitors sulbactam and tazobactam. Therefore, some amino acid substitutions have differing effects on inhibition by clavulanic acid compared with the penicillanic acid sulfones. Met69 may play a more structural role in beta-lactam positioning within the oxyanion hole.

6-(1-Hydroxyalkyl)penam sulfone derivatives as inhibitors of class A and class C beta-lactamases I.

Five 6-(1-hydroxyalkyl)penam sulfone derivatives and two 6-(hydroxymethyl)penams were synthesized for beta-lactamase inhibitor screens. The substituent effects and stereochemical requirements of 6alpha- and 6beta-(1-hydroxyalkyl) groups for the biological activity of penam sulfone derivatives were investigated. Of these substituents, only the 6beta-hydroxymethyl group of 15 improved the activity of sulbactam against both TEM-1 and AmpC beta-lactamases. The sulfone moiety is required for the enhancement of the beta-lactamase inhibitory activity. 6Beta-hydroxymethylsulbactam (15) was able to restore the activity of piperacillin in vitro and in vivo against various beta-lactamase producing microorganisms.

Kinetic properties and metal content of the metallo-beta-lactamase CcrA harboring selective amino acid substitutions.

The crystal structure of the metallo-beta-lactamase CcrA3 indicates that the active site of this enzyme contains a binuclear zinc center. To aid in assessing the involvement of specific residues in beta-lactam hydrolysis and susceptibility to inhibitors, individual substitutions of selected amino acids were generated. Substitution of the zinc-ligating residue Cys181 with Ser (C181S) resulted in a significant reduction in hydrolytic activity; kcat values decreased 2-4 orders of magnitude for all substrates. Replacement of His99 with Asn (H99N) significantly reduced the hydrolytic activity for penicillin and imipenem. Replacement of Asp103 with Asn (D103N) showed reduced hydrolytic activity for cephaloridine and imipenem. Deletion of amino acids 46-51 dramatically reduced both the hydrolytic activity and affinity for all beta-lactams. The metal binding capacity of each mutant enzyme was examined using nondenaturing electrospray ionization mass spectrometry. Two zinc ions were observed for the wild-type enzyme and most of the mutant enzymes. However, for the H99N, C181S, and D103N enzymes, three different zinc content patterns were observed. These enzymes contained two zinc molecules, one zinc molecule, and a mixture of one or two zinc molecules/enzyme molecule, respectively. Two enzymes with substitutions of Cys104 or Cys104 and Cys155 were also composed of mixed enzyme populations.

Structural consequences of the active site substitution Cys181 ==> Ser in metallo-beta-lactamase from Bacteroides fragilis.

The metallo-beta-lactamases require divalent cations such as zinc or cadmium for hydrolyzing the amide bond of beta-lactam antibiotics. The crystal structure of the Zn2+ -bound enzyme from Bacteroides fragilis contains a binuclear zinc center in the active site. A hydroxide, coordinated to both zinc atoms, is proposed as the moiety that mounts the nucleophilic attack on the carbonyl carbon atom of the beta-lactam bond of the substrate. It was previously reported that the replacement of the active site Cys181 by a serine residue severely impaired catalysis while atomic absorption measurements indicated that binding of the two zinc ions remained intact. Contradicting data emerge from recent mass spectrometry results, which show that only a single zinc ion binds to the C181S metallo-beta-lactamase. In the current study, the C181S mutant enzyme was examined at the atomic level by determining the crystal structure at 2.6 A resolution. The overall structure of the mutant enzyme is the same as that of the wild-type enzyme. At the mutation site, the side chain of Ser181 occupies the same position as that of the side chain of Cys181 in the wild-type protein. One zinc ion, Zn1, is present in the crystal structure; however, the site of the second zinc ion, Zn2 is unoccupied. A water molecule is associated with Zn1, reminiscent of the hydroxide seen in the structure of the wild-type enzyme but farther from the metal. The position of the water molecule is off the plane of the carboxylate group of Asp103; therefore, the water molecule may be less nucleophilic than a water molecule which is coplanar with the carboxylate group.

Expression of the AsbA1, OXA-12, and AsbM1 beta-lactamases in Aeromonas jandaei AER 14 is coordinated by a two-component regulon.

Aeromonas jandaei AER 14 (formerly Aeromonas sobria AER 14) expresses three inducible beta-lactamases, AsbA1, OXA-12 (AsbB1), and AsbM1. Mutant strains that constitutively overexpress all three enzyme simultaneously, suggesting that they share a common regulatory pathway, have been isolated. Detectable expression of the cloned genes of AsbA1 and OXA-12 in some Escherichia coli K-12 laboratory strains is achieved only in the presence of a blp mutation. These mutations map to the cre operon at 0 min, which encodes a classical two-component regulatory system of unknown function. Two regulatory elements from A. jandaei which permit high-level constitutive expression of OXA-12 in E. coli were cloned. Both loci encode proteins with characteristics of response regulator proteins of two-component regulatory systems. One of these loci, designated blrA, bestowed constitutive expression of all three beta-lactamases in A. jandaei AER 14 when present on a multicopy plasmid, confirming its role in the regulatory pathway of beta-lactamase production in this organism.

Antimicrobial resistance in anaerobes.

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.

Crystal structures of the cadmium- and mercury-substituted metallo-beta-lactamase from Bacteroides fragilis.

The metallo-beta-lactamases require zinc or cadmium for hydrolyzing beta-lactam antibiotics and are inhibited by mercurial compounds. To data, there are no clinically useful inhibitors of this class of enzymes. The crystal structure of the Zn(2+)-bound enzyme from Bacteroides fragilis contains a binuclear zinc center in the active site. A hydroxide, coordinated to both zinc atoms, is proposed as the moiety that mounts the nucleophilic attack on the carbonyl carbon atom of the beta-lactam ring. To study the metal coordination further, the crystal structures of a Cd(2+)-bound enzyme and of an Hg(2+)-soaked zinc-containing enzyme have been determined at 2.1 A and 2.7 A, respectively. Given the diffraction resolution, the Cd(2+)-bound enzyme exhibits the same active-site architecture as that of the Zn(2+)-bound enzyme, consistent with the fact that both forms are enzymatically active. The 10-fold reduction in activity of the Cd(2+)-bound molecule compared with the Zn(2+)-bound enzyme is attributed to fine differences in the charge distribution due to the difference in the ionic radii of the two metals. In contrast, in the Hg(2+)-bound structure, one of the zinc ions, Zn2, was ejected, and the other zinc ion, Zn1, remained in the same site as in the 2-Zn(2+)-bound structure. Instead of the ejected zinc, a mercury ion binds between Cys 104 and Cys 181, 4.8 A away from Zn1 and 3.9 A away from the site where Zn2 is located in the 2-Zn(2+)-bound molecule. The perturbed binuclear metal cluster explains the inactivation of the enzyme by mercury compounds.

Characterization of IMI-1 beta-lactamase, a class A carbapenem-hydrolyzing enzyme from Enterobacter cloacae.

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.

Crystal structure of the wide-spectrum binuclear zinc beta-lactamase from Bacteroides fragilis.

BACKGROUND: The metallo-beta-lactamase from Bacteroides fragilis hydrolyzes a wide range of beta-lactam antibiotics, and is not clinically susceptible to any known beta-lactamase inhibitors. B. fragilis is associated with post-surgery hospital infections, and there has been a recent report of plasmid-mediated dissemination of the enzyme. Effective inhibitors are therefore urgently needed. Knowledge of the three-dimensional structure will aid in the drug design effort. RESULTS: The crystal structure of the enzyme has been determined by using multiwavelength anomalous diffraction at the zinc absorption edge and refined to 1.85 A resolution. The structure is a four-layer alpha/beta/beta/alpha molecule. The active site, found at the edge of the beta sandwich contains a binuclear zinc center with several novel features. One zinc is tetrahedrally coordinated, the other has a trigonal bipyramidal coordination; a water/hydroxide molecule serves as a ligand for both metals. The residues that coordinate the two zincs are invariant in all metallo-beta-lactamases that have been sequenced, except for two conservative replacements. Despite the existence of the pattern for binuclear zinc binding, the reported structure of the Bacillus cereus enzyme contains only a single zinc. CONCLUSIONS: 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.

Dsb-insensitive expression of CcrA, a metallo-beta-lactamase from Bacteroides fragilis, in Escherichia coli after amino acid substitution at two cysteine residues within CcrA.

It has previously been shown that functional expression of CcrA, a metallo-beta-lactamase from Bacteroides fragilis, in Escherichia coli requires a mutation in either dsbA or dsbB, components of a periplasmic disulfide bond-catalyzing system. Site-directed mutagenesis resulting in the substitution of various amino acids for two of the three cysteine residues within CcrA allowed the expression of CcrA in a dsb+ background. This finding supports the hypothesis that DsbA creates aberrant disulfide bonds involving the Cys residues of CcrA.

SHV-7, a novel cefotaxime-hydrolyzing beta-lactamase, identified in Escherichia coli isolates from hospitalized nursing home patients.

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.

A mutation in either dsbA or dsbB, a gene encoding a component of a periplasmic disulfide bond-catalyzing system, is required for high-level expression of the Bacteroides fragilis metallo-beta-lactamase, CcrA, in Escherichia coli.

The metallo-beta-lactamase gene, ccrA, from Bacteroides fragilis is functionally expressed in Escherichia coli only in the presence of a genomic mutation in iarA or iarB (increased ampicillin resistance), identified in this study as dsbA or dsbB, respectively. DsbA and DsbB are components of a periplasmic protein disulfide bond-catalyzing system. Data indicated that DsbA interacted with CcrA, creating aberrant disulfide bond linkages that render CcrA proteolytically unstable. Mutations in dsbA or dsbB permissive for CcrA expression eliminated or greatly reduced DsbA activity, allowing CcrA to assume a disulfide bond-free and proteolytically stable conformation.

Cloning and expression of a cloxacillin-hydrolyzing enzyme and a cephalosporinase from Aeromonas sobria AER 14M in Escherichia coli: requirement for an E. coli chromosomal mutation for efficient expression of the class D enzyme.

Two beta-lactamase genes, asbA1 and asbB1, encoding AsbA1 and AsbB1, respectively, have been cloned from Aeromonas sobria AER 14M into Escherichia coli. AsbA1 was expressed at low but detectable levels in all E. coli laboratory cloning strains tested. AsbB1 was expressed well in the E. coli cloning strain DH5 alpha. However, no enzyme activity could be detected from the same clone when placed in E. coli MC1061. Ampicillin-resistant mutants of E. coli MC1061 were obtained that expressed high levels of enzymatically active AsbB1. Four independent mutants were examined. All four mutations mapped to one locus, designated blpA (beta-lactamase permissive). The blpA locus was distinct from other known loci that play a role in beta-lactamase expression, i.e., the two loci that affect expression of the Bacteroides fragilis metallo-beta-lactamase and the ampC regulatory genes, ampD, ampE, and ampG. Sequence analysis of asbA1 and asbB1 revealed that AsbA1 was a class C beta-lactamase most closely related to the Pseudomonas aeruginosa chromosomal cephalosporinase and probably represents the common A. sobria cephalosporinase. AsbB1 was a class D enzyme most closely related to the oxacillin-hydrolyzing enzyme OXA-1, with 34% amino acid sequence identity. Purified AsbA1 was a typical cephalosporinase with a substrate profile that reflected high rates of hydrolysis of cephaloridine compared with benzylpenicillin. Purified AsbB1 showed strong penicillinase activity, with hydrolysis rates for carbenicillin and cloxacillin 2 to 2.5 times that for benzylpenicillin. Hydrolysis of imipenem was < or = 1% of that for benzylpenicillin. Both clavulanic acid and tazobactam strongly inhibited AsbB1, while sulbactam inhibited the AsbB1 enzyme less effectively. None of the inhibitors worked well against the AsbA1 enzyme. The chelators EDTA and 1,10-o-phenanthroline did not affect the activity of either enzyme. A. sobria AER 14M was found to produce both a group 1 cephalosporinase and a novel group 2d cloxacillin-hydrolyzing beta-lactamase that has been designated here OXA-12.

Contribution of enzymatic properties, cell permeability, and enzyme expression to microbiological activities of beta-lactams in three Bacteroides fragilis isolates that harbor a metallo-beta-lactamase gene.

The metallo-beta-lactamase gene, ccrA, has been cloned from three clinical isolates of Bacteroides fragilis, TAL3636, QMCN3, and QMCN4. Although all three isolates harbored a gene encoding a potent beta-lactamase, the MICs of benzylpenicillin, piperacillin, cefotaxime, ceftazidime, imipenem, and biapenem for the three isolates varied from 4- to > 128-fold. QMCN4 was the most susceptible of the three isolates, followed by QMCN3. TAL3636 was resistant to all of the beta-lactams. Previous DNA sequence analysis of the three ccrA genes revealed that the enzymes differed at 5 amino acid residues (B. A. Rasmussen, Y. Gluzman, and F. P. Tally, Mol. Microbiol. 5:1211-1219, 1991). Biochemical characterization of the three enzymes revealed only small differences in kcat and Km values for the majority of beta-lactams tested. Thus, the 5 amino acid substitutions affected the hydrolyzing activity of the enzymes only modestly. Crypticity differences between the three isolates showed that QMCN4 was the least permeable of the isolates to cephaloridine, followed by TAL3636, and that QMCN3 was highly permeable to cephaloridine. Therefore, neither catalytic activity nor permeability was a major contributor to the dramatic differences in the MICs. Instead, microbiological susceptibility was closely related to the level of metallo-beta-lactamase present in each isolate. Both biochemical and physical studies indicated that TAL3636 produced 5- to 10-fold and 50- to 100-fold more metallo-beta-lactamase than QMCN3 and QMCN4, respectively. Therefore, the level of CcrA enzyme production is the dominant contributing factor to high-level resistance among strains harboring a ccrA gene.

Inhibition of protein synthesis occurring on tetracycline-resistant, TetM-protected ribosomes by a novel class of tetracyclines, the glycylcyclines.

One of the two major mechanisms of tetracycline resistance is ribosomal protection. Of this resistance type, tet(M) is the best characterized. Although the mechanism of tet(M) resistance has not yet been fully elucidated, it has been demonstrated that ribosomes isolated from a tet(M) strain are resistant to inhibition of protein synthesis by tetracycline. A new generation of tetracycline compounds, the glycylcyclines, that are able to inhibit protein synthesis occurring on tetracycline-resistant, TetM-protected ribosomes, as well as wild-type, tetracycline-sensitive ribosomes, have been identified.

Multiply resistant Klebsiella pneumoniae strains from two Chicago hospitals: identification of the extended-spectrum TEM-12 and TEM-10 ceftazidime-hydrolyzing beta-lactamases in a single isolate.

Ceftazidime-resistant Klebsiella pneumoniae strains began to appear when ceftazidime usage was increased in two unrelated Chicago hospitals. These strains produced a beta-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 beta-lactamase with a pI of 5.2 (RP-5.2) was coproduced in two representative strains. The second beta-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 beta-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 beta-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 beta-lactamases can occur sequentially in the same organism, with the genes encoding both enzymes maintained stably.