Ceftibuten stability to active-site serine and metallo-beta-lactamases.

Ceftibuten is an oral third-generation cephalosporin active against a wide range of bacteria and shows an improved stability to hydrolysis by several beta-lactamases because of the carboxyethilidine moiety at position 7 of the ss-acyl side chain. The kinetic interactions between ceftibuten and active-site serine and metallo-ss-lactamases were investigated. The activity of several TEM-derived extended spectrum beta-lactamases (ESbetaLs) against ceftibuten, cefotaxime and ceftazidime was compared using K(m), K(cat) and K(cat)/K(m). Ceftibuten behaved as a poor substrate for class A and B beta-lactamases compared with cefotaxime. The chromosomal class C beta-lactamase from Enterobacter cloacae 908R gave a high K(cat) value (21 s(-1)), whereas there was poor activity with enzymes from Acinetobacter baumannii and Morganella morganii and ceftibuten. Ceftibuten resists hydrolysis in the presence of typical respiratory or urogenital-tract pathogens producing beta-lactamases.

Purification and biochemical characterization of the VIM-1 metallo-beta-lactamase.

VIM-1 is a new group 3 metallo-beta-lactamase recently detected in carbapenem-resistant nosocomial isolates of Pseudomonas aeruginosa from the Mediterranean area. In this work, VIM-1 was purified from an Escherichia coli strain carrying the cloned bla(VIM-1) gene by means of an anion-exchange chromatography step followed by a gel permeation chromatography step. The purified enzyme exhibited a molecular mass of 26 kDa in sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and an acidic pI of 5.1 in analytical isoelectric focusing. Amino-terminal sequencing showed that mature VIM-1 results from the removal of a 26-amino-acid signal peptide from the precursor. VIM-1 hydrolyzes a broad array of beta-lactam compounds, including penicillins, narrow- to expanded-spectrum cephalosporins, carbapenems, and mechanism-based serine-beta-lactamase inactivators. Only monobactams escape hydrolysis. The highest catalytic constant/K(m) ratios (>10(6) M(-1). s(-1)) were observed with carbenicillin, azlocillin, some cephalosporins (cephaloridine, cephalothin, cefuroxime, cefepime, and cefpirome), imipenem, and biapenem. Kinetic parameters showed remarkable variability with different beta-lactams and also within the various penam, cephem, and carbapenem compounds, resulting in no clear preference of the enzyme for any of these beta-lactam subfamilies. Significant differences were observed with some substrates between the kinetic parameters of VIM-1 and those of other metallo-beta-lactamases. Inactivation assays carried out with various chelating agents (EDTA, 1,10-o-phenanthroline, and pyridine-2,6-dicarboxylic acid) indicated that formation of a ternary enzyme-metal-chelator complex precedes metal removal from the zinc center of the protein and revealed notable differences in the inactivation parameters of VIM-1 with different agents.

Characterization of the metallo-beta-lactamase determinant of Acinetobacter baumannii AC-54/97 reveals the existence of bla(IMP) allelic variants carried by gene cassettes of different phylogeny.

The metallo-beta-lactamase determinant of Acinetobacter baumannii AC-54/97, a clinical isolate from Italy that was previously shown to produce an enzyme related to IMP-1, was isolated by means of a PCR methodology which targets amplification of gene cassette arrays inserted into class 1 integrons. Sequencing revealed that this determinant was an allelic variant (named bla(IMP-2)) of bla(IMP) found in Japanese isolates and that it was divergent from the latter by 12% of its nucleotide sequence, which evidently had been acquired independently. Similar to bla(IMP), bla(IMP-2) was also carried by an integron-borne gene cassette. However, the 59-base element of the bla(IMP-2) cassette was unrelated to those of the bla(IMP) cassettes found in Japanese isolates, indicating a different phylogeny for the gene cassettes carrying the two allelic variants. Expression of the integron-borne bla(IMP-2) gene in Escherichia coli resulted in a significant decrease in susceptibility to a broad array of beta-lactams (ampicillin, carbenicillin, cephalothin, cefoxitin, ceftazidime, cefepime, and carbapenems). The IMP-2 enzyme was purified from an Escherichia coli strain carrying the cloned determinant, and kinetic parameters were determined with several beta-lactam substrates. Compared to IMP-1, the kinetic parameters of IMP-2 were similar overall with some beta-lactam substrates (cefoxitin, ceftazidime, cefepime, and imipenem) but remarkably different with others (ampicillin, carbenicillin, cephaloridine, and meropenem), revealing a functional significance of at least some of the mutations that differentiate the two IMP variants. Present findings suggest that the environmental reservoir of bla(IMP) alleles could be widespread and raise a question about the global risk of their transfer to clinically relevant species.

Cloning of a Chryseobacterium (Flavobacterium) meningosepticum chromosomal gene (blaA(CME)) encoding an extended-spectrum class A beta-lactamase related to the Bacteroides cephalosporinases and the VEB-1 and PER beta-lactamases.

In addition to the BlaB metallo-beta-lactamase, Chryseobacterium (Flavobacterium) meningosepticum CCUG 4310 (NCTC 10585) constitutively produces a 31-kDa active-site serine beta-lactamase, named CME-1, with an alkaline isoelectric pH. The blaA(CME) gene that encodes the latter enzyme was isolated from a genomic library constructed in the Escherichia coli plasmid vector pACYC184 by screening for cefuroxime-resistant clones. Sequence analysis revealed that the CME-1 enzyme is a new class A beta-lactamase structurally divergent from the other members of this class, being most closely related to the VEB-1 (also named CEF-1) and PER beta-lactamases and the Bacteroides chromosomal cephalosporinases. The blaA(CME) determinant is located on the chromosome and exhibits features typical of those of C. meningosepticum resident genes. The CME-1 protein was purified from an E. coli strain that overexpresses the cloned gene via a T7-based expression system by means of an anion-exchange chromatography step followed by a gel permeation chromatography step. Kinetic parameters for several substrates were determined. CME-1 is a clavulanic acid-susceptible extended-spectrum beta-lactamase that hydrolyzes most cephalosporins, penicillins, and monobactams but that does not hydrolyze cephamycins and carbapenems. The enzyme exhibits strikingly different kinetic parameters for different classes of beta-lactams, with both K(m) and k(cat) values much higher for cephalosporins than for penicillins and monobactams. However, the variability of both kinetic parameters resulted in overall similar acylation rates (k(cat)/K(m) ratios) for all types of beta-lactam substrates.