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Burkholderia pseudomallei class a beta-lactamase mutations that confer selective resistance against ceftazidime or clavulanic acid inhibition.

ABSTRACT: Burkholderia pseudomallei, the causative agent of melioidosis, is inherently resistant to a variety of antibiotics including aminoglycosides, macrolides, polymyxins, and beta-lactam antibiotics. Despite resistance to many beta-lactams, ceftazidime and beta-lactamase inhibitor-beta-lactam combinations are commonly used for treatment of melioidosis. Here, we examine the enzyme kinetics of beta-lactamase isolated from mutants resistant to ceftazidime and clavulanic acid inhibition and describe specific mutations within conserved motifs of the beta-lactamase enzyme which account for these resistance patterns. Sequence analysis of regions flanking the B. pseudomallei penA gene revealed a putative regulator gene located downstream of penA. We have cloned and sequenced the penA gene from B. mallei and found it to be identical to penA from B. pseudomallei.

SUBMITTER: Tribuddharat C

PROVIDER: S-EPMC161859 | biostudies-literature | 2003 Jul

REPOSITORIES: biostudies-literature

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Burkholderia pseudomallei class a beta-lactamase mutations that confer selective resistance against ceftazidime or clavulanic acid inhibition.

Tribuddharat Chanwit C Moore Richard A RA Baker Patricia P Woods Donald E DE

Antimicrobial agents and chemotherapy 20030701 7

Burkholderia pseudomallei, the causative agent of melioidosis, is inherently resistant to a variety of antibiotics including aminoglycosides, macrolides, polymyxins, and beta-lactam antibiotics. Despite resistance to many beta-lactams, ceftazidime and beta-lactamase inhibitor-beta-lactam combinations are commonly used for treatment of melioidosis. Here, we examine the enzyme kinetics of beta-lactamase isolated from mutants resistant to ceftazidime and clavulanic acid inhibition and describe spec ...[more]

PMID: 12821450

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Project description:Around the world, Burkholderia spp. are emerging as pathogens highly resistant to ?-lactam antibiotics, especially ceftazidime. Clinical variants of Burkholderia pseudomallei possessing the class A ?-lactamase PenI with substitutions at positions C69 and P167 are known to demonstrate ceftazidime resistance. However, the biochemical basis for ceftazidime resistance in class A ?-lactamases in B. pseudomallei is largely undefined. Here, we performed site saturation mutagenesis of the C69 position and investigated the kinetic properties of the C69F variant of PenI from B. pseudomallei that results in a high level of ceftazidime resistance (2 to 64 mg/liter) when expressed in Escherichia coli. Surprisingly, quantitative immunoblotting showed that the steady-state protein levels of the C69F variant ?-lactamase were ?4-fold lower than those of wild-type PenI (0.76 fg of protein/cell versus 4.1 fg of protein/cell, respectively). However, growth in the presence of ceftazidime increases the relative amount of the C69F variant to greater than wild-type PenI levels. The C69F variant exhibits a branched kinetic mechanism for ceftazidime hydrolysis, suggesting there are two different conformations of the enzyme. When incubated with an anti-PenI antibody, one conformation of the C69F variant rapidly hydrolyzes ceftazidime and most likely contributes to the higher levels of ceftazidime resistance observed in cell-based assays. Molecular dynamics simulations suggest that the electrostatic characteristics of the oxyanion hole are altered in the C69F variant. When ceftazidime was positioned in the active site, the C69F variant is predicted to form a greater number of hydrogen-bonding interactions than PenI with ceftazidime. In conclusion, we propose "a new twist" for enhanced ceftazidime resistance mediated by the C69F variant of the PenI ?-lactamase based on conformational changes in the C69F variant. Our findings explain the biochemical basis of ceftazidime resistance in B. pseudomallei, a pathogen of considerable importance, and suggest that the full repertoire of conformational states of a ?-lactamase profoundly affects ?-lactam resistance.

Dataset Information

Burkholderia pseudomallei class a beta-lactamase mutations that confer selective resistance against ceftazidime or clavulanic acid inhibition.

Publications

Burkholderia pseudomallei class a beta-lactamase mutations that confer selective resistance against ceftazidime or clavulanic acid inhibition.

Similar Datasets

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