Construction and Characterization of Mutants of the TEM-1 b-Lactamase Containing Amino Acid Substitutions Associated with both Extended-Spectrum Resistance and Resistance to b-Lactamase Inhibitors

Stapleton, P.D., Shannon, K.P. and French, G.L. (1999) Construction and Characterization of Mutants of the TEM-1 b-Lactamase Containing Amino Acid Substitutions Associated with both Extended-Spectrum Resistance and Resistance to b-Lactamase Inhibitors. Antimicrobial Agents and Chemotherapy, 43 (8). pp. 1881-1887.

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Abstract

Extended-spectrum TEM b-lactamases (ESBLs) do not usually confer resistance to b-lactamase inhibitors such as clavulanate or tazobactam. To investigate the compatibility of the two phenotypes we used site-directed mutagenesis of the blaTEM-1 gene to introduce into the TEM-1 b-lactamase amino acid substitutions that confer the ESBL phenotype: TEM-12 (Arg1643Ser), TEM-26 (Arg1643Ser plus Glu1043Lys), TEM-19 (Gly2383Ser), and TEM-15 (Gly2383Ser plus Glu1043Lys). These were combined with three sets of substitutions that confer inhibitor resistance: TEM-31 (Arg2443Cys), TEM-33 (Met693Leu), and TEM-35 (Met693Leu and Asn2763Asp). Introduction of the Arg2443Cys substitution gave rise to inhibitor-resistant hybrid enzymes that either lost ESBL activity (TEM-12, TEM-15, and TEM-19) or had reduced activity (TEM-26) against ceftazidime. In contrast, the introduction of Met693Leu or Met693Leu plus Asn2763Asp substitutions did not significantly affect the abilities of the enzymes to confer resistance to ceftazidime, although increased susceptibility to cefotaxime was observed with Escherichia coli strains that expressed the TEM-19 and TEM-26 b-lactamases. With the exception of the TEM-12 b-lactamase, introduction of the Met693Leu substitution did not give rise to enzymes with increased resistance to clavulanate compared to that of the TEM-1 b-lactamase. However, introduction of the double substitution Met693Leu plus Asn2763Asp in the ESBLs did give rise to low-level (TEM-19, TEM-15, and TEM-26) or moderate-level (TEM-12) clavulanate resistance. None of the hybrid enzymes were as resistant to clavulanate as the corresponding inhibitorresistant TEM b-lactamase mutant, suggesting that active-site configuration in the ESBLs limits the degree of clavulanate resistance conferred.

Item Type:Article
Departments, units and centres:Department of Pharmaceutical and Biological Chemistry > Department of Pharmaceutical and Biological Chemistry
ID Code:2471
Journal or Publication Title:Antimicrobial Agents and Chemotherapy
Deposited By:Library Staff
Deposited On:18 Nov 2011 12:59
Last Modified:18 Nov 2011 12:59

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