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Antimicrobial Agents and Chemotherapy, August 1999, p. 1881-1887, Vol. 43, No. 8
0066-4804/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.

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

Paul D. Stapleton,* Kevin P. Shannon, and Gary L. French

Department of Microbiology, The Guy's, King's & St. Thomas' School of Medicine, St. Thomas' Campus, London SE1 7EH, United Kingdom

Received 5 October 1998/Returned for modification 25 January 1999/Accepted 12 May 1999

Extended-spectrum TEM beta -lactamases (ESBLs) do not usually confer resistance to beta -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 beta -lactamase amino acid substitutions that confer the ESBL phenotype: TEM-12 (Arg164right-arrowSer), TEM-26 (Arg164right-arrowSer plus Glu104right-arrowLys), TEM-19 (Gly238right-arrowSer), and TEM-15 (Gly238right-arrowSer plus Glu104right-arrowLys). These were combined with three sets of substitutions that confer inhibitor resistance: TEM-31 (Arg244right-arrowCys), TEM-33 (Met69right-arrowLeu), and TEM-35 (Met69right-arrowLeu and Asn276right-arrowAsp). Introduction of the Arg244right-arrowCys 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 Met69right-arrowLeu or Met69right-arrowLeu plus Asn276right-arrowAsp 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 beta -lactamases. With the exception of the TEM-12 beta -lactamase, introduction of the Met69right-arrowLeu substitution did not give rise to enzymes with increased resistance to clavulanate compared to that of the TEM-1 beta -lactamase. However, introduction of the double substitution Met69right-arrowLeu plus Asn276right-arrowAsp 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 inhibitor-resistant TEM beta -lactamase mutant, suggesting that active-site configuration in the ESBLs limits the degree of clavulanate resistance conferred.

* Corresponding author. Mailing address: Department of Biology (Darwin Building), University College London, Gower Street, London WC1E 6BT, United Kingdom. Phone: 44 171 504 2934. Fax: 44 171 380 7098. E-mail: p.stapleton{at}ucl.ac.uk.

Antimicrobial Agents and Chemotherapy, August 1999, p. 1881-1887, Vol. 43, No. 8
0066-4804/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.


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