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Antimicrobial Agents and Chemotherapy, February 2000, p. 362-367, Vol. 44, No. 2
0066-4804/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.

High-Level Expression of Chromosomally Encoded SHV-1 beta -Lactamase and an Outer Membrane Protein Change Confer Resistance to Ceftazidime and Piperacillin- Tazobactam in a Clinical Isolate of Klebsiella pneumoniae

Louis B. Rice,1,2,* Lenore L. Carias,2 Andrea M. Hujer,3 Mary Bonafede,4 Rebecca Hutton,3 Claudia Hoyen,4 and Robert A. Bonomo1,2

Medical1 and Research3 Services, Department of Veterans Affairs Medical Center, and Departments of Medicine2 and Pediatrics,4 Case Western Reserve University School of Medicine, Cleveland, Ohio

Received 15 March 1999/Returned for modification 23 June 1999/Accepted 9 November 1999

We describe Klebsiella pneumoniae 15571, a clinical isolate resistant to ceftazidime MIC = 32 µg/ml) and piperacillin-tazobactam (MICs = 1,024 and 128 µg/ml). K. pneumoniae 15571 expresses a single beta -lactamase with a pI of 7.6. However, when cloned in a high-copy-number vector in Escherichia coli, this blaSHV-1 gene did not confer resistance to ceftazidime, a spectrum consistent with the nucleotide sequence, which was nearly identical to those of previously described blaSHV-1 genes. Outer membrane protein (OMP) analysis of K. pneumoniae 15571 revealed a decrease in the quantity of a minor 45-kDa OMP in comparison to that in K. pneumoniae 44NR, a low-level ampicillin-resistant strain that also expresses a chromosomally determined blaSHV-1. Crude beta -lactamase enzyme extracts from K. pneumoniae 15571 produced roughly 200-fold more beta -lactamase activity than K. pneumoniae 44NR. Northern hybridization analysis revealed that this difference was explainable by quantifiable differences in transcription of the blaSHV-1 gene in the two strains. Primer extension analysis of blaSHV-1 mRNA from K. pneumoniae 15571 and 44NR indicated that the transcriptional start sites were identical in the two strains. DNA sequencing of the promoter regions upstream of the of blaSHV-1 open reading frames in the two K. pneumoniae strains revealed an Aright-arrowC change in the second position of the -10 region in K. pneumoniae 44NR compared to that in 15571. Site-directed mutagenesis of the cloned K. pneumoniae 15571 blaSHV-1, in which the A in the second position of the 15571 -10 region was changed to a C, resulted in a substantial lowering of the MIC of ampicillin. When the levels of beta -lactamase enzyme expression in E. coli were compared, the blaSHV-1 downstream of the altered -10 region produced 17-fold less beta -lactamase enzyme. These results indicate that elevated levels of ceftazidime resistance can result from a combination of increased enzyme production and minor OMP changes and that levels of chromosomally encoded SHV-1 beta -lactamase production can vary substantially with a single-base-pair change in promoter sequence.

* Corresponding author. Mailing address: Medical Service 111(W), VA Medical Center, 10701 East Blvd., Cleveland, OH 44106. Phone: (216) 791-3800, ext. 4800. Fax: (216) 231-3289. E-mail: louis.rice{at}med.va.gov.

Antimicrobial Agents and Chemotherapy, February 2000, p. 362-367, Vol. 44, No. 2
0066-4804/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.


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