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

Antimicrob Agents Chemother. 2000 Feb;44(2):362-7. doi: 10.1128/AAC.44.2.362-367.2000.

Abstract

We describe Klebsiella pneumoniae 15571, a clinical isolate resistant to ceftazidime MIC = 32 microg/ml) and piperacillin-tazobactam (MICs = 1,024 and 128 microg/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 bla(SHV-1) gene did not confer resistance to ceftazidime, a spectrum consistent with the nucleotide sequence, which was nearly identical to those of previously described bla(SHV-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 bla(SHV-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 bla(SHV-1) gene in the two strains. Primer extension analysis of bla(SHV-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 bla(SHV-1) open reading frames in the two K. pneumoniae strains revealed an A-->C 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 bla(SHV-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 bla(SHV-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.

Publication types

  • Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

  • Bacterial Outer Membrane Proteins / biosynthesis*
  • Bacterial Outer Membrane Proteins / metabolism
  • Base Sequence
  • Biological Transport
  • Ceftazidime / pharmacology*
  • Cephalosporins / pharmacology
  • Chromosomes
  • Cloning, Molecular
  • DNA, Bacterial / analysis
  • Drug Resistance, Microbial / genetics
  • Drug Resistance, Microbial / physiology
  • Enzyme Inhibitors / pharmacology
  • Gene Expression Regulation, Bacterial / drug effects
  • Humans
  • Klebsiella pneumoniae / drug effects*
  • Klebsiella pneumoniae / genetics
  • Klebsiella pneumoniae / metabolism
  • Microbial Sensitivity Tests
  • Molecular Sequence Data
  • Nucleic Acid Hybridization
  • Penicillanic Acid / analogs & derivatives*
  • Penicillanic Acid / pharmacology
  • Penicillins / pharmacology
  • Piperacillin / pharmacology*
  • Promoter Regions, Genetic / genetics
  • Sequence Homology, Nucleic Acid
  • Tazobactam
  • beta-Lactamases / biosynthesis*
  • beta-Lactamases / genetics

Substances

  • Bacterial Outer Membrane Proteins
  • Cephalosporins
  • DNA, Bacterial
  • Enzyme Inhibitors
  • Penicillins
  • Penicillanic Acid
  • Ceftazidime
  • beta-lactamase PIT-2
  • beta-Lactamases
  • Tazobactam
  • Piperacillin