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

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 β-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 β-lactamase enzyme extracts from K. pneumoniae 15571 produced roughly 200-fold more β-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 β-lactamase enzyme expression in E. coli were compared, the bla(SHV-1) downstream of the altered −10 region produced 17-fold less β-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 β-lactamase production can vary substantially with a single-base-pair change in promoter sequence.