High-level expression of chromosomally encoded SHV-1 beta-lactamase and anouter 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 ce ftazidime MIC = 32 mu g/ml) and piperacillin-tazobactam (MICs = 1,024 and 1 28 mu g/ml). IC. 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 spe ctrum consistent with the nucleotide sequence, which was nearly identical t o those of previously described bla(SHV-1) genes. Outer membrane protein (O MP) analysis of ii, pneumoniae 15571 revealed a decrease in the quantity of a minor 45-kDa OMP in comparison to that in ii. pneumoniae 44NR, a Low-lev el ampicillin-resistant strain that also expresses a chromosomally determin ed bla(SHV-1). Crude beta-lactamase enzyme extracts from K. pneumoniae 1557 1 produced roughly 200-fold more beta-lactamase activity than K, pneumoniae 34NR. Northern hybridization analysis revealed that this difference was ex plainable by quantifiable differences in transcription of the bla(SHV-1) ge ne in the two strains. Primer extension analysis of bla(SHV-1) mRNA from K, pneumoniae 15571 and 44NR indicated that the transcriptional start sites w ere identical in the two strains. DNA sequencing of the promoter regions up stream of the of bla(SHV-1) open reading frames in the two K, pneumoniae st rains revealed an A-->C change in the second position of the -10 region in K. pneumoniae 34NR compared to that in 15571, Site-directed mutagenesis of the cloned Ii, pneumoniae 15571 bla(SHV-1) in which the A in the second pos ition 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 enzym e expression in E. coli were compared, the bla(SHV-1) downstream of the alt ered -10 region produced 17-fold less beta-lactamase enzyme. These results indicate that elevated levels of ceftazidime resistance can result from a c ombination of increased enzyme production and minor OMP changes and that le vels of chromosomally encoded SHV-1 beta-lactamase production can vary subs tantially with a single-base-pair change in promoter sequence.