title: 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.
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.
authors.affiliation: Medical Service, Department of Veterans Affairs Medical Center, Cleveland, OH 44106, USA. louis.rice@med.va.gov
authors.forename: L B
authors.initials: LB
authors.lastname: Rice
authors.forename: L L
authors.initials: LL
authors.lastname: Carias
authors.forename: A M
authors.initials: AM
authors.lastname: Hujer
authors.forename: M
authors.initials: M
authors.lastname: Bonafede
authors.forename: R
authors.initials: R
authors.lastname: Hutton
authors.forename: C
authors.initials: C
authors.lastname: Hoyen
authors.forename: R A
authors.initials: RA
authors.lastname: Bonomo
mesh_terms: D001425:Bacterial Outer Membrane Proteins / Q000096:biosynthesis* / Q000378:metabolism; D001483:Base Sequence; D001692:Biological Transport; D002442:Ceftazidime / Q000494:pharmacology*; D002511:Cephalosporins / Q000494:pharmacology; D002875:Chromosomes; D003001:Cloning, Molecular; D004269:DNA, Bacterial / Q000032:analysis; D004352:Drug Resistance, Microbial / Q000235:genetics / Q000502:physiology; D004791:Enzyme Inhibitors / Q000494:pharmacology; D015964:Gene Expression Regulation, Bacterial / Q000187:drug effects; D006801:Humans; D007711:Klebsiella pneumoniae / Q000187:drug effects* / Q000235:genetics / Q000378:metabolism; D008826:Microbial Sensitivity Tests; D008969:Molecular Sequence Data; D009693:Nucleic Acid Hybridization; D010397:Penicillanic Acid / Q000031:analogs & derivatives* / Q000494:pharmacology; D010406:Penicillins / Q000494:pharmacology; D010878:Piperacillin / Q000494:pharmacology*; D011401:Promoter Regions, Genetic / Q000235:genetics; D012689:Sequence Homology, Nucleic Acid; D000078142:Tazobactam; D001618:beta-Lactamases / Q000096:biosynthesis* / Q000235:genetics
chemical_list: D001425:Bacterial Outer Membrane Proteins; D002511:Cephalosporins; D004269:DNA, Bacterial; D004791:Enzyme Inhibitors; D010406:Penicillins; D010397:Penicillanic Acid; D002442:Ceftazidime; C054882:beta-lactamase PIT-2; D001618:beta-Lactamases; D000078142:Tazobactam; D010878:Piperacillin