Affiliation:
1. Medical1 and
2. Medicine2and
3. Research3 Services, Department of Veterans Affairs Medical Center, and Departments of
4. Pediatrics,4 Case Western Reserve University School of Medicine, Cleveland, Ohio
Abstract
ABSTRACT
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.
Publisher
American Society for Microbiology
Subject
Infectious Diseases,Pharmacology (medical),Pharmacology
Cited by
110 articles.
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