This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow E-mail this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowReprints and Permissions
Right arrow Copyright Information
Right arrow Books from ASM Press
Right arrow MicrobeWorld
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Rice, L. B.
Right arrow Articles by Bonomo, R. A.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Rice, L. B.
Right arrow Articles by Bonomo, R. A.

 Previous Article  |  Next Article 

Antimicrobial Agents and Chemotherapy, February 2000, p. 362-367, Vol. 44, No. 2
0066-4804/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.

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

Louis B. Rice,1,2,* Lenore L. Carias,2 Andrea M. Hujer,3 Mary Bonafede,4 Rebecca Hutton,3 Claudia Hoyen,4 and Robert A. Bonomo1,2

Medical1 and Research3 Services, Department of Veterans Affairs Medical Center, and Departments of Medicine2 and Pediatrics,4 Case Western Reserve University School of Medicine, Cleveland, Ohio

Received 15 March 1999/Returned for modification 23 June 1999/Accepted 9 November 1999


    ABSTRACT
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References

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 beta -lactamase with a pI of 7.6. However, when cloned in a high-copy-number vector in Escherichia coli, this blaSHV-1 gene did not confer resistance to ceftazidime, a spectrum consistent with the nucleotide sequence, which was nearly identical to those of previously described blaSHV-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 blaSHV-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 blaSHV-1 gene in the two strains. Primer extension analysis of blaSHV-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 blaSHV-1 open reading frames in the two K. pneumoniae strains revealed an Aright-arrowC 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 blaSHV-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 blaSHV-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.


    INTRODUCTION
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References

Resistance to ceftazidime in Klebsiella pneumoniae is most commonly attributed to the production of plasmid-mediated extended-spectrum beta -lactamases (ESBLs). These enzymes are frequently encoded by multidrug resistance conjugative plasmids and evolve from the more common TEM-1 and SHV-1 penicillinases through point mutations in regions important for beta -lactam binding and/or hydrolysis (10). Conjugative dissemination of ESBL-encoding plasmids is thought to facilitate the spread of resistance in the clinical setting. In rare cases, resistance to ceftazidime has been attributed solely to increased production of SHV-1, although MICs of ceftazidime for these strains are only mildly increased (ca. 8 µg/ml) (15).

Resistance to beta -lactam-beta -lactamase inhibitor combinations occurs most commonly in gram-negative bacilli through the overproduction of common plasmid-mediated enzymes (20). Overproduction has been attributed to altered promoter sequences, gene duplication, or the presence of an ESBL gene on a multicopy plasmid (15, 20, 22). Rarely, a derivative of TEM-1 or SHV-1 is found that confers high-level resistance to both ceftazidime and beta -lactam-beta -lactamase inhibitor combinations (TEM-50) (21). This phenotype can be achieved by other means, including the overproduction of an ESBL, the coexistence of an ESBL with an SHV-1 enzyme produced in large quantities, or the acquisition of a plasmid-encoded ampC-type gene (3, 15, 17).

A single point mutation 177 bp upstream of the TEM initiation codon that results in the creation of dual overlapping promoters is the most commonly described promoter mutation resulting in overproduction of TEM-type enzymes (4). Transcription from these two new promoters results in TEM production that is roughly 10-fold higher than with the standard TEM-1 promoter sequence. For reasons that remain unclear, a high percentage of TEM-type ESBL genes have been identified downstream of this more active promoter (8).

Although SHV-1 was originally characterized as a plasmid-mediated beta -lactamase, recent data suggest that its production may be intrinsic to K. pneumoniae, since it appears that many clinical K. pneumoniae strains (more than 90% of one series) encode SHV-1 beta -lactamase production from their chromosomes (11). In most cases, beta -lactamase production encoded by these chromosomal genes is at very low levels, resulting in relatively low levels of resistance to ampicillin (MIC, ca. 8 to 64 µg/ml). The product of the first sequenced chromosomal beta -lactamase gene from K. pneumoniae was designated LEN-1 (1). The nucleotide sequence of the gene encoding LEN-1 is 85% identical to that of the gene encoding SHV-1, suggesting a close evolutionary relationship between the two enzymes (13). Recent data suggest that chromosomally encoded SHV-1 may be more prevalent than LEN-1 in clinical K. pneumoniae isolates (11).

Promoter mutations that are associated with increased production of SHV-1 are less well described than those for TEM-type genes. Original reports identified putative -35 and -10 promoter sequences upstream of the SHV-1 gene based on sequence similarity to Escherichia coli consensus promoter sequences (13). A more recent report identified the specific transcriptional start site of the SHV-1 gene as 50 bp upstream of the previously presumed transcriptional start site (16). In this report, a more active promoter sequence was identified upstream of an ESBL gene (blaSHV-2a) resulting from the insertion of IS15 upstream of the gene (17). This promoter change was associated with a fivefold increase in the synthesis of SHV-2a mRNA compared to that observed with the traditional promoter. The extent to which extended-spectrum SHV-related beta -lactamase genes are preceded by more active promoter sequences is unknown.

We report a K. pneumoniae strain that expresses in vitro resistance to both ceftazidime and beta -lactam-beta -lactamase inhibitor combinations. The sole enzyme expressed by this strain is a chromosomally encoded SHV-1 that is produced in large amounts, partially accounting for the increased level of ceftazidime resistance. This strain also exhibited decreased production of a minor 45-kDa outer membrane protein that may be involved in the transport of antimicrobial agents. We present evidence that this chromosomal blaSHV-1 gene is encoded by an open reading frame downstream of what is considered to be the typical blaSHV-1 promoter sequence. This promoter is associated with 200-fold greater SHV-1 production than an SHV-1-like enzyme produced by a second K. pneumoniae strain in which the promoter differs by only 1 nucleotide in the -10 region. We also present confirmatory evidence that this single-nucleotide change is responsible for most, if not all, of the increased beta -lactamase production observed in this strain.


    MATERIALS AND METHODS
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References

Bacterial strains and plasmids. The bacterial strains and recombinant plasmids used and analyzed in these studies are described in Table 1. K. pneumoniae 15571 was a clinical isolate from the urinary tract of an infected adult patient. K. pneumoniae 44NR and 44NRF have been described previously (18). In the prior publication, K. pneumoniae 44NR was described in the text as producing "no beta -lactamase." In Table 1 from that article, however, it is clear that the strain produced a small but detectable amount of beta -lactamase. The present work confirms that this strain does in fact produce beta -lactamase, but at very low levels.

                              
View this table:
[in this window]
[in a new window]
  		TABLE 1.   Bacterial strains and plasmids

Microbiological techniques. MICs of different antibiotics were determined by standard agar dilution (14) techniques, except that Luria-Bertani (LB) agar was used instead of Mueller-Hinton agar and we used a fixed ratio of piperacillin-tazobactam (8:1) rather than a constant inhibitor concentration of 4 µg/ml.

Conjugation experiments. Matings between K. pneumoniae 15571 and E. coli J53-2 (Rifr) were carried out overnight at 37°C on sterile nitrocellulose filters as previously described (17).

beta -Lactamase assays. Crude beta -lactamase extracts were prepared from 5-ml overnight cultures. K. pneumoniae and E. coli cells containing beta -lactamase enzymes were grown to mid-log phase (optical density at 600 nm, 0.5) in LB broth containing 100 µg of ampicillin/ml. The cells were pelleted, washed, and resuspended in 500 µl of 50 mM Tris-HCl buffer, pH 7.4. A 40-mg/ml stock solution of freshly prepared lysozyme (Sigma, St. Louis, Mo.) in Tris-HCl buffer was added to a final concentration of 10 µg/ml, and bacterial cells were incubated for 15 min at room temperature. EDTA was added to a final concentration of 1 mM, and the mixture was gently shaken for 10 min. The cell suspension was clarified by centrifugation at 14,000 × g for 15 min, and the supernatant was collected. beta -Lactamase activity in the supernatant was measured with the chromogenic cephalosporin nitrocefin (Becton Dickinson, Cockeysville, Md.). The amount of protein in each beta -lactamase preparation was determined by the Bio-Rad (Hercules, Calif.) protein assay. The specific activity present in each sample was estimated by measuring the hydrolysis of 100 µM nitrocefin at 25°C in a Hewlett-Packard 8452 diode array spectrophotometer (lambda  = 482 nM; Delta varepsilon  = 17,400 M-1 cm-1). The initial (maximal) velocity was used to determine specific activity. Specific activity was defined as U/mg of protein. One unit was defined as the amount of nitrocefin hydrolyzed (micromolar) per minute.

aIEF. Analytical isoelectric focusing studies (aIEF) were performed with a Multiphor isoelectric focusing apparatus (Pharmacia, Piscataway, N.J.) according to a modification of the method of Vecoli et al. (25). The crude beta -lactamase preparations and prestained standards, pI 4.5 to 9.5, were directly applied to Ampholine PAGplates, pI 3.5 to 9.5 (Pharmacia). beta -Lactamase bands were identified with an overlay of 0.5 mg of nitrocefin (100 µM dilute solution)/ml.

Outer membrane protein analysis. Outer membrane proteins from three clinical strains of K. pneumoniae (15571, 44NR, and 44NRF) were isolated according to the method of Spratt (23) with modifications employed by Gutmann et al. (9).

Molecular techniques. Plasmid DNA was extracted from clinical strains by a technique described by Takahashi and Nagano (24). Genomic DNA was extracted as previously described (19) and digested with restriction enzymes (Promega, Madison, Wis.) as recommended by the manufacturer. Digested DNA was separated on agarose gels and transferred to nylon membranes with a negative-pressure transfer apparatus (Pharmacia). An internal probe for the blaSHV-1 gene was generated by PCR with primers designed to amplify a fragment internal to the blaSHV-1 gene (SHV-238, 5'-CCGCGTAGGCATGATAGAAA-3', and SHV-894, 5'-TCCCGCAGATAAA-3'). The template for the PCR was a cloned blaSHV-1 gene (recombinant plasmid pCWR101) previously described (17). The probes were labeled with digoxigenin (Boehringer Mannheim) according to the specifications of the manufacturer. Hybridization occurred overnight at 68°C, after which the membranes were washed under conditions of high stringency. Labeled fragments were detected with an anti-digoxigenin-alkaline phosphatase conjugate and chromogenic enzyme substrate.

Cloning and sequencing techniques. SHV-1-encoding BamHI fragments (3.6 kb) were identified in K. pneumoniae 15571 and K. pneumoniae 44NR by Southern hybridization with the PCR-generated internal blaSHV-1 probe. Gel slices containing these fragments were excised and ligated to BamHI-digested pBC SK(-) (Stratagene, La Jolla, Calif.). Commercially purchased electrocompetent E. coli DH10B cells were transformed with the ligation mixture by electroporation, and transformed colonies were selected on LB agar plates containing chloramphenicol (20 µg/ml) and ampicillin (100 µg/ml). The resultant recombinant plasmids were designated pCWR338 (from 15571) and pCWR498 (from 44NR). pCWR338 was further subcloned with the restriction enzymes ScaI and ClaI. The complete nucleotide sequence of the cloned blaSHV-1 gene from K. pneumoniae 15571 was determined on both strands with forward and reverse primers as well as commercially synthesized primers internal to the blaSHV-1 open reading frame. Sequencing reactions were performed with the Cy-5 Thermosequenase dye terminator kit (Pharmacia) with unlabeled primers or primers were end-labeled with Cy-5 and sequencing was carried out with the Thermosequenase fluorescent-labeled primer cycle-sequencing kit (Pharmacia). Sequences were determined with the A.L.F. Express automated sequencer (Pharmacia), and analysis was carried out with either the MacDNAsis analysis program (Hitachi) or the Lasergene Navigator (DNAStar) analysis program.

Northern hybridization. Total cellular RNA was extracted from the strains in this study with the Qiagen (Valencia, Calif.) RNeasy miniprep kit according to a protocol supplied by the manufacturer. RNA concentrations were measured spectrophotometrically, and equal amounts of RNA were separated on 1.2% agarose gels containing formaldehyde. The transfer of RNA from gels to nylon membranes was accomplished by capillary transfer. The digoxigenin-labeled internal blaSHV-1 probe was generated as described above and used to hybridize the RNA fixed to the nylon membrane. The hybridized membranes were washed under high-stringency conditions, and hybridized RNA fragments were detected by a chemiluminescence technique with disodium 3-(4-methoxyspiro [1,2-dioxetane-3,2'-(5'-chloro)tricyclo[3.3.1.13-7]decan]-4-yl)phenyl phosphate (Boehringer Mannheim) as the reagent.

Primer extension analysis. RNA was extracted and purified according to a commercial protocol (Rneasy Miniprep kit, Qiagen). Primer extension analysis was carried out with Cy-labeled primers designed to direct synthesis from the 3' to the 5' end of the open reading frame. The technique employed high temperatures to minimize complications resulting from secondary-structure formation, essentially as described by Yamada et al. (27).

Site-directed mutagenesis. In vitro site-directed mutagenesis was performed with the QuikChange site-directed mutagenesis kit manufactured by Stratagene. Two complementary mutagenic oligonucleotide primers were constructed at the Case Western Reserve University Molecular Biology Core Laboratory with the following restrictions: a melting temperature higher than 78°C, a G+C content greater than 40%, and termination in a C or G nucleotide. To fulfill these requirements, we designed a 43-mer due to the high AT content of the promoter region. The primers were nonphosphorylated and high-performance liquid chromatography purified. Mutagenesis was performed with Pfu polymerase supplied with the kit. The plasmid pBC SK(-), prepared with the Promega Miniprep Wizard kit and containing blaSHV-1, was used as the DNA template for the mutagenic PCR. The cycling parameters for mutagenesis were 95°C for 30 s, 55°C for 1 min, and 68°C for 10 min for a total of 16 cycles. DpnI was added after amplification to digest methylated (parental) DNA. Epicurean coli (E. coli) XL1-Blue supercompetent cells (Stratagene) were transformed with mutagenic DNA by heat pulse for 45 s at 42°C and then placed on ice for 2 min. The transformed cells were next incubated at 37°C for 1 h and plated, and single-colony transformants were selected the next day.

Nucleotide sequence accession number. The nucleotide sequence for the blaSHV-1 gene from K. pneumoniae 15571 has been entered in GenBank under accession no. AF124984.


    RESULTS
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References

Characterization of K. pneumoniae 15571. The MICs for ceftazidime and piperacillin-tazobactam were 32 µg/ml and 1024 and 128 µg/ml, respectively, against K. pneumoniae 15571 (Table 2). Repeated conjugation experiments failed to result in the transfer of ampicillin or ceftazidime resistance to E. coli J53-2 when ampicillin (100 µg/ml) was used for selection (data not shown). In addition, plasmid analysis revealed several plasmids in this strain, none of which hybridized to the internal blaSHV-1 probe (data not shown). Hybridization of K. pneumoniae 15571 genomic DNA digested individually with five different restriction enzymes revealed a single blaSHV-1-hybridizing genomic band with each digestion (data not shown).

                              
View this table:
[in this window]
[in a new window]
  		TABLE 2.   Susceptibilities and beta -lactamase activities of different strains

aIEF. aIEF of crude enzyme extracts from K. pneumoniae 15571 indicated the production of a single beta -lactamase with a pI of 7.6.

Outer membrane protein analysis. Outer membrane protein analysis (Fig. 1) suggested a reduction in a minor (ca. 45-kDa) outer membrane protein in strain 15571 in comparison to that in K. pneumoniae 44NR (Fig. 1).


View larger version (90K):
[in this window]
[in a new window]
  		FIG. 1.   Outer membrane proteins were separated on a sodium dodecyl sulfate-containing polyacrylamide gel (acrylamide/bisacrylamide ratio, 29/1; 5% stacking gel with a 12% separating gel). Lane 1, molecular mass marker; lane 2, K. pneumoniae 15571; lane 3, K. pneumoniae 44NR; lane 4, K. pneumoniae 44NRF. Note the diminished quantity of 45-kDa protein in lane 2 versus lane 3 (arrow).

Northern hybridization. In order to compare transcription of the blaSHV-1 genes in K. pneumoniae 15571 and 44NR, we performed Northern hybridizations of equal amounts of mRNA extracted from the two strains, using the internal blaSHV-1 fragment as a probe. The results are shown in Fig. 2. The hybridization signal from K. pneumoniae 15571 was dramatically greater than that in K. pneumoniae 44NR or its outer membrane protein-deficient, isogenic mutant, 44NRF. These data suggested that the differences in MICs were the result of marked differences in transcription of the genes in the two strains.


View larger version (48K):
[in this window]
[in a new window]
  		FIG. 2.   Northern hybridization of mRNAs extracted from K. pneumoniae 15571 and 44NR. Note the substantially greater levels of mRNA in K. pneumoniae 15571 compared to those in K. pneumoniae 44NR (44). K. pneumoniae 44NRFox (44 Fox) is a clinical strain with a missing outer membrane protein.

Cloning and characterization of the K. pneumoniae 15571 chromosomal beta -lactamase gene. The 3.6-kb blaSHV-1-hybridizing BamHI restriction fragment from K. pneumoniae 15571 was ligated to pBC SK(-) and transformed into E. coli DH10B. The recombinant plasmid, designated pCWR338, was further digested with ScaI and ClaI, resulting in a reduction of the insert size to 1,342 nucleotides, and ligated to EcoRV-ClaI-digested pBC SK(-), resulting in recombinant plasmid pCWR366. E. coli DH10B(pCWR366) expressed high levels of ampicillin resistance (MIC, >16,000 µg/ml) but lower levels of ceftazidime resistance than the K. pneumoniae 15571 parent strain, implying a contribution of the outer membrane protein change to ceftazidime resistance. The entire blaSHV-1 open reading frame was sequenced and found to be nearly identical to previously described blaSHV-1 open reading frames (2). Specifically, no amino acid changes were noted when comparison was made to the previously reported mature enzyme. In addition, the area upstream of the open reading frame that was reported to contain the promoter region was sequenced and found to be identical to previously reported regions for blaSHV-1 and blaSHV-2. Importantly, sequence alterations previously reported to be associated with increased expression of blaSHV-2a were not identified (16).

Promoter analysis. In order to further analyze the effect of promoter changes on the expression of beta -lactamase in K. pneumoniae 15571 in comparison to that in K. pneumoniae 44NR, we pursued a detailed comparison of the blaSHV-1 promoter regions in the two strains. The MIC of ampicillin for K. pneumoniae 44NR was much lower than that for K. pneumoniae 15571, and 44NR expressed at least 200-fold less beta -lactamase activity (Table 2). To ensure that this difference was not attributable to the SHV-1 protein itself, we cloned and sequenced the SHV-1 gene from the 44NR chromosome. A total of seven nucleotide changes were observed when the sequence of the 44NR beta -lactamase gene was compared with that of 15571. Five of these changes were silent in that they conferred no change in the amino acid sequence. The other two led to amino acid changes. Using the adenine of the ATG start codon as nucleotide 1, we observed an Aright-arrowG change at nucleotide 8 and a Tright-arrowA change at nucleotide 9, which together resulted in a Tyrright-arrowLeu substitution at the third amino acid. The amino acid change at position 3 is removed by the signal peptidases and is not part of the mature enzyme. Therefore, it is extremely unlikely to confer significant changes to the enzymatic activity of the beta -lactamase. The silent nucleotide changes occurred at nucleotides 324 (Cright-arrowT), 402 (Aright-arrowG), 633 (Gright-arrowA), 705 (Gright-arrowA), and 762 (Tright-arrowC).

That the difference between the MICs for 15571 and 44NR was explainable by differences in transcription was confirmed by Northern hybridization of mRNAs from the two strains, indicating a substantial increase in blaSHV-1 mRNA in K. pneumoniae 15571 in comparison to K. pneumoniae 44NR (Fig. 2). Analysis of the promoter region in this insert revealed two potentially important changes relative to the 15571 promoter (Fig. 3). The nucleotide that lies 4 bp upstream of the -35 region differs in K. pneumoniae 44 (T), K. pneumoniae 15571 and blaSHV-2 (G), and blaLEN-1 (A). In addition, the second nucleotide of the -10 region is a C in K. pneumoniae 44NR and blaLEN-1 while it is an A in K. pneumoniae 15571 and blaSHV-2. These differences suggest that the differences among the ampicillin MICs for K. pneumoniae LEN-1 (reported to be 7.8 µg/ml), K. pneumoniae 44 (256 µg/ml), and K. pneumoniae 15571 (>16,000 µg/ml) may be explainable, at least in part, by differences in the region upstream of the -35 region and internal to the -10 region.


View larger version (15K):
[in this window]
[in a new window]
  		FIG. 3.   Comparison of promoter sequences upstream of different SHV-type genes. K. p. LEN-1 denotes the sequence upstream of the chromosomal beta -lactamase gene of K. pneumoniae LEN-1 (ampicillin MIC, 7.8 µg/ml). K. p. 44 denotes the sequence of the chromosomal beta -lactamase gene of K. pneumoniae 44NR (ampicillin MIC, 128 to 256 µg/ml [see the text]). K. p. 15571 denotes the sequence upstream of the chromosomal beta -lactamase gene of K. pneumoniae 15571 (ampicillin MIC, 32,000 µg/ml). The published sequences of the promoter regions of the blaSHV-2 and blaSHV-2a genes are also shown. The boxes mark the different -35 and -10 regions. The horizontal arrows indicate a direct repeat within the typical -10 region. The vertical arrow indicates the nucleotide upstream of the -35 region that differs in three of the four sequences. The asterisks indicate the transcriptional start sites as determined by primer extension analysis of K. pneumoniae 44NR and 15571.

In order to further explore this possibility, we first sought to confirm that the transcriptional start sites for the blaSHV-1 message for K. pneumoniae 15571 and K. pneumoniae 44NR were the same and that these start sites were identical to those previously reported (16). Primer extension analysis confirmed that the transcriptional start site was identical to that previously reported. We next performed site-directed mutagenesis of the -10 region of the blaSHV-1 gene in recombinant plasmid pCWR366, changing the A in the second position to a C. The mutant plasmid was transformed into E. coli DH10B, after which ampicillin MICs were determined and Northern hybridizations were performed. The Aright-arrowC change was associated with a marked decrease in the ampicillin MIC from >16,000 to 2,000 µg/ml. This decrease in the MIC was associated with a decrease in the amount of blaSHV-1 mRNA detectable by Northern hybridization (data not shown). These data suggest that the nucleotide located at the second position of the -10 region of the blaSHV-1 promoter influences the expression of the enzyme and the ultimate level of ampicillin resistance.


    DISCUSSION
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References

Levels of antibiotic resistance expressed by beta -lactamase-producing bacteria can be affected by several different factors, including the affinity of the enzyme for the antibiotic, the amount of beta -lactamase produced, and the ease with which the antibiotic gains access to the periplasmic space. In this report, we describe a K. pneumoniae strain that expresses clinically important levels of resistance to both ceftazidime and the beta -lactam-beta -lactamase inhibitor combinations. Our data suggest this phenotype results from a combination of factors, most prominently increased production of SHV-1 enzyme and the reduction in a minor outer membrane protein that probably contributes to the entry of ceftazidime (and perhaps piperacillin and tazobactam) into the periplasmic space.

That increased production of the SHV-1 enzyme is not the sole explanation for ceftazidime resistance in K. pneumoniae 15571 is evidenced by the fact that the 15571 blaSHV-1 gene, when cloned into a high-copy-number vector in E. coli, expresses levels of ceftazidime resistance of only 2 µg/ml. This level of resistance is expressed despite the fact that, by Northern analysis, E. coli DH10B(pCWR366) produces dramatically more blaSHV-1 message than does 15571 (data not shown).

The loss of outer membrane proteins in clinical strains of K. pneumoniae has been repeatedly observed (3, 5, 12). Previously published analyses of the outer membrane proteins of K. pneumoniae have shown that the major proteins are the outer membrane doublet at ca. 35 to 40 kDa. A recent paper identified a third porin, OMPK37, which appears to be involved in resistance to beta -lactam antibiotics (7). Based on size comparison, the diminished outer membrane protein seen in K. pneumoniae 15571 was not any of these other porins. Our gel demonstrates the loss of a minor protein also identified by others that migrates more slowly than the doublet (9). The presence of porin reduction and ceftazidime resistance has also been described for E. coli by Weber et al. (26).

One recently published study suggests that most K. pneumoniae clinical isolates produce a beta -lactamase similar to SHV-1 and that in most cases these enzymes are chromosomally encoded (11). In the majority of cases, the level of enzyme production appears to be low. Our data suggest that this low level of expression is due to the activity of the promoter upstream of the gene. The observation that lower-level expression of beta -lactamase in K. pneumoniae 44NR is due to small differences between the promoter sequences in the two isolates was confirmed by site-directed mutagenesis. We changed A to C in the second position of the -10 sequence and noted a marked lowering of the ampicillin MIC. Our data do not allow us to exclude the possibility that a repressor or activator interacts with the promoter sequence to inhibit or enhance transcription. Further studies are planned to specifically investigate the possibility of repressor activity, as well as to sequence promoter regions from a variety of clinical K. pneumoniae strains in an effort to correlate, if possible, the content of promoter sequences with the level of ampicillin resistance on a broader scale.


    ACKNOWLEDGMENTS

This study was supported by the Office of Research and Development, Medical Research Service of the Department of Veterans Affairs (L.B.R. and R.A.B).

We are grateful to Kristine M. Hujer for her assistance with sequencing.


    FOOTNOTES

* Corresponding author. Mailing address: Medical Service 111(W), VA Medical Center, 10701 East Blvd., Cleveland, OH 44106. Phone: (216) 791-3800, ext. 4800. Fax: (216) 231-3289. E-mail: louis.rice{at}med.va.gov.


    REFERENCES
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References

1. Arakawa, Y., M. Ohta, N. Kido, Y. Fujii, T. Komatsu, and N. Kato. 1986. Close evolutionary relationship between the chromosomally encoded beta -lactamase gene of Klebsiella pneumoniae and the TEM beta -lactamase gene mediated by R plasmids. FEBS Lett. 207:69-74[CrossRef][Medline].
2. Barthelemy, M., J. Peduzzi, and R. Labia. 1988. Complete amino acid sequence of p453-plasmid-mediated PIT-2 beta-lactamase (SHV-1). Biochem. J. 251:73-79[Medline].
3. Bradford, P. A., C. Urban, N. Mariano, S. J. Projan, J. J. Rahal, and K. Bush. 1997. Imipenem resistance in Klebsiella pneumoniae is associated with the combination of ACT-1, a plasmid-mediated AmpC beta-lactamase, and the loss of an outer membrane protein. Antimicrob. Agents Chemother. 41:563-569[Abstract/Free Full Text].
4. Chen, S.-T., and R. C. Clowes. 1984. Two improved promoter sequences for the beta -lactamase expression arising from a single base-pair substitution. Nucleic Acids Res. 12:3219-3235[Abstract/Free Full Text].
5. Chen, Y., and D. M. Livermore. 1993. Activity of cefepime and other beta-lactam antibiotics against permeability mutants of Escherichia coli and Klebsiella pneumoniae. J. Antimicrob. Chemother. 32(Suppl. B):63-74[Abstract/Free Full Text].
6. Coetzee, J. N., N. Datta, and R. W. Hedges. 1972. R factors from Proteus rettgeri. J. Gen. Microbiol. 72:543-552[Abstract/Free Full Text].
7. Domenech-Sanchez, A., S. Hernandez-Alles, L. Martinez-Martinez, V. J. Benedi, and S. Alberti. 1999. Identification and characterization of a new porin gene of Klebsiella pneumoniae: its role in beta -lactam antibiotic resistance. J. Bacteriol. 181:2726-2732[Abstract/Free Full Text].
8. Goussard, S., and P. Courvalin. 1999. Updated sequence information for TEM beta -lactamase genes. Antimicrob. Agents Chemother. 43:367-370[Abstract/Free Full Text].
9. Gutmann, L., R. Williamson, N. Moreau, M.-D. Kitzis, E. Collatz, J. F. Acar, and F. W. Goldstein. 1985. Cross-resistance to nalidixic acid, trimethoprim, and chloramphenicol associated with alterations in outer membrane proteins of Klebsiella, Enterobacter and Serratia. J. Infect. Dis. 151:501-507[Medline].
10. Jacoby, G. A., and A. A. Medeiros. 1991. More extended-spectrum beta -lactamases. Antimicrob. Agents Chemother. 35:1697-1704[Free Full Text].
11. Leung, M., K. Shannon, and G. French. 1997. Rarity of transferable beta -lactamase production by Klebsiella species. J. Antimicrob. Chemother. 39:737-745[Abstract/Free Full Text].
12. Martinez-Martinez, L., S. Hernandez-Alles, S. Alberti, J. M. Tomas, V. J. Benedi, and G. A. Jacoby. 1996. In vivo selection of porin-deficient mutants of Klebsiella pneumoniae with increased resistance to cefoxitin and expanded-spectrum cephalosporins. Antimicrob. Agents Chemother. 40:342-348[Abstract/Free Full Text].
13. Mercier, J., and R. C. Levesque. 1990. Cloning of SHV-2, OHIO-1, and OXA-6 beta -lactamases and cloning and sequencing of SHV-1 beta -lactamase. Antimicrob. Agents Chemother. 34:1577-1583[Abstract/Free Full Text].
14. National Committee for Clinical Laboratory Standards. 1993. Methods for dilution antimicrobial susceptibility tests for bacteria that grow aerobically, 3rd ed., p. M7-A3. National Committee for Clinical Laboratory Standards, Wayne, Pa.
15. Petit, A., H. B. Yaghlane-Bouslama, L. Sofer, and R. Labia. 1992. Does high level production of SHV-type penicillinase confer resistance to ceftazidime in Enterobacteriaciae? FEMS Microbiol. Lett. 92:89-94[CrossRef].
16. Podbielski, A., A. Schonling, B. Melzer, K. Warnatz, and H.-J. Leusch. 1991. Molecular characterization of a new plasmid-encoded SHV-type beta -lactamase (SHV-2 variant) conferring high-level cefotaxime resistance upon Klebsiella pneumoniae. J. Gen. Microbiol. 137:569-578[Abstract/Free Full Text].
17. Rice, L. B., L. L. Carias, R. A. Bonomo, and D. M. Shlaes. 1996. Molecular genetics of resistance to both ceftazidime and beta -lactam-beta -lactamase inhibitor combinations in Klebsiella pneumoniae and in vivo response to beta -lactam therapy. J. Infect. Dis. 173:151-158[Medline].
18. Rice, L. B., L. L. Carias, L. Etter, and D. M. Shlaes. 1993. Resistance to cefoperazone-sulbactam in Klebsiella pneumoniae: evidence for enhanced resistance resulting from the coexistence of two different resistance mechanisms. Antimicrob. Agents Chemother. 37:1061-1064[Abstract/Free Full Text].
19. Rice, L. B., S. H. Willey, G. A. Papanicolaou, A. A. Medeiros, G. M. Eliopoulos, R. C. Moellering, Jr., and G. A. Jacoby. 1990. Outbreak of ceftazidime resistance caused by extended-spectrum beta -lactamases at a Massachusetts chronic care facility. Antimicrob. Agents Chemother. 34:2193-2199[Abstract/Free Full Text].
20. Shannon, K., H. Williams, A. King, and I. Phillips. 1990. Hyperproduction of TEM-1 beta-lactamase in clinical isolates of Escherichia coli serotype O15. FEMS Microbiol. Lett. 67:319-324[CrossRef].
21. Sirot, D., C. Recule, E. B. Chaibi, L. Bret, J. Croize, C. Chanal-Claris, R. Labia, and J. Sirot. 1997. A complex mutant of TEM-1 beta-lactamase with mutations encountered in both IRT-4 and extended-spectrum TEM-15, produced by an Escherichia coli clinical isolate. Antimicrob. Agents Chemother. 41:1322-1325[Abstract/Free Full Text].
22. Siu, L. K., P. L. Ho, K. Y. Yuen, S. S. Y. Wong, and P. Y. Chau. 1997. Transferable hyperproduction of TEM-1 beta -lactamase in Shigella flexneri due to a point mutation in the Pribnow box. Antimicrob. Agents Chemother. 41:468-470[Abstract/Free Full Text].
23. Spratt, B. G. 1977. Properties of the penicillin-binding proteins of Escherichia coli K-12. Eur. J. Biochem. 72:341-352[Medline].
24. Takahashi, S., and Y. Nagano. 1984. Rapid procedure for isolation of plasmid DNA. J. Clin. Microbiol. 20:608-613[Abstract/Free Full Text].
25. Vecoli, C., F. E. Prevost, J. J. Ververis, A. A. Medeiros, and G. P. O'Leary, Jr. 1983. Comparison of polyacrylamide and agarose gel thin-layer isoelectric focusing for the characterization of beta -lactamases. Antimicrob. Agents Chemother. 24:186-189[Abstract/Free Full Text].
26. Weber, D. A., C. C. Sanders, J. S. Bakken, and J. P. Quinn. 1990. A novel chromosomal TEM derivative and alterations in outer membrane proteins together mediate selective ceftazidime resistance in Escherichia coli. J. Infect. Dis. 162:460-465[Medline].
27. Yamada, M., H. Izu, T. Nitta, K. Kurihara, and T. Sakurai. 1998. High-temperature, nonradioactive primer extension assay for determination of a transcription initiation site. BioTechniques 25:72-75[Medline].

Antimicrobial Agents and Chemotherapy, February 2000, p. 362-367, Vol. 44, No. 2
0066-4804/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.


This article has been cited by other articles:

  • Turner, M. S., Andersson, P., Bell, J. M., Turnidge, J. D., Harris, T., Giffard, P. M. (2009). Plasmid-borne blaSHV genes in Klebsiella pneumoniae are associated with strong promoters. J Antimicrob Chemother 64: 960-964 [Abstract] [Full Text]  
  • Pattanaik, P., Bethel, C. R., Hujer, A. M., Hujer, K. M., Distler, A. M., Taracila, M., Anderson, V. E., Fritsche, T. R., Jones, R. N., Pagadala, S. R. R., van den Akker, F., Buynak, J. D., Bonomo, R. A. (2009). Strategic Design of an Effective {beta}-Lactamase Inhibitor: LN-1-255, A 6-ALKYLIDENE-2'-SUBSTITUTED PENICILLIN SULFONE. J. Biol. Chem. 284: 945-953 [Abstract] [Full Text]  
  • Dubois, V., Poirel, L., Demarthe, F., Arpin, C., Coulange, L., Minarini, L. A. R., Bezian, M.-C., Nordmann, P., Quentin, C. (2008). Molecular and Biochemical Characterization of SHV-56, a Novel Inhibitor-Resistant {beta}-Lactamase from Klebsiella pneumoniae. Antimicrob. Agents Chemother. 52: 3792-3794 [Abstract] [Full Text]  
  • Yang, K., Guglielmo, B J. (2007). Diagnosis and Treatment of Extended-Spectrum and AmpC {beta}-Lactamase-Producing Organisms. The Annals of Pharmacotherapy 41: 1427-1435 [Abstract] [Full Text]  
  • Thomson, J. M., Prati, F., Bethel, C. R., Bonomo, R. A. (2007). Use of Novel Boronic Acid Transition State Inhibitors To Probe Substrate Affinity in SHV-Type Extended-Spectrum {beta}-Lactamases. Antimicrob. Agents Chemother. 51: 1577-1579 [Abstract] [Full Text]  
  • Tofteland, S., Haldorsen, B., Dahl, K. H., Simonsen, G. S., Steinbakk, M., Walsh, T. R., Sundsfjord, A., the Norwegian ESBL Study Group, (2007). Effects of Phenotype and Genotype on Methods for Detection of Extended-Spectrum-{beta}-Lactamase-Producing Clinical Isolates of Escherichia coli and Klebsiella pneumoniae in Norway. J. Clin. Microbiol. 45: 199-205 [Abstract] [Full Text]  
  • Bethel, C. R., Hujer, A. M., Hujer, K. M., Thomson, J. M., Ruszczycky, M. W., Anderson, V. E., Pusztai-Carey, M., Taracila, M., Helfand, M. S., Bonomo, R. A. (2006). Role of Asp104 in the SHV {beta}-Lactamase. Antimicrob. Agents Chemother. 50: 4124-4131 [Abstract] [Full Text]  
  • Hujer, K. M., Hujer, A. M., Hulten, E. A., Bajaksouzian, S., Adams, J. M., Donskey, C. J., Ecker, D. J., Massire, C., Eshoo, M. W., Sampath, R., Thomson, J. M., Rather, P. N., Craft, D. W., Fishbain, J. T., Ewell, A. J., Jacobs, M. R., Paterson, D. L., Bonomo, R. A. (2006). Analysis of Antibiotic Resistance Genes in Multidrug-Resistant Acinetobacter sp. Isolates from Military and Civilian Patients Treated at the Walter Reed Army Medical Center. Antimicrob. Agents Chemother. 50: 4114-4123 [Abstract] [Full Text]  
  • Thomson, J. M., Distler, A. M., Prati, F., Bonomo, R. A. (2006). Probing Active Site Chemistry in SHV beta-Lactamase Variants at Ambler Position 244: UNDERSTANDING UNIQUE PROPERTIES OF INHIBITOR RESISTANCE. J. Biol. Chem. 281: 26734-26744 [Abstract] [Full Text]  
  • Reynolds, K. A., Thomson, J. M., Corbett, K. D., Bethel, C. R., Berger, J. M., Kirsch, J. F., Bonomo, R. A., Handel, T. M. (2006). Structural and Computational Characterization of the SHV-1 beta-Lactamase-beta-Lactamase Inhibitor Protein Interface. J. Biol. Chem. 281: 26745-26753 [Abstract] [Full Text]  
  • Spanu, T., Sanguinetti, M., Tumbarello, M., D'Inzeo, T., Fiori, B., Posteraro, B., Santangelo, R., Cauda, R., Fadda, G. (2006). Evaluation of the New VITEK 2 Extended-Spectrum Beta-Lactamase (ESBL) Test for Rapid Detection of ESBL Production in Enterobacteriaceae Isolates. J. Clin. Microbiol. 44: 3257-3262 [Abstract] [Full Text]  
  • Paterson, D. L., Bonomo, R. A. (2005). Extended-Spectrum {beta}-Lactamases: a Clinical Update. Clin. Microbiol. Rev. 18: 657-686 [Abstract] [Full Text]  
  • Furushita, M., Okamoto, A., Maeda, T., Ohta, M., Shiba, T. (2005). Isolation of Multidrug-Resistant Stenotrophomonas maltophilia from Cultured Yellowtail (Seriola quinqueradiata) from a Marine Fish Farm. Appl. Environ. Microbiol. 71: 5598-5600 [Abstract] [Full Text]  
  • Siebor, E., Pechinot, A., Duez, J.-M., Neuwirth, C. (2005). One New LEN Enzyme and Two New OKP Enzymes in Klebsiella pneumoniae Clinical Isolates and Proposed Nomenclature for Chromosomal {beta}-Lactamases of This Species. Antimicrob. Agents Chemother. 49: 3097-3098 [Full Text]  
  • Fang, H., Lundberg, C., Olsson-Liljequist, B., Hedin, G., Lindback, E., Rosenberg, A., Struwe, J. (2004). Molecular Epidemiological Analysis of Escherichia coli Isolates Producing Extended-Spectrum {beta}-Lactamases for Identification of Nosocomial Outbreaks in Stockholm, Sweden. J. Clin. Microbiol. 42: 5917-5920 [Abstract] [Full Text]  
  • Dubois, V., Poirel, L., Arpin, C., Coulange, L., Bebear, C., Nordmann, P., Quentin, C. (2004). SHV-49, a Novel Inhibitor-Resistant {beta}-Lactamase in a Clinical Isolate of Klebsiella pneumoniae. Antimicrob. Agents Chemother. 48: 4466-4469 [Abstract] [Full Text]  
  • Walckenaer, E., Poirel, L., Leflon-Guibout, V., Nordmann, P., Nicolas-Chanoine, M.-H. (2004). Genetic and Biochemical Characterization of the Chromosomal Class A {beta}-Lactamases of Raoultella (formerly Klebsiella) planticola and Raoultella ornithinolytica. Antimicrob. Agents Chemother. 48: 305-312 [Abstract] [Full Text]  
  • Helfand, M. S., Bethel, C. R., Hujer, A. M., Hujer, K. M., Anderson, V. E., Bonomo, R. A. (2003). Understanding Resistance to {beta}-Lactams and {beta}-Lactamase Inhibitors in the SHV {beta}-Lactamase: LESSONS FROM THE MUTAGENESIS OF SER-130. J. Biol. Chem. 278: 52724-52729 [Abstract] [Full Text]  
  • Babini, G. S., Yuan, M., Hall, L. M. C., Livermore, D. M. (2003). Variable susceptibility to piperacillin/tazobactam amongst Klebsiella spp. with extended-spectrum {beta}-lactamases. J Antimicrob Chemother 51: 605-612 [Abstract] [Full Text]  
  • Hujer, A. M., Hujer, K. M., Helfand, M. S., Anderson, V. E., Bonomo, R. A. (2002). Amino Acid Substitutions at Ambler Position Gly238 in the SHV-1 {beta}-Lactamase: Exploring Sequence Requirements for Resistance to Penicillins and Cephalosporins. Antimicrob. Agents Chemother. 46: 3971-3977 [Abstract] [Full Text]  
  • Corvec, S., Caroff, N., Espaze, E., Marraillac, J., Reynaud, A. (2002). -11 Mutation in the ampC Promoter Increasing Resistance to {beta}-Lactams in a Clinical Escherichia coli Strain. Antimicrob. Agents Chemother. 46: 3265-3267 [Abstract] [Full Text]  
  • Hujer, A. M., Page, M. G. P., Helfand, M. S., Yeiser, B., Bonomo, R. A. (2002). Development of a Sensitive and Specific Enzyme-Linked Immunosorbent Assay for Detecting and Quantifying CMY-2 and SHV {beta}-Lactamases. J. Clin. Microbiol. 40: 1947-1957 [Abstract] [Full Text]  
  • Howard, C., van Daal, A., Kelly, G., Schooneveldt, J., Nimmo, G., Giffard, P. M. (2002). Identification and Minisequencing-Based Discrimination of SHV {beta}-Lactamases in Nosocomial Infection-Associated Klebsiella pneumoniae in Brisbane, Australia. Antimicrob. Agents Chemother. 46: 659-664 [Abstract] [Full Text]  
  • Bradford, P. A. (2001). Extended-Spectrum {beta}-Lactamases in the 21st Century: Characterization, Epidemiology, and Detection of This Important Resistance Threat. Clin. Microbiol. Rev. 14: 933-951 [Abstract] [Full Text]  
  • Chaves, J., Ladona, M. G., Segura, C., Coira, A., Reig, R., Ampurdanes, C. (2001). SHV-1 {beta}-Lactamase Is Mainly a Chromosomally Encoded Species-Specific Enzyme in Klebsiella pneumoniae. Antimicrob. Agents Chemother. 45: 2856-2861 [Abstract] [Full Text]  
  • Arpin, C., Labia, R., Andre, C., Frigo, C., El Harrif, Z., Quentin, C. (2001). SHV-16, a {beta}-Lactamase with a Pentapeptide Duplication in the Omega Loop. Antimicrob. Agents Chemother. 45: 2480-2485 [Abstract] [Full Text]  
  • Steward, C. D., Rasheed, J. K., Hubert, S. K., Biddle, J. W., Raney, P. M., Anderson, G. J., Williams, P. P., Brittain, K. L., Oliver, A., McGowan, J. E. Jr., Tenover, F. C. (2001). Characterization of Clinical Isolates of Klebsiella pneumoniae from 19 Laboratories Using the National Committee for Clinical Laboratory Standards Extended-Spectrum {beta}-Lactamase Detection Methods. J. Clin. Microbiol. 39: 2864-2872 [Abstract] [Full Text]  
  • Wu, T.-L., Siu, L. K., Su, L.-H., Lauderdale, T. L., Lin, F. M., Leu, H.-S., Lin, T.-Y., Ho, M. (2001). Outer membrane protein change combined with co-existing TEM-1 and SHV-1 {beta}-lactamases lead to false identification of ESBL-producing Klebsiella pneumoniae. J Antimicrob Chemother 47: 755-761 [Abstract] [Full Text]  

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow E-mail this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowReprints and Permissions
Right arrow Copyright Information
Right arrow Books from ASM Press
Right arrow MicrobeWorld
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Rice, L. B.
Right arrow Articles by Bonomo, R. A.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Rice, L. B.
Right arrow Articles by Bonomo, R. A.

Copyright © 2010 by the American Society for Microbiology.   For an alternate route to Journals.ASM.org, visit: http://intl-journals.asm.org | More Info»