Outer membrane protein alterations and blaTEM-1 variants: their role in ß-lactam resistance in Klebsiella pneumoniae

E. C. Nelson1, Heidi Segal1 and B. Gay Elisha1,2,*

1 Department of Medical Microbiology, University of Cape Town, Cape Town; 2 National Health Laboratory Service, Cape Town, South Africa

Received 28 August 2003; returned 15 September 2003; revised 24 September 2003; accepted 27 September 2003


    Abstract
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Objectives: The aim of the study was to characterize the genetic basis of resistance to selected ß-lactam antibiotics in two clinical isolates of Klebsiella pneumoniae.

Methods and results: K. pneumoniae strains were isolated from two hospitalized patients. One of the strains was resistant to amoxicillin, co-amoxiclav, cefuroxime, piperacillin and cefoxitin but susceptible to all the other cephalosporins tested. The second strain displayed a similar phenotype except that it was resistant to piperacillin/tazobactam and susceptible to cefoxitin. PCR assays and DNA sequencing showed that the cefoxitin-susceptible strain contained a novel blaTEM-1 variant downstream of the strong Pa/Pb promoter. SDS–PAGE analysis of the outer membrane proteins (OMPs) did not identify OmpK35 and suggested reduced expression of OmpK36 in this strain. Following passage in non-selective media, expression of OmpK36 was restored with a concomitant increase in cefuroxime susceptibility. A similar experimental approach identified blaTEM-1C in the cefoxitin-resistant K. pneumoniae strain. This strain was deficient in OmpK35 and OmpK36; absence of the latter protein was due to the presence of IS1 in the ompK36 regulatory region.

Conclusions: Resistance to selected ß-lactams in two clinical isolates of K. pneumoniae was due to interplay between the expression of OMPs and TEM-1.

Keywords: cefuroxime, ß-lactamase, IS1, Pa/Pb


    Introduction
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Klebsiella pneumoniae is an important cause of serious nosocomial infections. Most K. pneumoniae strains carry a chromosomal class A SHV-1 ß-lactamase gene; the enzyme has limited activity against ampicillin, but it does not hydrolyse the newer extended-spectrum antibiotics.1 Over the last decade, K. pneumoniae has become increasingly resistant to frequently used ß-lactams, including the ß-lactam–inhibitor combination, co-amoxiclav, and the extended-spectrum ß-lactams. Resistance to co-amoxiclav has been shown to be associated with the production of an inhibitor-resistant TEM (IRT),2 or the overproduction of TEM-1.3 A plethora of extended-spectrum ß-lactamases (ESBLs) has been identified in K. pneumoniae, which account for resistance to the newer oxyiminocephalosporins in this organism.4 These enzymes do not hydrolyse cephamycins, such as cefoxitin, and resistance to these antibiotics is associated with the production of cephamycinases,57 or with outer membrane protein deficiency in K. pneumoniae.8,9

In 1999, K. pneumoniae strains were isolated from two patients in Groote Schuur Hospital (GSH), Cape Town. One of the strains was resistant to amoxicillin, co-amoxiclav, cefuroxime, piperacillin and cefoxitin, but susceptible to all of the other cephalosporins tested, including piperacillin/tazobactam. The second strain displayed a similar phenotype except that it was resistant to piperacillin/tazobactam and susceptible to cefoxitin. These were unusual resistance phenotypes for K. pneumoniae strains from GSH, Cape Town. Molecular characterization of the resistance identified a novel blaTEM-1 gene in one of the strains, and the role of porins in the resistance phenotype was demonstrated in both strains.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Bacterial strains, antibiotic susceptibility testing and detection of ESBLs

K. pneumoniae strains were isolated from two patients in GSH, Cape Town, South Africa. The isolates, designated strain ENG and strain MAJ, were isolated from blood and urine, respectively. K. pneumoniae (strain KAN), isolated from ascitic fluid, was used as a susceptible control for MIC and outer membrane protein (OMP) comparisons. MIC determinations were performed on Mueller–Hinton agar using Etest strips (AB Biodisk, Solna, Sweden) and interpreted using NCCLS guidelines.10 The double disc diffusion test was used to determine ESBL production. Briefly, a disc containing co-amoxiclav was placed between ceftazidime and ceftriaxone discs; ESBL activity was inferred if a dumb-bell-shaped extension of the zone of inhibition was observed around either of these discs towards the clavulanate disc. Escherichia coli ATCC 25922 was used as the control in these assays.

PCR assay for the detection of blaTEM genes

Bacterial genomic DNA was extracted11 and functional TEM genes were amplified using a primer set consisting of 5'-ATGAGTAAACTTGGTCTGAC-3'12 and 5'-AGGAAGCAAAGCTGAAAGGAATCAAATTTGG-3'. The PCR was performed in a thermocycler (Gene Amp 2400; Perkin Elmer). PCR products were separated by agarose gel electrophoresis, excised and extracted from the agarose gel using a Qiagen gel purification kit (Qiagen, Southern Cross Biotechnology) and sequenced directly.

DNA sequencing

DNA sequencing was carried out using automated sequencing in either the core sequencing facility, University of Stellenbosch, South Africa, or the sequencing facility, University of Dundee, Dundee, UK. DNA sequences were analysed with DNAMAN (version 4.0; Lynnon Biosoft, Vandreuil, Quebec, Canada). BLAST13 was used to search databases in GenBank for nucleic acid similarity, and to deduce amino acid sequence similarity with existing sequences.

PCR amplification of ompK36

Genomic DNA was isolated11 and primers U228 and L1370 were used to amplify the ompK36 functional gene.14 PCR products were separated by agarose gel electrophoresis, excised, extracted from the agarose gel using a Qiagen gel purification kit (Qiagen, Southern Cross Biotechnology), and cloned in pGEM-T Easy (Promega). Recombinant plasmids were introduced into E. coli JM109 cells,15 which were made competent for DNA uptake using the CaCl2 shock procedure.16 Recombinant plasmid DNA was extracted using the method described for small-scale plasmid extraction.17

Extraction of outer membrane proteins and SDS–PAGE

Bacterial cells were harvested from overnight cultures grown in 2x yeast tryptone broth (2YT), which has low osmolarity, containing cefuroxime (30 mg/L), resuspended in 30 mM Tris–HCl pH 8.0 and disrupted with a French pressure cell. Following removal of cell debris by centrifugation, cleared lysates were centrifuged at 40 000 rpm for 1 h at ambient temperature to pellet the proteins. Protein pellets were washed with 0.5% SDS for 30 min at 32°C and harvested by centrifugation. The wash was repeated and the final pellet was resuspended in 200 µL of 3% SDS buffer (1% glycerol, 6.25 mM Tris, pH 6.8). The protein content of the sonicate was determined with a commercial assay reagent (Bio-Rad). Prior to SDS–PAGE, the OMPs were denatured by the addition of 3% SDS– 5% ß-mercaptoethanol and boiled for 5 min. OMPs (10 mg) were separated by electrophoresis on 10% acrylamide–0.26% bis-acrylamide gels and stained with Coomassie Blue.

Expression of OMPs following passage in non-selective media

A volume of an overnight culture (100 µL) of strain ENG was inoculated into 10 mL of 2YT broth and incubated at 37°C for 18 h without shaking. From this culture, 100 µL was removed and inoculated into 10 mL of fresh 2YT broth and incubated at 37°C without shaking. This serial transfer was continued for 5 days at which point antibiotic susceptibility testing was carried out and the broth culture (10 mL) was used to inoculate 200 mL of fresh 2YT. Cells were cultured overnight at 37°C, and OMPs were extracted and analysed by SDS–PAGE.

MALDI-TOF mass spectrometry analysis

SDS–PAGE gels containing the OMPs of interest were submitted to the Department of Molecular and Cell Biology, University of Cape Town, for MALDI-TOF analysis. The protein bands of interest were excised from the PAGE gels and the protein in the gel was digested with trypsin.18 A list of peptide masses was obtained for each digest and the peptide mass fingerprint was analysed using software to identify the protein (PROFOUND, available at http://prowl.rockefeller.edu/; MS-FIT, available at http://prospector.ucsf.edu/; EMBL Bioanalytical Research Group, available at http://www.narrador.embl-heidelberg.de/GroupPages/Homepage.html).


    Results
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Antibiotic susceptibility testing and ESBL determinations

MICs of ß-lactam antibiotics for strains ENG and MAJ are presented in Table 1. Both strains were resistant to amoxicillin, co-amoxiclav, cefuroxime and piperacillin. In addition, strain ENG was resistant to piperacillin/tazobactam and strain MAJ was resistant to cefoxitin. Compared with the cefotaxime, ceftazidime and cefepime MICs for strain KAN, susceptibility to these antibiotics was reduced in strains ENG and MAJ (Table 1). Using the double disc technique, ESBL production was not detected in the two strains.


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Table 1. MIC determinations (mg/L) of ß-lactam antibiotics for Klebsiella pneumoniae strains
 
PCR amplification of blaTEM

PCR products of the expected size (1.2 kb), obtained from strains MAJ and ENG, were purified from the agarose gel and sequenced directly. An alignment of the nucleotide sequences with corresponding blaTEM gene sequences showed that the blaTEM structural gene from strain MAJ is identical to blaTEM-1C and downstream of the weak P3 promoter (Table 2). The blaTEM gene from strain ENG contains a C->T substitution at position 32, generating Pa/Pb. The coding sequence of the bla gene is similar to blaTEM-1E except for the silent mutation T682C, previously described in blaTEM-1F and blaTEM-2 (Table 2).


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Table 2. Nucleotide mutations in blaTEM gene frameworks
 
Expression of OMPs

OMPs were isolated from strains MAJ and ENG cultured in the presence of cefuroxime (30 mg/L). A comparison of the OMP profile from strain ENG with the corresponding profile from the susceptible strain KAN identified a band of ~40 kDa, migrating above the constitutively expressed OmpK34 (OmpA),9 in each of the strains (Figure 1). Compared with the 40 kDa protein band in strain KAN, the level of expression of its counterpart in strain ENG was reduced. The 40 kDa protein band is not present in strain MAJ (Figure 1). The 40 kDa bands from strain ENG and strain KAN were excised from the gel and identified as OmpK36, following analyses of their mass peptide fingerprints generated by MALDI-TOF mass spectrometry.



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Figure 1. OMPs of K. pneumoniae strains. Lane 1, molecular weight protein marker (Pharmacia Biotech); lanes 2 and 3, strains MAJ and ENG cultured in the presence of cefuroxime; lane 4, susceptible strain KAN; lane 5, strain ENG following sequential passage in antibiotic-free broth.

 
To test whether the reduced expression of OmpK36 was influenced by the presence of cefuroxime, strain ENG was passaged through five successive overnight cultures in antibiotic-free nutrient broth. Subsequently, OMPs were extracted and antibiotic susceptibility testing was carried out. As shown in Figure 1, the level of expression of OmpK36 in the passaged strain (strain ENGP) was similar to that in strain KAN, indicating restoration of OmpK36. MICs of cefuroxime (6 mg/L), co-amoxiclav (8 mg/L) and piperacillin/tazobactam (32 mg/L) for strain ENGP were reduced, compared with the corresponding MICs for the parent strain (Table 1).

Molecular basis of porin loss in strain MAJ

Studies to identify the mechanism responsible for the loss of OmpK36 in strain MAJ were carried out. Using primers designed to anneal to functional ompK36 sequences,14 products of 2.2 and 1.5 kb were obtained from strain MAJ and the control strain, respectively. The size of the fragment from strain MAJ suggested that ompK36 from this strain contained an insertion. The 2.2 kb PCR product was cloned in pGEM-T Easy (Promega), generating pJAM100 and sequenced. Analysis of the sequencing data identified IS1 10 bp upstream of the ompK36 ATG start codon. The element generated a 9 bp duplication of the target sequence; it is bounded by imperfect terminal inverted repeats of 23 bp and contains two open reading frames (insA and insB'), typical of IS1 elements.23


    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Extensive nucleotide sequencing of blaTEM-related genes has identified a wide variety of structural genes. Transcription of these genes proceeds from one or more of four promoters: the weak P3 promoter and the strong promoters, Pa/Pb, P4 and P5.24 Based on the recent TEM gene nomenclature, which takes into account the nucleotide sequence of the structural gene and the promoter, also called sequence framework, seven blaTEM-1 genes have been described.2022 Most blaTEM genes are expressed from P3;22 transcription from P4, Pa/Pb or P5 results in an increase in production of TEM-1 and concomitant resistance to co-amoxiclav in E. coli.24 Overproduction of TEM-1 is frequently the mechanism of resistance to co-amoxiclav in this organism.2527 Much less is known about the role of overproduction of TEM-1 in resistance to co-amoxiclav in K. pneumoniae.3 The promoter region of the blaTEM structural gene from strain ENG contains a C32T substitution, generating the strong Pa/Pb promoter (Table 2), previously described for blaTEM-2 and blaTEM-1B.21 The coding sequence of the bla gene is identical to blaTEM-1E except for the silent mutation T682C previously identified in blaTEM-1F and blaTEM-2;21 it is therefore a new blaTEM gene framework, which we have designated blaTEM-1H (GenBank accession number AY394610).

The role of porins in ß-lactam resistance in K. pneumoniae has been established. Strains deficient in the outer membrane 40 kDa doublet, OmpK35 and OmpK36, have increased resistance to a number of ß-lactams, including cefoxitin.8,9 The MICs of most ß-lactams for strains deficient in either OmpK35 or OmpK36 are the same as those for strains expressing both porins, indicating that increased resistance is dependent on the absence or reduced expression of both porins.9 With respect to strain ENG, expression of OmpK36 was reduced compared with its counterpart in the susceptible strain KAN, and a band corresponding to OmpK35 was not observed in either of the strains (Figure 1). The presence of a single protein is not uncommon; in one study, the majority of K. pneumoniae strains investigated were shown to express only one porin.28 Following sequential culture of strain ENG in non-selective media, OmpK36 proficiency was restored and the passaged strain (strain ENGP) was susceptible to cefuroxime (Table 1). In addition, strain ENGP had lost resistance to co-amoxiclav and piperacillin/tazobactam and, compared with the parent strain, it had increased susceptibility to cefotaxime and ceftazidime (Table 1). PCR assays confirmed the presence of blaTEM in strain ENGP (data not shown), suggesting that overproduction of TEM-1 is not solely responsible for co-amoxiclav and piperacillin/tazobactam resistance in strain ENG. Rather, resistance to these combinations is the result of interplay between uptake and TEM-1 activity.

K. pneumoniae strain MAJ was resistant to co-amoxiclav, cefuroxime and cefoxitin (Table 1). As strain MAJ was susceptible to ceftazidime and cefotaxime, the role of cephamycinases, which have activity against these antibiotics as well as cefoxitin,57 was considered unlikely. OmpK35 and OmpK36 were not identified in preparations from strain MAJ (Figure 1), supporting the findings of others that cefoxitin resistance is pursuant on the loss of these porins.8,9 It follows that, as with strain ENG, resistance to cefuroxime in strain MAJ is due to the alterations in the outer membrane. Whether the absence of OmpK35 and OmpK36 is solely responsible for the co-amoxiclav-resistant phenotype of strain MAJ is not known. In some studies, porin-deficient K. pneumoniae strains could be either resistant or susceptible to this combination, supporting the notion that the relationship between outer membrane disturbances and resistance are ‘neither simple nor universal’.29 blaTEM-1C, albeit expressed from a weak promoter (P3), was identified in strain MAJ (Table 2); thus, the combined activity of TEM-1 and alterations in the OMPs may account for resistance to co-amoxiclav in this strain.

The absence of OmpK36 has been shown to be due to the insertion of IS102 and an IS5-like element into structural ompK36 genes in clinical isolates of K. pneumoniae.14 In strain MAJ, IS1 was found to be inserted 10 bp upstream of the ompK36 ATG start codon. The patient from whom strain MAJ was isolated was given a single, prophylactic dose of cefazolin, which may have influenced the selection of porin-deficient strains.


    Acknowledgements
 
We thank Iva Shankland for carrying out the MIC determinations. This work was supported by a grant from the University of Cape Town to B.G.E. E.C.N. is a recipient of a Sainsbury Scholarship.


    Footnotes
 
* Correspondence address. Department of Medical Microbiology, Medical School, University of Cape Town, Anzio Road, Observatory 7925, Cape Town, South Africa. Tel: +27-21-4066378; Fax: +27-21-4488153; E-mail: gelisha{at}curie.uct.ac.za Back


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 Introduction
 Materials and methods
 Results
 Discussion
 References
 
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