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JAC Advance Access published online on June 26, 2008
Journal of Antimicrobial Chemotherapy, doi:10.1093/jac/dkn257
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© The Author 2008. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved. For Permissions, please e-mail: journals.permissions@oxfordjournals.org

Original research

The AmpC phenotype in Norwegian clinical isolates of Escherichia coli is associated with an acquired ISEcp1-like ampC element or hyperproduction of the endogenous AmpC

Bjørg Haldorsen1, Bettina Aasnaes1, Kristin H. Dahl1,2, Anne-Merethe Hanssen2, Gunnar S. Simonsen1,–,3, Timothy R. Walsh4, Arnfinn Sundsfjord1,2,* and Eirik W. Lundblad1

1 Reference Centre for Detection of Antimicrobial Resistance, Department of Microbiology and Infection Control, University Hospital of North-Norway, Tromsø, Norway 2 Department of Microbiology and Virology, University of Tromsø, Tromsø, Norway 3 Division of Infection Control, Norwegian Institute of Public Health, Oslo, Norway 4 University of Cardiff, Cardiff, UK

* Correspondence address. Department of Microbiology and Virology, Institute of Medical Biology, Faculty of Medicine, University of Tromsø, 9038 Tromsø, Norway. Tel: +47-90616118; Fax: +47-77645350; E-mail: arnfinn.sundsfjord{at}fagmed.uit.no

Received 31 March 2008; returned 22 April 2008; revised 25 May 2008; accepted 29 May 2008


   Abstract
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Objectives: The aim of the study was to examine resistance mechanisms associated with an AmpC phenotype in Norwegian clinical isolates of Escherichia coli.

Methods: Clinical E. coli isolates (n = 106) with reduced susceptibility to third-generation cephalosporins without clavulanic acid synergy were collected from 12 Norwegian laboratories from 2003 to 2005. Twenty-two isolates with an AmpC phenotype were selected for further characterization by PFGE, isoelectric focusing, different PCR-based techniques, DNA sequencing, AmpC qRT–PCR, transfer studies and plasmid analyses.

Results: The 22 isolates were not clonally related by the PFGE analysis. All isolates expressed a β-lactamase with a pI of 9.0–9.2. Ten isolates contained a blaCMY gene, which was linked to an ISEcp1-like element in all cases. Twelve isolates had mutations or insertions in the promoter or the attenuator regions, leading to increased expression of the chromosomal ampC gene. One of these isolates had an ISEc10 element inserted upstream of the chromosomal ampC gene.

Conclusions: This is the first molecular study of Norwegian clinical E. coli isolates with an AmpC phenotype. Resistance was mediated either by expression of blaCMY from acquired ISEcp1-like-blaCMY elements, or by mutations or insertions in the chromosomal ampC gene control region leading to hyperproduction of the endogenous AmpC enzyme. There was no correlation between the level of ampC mRNA and the MICs of cephalosporins.

Key Words: β-lactamases , cephalosporin resistance , insertion sequences , promoter mutations


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AmpC β-lactamases represent a group of clinically important β-lactamases that are naturally produced by a variety of Gram-negative bacteria. When overexpressed, they confer clinical resistance to penicillins and cephalosporins, except cefpirome and cefepime. In combination with porin loss, they may also mediate resistance to carbapenems.1,2 AmpC enzymes are, with a few exceptions, not inhibited by β-lactamase inhibitors.3 The chromosomal ampC gene of Escherichia coli has a weak promoter and a transcriptional attenuator.4 E. coli strains containing the wild-type gene produce a low basal level of the enzyme and are susceptible to cephalosporins, but mutations in the promoter or in the attenuator regions can lead to hyperproduction of the enzyme and cephalosporin resistance. The most common mutations change the promoter to resemble the E. coli consensus promoter or weaken the attenuator.5–13 Plasmid-mediated ampC genes were first detected in 1988.14 They are thought to have originated from their chromosomal location in organisms such as Enterobacter spp., Citrobacter freundii, Morganella morganii, Hafnia alvei and Aeromonas spp.15 These genes are of special interest, as plasmids are able to spread efficiently between species. They have been classified into six genetic clusters, which can be identified by a family-specific multiplex PCR.16 The blaCMY-2 gene is the most prevalent and has been reported worldwide. Interestingly, blaCMY-2 is found in many different plasmid backbones, suggesting that it can be mobilized as a part of a smaller transferable fragment.17 Recent data have shown that plasmid-mediated blaCMY-2 genes in E. coli and Salmonella enterica are linked to ISEcp1-like elements, which probably mobilize blaCMY-2 as part of a composite transposon.17

The purpose of this study was to examine Norwegian clinical isolates of E. coli with an AmpC phenotype for genetic alterations leading to increased expression of the chromosomal ampC gene, as well as acquired plasmid-mediated ampC genes and their genetic support.


   Materials and methods
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Bacterial strains and antimicrobial susceptibility testing

From March 2003 to December 2005, the Norwegian Reference Centre for Detection of Antimicrobial Resistance (K-res) received 106 clinical isolates of E. coli, with reduced susceptibility to third-generation cephalosporins without clavulanic acid synergy. Initial antimicrobial susceptibility testing and interpretations were performed in each laboratory by their routine methods in agreement with the Norwegian Working Group on Antibiotics guidelines and the recommendations of the European Committee for Antimicrobial Susceptibility Testing.18 The isolates were examined at K-res using the following panel of β-lactam Etests (AB Biodisk, Solna, Sweden), according to the manufacturer's instructions: ampicillin (AMP), amoxicillin/clavulanic acid (AMC), piperacillin (PIP), piperacillin/tazobactam (TZP), cefoxitin (FOX), cefpodoxime (CPD), cefotaxime (CTX), ceftazidime (CAZ), cefepime (FEP), aztreonam (ATM), imipenem (IMP) and meropenem (MEM). Final bacterial identification was performed at K-res using the Vitek2 ID-GNB system (bioMérieux, Marcy l’Étoile, France) or API ID32E (bioMérieux) and/or 16S rDNA-sequence typing in cases of low discrimination, as described previously.19 Vitek2 ASTN023 (bioMérieux) was used to determine susceptibility to non-β-lactam antibiotics. Tests for synergy between cephalosporins and clavulanic acid were performed by ESBL Etests (AB Biodisk), including cefotaxime/clavulanic acid, ceftazidime/clavulanic acid and cefepime/clavulanic acid. The Etest selection criteria for further molecular analysis were: (i) reduced susceptibility to third-generation cephalosporins (cefpodoxime, cefotaxime and ceftazidime MICs ≥4 mg/L) without clavulanic acid synergy; (ii) cefoxitin MIC > 16 mg/L; (iii) susceptibility or intermediate susceptibility to cefepime; and (iv) susceptibility to carbapenems. A total of 22 out of 106 isolates isolated at 12 laboratories fulfilled the selection criteria (urine n = 14, blood n = 4 and others n = 4) and were selected for molecular analysis. Control strains are presented in Table S1 [available as Supplementary data at JAC Online (http://jac.oxfordjournals.org/)]. The rifampicin-resistant E. coli A53 (J53-2) was used as a recipient strain in filter-mating experiments. Antimicrobial susceptibility testing of control strains was performed by Vitek II AST-029 (bioMérieux).

Isoelectric focusing (IEF) of β-lactamases

Analytic IEF of crude cell extracts was performed in precast Ampholine PAGplate polyacrylamide gels with a pH range of 3.5–9.5 (GE Healthcare, Oslo, Norway), using a Multiphor II Apparatus (GE Healthcare). β-Lactamase activity was detected by staining the gels with nitrocefin solution (0.5 g/L). The isoelectric points (pIs) of the studied β-lactamases were determined by comparison with reference β-lactamases blaTEM-1 (pI 5.4) and blaSHV-1 (pI 7.6), as well as with naturally coloured IEF Protein Standards pI 4.45–9.6 (Bio-Rad Laboratories, Hemel Hempstead, UK).

PCR amplification and DNA sequencing

Bacterial DNA extraction and consensus PCRs for the most prevalent Ambler class A β-lactamase genes (blaTEM, blaSHV and blaCTX-M) were performed, as described previously.19 A multiplex PCR covering the six families of ampC genes (ACC, CIT, DHA, EBC, FOX and MOX)16 and a specific blaCMY PCR were performed. blaCMY PCR products were sequenced. Chromosomal ampC promoter mutations and insertions were examined by PCR and sequencing. Genetic linkage between the insertion sequence ISEcp1 and blaCMY was examined by an ISEcp1-blaCMY linkage PCR. PCR primers are listed in Table 1. PCR amplification and bidirectional DNA sequencing were performed as described previously.19


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  		Table 1. Primers and probes used in this study

 
Molecular typing by PFGE

Possible genomic relatedness of strains was analysed by PFGE following a standard protocol.20 Briefly, agarose plugs with extracted DNAs were digested with XbaI (New England BioLabs, Beverly, USA). Agarose plugs were stored in 50 mM thiourea (Sigma-Aldrich Co., St Louis, MO, USA) before XbaI digestion to inhibit DNase activity. PFGE gels were run by multidirectional gel electrophoresis using the Chef-DR®III System (Bio-Rad Laboratories). Electrophoresis was run at 12°C with a pulse time of 1–20 s at 6 V/cm on a 120° angle in 0.5x TBE buffer for 21 h. DNA relatedness was interpreted by the criteria of Tenover et al.21

RNA isolation and cDNA synthesis

Bacterial isolates were cultured overnight in Luria–Bertani (LB) broth at 37°C and diluted 1:300 in fresh LB broth and grown to a mid-log growth phase. Total RNA was isolated using RNeasy kit (Qiagen, Oslo, Norway) and RNAprotect Bacteria Reagent (Qiagen), according to the manufacturer's instructions. Genomic DNA was removed by RNase-free DNase (Qiagen). Purified RNA was stored in RNase-free water at –80°C. RNA concentrations were determined using a NanoDrop ND-1000 spectrophotometer (Thermo Scientific, Wilmington, DE, USA). RNA integrity was checked in a FlashGel System (CAMBREX, ME, USA). cDNA syntheses were performed in triplicate using 400 ng of RNA per 20 µL reaction according to the manufacturer's instructions (High capacity cDNA Reverse Transcription Kit, Applied Biosystems, Oslo, Norway).

Real-time qRT–PCR

Real-time qRT–PCR experiments were performed using qPCR Mastermix Plus (Eurogenetec). Synthesized cDNAs were diluted 1:10 and amplified in triplicate. Amplifications were also performed on cDNA synthesis run without reverse transcriptase to confirm the absence of DNA in the RNA samples. The ampC target gene was normalized against the reference gene glyceraldehyde 3-phosphate dehydrogenase (gapA)13,22 using Q-Gene.23,24 The ampC mRNA mean normalized expression level was calibrated as fold differences using the mean normalized expression level of ATCC 25922 as 1.0. Standard errors were calculated for the fold differences according to the standard errors for the ampC mRNA mean normalized expression levels.

Plasmid analyses and hybridization

Filter-mating experiments were performed on LB agar plates. The donor/recipient ratio was 1:1. Filter-mating plates were incubated at 37°C for 16 h. E. coli transconjugants were selected on LB agar containing cefoxitin 32 mg/L and rifampicin 100 mg/L. An S1 nuclease plasmid assay was used for the detection and size determination of circular DNA.25 Briefly, the procedure includes lysis of bacterial cells embedded in agarose plugs,20 digestion with 50 U of S1 nuclease (Promega, Madison, WI, USA) at 37°C for 25 min, and finally PFGE at 14°C with a pulse time of 1–25 s at 6 V/cm on a 120° angle in 0.5x TBE buffer for 20 h. Southern transfer of S1-nuclease-digested DNAs from PFGE gels to positively charged nylon membranes (Roche Applied Science, Penzberg, Germany) was performed by vacuum blotting (Vacugene XL system, GE Healthcare). DNA was fixed to the nylon membrane by UV cross-linking. A blaCMY-2 probe was constructed by labelling a blaCMY-2 PCR product using the PCR DIG Probe Synthesis Kit (Roche Applied Science). The labelled PCR product was purified by agarose gel electrophoresis followed by extraction using the QIAquick Gel Extraction Kit (Qiagen). Hybridization was carried out at 68°C, and detection was performed using the DIG Luminescent Detection Kit (Roche Applied Science). All protocols were performed according to the manufacturer's instructions.

Computer analyses of DNA sequences

Analyses and alignments of DNA sequences were performed using the DNAStar software package (DNAStar Inc., Madison, WI, USA). Nucleotide sequences were compared with sequences in the NCBI GenBank database (http://www.ncbi.nlm.nih.gov/Genbank/index.html) using the Basic Local Alignment Search Tool (BLAST) BLASTn.26


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Norwegian clinical E. coli strains with an AmpC phenotype are genetically unrelated

No clonal relatedness between strains was detected by XbaI-PFGE analysis (data not shown). One strain (K26-21) was non-typeable by PFGE due to high DNase activity. The results indicate that the appearance of clinical E. coli isolates with an AmpC phenotype in Norway is not due to dissemination of specific clones.

Two different AmpC resistance mechanisms in clinical E. coli isolates in Norway

IEF analyses revealed that all E. coli isolates with an AmpC phenotype expressed β-lactamases with a pI of approximately 9.0–9.2 (Table 2), corresponding to the alkaline profile of AmpC β-lactamases (discussed subsequently). In addition, other β-lactamase bands with lower pIs were also detected in some isolates. These included bands with pIs around 5.5, corresponding to blaTEM-1 β-lactamases (Table 2). Importantly, control strains A2-6 and A2-7, expressing blaTEM-1 or blaSHV-1, respectively, had bands at pI 5.4 or 7.6 exclusively, as expected.


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  		Table 2. Summary of results for E. coli isolates: fold AmpC expression, Etest MICs, PCR, sequence typing and IEF

 
The presence of acquired AmpC β-lactamase genes was examined by multiplex PCR for ampC-type genes and showed blaCIT in 10 of 22 isolates (Table 2). A blaCMY-specific PCR and DNA sequence analysis revealed blaCMY-7 (n = 1) and blaCMY-2 (n = 9) (Table 2). blaTEM, blaSHV and blaCTX-M consensus PCRs identified nine blaTEM-positive strains (Table 2). Seven isolates had both blaTEM and blaCMY genes. All strains were negative for blaSHV and blaCTX-M.

All 22 isolates had a β-lactamase band with a pI of 9.0–9.2 on nitrocefin-stained IEF gels, but 12 of these were negative by ampC multiplex PCR (Table 2), suggesting increased expression of the chromosomal ampC gene. PCR and sequencing of the ampC control regions (promoters and attenuators) were performed, and the sequences were compared with the ampC control region of E. coli K12 strain (GenBank accession number NC_000913 [GenBank] ) by nucleotide alignment (Figure 1b). All ampC multiplex PCR-negative strains with an AmpC phenotype had mutations or insertions in their control regions, which could explain hyperproduction of the chromosomal AmpC enzyme (Figure 1b). Three different mechanisms for increased expression of chromosomal ampC genes were identified. (i) A major group of nine strains had mutations at positions –88 (C->T), –82 (A->G), –42 (C->T), –18 (G->A), –1 (C->T) and +58 (C->T). The –42 (C->T) mutation seems critical for resistance in this context as similar promoters without this mutation are susceptible to cephalosporins (Figure 1b). (ii) Two strains (K8-2 and K25-65) did not have the same mutations as the major group. These showed different single nucleotide insertions in the spacer regions between the –35 and –10 boxes, shifting the length of the spacer region to the optimal 17 bp (Figure 1b). In addition, both these strains showed mutations in the attenuator region. (iii) One isolate (K15-8) had a large insertion in the chromosomal ampC promoter region between the –35 and –10 boxes in position –17/–18 (Figure 1a and b). Sequence analyses and nucleotide BLAST searches demonstrated that the insertion element was identical to the putative IS element ISEc10 from E. coli CFT073 (GenBank accession number AE014075 [GenBank] ). The nucleotide sequence alignment of target sequences for ISEc10 insertions showed that the ISEc10 element generates a 5 or 6 bp direct repeat of the target gene region (Figure 2). A direct repeat is also found for the ampC promoter target sequence (Figure 2), suggesting that ISEc10 behaves as an IS element during transfer. The target sequence for ISEc10 does not seem to be conserved (Figure 2). The most conserved position is immediately 5' of the insertion element, cytosine (6/8)/pyrimidine (7/8) (Figure 2).


Figure 1
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  		Figure 1. The ampC control region. (a) Schematic representation of the ampC control region. The insertion element ISEc10 is located between the –35 and –10 boxes (black boxes) in strain K15-8. The stem of the attenuator region (grey with inverted arrows) is located downstream of the transcription initiation site (+1). Primers ampC F and ampC R used for the amplification of the ampC control regions are shown. The elements in the figure are not to scale. (b) Nucleotide sequence alignment of ampC control regions from E. coli K12, ampC multiplex PCR-negative strains (I), ampC multiplex PCR-positive strains (II) and control strains (III). The –35 and –10 promoter sequences are underlined. The new –35 and –10 promoter sequences generated by the –42 and –18 mutations, respectively, are shown as dashed lines. The insertion site for ISEc10 (in strain K15-8) is shown by a filled black triangle. Nucleotides are numbered according to the transcription initiation site (+1). Horizontal arrows mark the stem of the ampC attenuator. The last nucleotide in the alignment (adenine) is the first nucleotide in the ATG start codon.

 


Figure 2
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  		Figure 2. Nucleotide sequence alignment of target sites for ISEc10 insertion in E. coli strains. GenBank accession numbers and positions of the ISEc10 elements in the GenBank sequences are shown. Duplicated target sequences are underlined. Thirteen of the 48 nucleotides of the ISEc10 imperfect inverted repeats are depicted and boxed. The first six nucleotides in ISEc10 IRR (TTGACG) are highlighted in grey. These nucleotides are believed to function as a new –35 box for the ampC gene.

 
Importantly, although our control E. coli strains (n = 18) also had several mutations in their ampC control regions compared with E. coli K12, none of these had the –42 mutation, a 1 nt spacer region insertion, critical mutations in their attenuator regions or IS elements inserted in their promoter regions (Figure 1b).

Increased expression of chromosomal ampC genes by mutations or insertions in the gene control regions

Isolates with alterations in the ampC gene control region were analysed for the expression of the chromosomal ampC gene by real-time qRT–PCR. All strains showed highly increased expression of the ampC gene compared with the control strain E. coli ATCC 25922 ranging from 27- to 380-fold (Table 2). Importantly, clinical control strains showed about similar expression of the ampC gene as E. coli ATCC 25922.

Acquired blaCMY genes

The 10 blaCMY-positive E. coli isolates were recovered in seven different laboratories and were clonally unrelated, thus indicating the spread of a common blaCMY-containing plasmid. Filter-mating experiments between the 10 blaCMY-positive E. coli isolates and an E. coli recipient strain resulted in exconjugants from all donors, suggesting that blaCMY genes were located on plasmids (data not shown). The plasmid contents of all the 22 E. coli isolates were analysed by PFGE of S1-nuclease-digested total DNAs. Plasmid profiling and subsequent hybridization with a DIG-labelled blaCMY-2 probe showed that the blaCMY genes were located on plasmids in the majority (K2-67, K5-20, K13-18, K20-72, K26-7, K26-8 and K26-35) of the strains; see Figure S1(a and b) [available as Supplementary data at JAC Online (http://jac.oxfordjournals.org/)]. The size of the plasmids ranged from ~87 to 282 kb. K5-41 and K5-63 DNAs supported blaCMY hybridization to both plasmid-located and chromosomally located bands and a chromosomally located band only, respectively [lanes 4 and 5, Figure S1(b)]. This may suggest that blaCMY could be located on a mobile genetic element on the chromosome. The result for strain K26-21 could not be interpreted due to high DNase activity. Importantly, none of the ampC multiplex PCR-negative strains had bands that hybridized to the blaCMY-2 probe [Figure S1(b) and data not shown].

blaCMY genes are linked to ISEcp1-like elements

It has previously been reported that β-lactamase genes of the blaCTX-M and blaCMY types can be genetically linked to ISEcp1.27–37 ISEcp1 has experimentally been shown to mediate transfer of blaCTX-M genes by using surrogate IRR sequences.33,37 A PCR with a forward primer in the transposase gene of the ISEcp1 element and a reverse primer in the blaCMY genes was performed. All strains with blaCMY genes produced an amplicon of the expected size (data not shown), suggesting that all blaCMY genes were linked to an ISEcp1-like element. PCR analysis using primers specific for C. freundii-related genes blc, sugE and dsbC gave PCR products of the anticipated size in accordance with those reported by Kang et al.,17 categorizing them as either type 1 or type 2. Sequencing a subset of these amplicons revealed 100% identity to previous reports.17,38


   Discussion
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This is the first molecular study of Norwegian clinical E. coli isolates with an AmpC phenotype. Two different mechanisms explaining AmpC hyperproduction were detected in the 22 strains. Hyperproduction of the chromosomal AmpC enzyme due to mutations (n = 11) or an ISEc10 insertion (n = 1) in the control region of the chromosomal ampC gene was found in 12 strains. An ISEcp1-like-blaCMY element was detected in 10 strains. PFGE analyses and examination of blaCMY-containing plasmids revealed genetically unrelated strains, suggesting that the emergence of Norwegian AmpC E. coli strains is not due to clonal dissemination or spread of a common plasmid between strains. It should be noted that the ACC-1 β-lactamase would escape our phenotypic screening due to susceptibility to cefoxitin.39

Nine of the strains with increased expression of the chromosomal ampC had identical mutations at positions –88, –82, –42, –18, –1 and +58. Similar mutations have previously been described in clinical E. coli strains.6,9–11 Two of the ampC multiplex PCR-negative strains showed mutations in the attenuator regions and single nucleotide insertions in the spacer regions between the –35 and the –10 boxes, shifting the spacer region to the optimal 17 bp. Previous observations of the ampC gene control region suggest that insertion of a single base pair between the –35 and the –10 boxes or mutations of the attenuator region are by themselves sufficient for increased transcription of the ampC gene.4,9,40

An insertion of the putative IS element ISEc10 in the chromosomal ampC promoter region was found in one isolate. ISEc10 belongs to the IS21 family of IS elements and harbours two open reading frames, encoding a putative transposase and a putative ATP-binding protein (http://www-is.biotoul.fr/, 7 May 2008, date last accessed). The ISEc10 element probably enhances transcription of the chromosomal ampC gene by inserting a new strong –35 box (TTGACG), located in the ISEc10 IRR, 17 bp upstream of the endogenous –10 box in the ampC promoter. This may account for the 54-fold increased expression of AmpC and resistant phenotype observed in this strain (Table 2). It has been shown that insertion of IS elements can increase the expression of downstream genes,41 and upstream insertion of IS911, IS10 and IS2 has been suggested to mediate increased transcription of the ampC gene in previous studies.9,42

All strains with mutations or insertions in the chromosomal ampC gene control region had at least 27-fold increased expression of the ampC gene compared with E. coli ATCC 25922. These results correlate well with previous results from Canadian E. coli clinical isolates.13 Our results show that although six of our isolates (K2-68, K4-30, K4-37, K9-66, K22-31 and K25-19) have similar mutations in the sequenced ampC gene control region, their increase of expression varies from 27- to 203-fold. This may indicate that strain-specific factors other than the nucleotide composition of the gene control region contribute to the level of AmpC production in the cell. We do not see a direct relationship between the level of increased ampC mRNA expression in the cephalosporin-resistant isolates and MICs of cephalosporins (Table 2), supporting the finding of Tracz et al.13 that other strain-specific factors seem to play a role in determining the MIC of cefoxitin.

The finding of blaCMY-2 as the most commonly acquired ampC gene is consistent with observations in other countries.9,43–46 S1-nuclease analysis and blaCMY-2 hybridization revealed plasmid heterogeneity, suggesting that the distribution of blaCMY genes in Norwegian clinical isolates of E. coli is not due to the transfer of the same R-plasmid between different strains. Both blaCMY-2 and blaCMY-7 genes were genetically linked to ISEcp1-like elements. This is in accordance with observations showing ISEcp1-like elements linked to blaCMY as well as blaCTX-M.27–37 Experimental evidence for ISEcp1-mediated mobilization of blaCTX-M genes has recently been provided.33,37 The DNA sequence of the ISEcp1-blaCMY region included the genes blc and sugE immediately downstream, indicating that the genetic context of blaCMY-2 is remarkably conserved in strains from the USA, Taiwan and now Norway.17,38 We have no information about the possible contribution from other resistance mechanisms such as porin defects or mutations within the chromosomal ampC genes,47,48 but our observations may suggest that increased expression of the endogenous chromosomal ampC gene is as efficient as plasmid-acquired ampC when it comes to clinically relevant resistance levels to extended-spectrum cephalosporins in E. coli. The diagnostic implication of this finding is that no phenotypic test will discriminate clearly between the two mechanisms mediating the AmpC phenotype.48,49 We will thus have to rely on genetic methods for their identification.50

In conclusion, the present study demonstrates that AmpC resistance in Norwegian E. coli is due to expression of blaCMY from an acquired ISEcp1-like-blaCMY element or alterations in the ampC control region causing increased expression of the endogenous ampC gene.


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This work was supported by a research grant from the Northern Norway Regional Health Authority Medical Research Programme.


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None to declare.


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Table S1 and Figure S1(a and b) are available as Supplementary data at JAC Online (http://jac.oxfordjournals.org/).


   Acknowledgements
 
We thank Lorentz Mandal and the rest of the staff at the medium production unit for technical assistance and Rolf Rødven for help with qRT–PCR statistical analyses. T. R. W. acknowledges the help of Dr M. A. Toleman and D. M. C. Bennett with sequence analysis.


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1 . Bradford PA, Urban C, Mariano N, et al. Imipenem resistance in Klebsiella pneumoniae is associated with the combination of ACT-1, a plasmid-mediated AmpC β-lactamase, and the loss of an outer membrane protein. Antimicrob Agents Chemother (1997) 41:563–9.[Abstract]

2 . Cao VT, Arlet G, Ericsson BM, et al. Emergence of imipenem resistance in Klebsiella pneumoniae owing to combination of plasmid-mediated CMY-4 and permeability alteration. J Antimicrob Chemother (2000) 46:895–900.[Abstract/Free Full Text]

3 . Philippon A, Arlet G, Jacoby GA. Plasmid-determined AmpC-type β-lactamases. Antimicrob Agents Chemother (2002) 46:1–11.[Free Full Text]

4 . Jaurin B, Grundstrom T, Edlund T, et al. The E. coli β-lactamase attenuator mediates growth rate-dependent regulation. Nature (1981) 290:221–5.[CrossRef][Medline]

5 . Caroff N, Espaze E, Berard I, et al. Mutations in the ampC promoter of Escherichia coli isolates resistant to oxyiminocephalosporins without extended spectrum β-lactamase production. FEMS Microbiol Lett (1999) 173:459–65.[Web of Science][Medline]

6 . Caroff N, Espaze E, Gautreau D, et al. Analysis of the effects of –42 and –32 ampC promoter mutations in clinical isolates of Escherichia coli hyperproducing ampC. J Antimicrob Chemother (2000) 45:783–8.[Abstract/Free Full Text]

7 . Corvec S, Caroff N, Espaze E, et al. –11 Mutation in the ampC promoter increasing resistance to β-lactams in a clinical Escherichia coli strain. Antimicrob Agents Chemother (2002) 46:3265–7.[Abstract/Free Full Text]

8 . Jaurin B, Grundstrom T, Normark S. Sequence elements determining ampC promoter strength in E. coli. EMBO J (1982) 1:875–81.[Web of Science][Medline]

9 . Mulvey MR, Bryce E, Boyd DA, et al. Molecular characterization of cefoxitin-resistant Escherichia coli from Canadian hospitals. Antimicrob Agents Chemother (2005) 49:358–65.[Abstract/Free Full Text]

10 . Nelson EC, Elisha BG. Molecular basis of AmpC hyperproduction in clinical isolates of Escherichia coli. Antimicrob Agents Chemother (1999) 43:957–9.[Abstract/Free Full Text]

11 . Olsson O, Bergstrom S, Normark S. Identification of a novel ampC β-lactamase promoter in a clinical isolate of Escherichia coli. EMBO J (1982) 1:1411–6.[Web of Science][Medline]

12 . Siu LK, Lu PL, Chen JY, et al. High-level expression of ampC β-lactamase due to insertion of nucleotides between –10 and –35 promoter sequences in Escherichia coli clinical isolates: cases not responsive to extended-spectrum-cephalosporin treatment. Antimicrob Agents Chemother (2003) 47:2138–44.[Abstract/Free Full Text]

13 . Tracz DM, Boyd DA, Hizon R, et al. ampC gene expression in promoter mutants of cefoxitin-resistant Escherichia coli clinical isolates. FEMS Microbiol Lett (2007) 270:265–71.[CrossRef][Web of Science][Medline]

14 . Bauernfeind A, Chong Y, Schweighart S. Extended broad spectrum β-lactamase in Klebsiella pneumoniae including resistance to cephamycins. Infection (1989) 17:316–21.[CrossRef][Web of Science][Medline]

15 . Barlow M, Hall BG. Origin and evolution of the AmpC β-lactamases of Citrobacter freundii. Antimicrob Agents Chemother (2002) 46:1190–8.[Abstract/Free Full Text]

16 . Perez-Perez FJ, Hanson ND. Detection of plasmid-mediated AmpC β-lactamase genes in clinical isolates by using multiplex PCR. J Clin Microbiol (2002) 40:2153–62.[Abstract/Free Full Text]

17 . Kang MS, Besser TE, Call DR. Variability in the region downstream of the blaCMY-2 β-lactamase gene in Escherichia coli and Salmonella enterica plasmids. Antimicrob Agents Chemother (2006) 50:1590–3.[Abstract/Free Full Text]

18 . NWGA. AFA brytningspunkter for bakteriers følsomhet—versjon 1.9. (NWGA Breakpoints for Susceptibilities to Bacteria—Version 1.9.) Arbeidsgruppen for Antibiotikaspørsmål. (2006) Oslo, Norway: Universitetssykehuset Nord-Norge. www.antibiotikaresistens.no (7 May 2008, date last accessed).

19 . Tofteland S, Haldorsen B, Dahl KH, et al. Effects of phenotype and genotype on methods for detection of extended-spectrum-β-lactamase-producing clinical isolates of Escherichia coli and Klebsiella pneumoniae in Norway. J Clin Microbiol (2007) 45:199–205.[Abstract/Free Full Text]

20 . Matushek MG, Bonten MJ, Hayden MK. Rapid preparation of bacterial DNA for pulsed-field gel electrophoresis. J Clin Microbiol (1996) 34:2598–600.[Abstract]

21 . Tenover FC, Arbeit RD, Goering RV, et al. Interpreting chromosomal DNA restriction patterns produced by pulsed-field gel electrophoresis: criteria for bacterial strain typing. J Clin Microbiol (1995) 33:2233–9.[Web of Science][Medline]

22 . Tracz DM, Boyd DA, Bryden L, et al. Increase in ampC promoter strength due to mutations and deletion of the attenuator in a clinical isolate of cefoxitin-resistant Escherichia coli as determined by RT–PCR. J Antimicrob Chemother (2005) 55:768–72.[Abstract/Free Full Text]

23 . Pfaffl MW. A new mathematical model for relative quantification in real-time RT–PCR. Nucleic Acids Res (2001) 29:e45.[Abstract/Free Full Text]

24 . Muller PY, Janovjak H, Miserez AR, et al. Processing of gene expression data generated by quantitative real-time RT–PCR. Biotechniques (2002) 32:1372–4. 1376, 1378–9.[Web of Science][Medline]

25 . Barton BM, Harding GP, Zuccarelli AJ. A general method for detecting and sizing large plasmids. Anal Biochem (1995) 226:235–40.[CrossRef][Web of Science][Medline]

26 . Altschul SF, Gish W, Miller W, et al. Basic Local Alignment Search Tool. J Mol Biol (1990) 215:403–10.[CrossRef][Web of Science][Medline]

27 . Bou G, Cartelle M, Tomas M, et al. Identification and broad dissemination of the CTX-M-14 β-lactamase in different Escherichia coli strains in the northwest area of Spain. J Clin Microbiol (2002) 40:4030–6.[Abstract/Free Full Text]

28 . D'Andrea MM, Nucleo E, Luzzaro F, et al. CMY-16, a novel acquired AmpC-type β-lactamase of the CMY/LAT lineage in multifocal monophyletic isolates of Proteus mirabilis from northern Italy. Antimicrob Agents Chemother (2006) 50:618–24.[Abstract/Free Full Text]

29 . Dutour C, Bonnet R, Marchandin H, et al. CTX-M-1, CTX-M-3, and CTX-M-14 β-lactamases from Enterobacteriaceae isolated in France. Antimicrob Agents Chemother (2002) 46:534–7.[Abstract/Free Full Text]

30 . Eckert C, Gautier V, Arlet G. DNA sequence analysis of the genetic environment of various blaCTX-M genes. J Antimicrob Chemother (2006) 57:14–23.[Abstract/Free Full Text]

31 . Eckert C, Gautier V, Saladin-Allard M, et al. Dissemination of CTX-M-type β-lactamases among clinical isolates of Enterobacteriaceae in Paris, France. Antimicrob Agents Chemother (2004) 48:1249–55.[Abstract/Free Full Text]

32 . Giles WP, Benson AK, Olson ME, et al. DNA sequence analysis of regions surrounding blaCMY-2 from multiple Salmonella plasmid backbones. Antimicrob Agents Chemother (2004) 48:2845–52.[Abstract/Free Full Text]

33 . Lartigue MF, Poirel L, Aubert D, et al. In vitro analysis of ISEcp1B-mediated mobilization of naturally occurring β-lactamase gene blaCTX-M of Kluyvera ascorbata. Antimicrob Agents Chemother (2006) 50:1282–6.[Abstract/Free Full Text]

34 . Lartigue MF, Poirel L, Nordmann P. Diversity of genetic environment of blaCTX-M genes. FEMS Microbiol Lett (2004) 234:201–7.[CrossRef][Web of Science][Medline]

35 . Literacka E, Empel J, Baraniak A, et al. Four variants of the Citrobacter freundii AmpC-type cephalosporinases, including novel enzymes CMY-14 and CMY-15, in a Proteus mirabilis clone widespread in Poland. Antimicrob Agents Chemother (2004) 48:4136–43.[Abstract/Free Full Text]

36 . Poirel L, Decousser JW, Nordmann P. Insertion sequence ISEcp1B is involved in expression and mobilization of a blaCTX-M β-lactamase gene. Antimicrob Agents Chemother (2003) 47:2938–45.[Abstract/Free Full Text]

37 . Poirel L, Lartigue MF, Decousser JW, et al. ISEcp1B-mediated transposition of blaCTX-M in Escherichia coli. Antimicrob Agents Chemother (2005) 49:447–50.[Abstract/Free Full Text]

38 . Su LH, Chen HL, Chia JH, et al. Distribution of a transposon-like element carrying blaCMY-2 among Salmonella and other Enterobacteriaceae. J Antimicrob Chemother (2006) 57:424–9.[Abstract/Free Full Text]

39 . Bauernfeind A, Schneider I, Jungwirth R, et al. A novel type of AmpC β-lactamase, ACC-1, produced by a Klebsiella pneumoniae strain causing nosocomial pneumonia. Antimicrob Agents Chemother (1999) 43:1924–31.[Abstract/Free Full Text]

40 . Forward KR, Willey BM, Low DE, et al. Molecular mechanisms of cefoxitin resistance in Escherichia coli from the Toronto area hospitals. Diagn Microbiol Infect Dis (2001) 41:57–63.[CrossRef][Web of Science][Medline]

41 . Mahillon J, Chandler M. Insertion sequences. Microbiol Mol Biol Rev (1998) 62:725–74.[Abstract/Free Full Text]

42 . Jaurin B, Normark S. Insertion of IS2 creates a novel ampC promoter in Escherichia coli. Cell (1983) 32:809–16.[CrossRef][Web of Science][Medline]

43 . Brinas L, Lantero M, de Diego I, et al. Mechanisms of resistance to expanded-spectrum cephalosporins in Escherichia coli isolates recovered in a Spanish hospital. J Antimicrob Chemother (2005) 56:1107–10.[Abstract/Free Full Text]

44 . Deshpande LM, Jones RN, Fritsche TR, et al. Occurrence of plasmidic AmpC type β-lactamase-mediated resistance in Escherichia coli: report from the SENTRY Antimicrobial Surveillance Program (North America, 2004). Int J Antimicrob Agents (2006) 28:578–81.[CrossRef][Web of Science][Medline]

45 . Lee K, Lee M, Shin JH, et al. Prevalence of plasmid-mediated AmpC β-lactamases in Escherichia coli and Klebsiella pneumoniae in Korea. Microb Drug Resist (2006) 12:44–9.[CrossRef][Web of Science][Medline]

46 . Wu SW, Dornbusch K, Kronvall G, et al. Characterization and nucleotide sequence of a Klebsiella oxytoca cryptic plasmid encoding a CMY-type β-lactamase: confirmation that the plasmid-mediated cephamycinase originated from the Citrobacter freundii AmpC β-lactamase. Antimicrob Agents Chemother (1999) 43:1350–7.[Abstract/Free Full Text]

47 . Mammeri H, Poirel L, Fortineau N, et al. Naturally occurring extended-spectrum cephalosporinases in Escherichia coli. Antimicrob Agents Chemother (2006) 50:2573–6.[Abstract/Free Full Text]

48 . Mammeri H, Eb F, Berkani A, et al. Molecular characterization of AmpC-producing Escherichia coli clinical isolates recovered in a French hospital. J Antimicrob Chemother (2008) 61:498–503.[Abstract/Free Full Text]

49 . Black JA, Moland ES, Thomson KS. AmpC disk test for detection of plasmid-mediated AmpC β-lactamases in Enterobacteriaceae lacking chromosomal AmpC β-lactamases. J Clin Microbiol (2005) 43:3110–3.[Abstract/Free Full Text]

50 . Sundsfjord A, Simonsen GS, Haldorsen BC, et al. Genetic methods for detection of antimicrobial resistance. APMIS (2004) 112:815–37.[CrossRef][Web of Science][Medline]


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