JAC Advance Access originally published online on March 10, 2005
Journal of Antimicrobial Chemotherapy 2005 55(5):768-772; doi:10.1093/jac/dki074
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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
1 National Microbiology Laboratory, Public Health Agency of Canada, 1015 Arlington St, Winnipeg, Manitoba, Canada, R3E 3R2; 2 Cadham Provincial Laboratory, Winnipeg, Manitoba, Canada
* Corresponding author. Tel: +1-204-789-2133; Fax: +1-204-789-5020; Email: michael_mulvey{at}phac-aspc.gc.ca
Received 9 December 2004; returned 18 January 2005; revised 25 January 2005; accepted 26 January 2005
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Abstract |
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 Top Abstract IntroductionMaterials and methodsResults and discussionReferences |
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Objectives: To characterize the mechanism of cefoxitin resistance in clinical isolate Escherichia coli N99-0001.
Methods: Plasmid analysis, PCR for ß-lactamases, and sequencing of the ampC genes was carried out. An RT–PCR method was developed to determine relative ampC expression.
Results: Analysis of the ampC promoter region of E. coli N99-0001 revealed a T
A mutation at –32, a CA mutation at –11, an insertion of a T between –20 and –21, and a 28 bp deletion including the entire attenuator. RT–PCR showed that ampC was expressed 140-fold higher in E. coli N99-0001 than in E. coli ATCC 25922.
Conclusions: Cefoxitin resistance in E. coli N99-0001 was due to overexpression of ampC caused by an increase in promoter strength.
Keywords: cefoxitin resistance , E. coli , RT–PCR
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Introduction |
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TopAbstractIntroductionMaterials and methodsResults and discussionReferences |
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In Escherichia coli, resistance provided by class C ß-lactamases can be plasmid-mediated or due to overexpression of the chromosomal ampC gene. Mutations in the promoter region of ampC have been described as the mechanism for AmpC hyper-production.1–9 The mutations are thought to generate promoters that more closely resemble the E. coli consensus, which leads to overexpression of the normally low level constitutively expressed ampC. Attenuator mutations are thought to destabilize the hairpin structure allowing for greater read-through. Here we report the unique structure of the ampC promoter region in clinical isolate E. coli N99-0001 and the development of an RT–PCR technique for the relative quantification of ampC gene expression.
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Materials and methods |
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TopAbstractIntroductionMaterials and methodsResults and discussionReferences |
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Patient, bacteria and antimicrobial susceptibility
The patient was an 81-year-old woman living in a personal care home, with underlying diagnoses of Alzheimer's disease, type 2 diabetes, chronic diarrhoea, and depression. She presented to the hospital with a complaint of diffuse abdominal pain and initial investigation revealed evidence of small bowel obstruction, and she was admitted for management of this condition. Urine collected from the emergency room revealed pyuria and a mixed growth of > 108 cfu/L of E. coli and > 108 cfu/L of group B Streptococcus. E. coli N99-0001 was isolated in pure culture from the urine and forwarded to Cadham Provincial Laboratory and the National Microbiology Laboratory for further characterization. The patient at that point was recovering without antibiotics and was discharged with an antibiotic, although which antibiotic was not documented. No concern was indicated on follow-up outpatient visits.
Identification was confirmed using the Vitek GNI card (bioMérieux, Hazelwood, MO, USA). E. coli C600N (nalidixic acid-resistant) was used as a recipient in conjugation experiments and E. coli ATCC 25922 was used as the wild-type ampC promoter control. Antimicrobial susceptibility testing was carried out by the broth microdilution method, as described by NCCLS.
DNA methodology and isoelectric focusing
The E. coli ampC gene and promoter region was amplified by PCR with primers AmpC1 (5'-AATGGGTTTTCTACGGTCTG–52) and COL-E (5'-AAGTGTAGATGACAGCAAG1241) (coordinate of last base is in relation to the +1 of the mRNA).1,2 PCR for CMY-2-type genes was carried out as previously described.9 PCR for TEM-type genes (primers TEM-1, 5'-ATAAAATTCTTGAAGAC; TEM-2, 5'-TTACCAATGCTTAATCA), SHV-type genes (primers SHV-up, 5'-CGCCGGGTTATTCTTATTTGTCGC; SHV-lo, 5'-TCTTTCCGATGCCGCCGCCAGTCA), and CTX-M-type genes (primers CTX-U1, 5'-ATGTGCAGYACCAGTAARGTKATGGC; CTX-U2, 5'-TGGGTRAARTARGTSACCAGAAYCAGCGG), were as for CMY-2-type genes with annealing temperatures of 42, 60 and 58 °C, respectively. Plasmid isolation was done using the QIAGEN Plasmid Midi Kit (Qiagen Inc., Mississauga, ON, Canada). DNA sequencing was done by dideoxy cycle sequencing as carried out by the DNA Core Facility of the National Microbiology Laboratory. Crude cell extracts were prepared by resuspending 2 mL of overnight culture in 0.2 mL of 1% glycine and sonicating twice for 30 s at maximum power with a Virsonic 100 and a Micro-Probe (SP Industries, Gardiner, NY, USA). Extracts were run on precast pH 3–10 Ready Gel IEF Gels (Bio-Rad, Mississauga, ON, Canada).
RT–PCR
Total cellular RNA was extracted from mid-log phase cells with a QIAGEN RNEasy RNAprotect Mini Kit (Qiagen Inc.), treated with RNAse-free DNAse DNA-free (Ambion Inc., Austin, TX, USA), and quantified by measuring absorbance at 260 nm. Absence of DNA was verified by control PCR reactions using the RNA as a template. For PCR we used the single fluorophore-labelled, self-quenched primers obtained from Invitrogen Corp. (Burlington, ON, Canada) and designed using the LUX Designer software at their web site (http://www.invitrogen.com/) using the genes sequences from E. coli K12 strain MG1655 (accession number U00096
[GenBank]
). Primers for ampC (57 bp product) were AmpC LUX353FL, 5'-gaccgCGTTGTTTGAGTTAGGTTCGGTC243-FAM (FAM is 6-carboxy-fluorescein and is attached to the second last base from the 3' end; bases in lower case are non-gene specific; coordinate within the gene of the last base is shown) and AmpC 353FL_361RU, 5'-CACCAAGCACGCCAGTAAATG251 (unlabelled), and for the housekeeping gene glyceraldehyde dehydrogenase gapA (100 bp product) were GapA LUX620FL, 5'-gagccCTTCCCAGAACATCATCCCGTC629-FAM, and GapA 620FL_671RU, 5'-CGCCATACCAGTCAGTTTGC680. The assay to semi-quantify specific mRNAs was carried out using the SuperScript III Platinum Two-step Quantitative RT–PCR system according to manufacturer's instructions (Invitrogen). Real time PCR reactions were carried out in the ABI Prism 7900HT Sequence Detection System (Applied Biosystems, Foster City, CA, USA). In the first step, cDNA was synthesized from 200 ng of RNA using M-MLV reverse transcriptase provided in the above kit. In the second step, individual reactions were set-up in triplicate containing 5 µL of the first-step reaction and 5 µM of each primer for either ampC or gapA. PCR reaction conditions were as follows: initial denaturation at 95 °C for 5 min, and PCR for 40 cycles at 95 °C for 15 s, 55°C for 30 s and 72 °C for 30 s. Data were analysed using Sequence Detection Software 2.0 (Applied Biosystems) and exported to Microsoft Excel. Relative quantification was carried out by using the 2–
CT method, also known as the delta-delta CT method, where the CT value was defined as the cycle at which the fluorescence exceeded a value of 0.2.10
E. coli ATCC 25922 ampC was the calibrator gene and by definition was given a 2–CT value of 1.00. Values for ampC were normalized to those of gapA. The amplification efficiencies of ampC and gapA were determined by amplification of a template dilution series and plotting the log cDNA dilution versus CT. A value of 0.1 was obtained for the slope indicating comparable amplification efficiencies.10
Nucleotide accession number
The nucleotide sequences of the E. coli N99-0001 and ATCC 25922 ampC genes have been deposited in the GenBank database under accession numbers AY843211 [GenBank] and AY899338 [GenBank] , respectively.
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Results and discussion |
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TopAbstractIntroductionMaterials and methodsResults and discussionReferences |
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Mechanism of cefoxitin resistance in E. coli N99-0001
The MIC for cefoxitin was > 256 mg/L for E. coli N99-0001. This strain also exhibited decreased susceptibility to other ß-lactams including cefotaxime (16 mg/L), ceftazidime ( > 16 mg/L), cefpodoxime ( > 16 mg/L) and aztreonam (16 mg/L), compared with E. coli ATCC 25922 (MICs < 1). In addition, there was little synergy with clavulanic acid in combination with cefotaxime or ceftazidime. PCR for TEM-, SHV-, CTX-M-, and CMY-2-type ß-lactamase genes was negative. Repeated attempts to isolate plasmid DNA were unsuccessful, as were attempts to transfer cefoxitin resistance to E. coli C600N by conjugation in liquid media or on solid media containing 50 mg/L nalidixic acid. Isoelectric focusing revealed a single band of ß-lactamase activity with a pI of > 8.5. Sequence analysis of the ampC promoter region showed three base substitutions, a single bp insertion, and a 28 bp deletion when compared with the corresponding E. coli ATCC 25922 sequence (Figure 1a). A transversion at position –32, TA, changed the wild-type –35 box, TTGTCA, to the consensus –35 box sequence, TTGACA (pertinent nucleotides are in boldface). This change has been linked with increased ampC expression in previous studies.1,2,4,5,7
A CA transversion at position –11 changed the wild-type –10 box, TACAAT, to TAAAAT, placing a purine where a T is found in the consensus –10 box, TATAAT. Two other changes were found in the spacer region between the –35 and –10 boxes, a GA transition at position –28, and an insertion of a T between position –21 and –20. Previous studies have reported nucleotide insertions that produce greater separation between the –10 and –35 boxes of the ampC promoter result in higher promoter efficiency.5,8
The insertion in E. coli N99-0001 resulted in a separation of 17 bp between the two conserved promoter regions, which is the optimal spacing distance for efficient gene expression from sigma70 promoters.1
Sequence analysis also revealed a 28 bp deletion that included the complete ampC attenuator region, and spanned positions +16 to +43 (Figure 1a). Mutations in the attenuator region are thought to contribute to AmpC over-production through the destabilization of the hairpin structure resulting in increased transcription.1
Direct confirmation of this has not yet been obtained due to the difficulty in introducing site-specific mutations in the attenuator presumably due to the hairpin structure. To our knowledge, this is the first description of an E. coli ampC promoter region with a deleted attenuator.
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Sequence analysis of the complete coding regions from E. coli N99-0001 and ATCC 25922 showed the two AmpC proteins were 99% identical with only four amino acid differences (Figure 1b). Three of the differences were conserved substitutions and none involved residues in or close to conserved motifs of class C ß-lactamases. It is unlikely that any of the changes in the E. coli N99-0001 AmpC confer an expanded spectrum to the ß-lactamase compared with the E. coli ATCC 25922 AmpC.
This, in combination with the changes described above that lead to a promoter more closely resembling the E. coli sigma70 consensus, and the lack of the attenuator, provide strong evidence that overexpression of ampC is the cause for cefoxitin resistance in E. coli N99-0001.
In a recent study of cefoxitin-resistant E. coli isolated from Canadian hospitals, we identified 49 novel ampC promoter sequences in 166 out of 183 unique strains analysed.9 No strains were found that had a promoter identical to E. coli N99-0001, though base substitutions were found at –32 and –11 in some promoter types, and some had increased spacer regions. The increased spacer regions were due to either a single or a dinucleotide insertion between positions –13 and –14 not between –20 and –21 as found in E. coli N99-0001. Interestingly, in the study mentioned above, the changes in the –10 and –35 boxes and the insertions between –13 and –14 were never found in the same promoter type.
Relative expression of ampC as determined by RT–PCR
To provide direct evidence of overexpression of the E. coli N99-0001 ampC, we developed a two-step RT–PCR assay to measure relative expression compared with that of ampC in E. coli ATCC 25922 (constitutive low level ampC expression). Results of three experiments are shown in Table 1. Data were analysed using the 2–CT method which allows relative gene expression data to be presented as a fold change in the target gene (E. coli N99-001 ampC) compared with the calibrator gene (E. coli ATCC 25922 ampC).10
Our results indicate ampC was expressed about 140-fold higher in E. coli N99-0001 than in E. coli ATCC 25922 (range 130- to 152-fold).
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Previous studies have employed different methods of quantifying ampC expression including measuring ß-lactamase levels and transcript analysis, northern blot analysis, gene reporter systems, and RT–PCR using SYBR Green I.1,3,4,6–8 None of the promoters studied was identical to the E. coli N99-0001 ampC promoter though some of the mutations in the N99-0001 promoter were found. The T
A mutation at position –32 has been shown to increase ampC expression 5.4-fold in the RT–PCR assay, 13-fold using a gene reporter system, and from 18- to 22-fold by measuring ß-lactamase and transcript levels.1,4,6
In two strains with either a single or dinucleotide insertion in the spacer region, northern blot analysis showed a 10-fold or six-fold increase in ampC mRNA levels, respectively.8
Thus, in E. coli N99-0001, the promoter changes including the CA transversion at position –11 and the complete deletion of the attenuator, would be expected to be additive, contributing to the high level of overexpression of ampC compared with that found in E. coli ATCC 25922. The RT–PCR method described here uses gene-specific primer sets in which only one primer is labelled with a fluorophore. A 5'-tail of 5 bp forms a hairpin with the 3'-end in unincorporated primer providing quenching of the fluorophore and negates the need for a separate quencher moiety. The potential for multiplexing exists if one primer is labelled with a different fluorophore, which is not possible with SYBR Green I DNA detection. In addition, the presence of the hairpin prevents primer–dimer formation and mispriming. Unlike the 5' nuclease assay, there is no need for a hybridization probe. SYBR Green methods are more difficult to optimize, and can suffer from non-specific binding of the dye creating false signals. We have found that our RT–PCR method is rapid, sensitive and reproducible. We are currently using this RT–PCR protocol to analyse ampC gene expression in the ampC promoter variants characterized from E. coli strains isolated from hospitalized patients in Canada.9
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Acknowledgements |
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We thank the DNA Core Facility of the National Microbiology Laboratory for DNA sequencing and oligonucleotide synthesis.
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References |
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TopAbstractIntroductionMaterials and methodsResults and discussionReferences |
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1 . Jaurin, B., Grundstrom, T. & Normark, S. (1982). Sequence elements determining ampC promoter strength in E. coli. EMBO Journal 1, 875–81.[Web of Science][Medline]
2 . Caroff, N., Espaze, E., Berard, I. et al. (1999). Mutations in the ampC promoter of Escherichia coli isolates to oxyiminocephalosporins without extended spectrum ß-lactamase production. FEMS Microbiology Letters 173, 459–65.[Web of Science][Medline]
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Corvec, S., Caroff, N., Espaze, E. et al. (2002). –11 mutation in the ampC promoter increasing resistance to ß-lactams in a clinical Escherichia coli strain. Antimicrobial Agents and Chemotherapy 46, 3265–7.
7 . Corvec, S., Caroff, N., Espaze, E. et al. (2003). Comparison of two RT–PCR methods for quantifying ampC specific transcripts in Escherichia coli strains. FEMS Microbiology Letters 228, 187–91.[CrossRef][Web of Science][Medline]
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Siu, L., Lu, P., Chen, J. et al. (2003). High-level expression of ampC ß-lactamase due to insertion of nts between –10 and –35 promoter sequences in Escherichia coli clinical isolates: cases not responsive to extended-spectrum-cephalosporin treatment. Antimicrobial Agents and Chemotherapy 47, 2138–44.
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Mulvey, M., Bryce, E., Boyd, D. et al. (2005). Molecular characterization of cefoxitin resistant Escherichia coli from Canadian hospitals. Antimicrobial Agents and Chemotherapy 49, 358–65.
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