1 National Microbiology Laboratory, Public Health Agency of Canada, 1015 Arlington St, Winnipeg, Manitoba, Canada, R3E 3R2; 2 Cadham Provincial Laboratory, Winnipeg, Manitoba, Canada
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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Methods: Plasmid analysis, PCR for ß-lactamases, and sequencing of the ampC genes was carried out. An RTPCR method was developed to determine relative ampC expression.
Results: Analysis of the ampC promoter region of E. coli N99-0001 revealed a TA mutation at 32, a C
A mutation at 11, an insertion of a T between 20 and 21, and a 28 bp deletion including the entire attenuator. RTPCR 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 , RTPCR
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Introduction |
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Materials and methods |
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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'-AATGGGTTTTCTACGGTCTG52) 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 310 Ready Gel IEF Gels (Bio-Rad, Mississauga, ON, Canada).
RTPCR
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). 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 RTPCR 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 2CT 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 and AY899338, respectively.
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Results and discussion |
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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 C
A 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 G
A 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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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 RTPCR
To provide direct evidence of overexpression of the E. coli N99-0001 ampC, we developed a two-step RTPCR 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 2CT 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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The RTPCR 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 primerdimer 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 RTPCR method is rapid, sensitive and reproducible. We are currently using this RTPCR 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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References |
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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, 35865.
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