a Shanghai Research Center of Biotechnology, SIBS, Chinese Academy of Sciences, 500 Cao Bao Road, Shanghai 200233; b Institute of Antibiotics, Medical Center of Fu Dan University, Shanghai, China
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Abstract |
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Introduction |
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Resistance to fluoroquinolones in E. coli has mostly been attributed to mutations in the genes encoding DNA gyrase and topoisomerase IV. DNA gyrase and topoisomerase IV are both enzymes composed of four subunits (two A and two B) encoded by gyrA and gyrB, and parC and parE, respectively. In E. coli, mutations in the quinolone resistance determining regions (QRDRs) of the gyrA and parC genes, at nucleotide positions 248 and 259/260 of gyrA resulting in Ser-83 and Asp-87 alterations and mutations at nucleotide position 238/239 and 250/251 of parC resulting in Ser-80 or Glu-84 changes, have been reported to be mainly responsible for fluoroquinolone resistance.35
Although several different methods, such as restriction fragment length polymorphism (RFLP), single-strand conformational polymorphism (SSCP) analysis and sequencing of the relevant gene regions, have been used to detect such mutations,6,7 the procedures are labour intensive and time consuming. In this study, we developed a simple, rapid PCR mismatch amplification mutation assay (MAMA PCR) to detect the significant mutations in both chromosomal gyrA and parC of E. coli isolates.
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Materials and methods |
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One hundred and twenty-one E. coli isolates collected from five general hospitals in Shanghai from 1998 to 2000 were used in the study. The MICs of ciprofloxacin (CIP) and other antimicrobial agents were determined by agar dilution testing according to the National Committee for Clinical Laboratory Standards (NCCLS) performance guidelines. E. coli ATCC 25922 and ATCC 35218 were employed as controls: each exhibited an MIC of CIP 0.06 mg/L.
Primer design and MAMA PCR protocol
The rationale behind MAMA PCR is that a single nucleotide mismatch at the 3' extremity of the annealed reverse primer renders Taq polymerase unable to extend the primer. So, the absence of the specific PCR product (coupled with a positive internal PCR control) reveals a deviation from the wild-type DNA sequence. In this study, we introduced another nucleotide alteration near the 3' end of the MAMA primer to enhance the 3' mismatch effect. The MAMA primers for mutation detection are shown in Figure 1. Other primers used are as follows: WPgyrA, 5'-GACCTTGCGAGAGAAATTACAC-3' (forward, position: 728); ControlgyrA, 5'-GATGTTGGTTGCCATACCTACG-3' (reverse, position: 546525); WPparC, 5'-CGGAAAACGCCTACTTAAACTA-3' (forward, position: 4162); and ControlparC, 5'-GTGCCGTTAAGCAAAATGT-3' (reverse, position: 506488). In each PCR, a forward primer and a MAMA primer were used in a PCR for mutation detection. These primers generate a short PCR product from the wild-type gene, but fail to produce a product from any gene with a mutation at the location covered by the mismatch positions on the MAMA primer. A third, control primer that is expected to anneal efficiently to all gene alleles was used in conjunction with the forward primer to generate a longer PCR product as an internal control.
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Template for PCR was prepared by the heat lysis method of Pitout et al.,8 except that bacteria were directly inoculated into 1.0 mL of LB broth in Eppendorf tubes for overnight culture. For each PCR, 1 µL of supernatant containing template DNA was added to a final volume of 50 µL containing: 0.35 µM forward primer, 0.25 µM MAMA primer, 0.10 µM control primer, 5 µL of 10 x Taq buffer, 200 µM of each deoxynucleotide triphosphate and 1.5 U of Taq DNA polymerase [Takara Taq; Takara Biotechnology (Dalian) Co. Ltd, Dalian, China]. Amplification was carried out on a DNA Thermolyne (Barnstead/Thermolyne, Dubeque, Iowa, USA) programmed as follows: initial denaturation at 94°C for 5 min and 35 cycles of denaturation at 94°C for 40 s, annealing at 54°C for 40 s and extension at 72°C for 40 s, with a final step of 72°C for 5 min. Large scale PCR was carried out on a DNA Multiblock System (Hybaid Ltd, Middlesex, UK) with the same programme. PCR products were visualized on horizontal 1.0% agarose gels in 0.5 x TBE buffer, loaded with 9 µL of reaction mix and stained with ethidium bromide after electrophoresis.
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Results and discussion |
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The method that has been developed is intended to detect the most common mutations in E. coli, in gyrA and parC, associated with fluoroquinolone resistance. The design of the MAMA protocol differs from others reported,10,11 in that it targets wild-type gene sequences rather than mutant ones. To avoid false negative results, a second reverse primer is employed to generate a product that serves as a positive control in a nested PCR. Other MAMA PCR protocols, designed to amplify mutant gene sequences, detect specific nucleotide changes at particular positions in the gene. Alternative changes are not detected. Mutations adjacent to the particular nucleotide of interest can also give rise to amino acid substitutions that alter the MIC of CIP and would be detected with our approach. For example, in gyrA, G259A results in an Asp87Asn substitution, whereas A260G generates a different change (Asp87Gly); both affect susceptibility to CIP. In parC, A238C causes a Ser80Arg substitution and G239T results in a Ser80Ile change. Targeting the wild-type sequence is a more comprehensive tactic than targeting a particular mutation. However, a warning is pertinent. Although the approach described in this paper will detect a number of different mutations in the wild-type sequence at the position of interest, it has limitations. First, it does not identify the nature of the mutation. Therefore, it is not a substitute for DNA sequence analysis. Secondly, changes at the third base position of a codon would be detected by our version of MAMA, but these will not necessarily result in an amino acid substitution in the gene product because of the degenerate nature of the genetic code. For example, any change at the third base position of gyrA codon 83 (TCG) would not alter the amino acid in GyrA, i.e. Ser-83. Similar considerations apply to other codons. Hence, detection of such silent mutations by our version of MAMA could, in principle, lead to wrong conclusions being drawn about amino acid substitution in the gene product. However, nucleotide changes at the third base positions of the four codons targeted in this study have, to our knowledge, not been reported. Therefore, the MAMA PCR method proposed reliably detects the mutations in gyrA and parC that are commonly responsible for resistance to fluoroquinolones displayed by E. coli, and the method is suitable for profiling and characterizing a large number of isolates in resistant outbreaks.
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Acknowledgements |
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Notes |
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References |
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2
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Zirnstein, G. W., Li, Y., Swaminathan, B. & Angulo, F. (1999). Ciprofloxacin resistance in Campylobacter jejuni isolates: detection of gyrA resistance mutations by MAMA PCR and DNA sequence analysis. Journal of Clinical Microbiology 37, 327680.
Received 20 June 2001; returned 17 August 2001; revised 27 November 2001; accepted 14 December 2001