Use of a rapid mismatch PCR method to detect gyrA and parC mutations in ciprofloxacin-resistant clinical isolates of Escherichia coli

Yan Zhi Qianga, Tong Qina, Wang Fub, Wu Pei Chengb, Yang Sheng Lia and Gong Yia,*

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


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 Acknowledgements
 References
 
Four amino acid substitutions, two in GyrA and two in ParC subunits of DNA gyrase and topoisomerase IV, respectively, are commonly responsible for fluoroquinolone resistance in Escherichia coli. In this study, an economical and time-efficient mismatch amplification mutation assay (MAMA) PCR was developed to detect mutations in the chromosomal gyrA and parC genes causing these substitutions. One hundred and twenty-one clinical E. coli isolates were tested by this assay, and the results confirmed that accumulation of amino acid alterations in GyrA and ParC correlates closely with stepwise increases in the MIC of ciprofloxacin.


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 Acknowledgements
 References
 
Escherichia coli is one of the most common causes of bacterial infections and, in China, increasing numbers of fluoroquinolone-resistant E. coli have been giving cause for concern since the late 1990s. Furthermore, the number of reports of fluoroquinolone-resistant E. coli in other countries continues to rise at the start of the new century.1,2

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.3–5

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.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 Acknowledgements
 References
 
Bacteria and MICs

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 1Go. Other primers used are as follows: WPgyrA, 5'-GACCTTGCGAGAGAAATTACAC-3' (forward, position: 7–28); ControlgyrA, 5'-GATGTTGGTTGCCATACCTACG-3' (reverse, position: 546–525); WPparC, 5'-CGGAAAACGCCTACTTAAACTA-3' (forward, position: 41–62); and ControlparC, 5'-GTGCCGTTAAGCAAAATGT-3' (reverse, position: 506–488). 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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Figure 1. Comparison of DNA sequence around the codons for amino acids of interest in GyrA and ParC in fluoroquinolone-susceptible and CIP-resistant isolates. The amino acids found in the native proteins are indicated above the corresponding nucleotide sequences and mutated nucleotides of resistant isolates are in bold type. MAMA primers used in this study are shown below the sequence. Underlined letters indicate the nucleotide alterations introduced to enhance the 3' mismatch effect.

 
PCR experiments

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.


    Results and discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 Acknowledgements
 References
 
Each MAMA primer is complementary to the corresponding sequence of the wild-type gene with one mismatch introduced at the third nucleotide from the 3' end of the primer. However, between the MAMA primer and mutant genes there are two mismatched nucleotides at the 3' end of the primer. A single mismatch at the third nucleotide from the 3' end of the MAMA primer has little influence on the yield of PCR products, whereas an additional mismatch at the 3' end of the primer inhibits the PCR. Strains with known mutations in the QRDRs of gyrA and parC were used as negative quality controls (Figure 2Go).



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Figure 2. Agarose gel of E. coli MAMA PCR products. Lanes 1, 3 and 4 indicate the 540 bp PCR product obtained from strains with C248T, G259A or A260G mutations in gyrA, respectively. Lanes 7, 8, 10 and 11 indicate strains with A238C, G239T, G250A or A251G mutations in parC generate only a 446 bp control fragment. Lanes 2, 5, 6 and 9 indicate that strains without corresponding mutations generate two fragments (540/259 bp, 540/274 bp, 446/217 bp, 446/238 bp, respectively). M, DNA molecular weight standard; {phi}X174–HincII digest [Takara Biotechnology (Dalian) Co. Ltd].

 
One hundred and twenty-one isolates were analysed by MAMA PCR. The results defined five groups according to the mutations in gyrA and parC. Group 1: 25 strains with CIP MICs of <=0.06 mg/L showed no changes in either gyrA or parC. Group II: 10 isolates with CIP MICs of 0.125– 0.5 mg/L have Ser-83 substitutions in GyrA. Group III: 15 strains with CIP MICs of 1–4 mg/L have two alterations, at Ser-83 in GyrA and at Ser-80 in ParC. Group IV: three amino acid substitutions, two in GyrA at Ser-83 and Asp-87 and a third at Ser-80 or Glu-84 in ParC, were found in most isolates (51 of 55) with CIP MICs of 8–64 mg/L. Group V: 14 of 16 isolates with CIP MICs >= 128 mg/L have four amino acid substitutions (two in GyrA, at Ser-83 and Asp-87, and two in ParC, at Ser-80 and Glu-84). These data demonstrate the strong correlation between the stepwise accumulation of mutations in gyrA and parC and increases in resistance to fluoroquinolones reported previously.9 However, there were exceptions. One isolate in Group IV (MIC 64 mg/L) has both sets of amino acid substitutions (two in GyrA and two in ParC) rather than the three expected. The resistance phenotype may reflect an increase in drug permeation or a decrease in drug efflux, as described by Vila et al.5 or could reflect a rare amino acid substitution at one of the sites. In the cases of the other five isolates (three in Group IV and two in Group V), each has fewer amino acid substitutions than would be predicted from its CIP MIC. However, these strains are also less susceptible to aminoglycosides and co-trimoxazole, indicating that active efflux and impermeability may also contribute to the fluoroquinolone resistance phenotype displayed by these isolates.

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.


    Acknowledgements
 Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 Acknowledgements
 References
 
This study was supported by a grant from the Science and Technology Commission of Shanghai Municipality. We thank Shanghai BioStar Genechip Inc. for providing the DNA Multiblock System (Hybaid Ltd, Middlesex, UK).


    Notes
 
* Corresponding author. Tel: +86-21-6436-9607; Fax: +86-21-6470-0244; E-mail: yigong{at}srcb.ac.cn Back


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 Acknowledgements
 References
 
1 . Iqbal, J., Rahman, M. & Kabir, M. S. (1997). Increasing ciprofloxacin resistance among prevalent urinary tract bacterial isolates in Bangladesh. Japanese Journal of Medical Science and Biology 50, 241–50.[Medline]

2 . Goettsch, W., van Pelt, W., Nagelkerke, N., Hendrix, M. G., Buiting, A. G., Petit, P. L. et al. (2000). Increasing resistance to fluoroquinolones in Escherichia coli from urinary tract infections in The Netherlands. Journal of Antimicrobial Chemotherapy 46, 223–8.[Abstract/Free Full Text]

3 . Cullen, M. E., Wyke, A. W., Kuroda, R. & Fisher, L. M. (1989). Cloning and characterization of a DNA gyrase A gene from Escherichia coli that confers clinical resistance to 4-quinolones. Antimicrobial Agents and Chemotherapy 33, 886–94.[ISI][Medline]

4 . Oram, M. & Fisher, L. M. (1991).4-Quinolone resistance mutations in the DNA gyrase of Escherichia coli clinical isolates identified by using the polymerase chain reaction. Antimicrobial Agents and Chemotherapy 35, 387–9.[ISI][Medline]

5 . Vila, J., Ruiz, J., Goñi, P. & Jimenez de Anta, M. T. (1996). Detection of mutations in parC in quinolone-resistance clinical isolates of Escherichia coli. Antimicrobial Agents and Chemotherapy 40, 491–3.[Abstract]

6 . Fisher, L. M., Lawrence, J. M., Josty, I. C., Hopewell, R., Margerrison, E. E. & Cullen, M. E. (1989). Ciprofloxacin and the fluoroquinolones. New concepts on the mechanism of action and resistance. American Journal of Medicine 87, S2–8.[Medline]

7 . Ouabdesselam, S., Hooper, D. C., Tankovic, J. & Soussy, C. J. (1995). Detection of gyrA and gryB mutations in quinolone-resistance clinical isolates of Escherichia coli by single-strand conformational polymorphism analysis and determination of levels of resistance conferred by two different single gyrA mutations. Antimicrobial Agents and Chemotherapy 39, 1667–70.[Abstract]

8 . Pitout, J. D. D., Thomson, K. S., Hanson, N. D., Ehrhardt, A. F., Coudron, P. & Sanders, C. C. (1998). Plasmid-mediated resistance to expanded-spectrum cephalosporins among Enterobacter aerogenes strains. Antimicrobial Agents and Chemotherapy 42, 596–600.[Abstract/Free Full Text]

9 . Webber, M. & Piddock, L. J. V. (2001). Quinolone resistance in Escherichia coli. Veterinary Research 32, 275–84.[ISI][Medline]

10 . Newton, C. R., Graham, A., Heptinstall, L. E., Powell, S. J., Summers, C., Kalsheker, N. et al. (1989). Analysis of any point mutation in DNA: The amplification refractory mutation system (ARMS). Nucleic Acids Research 17, 2503–16.[Abstract]

11 . 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, 3276–80.[Abstract/Free Full Text]

Received 20 June 2001; returned 17 August 2001; revised 27 November 2001; accepted 14 December 2001