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High resolution melting analysis for rapid mutation screening in gyrase and Topoisomerase IV genes in quinolone-resistant Salmonella enterica.

Ngoi ST, Thong KL - Biomed Res Int (2014)

Bottom Line: Mutations were detected in QRDR of gyrA (n = 52; S83F, S83Y, S83I, D87G, D87Y, and D87N) and parE (n = 1; M438I).Salmonella strains with mutations within QRDR of gyrA are generally more resistant to nalidixic acid (MIC 16 > 256 μg/mL).In conclusion, HRM analysis allows for rapid screening for mutations at the QRDRs of gyrase and topoisomerase IV genes in Salmonella.

View Article: PubMed Central - PubMed

Affiliation: Institute of Biological Sciences, Faculty of Science, University of Malaya, 50603 Kuala Lumpur, Malaysia ; Laboratory of Biomedical Science and Molecular Microbiology, Institute of Graduate Studies, University of Malaya, 50603 Kuala Lumpur, Malaysia.

ABSTRACT
The increased Salmonella resistance to quinolones and fluoroquinolones is a public health concern in the Southeast Asian region. The objective of this study is to develop a high resolution melt curve (HRM) assay to rapidly screen for mutations in quinolone-resistant determining region (QRDR) of gyrase and topoisomerase IV genes. DNA sequencing was performed on 62 Salmonella strains to identify mutations in the QRDR of gyrA, gyrB, parC, and parE genes. Mutations were detected in QRDR of gyrA (n = 52; S83F, S83Y, S83I, D87G, D87Y, and D87N) and parE (n = 1; M438I). Salmonella strains with mutations within QRDR of gyrA are generally more resistant to nalidixic acid (MIC 16 > 256 μg/mL). Mutations were uncommon within the QRDR of gyrB, parC, and parE genes. In the HRM assay, mutants can be distinguished from the wild-type strains based on the transition of melt curves, which is more prominent when the profiles are displayed in difference plot. In conclusion, HRM analysis allows for rapid screening for mutations at the QRDRs of gyrase and topoisomerase IV genes in Salmonella. This assay markedly reduced the sequencing effort involved in mutational studies of quinolone-resistance genes.

No MeSH data available.


Related in: MedlinePlus

Representative HRM aligned melting curves (a) and difference plot (b) for mutations in parE QRDR. The reference strain is indicated by the horizontal black line in difference plot. Wild-type samples are indicated by green curves and mutants are indicated by red curves. All mutant strains contain similar nucleotide changes in the QRDR, resulting in tightly clustered melting curves.
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fig4: Representative HRM aligned melting curves (a) and difference plot (b) for mutations in parE QRDR. The reference strain is indicated by the horizontal black line in difference plot. Wild-type samples are indicated by green curves and mutants are indicated by red curves. All mutant strains contain similar nucleotide changes in the QRDR, resulting in tightly clustered melting curves.

Mentions: The melting temperature of wild-type gyrA allele is 82.5–82.8°C, while that of the mutants (D87Y, D87N, S83F, and S83Y) is at 82.0–82.4°C. Interestingly, Salmonella strains with gyrA D87G mutation produced a distinct melting curve compared to other mutants, at slightly higher melting temperature (82.9–83.5°C). The differences in the melting curves of the wild type versus mutants were distinguishable in both aligned and difference plot (Figure 1). The melting temperature for gyrB wild-type allele is 86.1°C–86.2°C, while that of the mutants is 86.2–86.7°C. One mutant (STM043/05) with three mutations in gyrB has a slightly lower melting temperature at 86.0°C. The aligned plot did not show any difference when comparing the melt curves of wild-type versus mutant gyrB alleles. However, they can be distinguished in difference plot (Figure 2). For parC gene, the melting temperature for wild-type allele is at 86.1–86.5°C (98% melted below 86.4°C) and the mutant at 86.4°C. Although the differences in the melting temperature were small, the mutant produced a unique melting curve in the difference plot when compared to the wild type (Figure 3). For parE gene, the melting temperature for wild-type allele is at 84.6–84.9°C, whereas that of the mutants containing multiple mutations is at 84.9–85.2°C. One mutant (STM018/03), which harbors a single mutation in parE QRDR, has a lower melting temperature at 83.9°C. The differences in wild-type versus mutant melt curves were apparent in both aligned and difference plot (Figure 4).


High resolution melting analysis for rapid mutation screening in gyrase and Topoisomerase IV genes in quinolone-resistant Salmonella enterica.

Ngoi ST, Thong KL - Biomed Res Int (2014)

Representative HRM aligned melting curves (a) and difference plot (b) for mutations in parE QRDR. The reference strain is indicated by the horizontal black line in difference plot. Wild-type samples are indicated by green curves and mutants are indicated by red curves. All mutant strains contain similar nucleotide changes in the QRDR, resulting in tightly clustered melting curves.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4209765&req=5

fig4: Representative HRM aligned melting curves (a) and difference plot (b) for mutations in parE QRDR. The reference strain is indicated by the horizontal black line in difference plot. Wild-type samples are indicated by green curves and mutants are indicated by red curves. All mutant strains contain similar nucleotide changes in the QRDR, resulting in tightly clustered melting curves.
Mentions: The melting temperature of wild-type gyrA allele is 82.5–82.8°C, while that of the mutants (D87Y, D87N, S83F, and S83Y) is at 82.0–82.4°C. Interestingly, Salmonella strains with gyrA D87G mutation produced a distinct melting curve compared to other mutants, at slightly higher melting temperature (82.9–83.5°C). The differences in the melting curves of the wild type versus mutants were distinguishable in both aligned and difference plot (Figure 1). The melting temperature for gyrB wild-type allele is 86.1°C–86.2°C, while that of the mutants is 86.2–86.7°C. One mutant (STM043/05) with three mutations in gyrB has a slightly lower melting temperature at 86.0°C. The aligned plot did not show any difference when comparing the melt curves of wild-type versus mutant gyrB alleles. However, they can be distinguished in difference plot (Figure 2). For parC gene, the melting temperature for wild-type allele is at 86.1–86.5°C (98% melted below 86.4°C) and the mutant at 86.4°C. Although the differences in the melting temperature were small, the mutant produced a unique melting curve in the difference plot when compared to the wild type (Figure 3). For parE gene, the melting temperature for wild-type allele is at 84.6–84.9°C, whereas that of the mutants containing multiple mutations is at 84.9–85.2°C. One mutant (STM018/03), which harbors a single mutation in parE QRDR, has a lower melting temperature at 83.9°C. The differences in wild-type versus mutant melt curves were apparent in both aligned and difference plot (Figure 4).

Bottom Line: Mutations were detected in QRDR of gyrA (n = 52; S83F, S83Y, S83I, D87G, D87Y, and D87N) and parE (n = 1; M438I).Salmonella strains with mutations within QRDR of gyrA are generally more resistant to nalidixic acid (MIC 16 > 256 μg/mL).In conclusion, HRM analysis allows for rapid screening for mutations at the QRDRs of gyrase and topoisomerase IV genes in Salmonella.

View Article: PubMed Central - PubMed

Affiliation: Institute of Biological Sciences, Faculty of Science, University of Malaya, 50603 Kuala Lumpur, Malaysia ; Laboratory of Biomedical Science and Molecular Microbiology, Institute of Graduate Studies, University of Malaya, 50603 Kuala Lumpur, Malaysia.

ABSTRACT
The increased Salmonella resistance to quinolones and fluoroquinolones is a public health concern in the Southeast Asian region. The objective of this study is to develop a high resolution melt curve (HRM) assay to rapidly screen for mutations in quinolone-resistant determining region (QRDR) of gyrase and topoisomerase IV genes. DNA sequencing was performed on 62 Salmonella strains to identify mutations in the QRDR of gyrA, gyrB, parC, and parE genes. Mutations were detected in QRDR of gyrA (n = 52; S83F, S83Y, S83I, D87G, D87Y, and D87N) and parE (n = 1; M438I). Salmonella strains with mutations within QRDR of gyrA are generally more resistant to nalidixic acid (MIC 16 > 256 μg/mL). Mutations were uncommon within the QRDR of gyrB, parC, and parE genes. In the HRM assay, mutants can be distinguished from the wild-type strains based on the transition of melt curves, which is more prominent when the profiles are displayed in difference plot. In conclusion, HRM analysis allows for rapid screening for mutations at the QRDRs of gyrase and topoisomerase IV genes in Salmonella. This assay markedly reduced the sequencing effort involved in mutational studies of quinolone-resistance genes.

No MeSH data available.


Related in: MedlinePlus