High resolution melting analysis for rapid mutation screening in gyrase and Topoisomerase IV genes in quinolone-resistant Salmonella enterica.
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.
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.
Soo Tein
0000-0001-8332-4833
ST
Ngoi
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.
Kwai Lin
KL
Thong
D027081:DNA Gyrase / Q000235:genetics*; D004252:DNA Mutational Analysis / Q000379:methods*; D027101:DNA Topoisomerase IV / Q000235:genetics*; D024881:Drug Resistance, Bacterial / Q000187:drug effects / Q000235:genetics*; D005798:Genes, Bacterial / Q000235:genetics; D009154:Mutation / Q000235:genetics*; D009691:Nucleic Acid Denaturation / Q000235:genetics*; D015363:Quinolones / Q000494:pharmacology*; D019779:Salmonella enterica / Q000201:enzymology / Q000235:genetics*; D013997:Time Factors
D015363:Quinolones; D027101:DNA Topoisomerase IV; D027081:DNA Gyrase