Rapid Identification of Mutations in Quinolone-Resistant Shigella Isolates by Scanning of gyrA and parC Genes using High-Resolution Melting Curve Analysis

• Main Authors: Mohammad Mehdi Soltan Dallal, Sajad Yaghoubi, Asiyeh Dezhkam, Shahriyar Yavari, Azad Jamee
• Format: Article
• Language: English
• Published: Light House Polyclinic Mangalore 2020-05-01
• Series: Online Journal of Health & Allied Sciences
• Subjects: shigellosis; dna gyrase; topoisomerase iv; high-resolution melting
• Online Access: https://www.ojhas.org/issue73/2020-1-4.html
• ISSN: 0972-5997; 0972-5997

Objectives: Mutations in the quinolone resistance–determining regions (QRDRs) of the topoisomerase genes are considered to be a primary mechanism of quinolone and fluoroquinolone resistance. The objective of this study was to identify the most common gyrA and parC mutations in Shigella spp., with the high-resolution melting (HRM) method. Methods: A total of 40 Shigella isolates analyzed for antibiotic susceptibility nalidoxic acid (NAL) and ciprofloxacin (CIP) by disc diffusion and minimum inhibitory concentration (MIC) method. Polymerase chain reaction (PCR) and high resolution melt curve analysis were done for detection of common mutation in gyrA and parC genes. Results: The MIC for 5 of the NAL and CIP-resistant isolates was ≥ 256, and the MIC in 30 NAL-resistant isolates was ≥ 8. The Tm of the wild-type gyrA allele was 83.5–83.8°C while that of the mutant strains (S83L and D87G) were at 83.9–84.2°C. In the parC gene, the Tm for the wild-type allele was at 85.5–85.8°C, and the mutant-type allele’s Tm was at 85.30–85.50°C. Conclusions: High-Resolution melting curve PCR analysis clearly decreases the sequencing labor involved in monitoring quinolone-resistance genes for mutations and is practicable for a wide-ranging quinolone-resistance monitoring program.