Detection of gyrA mutations in quinolone-resistant Salmonella enterica by denaturing high-performance liquid chromatography

Abstract

Denaturing high-performance liquid chromatography (DHPLC) was evaluated as a rapid screening and identification method for DNA sequence variation detection in the quinolone resistance-determining region of gyrA from Salmonella serovars. A total of 203 isolates of Salmonella were screened using this method. DHPLC analysis of 14 isolates representing each type of novel or multiple mutations and the wild type were compared with LightCycler-based PCR-gyrA hybridization mutation assay (GAMA) and single-strand conformational polymorphism (SSCP) analyses. The 14 isolates gave seven different SSCP patterns, and LightCycler detected four different mutations. DHPLC detected 11 DNA sequence variants at eight different codons, including those detected by LightCycler or SSCP. One of these mutations was silent. Five isolates contained multiple mutations, and four of these could be distinguished from the composite sequence variants by their DHPLC profile. Seven novel mutations were identified at five different loci not previously described in quinolone-resistant salmonella. DHPLC analysis proved advantageous for the detection of novel and multiple mutations. DHPLC also provides a rapid, high-throughput alternative to LightCycler and SSCP for screening frequently occurring mutations.

FIG. 1.

DHPLC analyses of S. enterica strains at 61 and 63°C. The DHPLC profiles of S. enterica strains (mutations shown in parentheses) are shown in panels a to k as follows: a, NCTC 74 (wild type); b, L378 and L575 (Ser83-Phe); c, L400 (Asp72-Gly, Ser83-Phe, and Ala119-Ser); d, L522 (Ser83-Phe and Glu139-Ala); e, L471, L607 (Ser83-Tyr), and L537 (Ser83-Tyr and Ala131-Ala); f, L21 (Asp87-Gly); g, L24 (Asp87-Gly and Asp144-Asp); h, L469 (Asp87-Gly and Ala131-Gly); i, L90 (Asp87-Tyr); j, L574 (Asp82-Asn); k, L4 (Ala119-Glu).

FIG. 2.

SSCP analysis of S. enterica strains. Lane 1, NCTC 74 (wild type); lane 2, L400 (pattern II); lane 3, L575 (pattern II); lane 4, L522 (pattern II); lane 5, L378 (pattern II); lane 6, L471 (pattern III); lane 7, L607 (pattern III); lane 8, L537 (pattern III); lane 9, L574 (pattern IV); lane 10, L21 (pattern V); lane 11, L24 (pattern V); lane 12, L469 (pattern V); lane 13, L90 (pattern VI); lane 14, L4 (pattern VII).