MUTATION IN QUINOLONE RESISTANCE DETERMINING REGIONS OF QUINOLONE TARGET GENES IN FLAVOBACTERIUM COLUMNARE ISOLATED FROM FRESHWATER FISH IN THAILAND

Abstract

Flavobacterium columnare is the causative agent of columnaris disease, which is seriously affected several freshwater fish species worldwide. Many kinds of antibiotics have been applied in fish farms for the treatment of columnaris disease in Thailand, especially quinolones. Thus, quinolone resistance (QR) in F. columnare should be monitored. The objectives of this study were to determine antimicrobial susceptibility and to detect the mutations in quinolone resistance-determining regions (QRDRs) of gyrA, gyrB, parC, and parE in F. columnare. Totally 50 F. columnare isolates from previous study (Dong et al., 2014) were examined. All isolates tested were sensitive to most routinely drugs used in aquaculture except 2 quinolones; nalidixic acid (NA) and oxolinic acid (OA), which performed the resistant results (14% for NA and 22% for OA). For minimum inhibitory concentration (MIC) of OA, out of 50 isolates, 9 were intermediate and 16 were resistant. The QRDRs of F. columnare were amplified by specific designed primers and sequenced. All OA-intermediate and -resistant isolates revealed novel double point mutations and amino acid substitutions in gyrA and parC: at position 83 in gyrA according to Escherichia coli system: Ser to Phe, Ser to Tyr (MIC=4 µg/ml), and Ser to Ala (MIC=8,16 µg/ml) while in parC at position 87: His to Tyr (MIC≥4 µg/ml). No mutation was detected in gyrB and parE. These results suggested that mutations in both gyrA and parC are the main QR mechanism and are considered as the major target of OA in F. columare. Morover, this is the first investigation of QR mechanism in F. columnare. However, other mechanisms should require further research.