Abstract
Flavobacterium columnare is a Gram-negative bacterium which causes columnaris disease in freshwater fish species worldwide. Antibiotics, especially quinolone, is a primary therapeutic option to mitigate the economic loss caused by this disease. Phenotypic characterization and susceptibility testing were performed to identify quinolone resistance (QR)-associated genes in F. columnare isolated from freshwater cultured Asian sea bass. A total of 15 F. columnare isolates were collected from moribund fish that exhibited columnaris-like clinical signs and lesions including gill necrosis, saddleback lesion, and fin erosion. Phylogenetic analysis based on 16S rRNA and dnaK revealed that these isolates belonged to genetic group (GG) 2 and 4. Disk diffusion test results indicated that the 15 isolates were susceptible to all tested antibiotics except quinolones. Five and six isolates were resistant to oxolinic (OA) (33.33%) and nalidixic acid (NA) (40%) respectively. Broth microdilution indicated that highest OA, NA and enrofloxacin (ENR) minimum inhibitory concentration (MIC) values were 16, 64 and 1 μg/mL respectively. The MIC level to quinolone of GG 2 was higher for the GG 4 counterparts. Sequence analysis revealed a serine:phenylalanine amino acid substitution at position 83 (S83F) and a glutamine:histidine amino acid substitution at position 458 (Q458H) of quinolone resistance-determining regions (QRDRs) of gyrA and gyrB respectively. The Q458H in gyrB was identified as a novel non-synonymous mutation of the Flavobacteriaceae family. A genome-scale resistome analysis revealed that connections between GG and QR level potentially correlated with the presence or absence of chromosomal antimicrobial resistance genes and accumulated mutations in QRDRs. This study provide a guidance for choosing antibiotics for treatment columnaris disease and point out the important quinolone resistance mechanisms in F. columnare.
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