Streptococcus spp.의 quinolone 내성유전자 클로닝과 특성 비교

Abstract

Quinolone is one of the current drugs of choice for treating fishes infected by Streptococcus spp. in Asian countries. To evaluate the importance of quinolone resistant gene, we cloned full length of genes encoding gyrA (2478 bp), gyrB (1953 bp), parC (2460 bp) and parE (1950 bp) were cloned from S. iniae by amplification with degenerate primers of the quinolone-resistance-determining region (QRDR), followed by DNA walking. Cloned those four genes, were preceded putative promoter, ribosome binding site. The two topoisomerase genes, parC/parE are contiguous in the chromosome of S. iniae and many other Gram positive bacteria. To investigate the mechanism of DNA gyrase and topoisomerase effects at the molecular levels, we examined point mutation of QRDR of quinolone-resistant isolates and in vitro mutants. Ciprofloxacin-resistant isolates of S. parauberis had mutations affecting amino acid residues of the quinolone resistance-determining region of parC. Mutations were found in residue positions equivalent to the serine at position 79 of the parC gene. The ciprofloxacin MICs of the isolates were 4 and 8 ㎍/㎖, respectively. However, altered QRDR region of parE(Arg449→Ser) followed parC(Ser79→Ile) mutation were detected in the mutants of S. parauberis selected in vitro with high concentration of ciprofloxacin. Additionally L. garvieae mutants resistant to ciprofloxacin carried parC mutations of Ser79 to Ile, Arg sequencely with gyrA mutations of Glu85 to Lys. None of the mutants exhibited mutations in gyrB, and could be suggested that parC and might be parE are involving more critically for quinolone resistance than gyrA, gyrB in these species. Thus, mutation of the parC or parE gene before change in the gyrase genes take place, suggesting that topoisomerase Ⅳ is the primary ciprofloxacin target in Streptococcus spp. Additionally in this study describes that HRM assay capable of detecting the dominant mutation confer ciprofloxacin resistance. These rapid and reliable assays may provide a convenient method to conduct surveillance for genetic mutations conferring antibiotic resistance.