국내에서 분리된 세균성 이질균에서의 표적효소 변이와 efflux pump 관련 유전자 발현 증강으로 인한 quinolone계 항균제 내성에 대한 연구

초록

Purpose : In this work, we have determined the mechanism of quinolone resistance and demonstrated the presence of multidrug resistance efflux systems in S. flexneri and S. sonnei strains isolated in Korea. Methods : Two S. flexneri strains were isolated as resistant to fluoroquinolones, among 5938 clinical Shigella spp. isolates, based on the minimal inhibitory concentration(MIC) against ciprofloxacin and other fluorquinolones. The QRDR of gyrA, gyrB, parC, and parE genes was amplified from the two fluoroquinolone resistant isolates by PCR and the amplified products were directly sequenced. Synergy experiments were performed using fluoroquinolones and the efflux pump inhibitor carbonyl cyanide-m-chlorophenylhydrazone(CCCP). Expression level of some efflux pump encoding genes were determined by real-time PCR quantification, which was based on the relative expression level of acrA, acrB, mdfA, tolC, and ydhE versus a reference gene gyrB. Results : S. flexneri 021787 contained three substitutions, one in GyrA(Ser83Leu) and two in ParC(Ser80Ile and Arg91Gln). S. flexneri 021895 exhibited four substitutions, two in GyrA(Ser83Leu and Asp87Gly) and two in ParC(Ser80Ile and Arg91Gln). Increased susceptibility of S. flexneri 021787 and S. flexneri 021895 to CIP, NOR, and OFX in the presence of the uncoupler CCCP implied that energy-dependent active efflux pumps contributed to the resistance against fluoroquinolones. Both S. flexneri 021787 and 021895 isolates were also induced to express tolC(encoding a resistance-nodulation-division(RND) transporter), mdfA(encoding a major facilitator superfamily(MFS) transporter), and ydhE(encoding a multidrug and toxic compound extrusion(MATE) transporter) in the presence of ciprofloxacin. Increased expression was also observed, by constructing mdfA mutant, the role of the MdfA efflux system of S. flexneri displaying fluoroquinolone resistance or not displaying fluoroquinolone resistance. The mdfA mutant increased CIP and NOR susceptibility 1.3 fold and 3 fold, respectively. Conclusions : these results indicated that chromosome-mediated fluoroquinolone resistance of S. flexneri 021787 and S. flexneri 021895 resulted from the combination of target site mutations and an enhanced expression of genes encoding efflux pumps.

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