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Spontaneous mutation frequency and molecular mechanisms of Shigella flexneri fluoroquinolone resistance under antibiotic selective stress.

Abstract：

:The incidence of fluoroquinolone-resistant Shigella strains has risen rapidly, presumably in response to ciprofloxacin antibiotic stress. Understanding the molecular mechanisms underlying this resistance phenotype is critical to developing novel and effective therapeutic strategies. In this study, the frequency of ciprofloxacin-induced mutation was measured in antibiotic resistance genes (gyrA, gyrB, parC, parE, marOR, and marA) of Shigella flexneri. The S. flexneri 2a strain 301 was cultured on Luria-Bertani agar plates containing one of seven different ciprofloxacin concentrations (range: 0.03125-2 μg mL(-1)). Resistant colonies were selected for gene-targeted sequencing analysis; the identified point mutations were subsequently confirmed by insertion into antibiotic cassette plasmids and growth under ciprofloxacin stress. The results demonstrated that the seven different ciprofloxacin concentrations produced dose-dependent frequencies of spontaneous mutations: 10(-8) (0.03125 and 0.0625 μg mL(-1)), 10(-9) (0.125 μg mL(-1)), and <10(-9) (0.25, 0.5, 1, 2 μg mL(-1)). PCR sequencing of the ten randomly selected resistant colonies (minimum inhibitory concentrations (MICs) of 0.125 μg mL(-1), n = 5 and 0.25 μg mL(-1), n = 5) revealed that all colonies had mutations in the gyrA gene at either codon 83 (Ser83 → Leu) or 87 (Asp87 → Tyr or → Gly), both of which were confirmed at MIC of 0.125 μg mL(-1). None of the spontaneous mutation colonies exhibited gyrB, parC, parE, marOR, or marA mutations. In conclusion, S. flexneri is normomutable under ciprofloxacin antibiotic stress and fluoroquinolone resistance by spontaneous mutation occurs at a low rate. Codon mutations gyrA 83 and/or gyrA 87 cause a 4-fold increase in the ciprofloxacin MIC, and may represent the natural mechanism of fluoroquinolone resistance.

journal_name

World J Microbiol Biotechnol

journal_title

World journal of microbiology & biotechnology

authors

Pu XY,Zhang Q,Pan JC,Shen Z,Zhang W

doi

10.1007/s11274-012-1190-3

subject

Has Abstract

pub_date

2013-02-01 00:00:00

pages

365-71

issue

eissn

0959-3993

issn

1573-0972

journal_volume

pub_type

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Spontaneous mutation frequency and molecular mechanisms of Shigella flexneri fluoroquinolone resistance under antibiotic selective stress.