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Spontaneous mutation frequency and molecular mechanisms of Shigella flexneri fluoroquinolone resistance under antibiotic selective stress.
MedLine Citation:
PMID:  23070800     Owner:  NLM     Status:  Publisher    
Abstract/OtherAbstract:
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.
Authors:
Xiao-Ying Pu; Qijing Zhang; Jing-Cao Pan; Zhangqi Shen; Wei Zhang
Publication Detail:
Type:  JOURNAL ARTICLE     Date:  2012-10-16
Journal Detail:
Title:  World journal of microbiology & biotechnology     Volume:  -     ISSN:  1573-0972     ISO Abbreviation:  World J. Microbiol. Biotechnol.     Publication Date:  2012 Oct 
Date Detail:
Created Date:  2012-10-16     Completed Date:  -     Revised Date:  -    
Medline Journal Info:
Nlm Unique ID:  9012472     Medline TA:  World J Microbiol Biotechnol     Country:  -    
Other Details:
Languages:  ENG     Pagination:  -     Citation Subset:  -    
Affiliation:
Microbiology Laboratory, Hangzhou Center for Disease Control and Prevention, Hangzhou, 310021, People's Republic of China, pxygym@126.com.
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