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Updated: Jul 19, 2026
Mutagenesis and Functional Selection Protocols for Directed Evolution of Proteins in E. coli
Published on: March 16, 2011
In vitro induction and selection of fluoroquinolone-resistant mutants of Streptococcus pyogenes strains with multiple
Dewan S Billal1, Daniel P Fedorko, S Steve Yan
1Department of Otolaryngology-Head and Neck Surgery, Wakayama Medical University, 811-1 Kimiidera, Wakayama, 641-8510, Japan.
Objectives:
To perform a systematic analysis of point mutations in the quinolone resistance determining regions (QRDRs) of the DNA gyrase and topoisomerase genes of emm type 6 and other emm types of Streptococcus pyogenes strains after in vitro exposure to stepwise increasing concentrations of levofloxacin.
Methods:
Twelve parent strains of S. pyogenes, each with a different emm type, were chosen for stepwise exposure to increasing levels of levofloxacin followed by selection of resistant mutants. The QRDRs of gyrA, gyrB, parC and parE correlating to mutants with increased MICs were analysed for point mutations.
Results:
Multiple mutants with significantly increased MICs were generated from each strain. The amino acid substitutions identified were consistent regardless of emm type and were similar to the mechanisms of resistance reported in clinical isolates of S. pyogenes. The number of induction/selection cycles required for the emergence of key point mutations in gyrA and parC was variable among strains. For each parent-mutant set, when MIC increased, serine-81 of gyrA and serine-79 of parC were the primary targets for amino acid substitutions. No point mutations were found in the QRDRs of gyrB and parE in any of the resistant mutants sequenced.
Conclusions:
Despite its intrinsic polymorphism in the QRDR of parC, emm type 6 is not more likely to develop high-level resistance to fluoroquinolones when compared with other emm types. All emm types seem equally inducible to high-level fluoroquinolone resistance.
Insights
Streptococcus pyogenes strains, regardless of emm type, develop fluoroquinolone resistance through mutations in gyrA and parC genes. All emm types showed similar inducibility to high-level resistance, including emm type 6.
Area of Science:
- Microbiology
- Genetics
- Molecular Biology
Background:
- Fluoroquinolone antibiotics are crucial for treating Streptococcus pyogenes infections.
- Understanding resistance mechanisms is vital for effective treatment strategies.
- emm typing is a key factor in differentiating S. pyogenes strains.
Purpose of the Study:
- To analyze point mutations in quinolone resistance-determining regions (QRDRs) of DNA gyrase and topoisomerase genes.
- To investigate resistance development in emm type 6 and other emm types of S. pyogenes after levofloxacin exposure.
- To compare the susceptibility of different emm types to fluoroquinolone resistance.
Main Methods:
- Twelve S. pyogenes strains with different emm types were exposed to increasing levofloxacin concentrations.
- Resistant mutants were selected, and QRDRs of gyrA, gyrB, parC, and parE were analyzed for point mutations.
- Minimum Inhibitory Concentrations (MICs) were determined for parent strains and resistant mutants.
Main Results:
- Multiple resistant mutants with significantly increased MICs were generated from all tested strains.
- Key amino acid substitutions occurred at serine-81 of gyrA and serine-79 of parC.
- No mutations were observed in gyrB and parE QRDRs; resistance mechanisms were consistent across emm types.
Conclusions:
- emm type 6 does not exhibit increased susceptibility to fluoroquinolone resistance compared to other emm types.
- All emm types of S. pyogenes appear equally inducible to high-level fluoroquinolone resistance.
- The study highlights conserved resistance mechanisms in S. pyogenes, irrespective of emm type.
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