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Molecular cloning and analysis of Staphylococcus aureus chromosomal aminoglycoside resistance genes
Abstract:
Most of the aminoglycoside resistant Staphylococcus aureus strains isolated in France are resistant to all the antibiotics belonging to this family. Two aminoglycoside-modifying enzymes were detected in the wild-type strains studied: an APH3'III and an AAC6'-APH2". These strains also carry two types of streptomycin resistance: high-level resistance due to chromosomal mutation(s) affecting ribosome affinity and low-level resistance, the mechanism of which was not characterized. All the aminoglycoside resistance genes were located on the chromosome. DNA fragments of 1.5 and 1.95 kb carrying the aphA and aacA genes, respectively, were isolated, by cloning, from the cellular DNA of a clinical isolate. When these genes were introduced into Escherichia coli and Bacillus subtilis strains, the enzymes synthesized were indistinguishable from those produced by the S. aureus strains. When the cellular DNAs of wild-type and resistant strains were hybridized with the cloned fragments, sequences homologous to the fragment carrying the aphA gene were found to be located at the same chromosomal site, while those hybridizing with the fragment carrying the aacA gene were at different chromosomal sites.
Insights
Most Staphylococcus aureus strains in France exhibit broad aminoglycoside resistance. Researchers identified specific resistance genes, aphA and aacA, located on the chromosome, contributing to this widespread antibiotic resistance.
Area of Science:
- Microbiology
- Molecular Biology
- Genetics
Background:
- Aminoglycoside antibiotics are crucial for treating Staphylococcus aureus infections.
- Widespread resistance to aminoglycosides in S. aureus poses a significant clinical challenge.
- Understanding the genetic basis of this resistance is essential for developing effective treatment strategies.
Purpose of the Study:
- To investigate the mechanisms of aminoglycoside resistance in clinical isolates of Staphylococcus aureus from France.
- To identify and characterize the aminoglycoside resistance genes present in these strains.
- To determine the genetic location and transferability of these resistance determinants.
Main Methods:
- Phenotypic characterization of aminoglycoside resistance in S. aureus isolates.
- Detection and molecular analysis of aminoglycoside-modifying enzymes (APH3'-III and AAC6'-APH2).
- Cloning of resistance genes (aphA and aacA) and their introduction into heterologous hosts (E. coli, B. subtilis).
- Chromosomal mapping using DNA hybridization techniques.
Main Results:
- Most French S. aureus isolates displayed resistance to all aminoglycosides.
- Two key aminoglycoside-modifying enzymes, APH3'-III and AAC6'-APH2, were identified.
- Streptomycin resistance involved both high-level (chromosomal mutation) and low-level mechanisms.
- Aminoglycoside resistance genes (aphA and aacA) were chromosomally located.
- Cloned aphA and aacA genes conferred resistance when expressed in E. coli and B. subtilis.
- The aphA gene was found at a conserved chromosomal site, while aacA was located at variable sites.
Conclusions:
- The study elucidates the genetic basis of extensive aminoglycoside resistance in French S. aureus strains.
- Chromosomal location and specific resistance genes (aphA, aacA) contribute to the observed resistance patterns.
- The findings highlight the importance of understanding resistance mechanisms for effective antibiotic stewardship.