Related Experiment Videos
High-level mupirocin resistance in Staphylococcus aureus: evidence for two distinct isoleucyl-tRNA synthetases
J Gilbart1, C R Perry, B Slocombe
1SmithKline Beecham Pharmaceuticals, Betchworth, Surrey, United Kingdom.
Abstract:
Mupirocin resistance in Staphylococcus aureus results from changes in the target enzyme, isoleucyl-tRNA synthetase (IRS). Twelve strains of S. aureus comprising four susceptible (MICs < or = 4 micrograms/ml), four intermediate level-resistant (MICs between 8 and 256 micrograms/ml), and four highly resistant (MICs > or = 512 micrograms/ml) isolates were examined for their IRS content and the presence of a gene known to encode high-level mupirocin resistance. Ion-exchange chromatography of cell extracts showed a single IRS active peak in mupirocin-susceptible strains, with 50% inhibitory concentrations (IC50s) of 0.7 to 3.0 ng of mupirocin per ml. In strains showing intermediate mupirocin resistance, similar single IRS activity peaks were observed, but these were less sensitive to inhibition, and the mupirocin IC50s for them were 19 to 43 ng/ml. Strains that were highly resistant to mupirocin displayed two distinct peaks; one was similar to that found with susceptible strains (IC50, 0.9 to 2.5 ng/ml), but an additional peak with an IC50 of 7,000 to 10,000 ng/ml was also observed. A strain cured of the plasmid encoding high-level mupirocin resistance lacked the resistant IRS peak. Restriction digests, produced by endonuclease NcoI, of total bacterial DNA isolated from the highly resistant strains hybridized with a mupirocin resistance gene probe, whereas DNA isolated from the intermediate level-resistant and susceptible strains did not. These results demonstrate that two different IRS enzymes were present in highly mupirocin-resistant S. aureus strains. In strains expressing intermediate levels of resistance, only a chromosomally encoded IRS which was inhibited less by mupirocin than IRS from fully susceptible strains was detected.
Insights
Mupirocin resistance in Staphylococcus aureus involves changes in the target enzyme, isoleucyl-tRNA synthetase (IRS). High resistance is linked to two IRS enzymes, while intermediate resistance shows a less sensitive chromosomally encoded IRS.
Area of Science:
- Microbiology
- Molecular Biology
- Biochemistry
Background:
- Mupirocin is a key antibiotic for treating Staphylococcus aureus infections.
- Increasing mupirocin resistance in S. aureus poses a significant clinical challenge.
- Resistance mechanisms often involve alterations in the target enzyme, isoleucyl-tRNA synthetase (IRS).
Purpose of the Study:
- To investigate the characteristics of isoleucyl-tRNA synthetase (IRS) in Staphylococcus aureus strains with varying levels of mupirocin resistance.
- To identify genetic elements associated with high-level mupirocin resistance in S. aureus.
Main Methods:
- Analysis of twelve S. aureus strains with susceptible, intermediate, and high mupirocin resistance.
- Ion-exchange chromatography to assess IRS content and activity.
- Enzyme inhibition assays using mupirocin to determine IC50 values.
- Plasmid curing experiments and DNA hybridization with a mupirocin resistance gene probe.
Main Results:
- Susceptible and intermediate-resistant strains exhibited a single IRS peak with varying sensitivity to mupirocin.
- Highly resistant strains displayed two distinct IRS peaks: one susceptible and one highly resistant (IC50 7,000-10,000 ng/ml).
- A plasmid-encoded resistant IRS was identified in highly resistant strains, absent in susceptible and intermediate strains.
- DNA hybridization confirmed the presence of a specific mupirocin resistance gene in highly resistant isolates only.
Conclusions:
- High-level mupirocin resistance in S. aureus is associated with the presence of two distinct IRS enzymes, one of which is plasmid-encoded and highly resistant.
- Intermediate mupirocin resistance is linked to a chromosomally encoded IRS with reduced sensitivity to the antibiotic.
- Understanding these resistance mechanisms is crucial for effective S. aureus infection management.