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Rifampin resistance in Neisseria meningitidis due to alterations in membrane permeability
F J Abadi1, P E Carter, P Cash
1Department of Medical Microbiology, University of Aberdeen Medical School, Scotland.
Abstract:
Rifampin-resistant (Rifr) Neisseria meningitidis strains are known to have single point mutations in the central conserved regions of the rpoB gene. We have demonstrated two distinct resistance phenotypes in strains with identical mutations in this region, an intermediate level of resistance in Rifr clinical isolates and a high level of resistance in mutants selected in vitro. The possible role of membrane permeability in the latter was investigated by measuring MICs in the presence of Tween 80; values for high-level-resistance mutants were reduced to intermediate levels, whereas those for intermediate-level-resistance strains were unaffected. The highly resistant mutants were also found to have increased resistance to Triton X-100 and gentian violet. Sequencing of the meningococcal mtrR gene and its promoter region (which determine resistance to hydrophobic agents in Neisseria gonorrhoeae) from susceptible or intermediate strains and highly resistant mutants generated from them showed no mutation within this region. Two-dimensional gel electrophoresis of two parent and Rif mutant strains showed identical shifts in the pI of one protein, indicating that differences between the parent and the highly Rifr mutant are not confined to the rpoB gene. These results indicate that both permeability and rpoB mutations play a role in determining the resistance of N. meningitidis to rifampin.
Insights
Rifampin resistance in Neisseria meningitidis involves both rpoB mutations and changes in membrane permeability. High-level resistance is linked to permeability, while intermediate resistance is primarily due to rpoB gene mutations.
Area of Science:
- Microbiology
- Molecular Biology
- Antimicrobial Resistance
Background:
- Rifampin-resistant (Rifr) Neisseria meningitidis strains typically exhibit point mutations in the rpoB gene.
- Distinct resistance levels (intermediate vs. high) were observed in clinical isolates and in vitro selected mutants with identical rpoB mutations.
Purpose of the Study:
- To investigate the mechanisms underlying differential rifampin resistance phenotypes in Neisseria meningitidis.
- To explore the role of membrane permeability and other genetic factors beyond rpoB in high-level rifampin resistance.
Main Methods:
- Minimum Inhibitory Concentration (MIC) assays with Tween 80 to assess membrane permeability effects.
- Testing resistance to other hydrophobic agents like Triton X-100 and gentian violet.
- Sequencing of the mtrR gene and its promoter region.
- Two-dimensional gel electrophoresis to analyze protein expression differences.
Main Results:
- Tween 80 reduced rifampin MICs in high-level resistant mutants to intermediate levels, suggesting a role for membrane permeability.
- High-level resistant mutants showed increased resistance to Triton X-100 and gentian violet.
- No mutations were found in the mtrR gene or its promoter region in highly resistant mutants.
- Two-dimensional gel electrophoresis revealed protein alterations beyond the rpoB gene in highly resistant mutants.
Conclusions:
- Both rpoB mutations and altered membrane permeability contribute to rifampin resistance in Neisseria meningitidis.
- Mechanisms conferring high-level resistance involve factors affecting membrane permeability, not solely rpoB mutations.