Acquired gentamicin resistance by permeability impairment in Enterococcus faecalis

Area of Science:

• Microbiology
• Molecular Biology
• Antimicrobial Resistance

Background:

• Enterococci exhibit intrinsic resistance to low aminoglycoside levels.
• Previous studies identified Enterococcus faecalis with intermediate gentamicin resistance.
• Synergism with cell-wall-active agents was maintained in previously selected mutants.

Purpose of the Study:

• To elucidate the resistance mechanism of a high-level gentamicin-resistant Enterococcus faecalis mutant (1688-G3).
• To investigate potential genetic mutations, enzyme activity, efflux pumps, and drug accumulation in the resistant strain.

Main Methods:

• Genomic analysis for mutations in ribosomal protein L6 and 16S rRNA genes.
• Phenotypic screening for aminoglycoside-modifying enzymes and efflux pump activity using ethidium bromide and pump inhibitors.
• Development and application of a fluorescent polarization immunoassay to quantify gentamicin accumulation.
• Assessment of FoF1-ATPase activity using N,N'-dicyclohexylcarbodiimide (DCCD) and analysis of atp gene expression.

Main Results:

• No mutations were found in L6 ribosomal protein or 16S rRNA genes.
• Aminoglycoside-modifying enzymes and efflux pumps were ruled out as primary resistance mechanisms.
• Gentamicin accumulation was significantly impaired in the 1688-G3 mutant compared to the wild-type strain.
• Impaired accumulation was partially reversible by DCCD, suggesting a potential role for the FoF1-ATPase, though its structural genes showed no mutations.

Conclusions:

• The primary mechanism for high-level gentamicin resistance in this Enterococcus faecalis mutant is impaired gentamicin uptake.
• Alterations in cellular processes, potentially related to the FoF1-ATPase, contribute to reduced drug accumulation.
• Findings highlight uptake impairment as a novel mechanism for aminoglycoside resistance in enterococci.