Acquired gentamicin resistance by permeability impairment in Enterococcus faecalis

Abstract

Enterococci are intrinsically resistant to low levels of aminoglycosides. We previously selected in vitro and in vivo Enterococcus faecalis with intermediate-level resistance to gentamicin that did not abolish synergism with a cell-wall-active agent (E. Aslangul et al., Antimicrob. Agents Chemother. 49:4144-4148, 2005). The aim of this study was to investigate the mechanism of resistance to gentamicin in the 1688-G3 third-step mutant (MIC, 512 microg/ml) of E. faecalis JH2-2. No mutations were found in the genes for L6 ribosomal protein and the four copies of 16S rRNA. Production of a known aminoglycoside-modifying enzyme was unlikely due to the distinct resistance phenotype and absence of the corresponding genes. Efflux was also unlikely since ethidium bromide MICs were similar for JH2-2 and 1688-G3 and since the pump inhibitors reserpine and verapamil had no effect on gentamicin resistance in both strains. To study gentamicin accumulation, we developed a nonisotopic method based on a fluorescent polarization immunoassay. Impaired gentamicin accumulation was observed in 1688-G3 compared to JH2-2 and was only partially reversible by the N,N'-dicyclohexylcarbodiimide (DCCD) uncoupler agent. The lower sensitivity of 1688-G3 to DCCD suggested alteration of the FoF1-ATPase. However, no mutations were detected in the structural genes (atp) for the Fo channel and no difference in transcript levels of atpB and atpE was found between 1688-G3 and JH2-2. Our data are compatible with acquisition of intermediate-level gentamicin resistance by uptake impairment in E. faecalis.

FIG. 1.

Gentamicin uptake by E. faecalis JH2-2 at a concentration of 32 (circles), 64 (squares), and 128 (triangles) μg/ml of gentamicin (A) and ¹²⁵I-gentamicin (B). The number of viable bacteria ranged from 7.5 × 10⁹ to 1.65 × 10¹⁰ for gentamicin at 32 μg/ml, from 2 × 10⁸ to 3.2 × 10⁹ for gentamicin at 64 μg/ml, and from 9.5 × 10⁹ to 1.55 × 10⁷ for gentamicin at 128 μg/ml.

FIG. 2.

Gentamicin uptake by E. faecalis JH2-2 and derivative E. faecalis 1688-G3 at various gentamicin concentrations (micrograms per milliliter) below the MIC of each strain.

FIG. 3.

Effect of 100 μM of DCCD on gentamicin uptake by E. faecalis JH2-2 (top) and mutant E. faecalis 1688-G3 (bottom) at a fixed gentamicin concentration of 64 μg/ml.