Open AccessAcquired Gentamicin Resistance by Permeability Impairment in Enterococcus faecalis
Author(s) -
E. Aslangul,
Laurent Massias,
A. Meulemans,
Françoise Chau,
Antoine Andremont,
Patrice Courvalin,
Bruno Fantin,
Raymond Ruimy
Publication year - 2006
Publication title -
antimicrobial agents and chemotherapy
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 2.07
H-Index - 259
eISSN - 1070-6283
pISSN - 0066-4804
DOI - 10.1128/aac.00390-06
Subject(s) - gentamicin , enterococcus faecalis , efflux , ethidium bromide , aminoglycoside , microbiology and biotechnology , biology , mutant , enterococcus , kanamycin , chemistry , biochemistry , escherichia coli , antibiotics , gene , dna
Enterococci are intrinsically resistant to low levels of aminoglycosides. We previously selected in vitro and in vivoEnterococcus 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 μg/ml) ofE. 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 theN ,N ′-dicyclohexylcarbodiimide (DCCD) uncoupler agent. The lower sensitivity of 1688-G3 to DCCD suggested alteration of the Fo F1 -ATPase. However, no mutations were detected in the structural genes (atp ) for the Fo channel and no difference in transcript levels ofatpB andatpE was found between 1688-G3 and JH2-2. Our data are compatible with acquisition of intermediate-level gentamicin resistance by uptake impairment inE. faecalis .