title: 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.
authors.affiliation: EA 3964, Faculté de Médecine de l'Université Paris 7, 46, rue Henri Huchard, 75870 Paris Cedex 18, France. elisabeth.aslangul@htd.aphp.fr
authors.forename: Elisabeth
authors.initials: E
authors.lastname: Aslangul
authors.forename: Laurent
authors.initials: L
authors.lastname: Massias
authors.forename: Alain
authors.initials: A
authors.lastname: Meulemans
authors.forename: Françoise
authors.initials: F
authors.lastname: Chau
authors.forename: Antoine
authors.initials: A
authors.lastname: Andremont
authors.forename: Patrice
authors.initials: P
authors.lastname: Courvalin
authors.forename: Bruno
authors.initials: B
authors.lastname: Fantin
authors.forename: Raymond
authors.initials: R
authors.lastname: Ruimy
mesh_terms: D000255:Adenosine Triphosphate / Q000235:genetics / Q000378:metabolism; D000617:Aminoglycosides / Q000378:metabolism; D000900:Anti-Bacterial Agents / Q000378:metabolism / Q000494:pharmacology*; D002463:Cell Membrane Permeability / Q000187:drug effects / Q000235:genetics*; D003001:Cloning, Molecular; D004269:DNA, Bacterial / Q000096:biosynthesis / Q000235:genetics; D004024:Dicyclohexylcarbodiimide / Q000494:pharmacology; D024881:Drug Resistance, Bacterial / Q000235:genetics / Q000502:physiology; D013293:Enterococcus faecalis / Q000187:drug effects* / Q000235:genetics* / Q000378:metabolism; D005839:Gentamicins / Q000378:metabolism / Q000494:pharmacology*; D007457:Iodine Radioisotopes; D007553:Isotope Labeling; D007700:Kinetics; D008826:Microbial Sensitivity Tests; D009154:Mutation; D006180:Proton-Translocating ATPases / Q000235:genetics; D012329:RNA, Bacterial / Q000096:biosynthesis / Q000235:genetics; D012336:RNA, Ribosomal, 16S / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction
chemical_list: D000617:Aminoglycosides; D000900:Anti-Bacterial Agents; D004269:DNA, Bacterial; D005839:Gentamicins; D007457:Iodine Radioisotopes; D012329:RNA, Bacterial; D012336:RNA, Ribosomal, 16S; D004024:Dicyclohexylcarbodiimide; D000255:Adenosine Triphosphate; D006180:Proton-Translocating ATPases