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Antimicrobial Agents and Chemotherapy, November 2006, p. 3615-3621, Vol. 50, No. 11
0066-4804/06/$08.00+0     doi:10.1128/AAC.00390-06
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

Acquired Gentamicin Resistance by Permeability Impairment in Enterococcus faecalis

Elisabeth Aslangul,^1* Laurent Massias,² Alain Meulemans,³ Françoise Chau,¹ Antoine Andremont,¹ Patrice Courvalin,⁴ Bruno Fantin,¹ and Raymond Ruimy¹

EA 3964, Faculté de Médecine de l'Université Paris 7, 75870 Paris Cedex 18,¹ Service de Pharmacie, Hôpital Bichat, 75018 Paris,² EA 3512, Faculté de Médecine de l'Université Paris 7, 75870 Paris Cedex 18,³ Unité des Agents Antibactériens, Institut Pasteur, 75724 Paris Cedex 15, France⁴

Received 30 March 2006/ Returned for modification 10 May 2006/ Accepted 31 July 2006

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 µg/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 F_oF₁-ATPase. However, no mutations were detected in the structural genes (atp) for the F_o 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.

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* Corresponding author. Mailing address: EA 3964, Faculté de Médecine de l'Université Paris 7, 46, rue Henri Huchard, 75870 Paris Cedex 18, France. Phone: 33 1 42 34 86 43. Fax: 33 1 42 34 88 85. E-mail: elisabeth.aslangul{at}htd.aphp.fr.

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Antimicrobial Agents and Chemotherapy, November 2006, p. 3615-3621, Vol. 50, No. 11
0066-4804/06/$08.00+0     doi:10.1128/AAC.00390-06
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

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