Intracellular accumulation of linezolid in Escherichia coli, Citrobacter freundii and Enterobacter aerogenes: role of enhanced efflux pump activity and inactivation.
OBJECTIVE
The oxazolidinone class of antibiotics such as linezolid have a narrow spectrum of activity that targets Gram-positive bacteria. We hypothesized that the poor activity of linezolid in Gram-negative bacteria is in part caused by relatively low intracellular concentration due to efflux.


METHODS
Using whole cell accumulation assays we estimated the intracellular concentration of linezolid in Escherichia coli and other Enterobacteriaceae. We included test strains with enhanced RND-type multidrug efflux pump activity and with genetic inactivation of the pump or functional inhibition by carbonyl cyanide m-chlorophenylhydrazone as inhibitor of the proton motive force or 1-(1-naphthylmethyl)-piperazine (NMP), an efflux pump inhibitor.


RESULTS
Consistent with susceptibility studies, enhanced pump activity caused decreased accumulation, and pump inactivation and inhibition caused increased accumulation, of linezolid. The accumulation levels in test strains of E. coli, Citrobacter freundii and Enterobacter aerogenes with functional pumps were lower than in control strains of Staphylococcus aureus and Enterococcus faecium, but were higher after pump inactivation and correlated with ethidium bromide and pyronin Y accumulation.


CONCLUSIONS
The intracellular concentration of linezolid is comparatively low owing to efficient efflux of the drug and could be increased substantially by inhibition of RND-type efflux pumps.
Schumacher
Anja
A
Center for Infectious Diseases and Travel Medicine, University Hospital Hugstetter Strasse 55, Freiburg, Germany.
Trittler
Rainer
R
Bohnert
Jürgen A
JA
Kümmerer
Klaus
K
Pagès
Jean-Marie
JM
Kern
Winfried V
WV
D018435:ATP Binding Cassette Transporter, Subfamily B / Q000037:antagonists & inhibitors* / Q000378:metabolism*; D000081:Acetamides / Q000378:metabolism*; D000900:Anti-Bacterial Agents / Q000378:metabolism* / Q000494:pharmacology*; D002258:Carbonyl Cyanide m-Chlorophenyl Hydrazone / Q000494:pharmacology; D002851:Chromatography, High Pressure Liquid; D016971:Citrobacter freundii / Q000378:metabolism*; D004396:Coloring Agents; D021902:Enterobacter aerogenes / Q000378:metabolism*; D004926:Escherichia coli / Q000378:metabolism*; D004996:Ethidium; D005470:Fluorometry; D000069349:Linezolid; D008826:Microbial Sensitivity Tests; D023303:Oxazolidinones / Q000378:metabolism*; D011754:Pyronine
D018435:ATP Binding Cassette Transporter, Subfamily B; D000081:Acetamides; D000900:Anti-Bacterial Agents; D004396:Coloring Agents; D023303:Oxazolidinones; D002258:Carbonyl Cyanide m-Chlorophenyl Hydrazone; D004996:Ethidium; D000069349:Linezolid; D011754:Pyronine