Skip Navigation


JAC Advance Access originally published online on September 13, 2006
Journal of Antimicrobial Chemotherapy 2007 59(6):1261-1264; doi:10.1093/jac/dkl380
This Article
Right arrow Abstract Freely available
Right arrow FREE Full Text (PDF) Freely available
Right arrow All Versions of this Article:
59/6/1261    most recent
dkl380v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in ISI Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Add to My Personal Archive
Right arrow Download to citation manager
Right arrow Search for citing articles in:
ISI Web of Science (2)
Right arrowRequest Permissions
Right arrow Disclaimer
Google Scholar
Right arrow Articles by Schumacher, A.
Right arrow Articles by Kern, W. V.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Schumacher, A.
Right arrow Articles by Kern, W. V.
Social Bookmarking
Add to CiteULike   Add to Connotea   Add to Del.icio.us  
What's this?

© The Author 2006. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved. For Permissions, please e-mail: journals.permissions@oxfordjournals.org

Special section: Efflux

Intracellular accumulation of linezolid in Escherichia coli, Citrobacter freundii and Enterobacter aerogenes: role of enhanced efflux pump activity and inactivation

Anja Schumacher1, Rainer Trittler2, Jürgen A. Bohnert1, Klaus Kümmerer3, Jean-Marie Pagès4 and Winfried V. Kern1,*

1 Center for Infectious Diseases and Travel Medicine, University Hospital Hugstetter Strasse 55, D-79106 Freiburg, Germany 2 Pharmacy Service, University Hospital Hugstetter Strasse 55, D-79106 Freiburg, Germany 3 Institute of Environmental Medicine and Hospital Epidemiology Breisacher Strasse 115B, D-79106 Freiburg, Germany 4 EA2197—Enveloppe Bactérienne, Perméabilité et Antibiotiques, IFR48, Faculté de Médecine, Université de la Méditerranée F-13385 Marseille Cédex 05, France

*Correspondence address. Medizinische Universitätsklinik, Hugstetter Strasse 55, D-79106 Freiburg, Germany. Tel: +49-761-270-1819; Fax: +49-761-270-1820; E-mail: kern{at}if-freiburg.de

Received 12 July 2006; returned 8 August 2006; revised 15 August 2006; accepted 21 August 2006


   Abstract
Top
 Abstract
Introduction
 Materials and methods
 Results and discussion
 Transparency declarations
 References
 
Objectives: 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.

Keywords: oxazolidinones , multidrug resistance , efflux pumps , Enterobacteriaceae


   Introduction
Top
 Abstract
 Introduction
Materials and methods
 Results and discussion
 Transparency declarations
 References
 
Linezolid is the first member of the oxazolidinone group of compounds, a class of antibiotics that are chemically unrelated to other currently used antibacterial drugs. Oxazolidinones target the 50S subunit of the prokaryotic ribosome and thereby prevent assembly of the initiation complex, which is a different mode of action from that of other inhibitors of protein synthesis such as chloramphenicol and macrolides. Although the ribosomes of Escherichia coli are as susceptible to linezolid as those of Gram-positive cocci, linezolid MICs for Gram-negative bacteria are higher than those for Gram-positive cocci and usually lie above the breakpoint for resistance.1 The activity of efflux pumps in Gram-negative bacteria appears to account for this finding: E. coli with inactivation of AcrAB, probably the most important RND (resistance–nodulation–cell division)-type multicomponent drug efflux pump in this species, is more susceptible to linezolid than cells with an intact AcrAB pump.2,3 A small number of non-lytic arylpiperidines have been shown to increase the accumulation of radiolabelled linezolid in wild-type E. coli cells, whereas such an effect was not seen with the putative efflux pump inhibitor phenylalanine-arginyl-ß-naphthylamide (PAßN) at a concentration that was effective in Pseudomonas aeruginosa.4 We recently demonstrated that 1-(1-naphthylmethyl)-piperazine (NMP), another putative efflux pump inhibitor, reduced the MIC of linezolid by ≥4-fold for E. coli, Citrobacter freundii, Enterobacter aerogenes and Acinetobacter baumannii.3,5–7 Although these findings suggest a role for RND-type efflux pumps in the susceptibility of Gram-negative bacteria to linezolid, confirmatory data showing corresponding differences in intracellular linezolid accumulation between Enterobacteriaceae with and without functional RND pump(s) and Gram-positive cocci are not available. In the present work, we used a specific HPLC assay to measure the intracellular linezolid concentration in E. coli, E. aerogenes and C. freundii strains with different efflux pump activity. The effects of NMP on the intracellular concentration of this drug were also assessed.


   Materials and methods
Top
 Abstract
 Introduction
 Materials and methods
Results and discussion
 Transparency declarations
 References
 
Bacterial strains

E. coli K-12 strains AG100, 3-AG100, 3-AG100MKX, 1-DC14PS and 2-DC14PS as well as E. aerogenes strains EAEP289 and EAEP298 have previously been described (Table 1).6,8–11 Reference strains of C. freundii (ATCC8090) and E. aerogenes (ATCC13048) were used in mutant selection experiments. After overnight incubation at 37°C in Luria–Bertani broth, cells were harvested and inocula of ~1010 cfu were plated on LB agar containing inhibitory concentrations (2–16 times the MIC) of levofloxacin. Plates were incubated for 24 h at 37°C. Three to six single colonies from each selecting concentration, if available, were purified on fluoroquinolone-supplemented agar plates and examined for antimicrobial susceptibility. We obtained C. freundii mutant 2-CF and E. aerogenes mutant 1-EA that showed a multidrug-resistant (MDR) phenotype not seen in the parental strains. 2-CF overexpressed acrB; 1-EA had an undefined acquired mechanism of MDR, presumably associated with increased efflux pump activity. We constructed CFASII0 by means of inactivation of acrB in 2-CF, using the {lambda}-based Red/ET homologous recombination system (Gene Bridges, Dresden, Germany).12


View this table:
[in this window]
[in a new window]

  		Table 1. Bacterial strains used in this study, with MICs of ethidium bromide (EtBr) and linezolid alone and after addition of NMP (100 mg/L); the results of pump gene expression studies [relative acrB or acrF (strain 2-DC14PS only) expression measured by quantitative RT–PCR, normalized for gapA expression] for selected strains are also shown

 
Susceptibility testing

Susceptibility to linezolid and ethidium bromide (EtBr) with or without NMP (final concentration, 100 mg/L) was studied by microbroth dilution with an inoculum of ~5 x 105 cfu/mL and overnight incubation (~18 h) at 37°C in LB broth.

Quantitative RT–PCR of acrB and acrF

Gene transcription studies were done as described previously with some modification.7 Cells were grown to mid-logarithmic phase, and RNA was isolated using the RNeasy Kit (Qiagen, Hilden, Germany). RNA samples were treated with DNase (Qiagen) on the column, quantified and stored at –80°C. An amount of 1 µg total RNA was reverse transcribed into cDNA using the iScriptTM cDNA Synthesis Kit (Bio-Rad, Hercules, CA, USA) with an incubation of 45 min at 42°C. cDNA was stored at –20°C. PCR was carried out with the LightCycler FastStart DNA Master SYBR Green I (Roche Diagnostics, Germany). Primers were designed with the webtool primer 3 (http://frodo.wi.mit.edu/cgi-bin/primer3/primer3_www.cgi). The primer sequences were: E. coli acrB, GAA CAA CTG GCG AGC AAA CT; E. coli acrF, AGT CTG AAA CCG TGG GAA GA; C. freundii acrB, TTA TCC CAA TGG CGT TCT TC; and E. aerogenes acrB, TCG CGT GAA GAA AGT GTA CG.

The 10 µL final volume contained 2.4 µL of MgCl2 (25 mM), 0.5 µM of each primer, 1 µL of cDNA extract and 1 µL of Lightcycler Mix including FastStart Taq DNA polymerase, reaction buffer, dNTP mix, SYBR Green I dye and 10 mM MgCl2. The PCR profile was as follows: denaturation at 95°C for 10 min and 30–40 cycles of 10 s at 95°C, 5 s at 55°C and 10 s at 72°C. Fluorescence was detected at the end of the 72°C segment in the PCR step (single mode). Expression of the housekeeping gene gapA (glyceraldehyde-3-phosphate dehydrogenase, forward primer sequence, CCA GAA CTT TGT TGG AGT AAC C; reverse primer sequence, AGC TTT AGC AGC ACC GGT A), was used as a relative standard.13 The results of at least two independent RNA extractions were interpreted using the Relative Expression Software Tool (REST).14

Whole cell drug accumulation assays

EtBr and pyronin Y accumulation was measured by spectrum fluorometry as described previously.5,6 For the estimation of the intracellular concentration of linezolid, cells were grown to logarithmic phase in LB broth at 37°C, washed in 50 mM sodium phosphate buffer (pH 7.0) and resuspended in the same buffer containing 0.4% glucose to an OD600 of 1.6–1.7. After an incubation of 10 min at 37°C, linezolid was added to a final concentration of 20 mg/L. At timed intervals, 1 mL samples were removed and centrifuged through silicone oil. The pellet was resuspended in 0.1 M glycine hydrochloride (pH 3.0). After overnight incubation at room temperature, samples were centrifuged and the supernatant was frozen until measurement. The HPLC analysis was performed following the method of Ehrlich et al.15 Column switching was not necessary. In case of interferences with the matrix, the peak identity was determined by liquid chromatography-tandem mass spectrometry (LC-MS/MS). Experiments included measurements after addition of carbonyl cyanide m-chlorophenylhydrazone (CCCP) (300 µM) or NMP (100 mg/L). For selected strains, the NMP concentration was doubled (200 mg/L). All tests were done in triplicate. Staphylococcus aureus ATCC 29123 and Enterococcus faecium ATCC 6057 were included as Gram-positive control strains in the linezolid accumulation assays.


   Results and discussion
Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
Transparency declarations
 References
 
MICs of linezolid (4–8 mg/L) and EtBr (2–16 mg/L) for efflux-deficient strains E. coli 1-DC14PS, E. aerogenes EAEP298 and C. freundii CFAS0 and CFASII0 were much lower than for wild-type strains or for strains overexpressing an RND-type efflux pump (Table 1). When NMP was added to either of the two drugs, MICs decreased ≥4-fold only for efflux-competent strains (Table 1). MICs in the presence of NMP were in all cases slightly higher than expected from efflux-deficient control strains. Thus, the transport inhibition by NMP at the concentration used was submaximal.

Accumulation data for the two dyes EtBr and pyronin Y and for linezolid are shown in Figures 1 and 2. Maximal accumulation of the three substances was observed in the efflux-incompetent strains, and lower intracellular concentrations were measured in the other test strains. E. coli wild-type strain AG100 accumulated less of the three substrates than the two other species, but in the case of linezolid and EtBr this did not perfectly correlate with the MICs. However, the relationship between accumulation in efflux-competent versus efflux-incompetent cells correlated well with the MICs for the test strains of a given species (Figures 1 and 2).


Figure 1
View larger version (36K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Figure 1. Whole cell accumulation of linezolid in E. coli, C. freundii and E. aerogenes with different levels of efflux pump activity. Linezolid (external concentration, 20 mg/L) was measured after 20 min (or 10 min after addition of NMP or CCCP) by an HPLC method. Shown are the means of triplicate experiments. Grey bars (first row) indicate accumulation without inhibitor, black bars (second row) represent accumulation levels in the presence of NMP and white bars (back row) represent accumulation levels in the presence of CCCP.

 


Figure 2
View larger version (42K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Figure 2. Whole cell accumulation of pyronin Y (upper panel) and EtBr (lower panel) in E. coli, C. freundii and E. aerogenes with different levels of efflux pump activity. Pyronin Y and EtBr were measured at 30 min by spectrum fluorimetry. The means of duplicate experiments are shown. Note that as intracellular pyronin Y accumulates and binds to RNA, the fluorescence is quenched; hence the fluorescence intensity inversely correlates with accumulation.

 
Addition of NMP and CCCP increased the intracellular concentration of linezolid (Figure 1). The increases with NMP were seen only in efflux-competent strains and were smaller than with CCCP, which is consistent with a submaximal effect of NMP, as seen in the MIC reduction assays. Alternatively, CCCP may have inhibited other pumps that depend on the proton-motive force and are able to bind and pump out linezolid, but are not inhibited as well by NMP. This is supported by the observation of linezolid accumulation levels increasing slightly with CCCP, even in efflux-‘incompetent’ cells. This effect was largest in Enterobacter. It is unknown whether the recently described eefABC efflux system which accommodates macrolides16 can explain this finding. In AG100 and 3-AG100 we used an NMP concentration of 200 mg/L, which resulted in a large increase (2-fold) in the linezolid accumulation (data not shown), but the levels were still slightly lower than those obtained after CCCP addition.

At an extracellular concentration of 20 mg/L the level of linezolid in the supernatant fluid after cell lysis ranged between 0.21 (E. coli 3-AG100 and 3-AG100MKX) and 0.35 mg/L (C. freundii CF). With the same assay conditions the intracellular linezolid concentration in the two Gram-positive control organisms was higher (0.41 mg/L in both S. aureus and E. faecium). Linezolid levels could be increased by pump inactivation or inhibition to >0.5 mg/L, which is beyond the levels probably typical for S. aureus and enterococci although the MICs did not fall to a level <4–8 mg/L. Thus, the correlation between the intracellular accumulation of and susceptibility to linezolid was not perfect; MICs were 0.5–2 mg/L for Gram-positive control strains. Currently, there is no plausible explanation for this finding.

Linezolid, like macrolides and the recently described peptide deformylase inhibitors, has a narrow spectrum of activity, typically including Gram-positive pathogens. The observation that intracellular levels of linezolid could be increased by efflux inhibition to levels higher than those found in the typical target organisms is, we believe, noteworthy. With the increasing understanding of the mechanism of pump action and substrate binding sites it might be possible to design molecules that are less sensitive to efflux and/or that more efficiently inhibit relevant efflux pumps in Gram-negative bacteria.17–19 Recycling of existing drugs, including narrow-spectrum drugs, for use in Gram-negative bacterial infections might then be a realistic goal.


   Transparency declarations
Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 Transparency declarations
References
 
None to declare.


   Acknowledgements
 
This study was supported in part by the Landesstiftung Baden-Württemberg. Pfizer provided a grant for HPLC measurements of linezolid.


   References
Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 Transparency declarations
 References
 
1 Livermore DM. Linezolid in vitro: mechanism and antibacterial spectrum. J Antimicrob Chemother (2003) 51(Suppl 2):ii9–16.[Abstract]

2 Buysse JM, Demyan WF, Dunyak DS, et al. Mutation of the AcrAB antibiotic efflux pump in Escherichia coli 12 confers susceptibility to oxazolidinone antibiotics. In: Program and Abstracts of the Thirty-sixth Interscience Conference on Antimicrobial Agents and Chemotherapy, 2003. Abstract C-42, p. 41. American Society for Microbiology, Washington, DC, USA.

3 Bohnert JA, Kern WV. Selected arylpiperazines are capable of reversing multidrug resistance in Escherichia coli overexpressing RND efflux pumps. Antimicrob Agents Chemother (2005) 49:849–52.[Abstract/Free Full Text]

4 Thorarensen A, Presley-Bodnar AL, Marotti KR, et al. 3-Arylpiperidines as potentiators of existing antibacterial agents. Bioorg Med Chem Lett (2001) 11:1903–6.[CrossRef][Medline]

5 Kern WV, Steinke P, Schumacher A, et al. Effect of 1-(1-naphthylmethyl)-piperazine, a novel putative efflux pump inhibitor, on antimicrobial drug susceptibility in clinical isolates of Escherichia coli. J Antimicrob Chemother (2006) 57:339–43.[Abstract/Free Full Text]

6 Schumacher A, Steinke P, Bohnert JA, et al. Effect of 1-(1-naphthylmethyl)-piperazine, a novel putative efflux pump inhibitor, on antimicrobial drug susceptibility in clinical isolates of Enterobacteriaceae other than Escherichia coli. J Antimicrob Chemother (2006) 57:344–8.[Abstract/Free Full Text]

7 Pannek S, Higgins PG, Steinke P, et al. Multidrug efflux inhibition in Acinetobacter baumannii: comparison between 1-(1-naphthylmethyl)-piperazine and phenyl-arginine-ß-naphthylamide. J Antimicrob Chemother (2006) 57:970–4.[Abstract/Free Full Text]

8 George AM, Levy SB. Amplifiable resistance to tetracycline, chloramphenicol, and other antibiotics in Escherichia coli: involvement of a non-plasmid-determined efflux of tetracycline. J Bacteriol (1983) 155:531–40.[Abstract/Free Full Text]

9 Kern WV, Oethinger M, Jellen-Ritter AS, et al. Non-target gene mutations in the development of fluoroquinolone resistance in Escherichia coli. Antimicrob Agents Chemother (2000) 44:814–20.[Abstract/Free Full Text]

10 Jellen-Ritter AS, Kern WV. Enhanced expression of the multidrug efflux pumps AcrAB and AcrEF associated with insertion element transposition in Escherichia coli mutants selected with a fluoroquinolone. Antimicrob Agents Chemother (2001) 45:1467–72.[Abstract/Free Full Text]

11 Pradel E, Pagès JM. The AcrAB-TolC efflux pump contributes to multidrug resistance in the nosocomial pathogen Enterobacter aerogenes. Antimicrob Agents Chemother (2002) 46:2640–3.[Abstract/Free Full Text]

12 Muyrers JPP, Zhang Y, Testa G, et al. Rapid modification of bacterial artificial chromosomes by ET-recombination. Nucleic Acids Res (1999) 27:1555–7.[Abstract/Free Full Text]

13 Wertz JE, Goldstone C, Gordon DM, et al. A molecular phylogeny of enteric bacteria and implications for a bacterial species concept. J Evol Biol (2003) 16:1236–48.[CrossRef][Web of Science][Medline]

14 Pfaffl MW, Horgan GW, Dempfle L. Relative expression software tool (REST) for group-wise comparison and statistical analysis of relative expression results in real-time PCR. Nucleic Acids Res (2002) 30:e36.[Abstract/Free Full Text]

15 Ehrlich M, Trittler R, Daschner FD, et al. A new and rapid method for monitoring the new oxazolidinone antibiotic linezolid in serum and urine by high performance liquid chromatography-integrated sample preparation. J Chromatogr B Biomed Sci Appl (2001) 755:373–7.[CrossRef][Medline]

16 Masi M, Pagès JM, Pradel E. Production of the cryptic EefABC efflux pump in Enterobacter aerogenes chloramphenicol-resistant mutants. J Antimicrob Chemother (2006) 57:1223–6.[Abstract/Free Full Text]

17 Piddock LJ. Clinically relevant chromosomally encoded multidrug resistance efflux pumps in bacteria. Clin Microbiol Rev (2006) 19:382–402.[Abstract/Free Full Text]

18 Pagès JM, Masi M, Barbe J. Inhibitors of efflux pumps in Gram-negative bacteria. Trends Mol Med (2005) 11:382–9.[CrossRef][Web of Science][Medline]

19 Lynch AS. Efflux systems in bacterial pathogens: an opportunity for therapeutic intervention? An industry view. Biochem Pharmacol (2006) 71:949–56.[CrossRef][Web of Science][Medline]


Add to CiteULike CiteULike   Add to Connotea Connotea   Add to Del.icio.us Del.icio.us    What's this?


This article has been cited by other articles:


Home page
 J Antimicrob ChemotherHome page
J. M. Sierra, M. Ortega, C. Tarrago, C. Albet, J. Vila, J. Terencio, and A. Guglietta
Decreased linezolid uptake in an in vitro-selected linezolid-resistant Staphylococcus epidermidis mutant
J. Antimicrob. Chemother., November 1, 2009; 64(5): 990 - 992.
[Abstract] [Full Text] [PDF]

Home page
 Antimicrob. Agents Chemother.Home page
K. L. LaPlante and G. Sakoulas
Evaluating Aztreonam and Ceftazidime Pharmacodynamics with Escherichia coli in Combination with Daptomycin, Linezolid, or Vancomycin in an In Vitro Pharmacodynamic Model
Antimicrob. Agents Chemother., October 1, 2009; 53(10): 4549 - 4555.
[Abstract] [Full Text] [PDF]

Home page
 J Antimicrob ChemotherHome page
L. Rodrigues, D. Wagner, M. Viveiros, D. Sampaio, I. Couto, M. Vavra, W. V. Kern, and L. Amaral
Thioridazine and chlorpromazine inhibition of ethidium bromide efflux in Mycobacterium avium and Mycobacterium smegmatis
J. Antimicrob. Chemother., May 1, 2008; 61(5): 1076 - 1082.
[Abstract] [Full Text] [PDF]

Home page
 Antimicrob. Agents Chemother.Home page
A. Perez, D. Canle, C. Latasa, M. Poza, A. Beceiro, M. del Mar Tomas, A. Fernandez, S. Mallo, S. Perez, F. Molina, et al.
Cloning, Nucleotide Sequencing, and Analysis of the AcrAB-TolC Efflux Pump of Enterobacter cloacae and Determination of Its Involvement in Antibiotic Resistance in a Clinical Isolate
Antimicrob. Agents Chemother., September 1, 2007; 51(9): 3247 - 3253.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow FREE Full Text (PDF) Freely available
Right arrow All Versions of this Article:
59/6/1261    most recent
dkl380v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in ISI Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Add to My Personal Archive
Right arrow Download to citation manager
Right arrow Search for citing articles in:
ISI Web of Science (2)
Right arrowRequest Permissions
Right arrow Disclaimer
Google Scholar
Right arrow Articles by Schumacher, A.
Right arrow Articles by Kern, W. V.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Schumacher, A.
Right arrow Articles by Kern, W. V.
Social Bookmarking
Add to CiteULike   Add to Connotea   Add to Del.icio.us  
What's this?