Skip to main content

Some NLM-NCBI services and products are experiencing heavy traffic, which may affect performance and availability. We apologize for the inconvenience and appreciate your patience. For assistance, please contact our Help Desk at info@ncbi.nlm.nih.gov.

Journal of Bacteriology logoLink to Journal of Bacteriology
. 1996 Jul;178(13):3791–3795. doi: 10.1128/jb.178.13.3791-3795.1996

Active efflux of fluoroquinolones in Mycobacterium smegmatis mediated by LfrA, a multidrug efflux pump.

J Liu 1, H E Takiff 1, H Nikaido 1
PMCID: PMC232638  PMID: 8682782

Abstract

The lfrA gene cloned from chromosomal DNA of quinolone-resistant Mycobacterium smegmatis mc2-552 conferred low-level resistance to fluoroquinolones when present on multicopy plasmids. Sequence analysis suggested that lfrA encodes a membrane efflux pump of the major facilitator family (H. E. Takiff, M. Cimino, M. C. Musso, T. Weisbrod, R. Martinez, M. B. Delgado, L Salazar, B. R. Bloom, and W. R. Jacbos, Jr., Proc. Natl. Acad. Sci. USA 93:362-366, 1996). In this work, we studied the role of LfrA in the accumulation of fluoroquinolones by M. smegmatis. The steady-state accumulation level of a hydrophilic quinolone, norfloxacin, by M. smegmatis harboring a plasmid carrying the lfrA gene was about 50% of that by the parent strain but was increased to the same level as that of the parent strain by addition of a proton conductor, carbonyl cyanide m-chorophenylhydrazone. Norfloxacin efflux mediated by LfrA was competed for strongly by ciprofloxacin but not by nalidixic acid. Furthermore, we showed that portions of norfloxacin accumulated by starved cells were pumped out upon reenergization of the cells, and the rates of this efflux showed evidence of saturation at higher intracellular concentrations of the drug. These results suggest that the LfrA polypeptide catalyzes the active efflux of several quinolones.

Full Text

The Full Text of this article is available as a PDF (260.6 KB).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Brennan P. J., Nikaido H. The envelope of mycobacteria. Annu Rev Biochem. 1995;64:29–63. doi: 10.1146/annurev.bi.64.070195.000333. [DOI] [PubMed] [Google Scholar]
  2. Bryan L. E., Bedard J. Impermeability to quinolones in gram-positive and gram-negative bacteria. Eur J Clin Microbiol Infect Dis. 1991 Apr;10(4):232–239. doi: 10.1007/BF01966995. [DOI] [PubMed] [Google Scholar]
  3. Cambau E., Sougakoff W., Besson M., Truffot-Pernot C., Grosset J., Jarlier V. Selection of a gyrA mutant of Mycobacterium tuberculosis resistant to fluoroquinolones during treatment with ofloxacin. J Infect Dis. 1994 Aug;170(2):479–483. doi: 10.1093/infdis/170.2.479. [DOI] [PubMed] [Google Scholar]
  4. Chamberland S., Bayer A. S., Schollaardt T., Wong S. A., Bryan L. E. Characterization of mechanisms of quinolone resistance in Pseudomonas aeruginosa strains isolated in vitro and in vivo during experimental endocarditis. Antimicrob Agents Chemother. 1989 May;33(5):624–634. doi: 10.1128/aac.33.5.624. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Cohen S. P., Hooper D. C., Wolfson J. S., Souza K. S., McMurry L. M., Levy S. B. Endogenous active efflux of norfloxacin in susceptible Escherichia coli. Antimicrob Agents Chemother. 1988 Aug;32(8):1187–1191. doi: 10.1128/aac.32.8.1187. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Frieden T. R., Sterling T., Pablos-Mendez A., Kilburn J. O., Cauthen G. M., Dooley S. W. The emergence of drug-resistant tuberculosis in New York City. N Engl J Med. 1993 Feb 25;328(8):521–526. doi: 10.1056/NEJM199302253280801. [DOI] [PubMed] [Google Scholar]
  7. Goble M., Iseman M. D., Madsen L. A., Waite D., Ackerson L., Horsburgh C. R., Jr Treatment of 171 patients with pulmonary tuberculosis resistant to isoniazid and rifampin. N Engl J Med. 1993 Feb 25;328(8):527–532. doi: 10.1056/NEJM199302253280802. [DOI] [PubMed] [Google Scholar]
  8. Grinius L. L., Goldberg E. B. Bacterial multidrug resistance is due to a single membrane protein which functions as a drug pump. J Biol Chem. 1994 Nov 25;269(47):29998–30004. [PubMed] [Google Scholar]
  9. Hashmi Z. S., Smith J. M. Outer membrane changes in quinolone resistant Pseudomonas aeruginosa. J Antimicrob Chemother. 1991 Sep;28(3):465–470. doi: 10.1093/jac/28.3.465. [DOI] [PubMed] [Google Scholar]
  10. Hooper D. C., Wolfson J. S., Souza K. S., Tung C., McHugh G. L., Swartz M. N. Genetic and biochemical characterization of norfloxacin resistance in Escherichia coli. Antimicrob Agents Chemother. 1986 Apr;29(4):639–644. doi: 10.1128/aac.29.4.639. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Ito H., Yoshida H., Bogaki-Shonai M., Niga T., Hattori H., Nakamura S. Quinolone resistance mutations in the DNA gyrase gyrA and gyrB genes of Staphylococcus aureus. Antimicrob Agents Chemother. 1994 Sep;38(9):2014–2023. doi: 10.1128/aac.38.9.2014. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Jacobs W. R., Jr, Kalpana G. V., Cirillo J. D., Pascopella L., Snapper S. B., Udani R. A., Jones W., Barletta R. G., Bloom B. R. Genetic systems for mycobacteria. Methods Enzymol. 1991;204:537–555. doi: 10.1016/0076-6879(91)04027-l. [DOI] [PubMed] [Google Scholar]
  13. Jarlier V., Nikaido H. Mycobacterial cell wall: structure and role in natural resistance to antibiotics. FEMS Microbiol Lett. 1994 Oct 15;123(1-2):11–18. doi: 10.1111/j.1574-6968.1994.tb07194.x. [DOI] [PubMed] [Google Scholar]
  14. Jarlier V., Nikaido H. Permeability barrier to hydrophilic solutes in Mycobacterium chelonei. J Bacteriol. 1990 Mar;172(3):1418–1423. doi: 10.1128/jb.172.3.1418-1423.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Levy S. B. Active efflux mechanisms for antimicrobial resistance. Antimicrob Agents Chemother. 1992 Apr;36(4):695–703. doi: 10.1128/aac.36.4.695. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Li X. Z., Livermore D. M., Nikaido H. Role of efflux pump(s) in intrinsic resistance of Pseudomonas aeruginosa: resistance to tetracycline, chloramphenicol, and norfloxacin. Antimicrob Agents Chemother. 1994 Aug;38(8):1732–1741. doi: 10.1128/aac.38.8.1732. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Liu J., Rosenberg E. Y., Nikaido H. Fluidity of the lipid domain of cell wall from Mycobacterium chelonae. Proc Natl Acad Sci U S A. 1995 Nov 21;92(24):11254–11258. doi: 10.1073/pnas.92.24.11254. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Masuda N., Sakagawa E., Ohya S. Outer membrane proteins responsible for multiple drug resistance in Pseudomonas aeruginosa. Antimicrob Agents Chemother. 1995 Mar;39(3):645–649. doi: 10.1128/AAC.39.3.645. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. McMurry L., Petrucci R. E., Jr, Levy S. B. Active efflux of tetracycline encoded by four genetically different tetracycline resistance determinants in Escherichia coli. Proc Natl Acad Sci U S A. 1980 Jul;77(7):3974–3977. doi: 10.1073/pnas.77.7.3974. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Mühlradt P. F., Menzel J., Golecki J. R., Speth V. Lateral mobility and surface density of lipopolysaccharide in the outer membrane of Salmonella typhimurium. Eur J Biochem. 1974 Apr 16;43(3):533–539. doi: 10.1111/j.1432-1033.1974.tb03440.x. [DOI] [PubMed] [Google Scholar]
  21. Nakamura S., Nakamura M., Kojima T., Yoshida H. gyrA and gyrB mutations in quinolone-resistant strains of Escherichia coli. Antimicrob Agents Chemother. 1989 Feb;33(2):254–255. doi: 10.1128/aac.33.2.254. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Neyfakh A. A., Borsch C. M., Kaatz G. W. Fluoroquinolone resistance protein NorA of Staphylococcus aureus is a multidrug efflux transporter. Antimicrob Agents Chemother. 1993 Jan;37(1):128–129. doi: 10.1128/aac.37.1.128. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Ng E. Y., Trucksis M., Hooper D. C. Quinolone resistance mediated by norA: physiologic characterization and relationship to flqB, a quinolone resistance locus on the Staphylococcus aureus chromosome. Antimicrob Agents Chemother. 1994 Jun;38(6):1345–1355. doi: 10.1128/aac.38.6.1345. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Nikaido H. Prevention of drug access to bacterial targets: permeability barriers and active efflux. Science. 1994 Apr 15;264(5157):382–388. doi: 10.1126/science.8153625. [DOI] [PubMed] [Google Scholar]
  25. Nikaido H., Thanassi D. G. Penetration of lipophilic agents with multiple protonation sites into bacterial cells: tetracyclines and fluoroquinolones as examples. Antimicrob Agents Chemother. 1993 Jul;37(7):1393–1399. doi: 10.1128/aac.37.7.1393. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Poole K., Krebes K., McNally C., Neshat S. Multiple antibiotic resistance in Pseudomonas aeruginosa: evidence for involvement of an efflux operon. J Bacteriol. 1993 Nov;175(22):7363–7372. doi: 10.1128/jb.175.22.7363-7372.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Robillard N. J., Scarpa A. L. Genetic and physiological characterization of ciprofloxacin resistance in Pseudomonas aeruginosa PAO. Antimicrob Agents Chemother. 1988 Apr;32(4):535–539. doi: 10.1128/aac.32.4.535. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Rouch D. A., Cram D. S., DiBerardino D., Littlejohn T. G., Skurray R. A. Efflux-mediated antiseptic resistance gene qacA from Staphylococcus aureus: common ancestry with tetracycline- and sugar-transport proteins. Mol Microbiol. 1990 Dec;4(12):2051–2062. doi: 10.1111/j.1365-2958.1990.tb00565.x. [DOI] [PubMed] [Google Scholar]
  29. Shen L. L., Mitscher L. A., Sharma P. N., O'Donnell T. J., Chu D. W., Cooper C. S., Rosen T., Pernet A. G. Mechanism of inhibition of DNA gyrase by quinolone antibacterials: a cooperative drug--DNA binding model. Biochemistry. 1989 May 2;28(9):3886–3894. doi: 10.1021/bi00435a039. [DOI] [PubMed] [Google Scholar]
  30. Sullivan E. A., Kreiswirth B. N., Palumbo L., Kapur V., Musser J. M., Ebrahimzadeh A., Frieden T. R. Emergence of fluoroquinolone-resistant tuberculosis in New York City. Lancet. 1995 May 6;345(8958):1148–1150. doi: 10.1016/s0140-6736(95)90980-x. [DOI] [PubMed] [Google Scholar]
  31. Takiff H. E., Cimino M., Musso M. C., Weisbrod T., Martinez R., Delgado M. B., Salazar L., Bloom B. R., Jacobs W. R., Jr Efflux pump of the proton antiporter family confers low-level fluoroquinolone resistance in Mycobacterium smegmatis. Proc Natl Acad Sci U S A. 1996 Jan 9;93(1):362–366. doi: 10.1073/pnas.93.1.362. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Takiff H. E., Salazar L., Guerrero C., Philipp W., Huang W. M., Kreiswirth B., Cole S. T., Jacobs W. R., Jr, Telenti A. Cloning and nucleotide sequence of Mycobacterium tuberculosis gyrA and gyrB genes and detection of quinolone resistance mutations. Antimicrob Agents Chemother. 1994 Apr;38(4):773–780. doi: 10.1128/aac.38.4.773. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Thanassi D. G., Suh G. S., Nikaido H. Role of outer membrane barrier in efflux-mediated tetracycline resistance of Escherichia coli. J Bacteriol. 1995 Feb;177(4):998–1007. doi: 10.1128/jb.177.4.998-1007.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Trias J., Benz R. Permeability of the cell wall of Mycobacterium smegmatis. Mol Microbiol. 1994 Oct;14(2):283–290. doi: 10.1111/j.1365-2958.1994.tb01289.x. [DOI] [PubMed] [Google Scholar]
  35. Yoshida H., Bogaki M., Nakamura S., Ubukata K., Konno M. Nucleotide sequence and characterization of the Staphylococcus aureus norA gene, which confers resistance to quinolones. J Bacteriol. 1990 Dec;172(12):6942–6949. doi: 10.1128/jb.172.12.6942-6949.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from Journal of Bacteriology are provided here courtesy of American Society for Microbiology (ASM)

RESOURCES