Skip to main content
PMC home page
Journal of Bacteriology logoLink to Journal of Bacteriology
. 1993 Nov;175(22):7363–7372. doi: 10.1128/jb.175.22.7363-7372.1993

Multiple antibiotic resistance in Pseudomonas aeruginosa: evidence for involvement of an efflux operon.

K Poole 1, K Krebes 1, C McNally 1, S Neshat 1
PMCID: PMC206881  PMID: 8226684

Abstract

An outer membrane protein of 50 kDa (OprK) was overproduced in a siderophore-deficient mutant of Pseudomonas aeruginosa capable of growth on iron-deficient minimal medium containing 2,2'-dipyridyl (0.5 mM). The expression of OprK in the mutant (strain K385) was associated with enhanced resistance to a number of antimicrobial agents, including ciprofloxacin, nalidixic acid, tetracycline, chloramphenicol, and streptonigrin. OprK was inducible in the parent strain by growth under severe iron limitation, as provided, for example, by the addition of dipyridyl or ZnSO4 to the growth medium. The gene encoding OprK (previously identified as ORFC) forms part of an operon composed of three genes (ORFABC) implicated in the secretion of the siderophore pyoverdine. Mutants defective in ORFA, ORFB, or ORFC exhibited enhanced susceptibility to tetracycline, chloramphenicol, ciprofloxacin, streptonigrin, and dipyridyl, consistent with a role for the ORFABC operon in multiple antibiotic resistance in P. aeruginosa. Sequence analysis of ORFC (oprK) revealed that its product is homologous to a class of outer membrane proteins involved in export. Similarly, the products of ORFA and ORFB exhibit homology to previously described bacterial export proteins located in the cytoplasmic membrane. These data suggest that ORFA-ORFB-oprK (ORFC)-dependent drug efflux contributes to multiple antibiotic resistance in P. aeruginosa. We propose, therefore, the designation mexAB (multiple efflux) for ORFAB.

Full text

PDF
7363

Images in this article

7365Image
on p.7365
7366Image
on p.7366
7369Image
on p.7369

Selected References

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

  1. Altschul S. F., Gish W., Miller W., Myers E. W., Lipman D. J. Basic local alignment search tool. J Mol Biol. 1990 Oct 5;215(3):403–410. doi: 10.1016/S0022-2836(05)80360-2. [DOI] [PubMed] [Google Scholar]
  2. Ankenbauer R., Hanne L. F., Cox C. D. Mapping of mutations in Pseudomonas aeruginosa defective in pyoverdin production. J Bacteriol. 1986 Jul;167(1):7–11. doi: 10.1128/jb.167.1.7-11.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Baev N., Endre G., Petrovics G., Banfalvi Z., Kondorosi A. Six nodulation genes of nod box locus 4 in Rhizobium meliloti are involved in nodulation signal production: nodM codes for D-glucosamine synthetase. Mol Gen Genet. 1991 Aug;228(1-2):113–124. doi: 10.1007/BF00282455. [DOI] [PubMed] [Google Scholar]
  4. Berry D., Kropinski A. M. Effect of lipopolysaccharide mutations and temperature on plasmid transformation efficiency in Pseudomonas aeruginosa. Can J Microbiol. 1986 May;32(5):436–438. doi: 10.1139/m86-082. [DOI] [PubMed] [Google Scholar]
  5. Bochner B. R., Huang H. C., Schieven G. L., Ames B. N. Positive selection for loss of tetracycline resistance. J Bacteriol. 1980 Aug;143(2):926–933. doi: 10.1128/jb.143.2.926-933.1980. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Chakrabarty A. M., Roy S. C. Effect of trace elements on the production of pigments by a pseudomonad. Biochem J. 1964 Nov;93(2):228–231. doi: 10.1042/bj0930228. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. 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]
  8. Cohen S. P., McMurry L. M., Hooper D. C., Wolfson J. S., Levy S. B. Cross-resistance to fluoroquinolones in multiple-antibiotic-resistant (Mar) Escherichia coli selected by tetracycline or chloramphenicol: decreased drug accumulation associated with membrane changes in addition to OmpF reduction. Antimicrob Agents Chemother. 1989 Aug;33(8):1318–1325. doi: 10.1128/aac.33.8.1318. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Cullmann W., Stieglitz M., Baars B., Opferkuch W. Comparative evaluation of recently developed quinolone compounds--with a note on the frequency of resistant mutants. Chemotherapy. 1985;31(1):19–28. doi: 10.1159/000238309. [DOI] [PubMed] [Google Scholar]
  10. Curtis N. A., Eisenstadt R. L., East S. J., Cornford R. J., Walker L. A., White A. J. Iron-regulated outer membrane proteins of Escherichia coli K-12 and mechanism of action of catechol-substituted cephalosporins. Antimicrob Agents Chemother. 1988 Dec;32(12):1879–1886. doi: 10.1128/aac.32.12.1879. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Daikos G. L., Lolans V. T., Jackson G. G. Alterations in outer membrane proteins of Pseudomonas aeruginosa associated with selective resistance to quinolones. Antimicrob Agents Chemother. 1988 May;32(5):785–787. doi: 10.1128/aac.32.5.785. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Darzins A., Casadaban M. J. In vivo cloning of Pseudomonas aeruginosa genes with mini-D3112 transposable bacteriophage. J Bacteriol. 1989 Jul;171(7):3917–3925. doi: 10.1128/jb.171.7.3917-3925.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Dean C. R., Poole K. Cloning and characterization of the ferric enterobactin receptor gene (pfeA) of Pseudomonas aeruginosa. J Bacteriol. 1993 Jan;175(2):317–324. doi: 10.1128/jb.175.2.317-324.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Fellay R., Frey J., Krisch H. Interposon mutagenesis of soil and water bacteria: a family of DNA fragments designed for in vitro insertional mutagenesis of gram-negative bacteria. Gene. 1987;52(2-3):147–154. doi: 10.1016/0378-1119(87)90041-2. [DOI] [PubMed] [Google Scholar]
  15. Feng D. F., Johnson M. S., Doolittle R. F. Aligning amino acid sequences: comparison of commonly used methods. J Mol Evol. 1984;21(2):112–125. doi: 10.1007/BF02100085. [DOI] [PubMed] [Google Scholar]
  16. Fukuda H., Hosaka M., Hirai K., Iyobe S. New norfloxacin resistance gene in Pseudomonas aeruginosa PAO. Antimicrob Agents Chemother. 1990 Sep;34(9):1757–1761. doi: 10.1128/aac.34.9.1757. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Glaser P., Sakamoto H., Bellalou J., Ullmann A., Danchin A. Secretion of cyclolysin, the calmodulin-sensitive adenylate cyclase-haemolysin bifunctional protein of Bordetella pertussis. EMBO J. 1988 Dec 1;7(12):3997–4004. doi: 10.1002/j.1460-2075.1988.tb03288.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Guthmiller J. M., Kraig E., Cagle M. P., Kolodrubetz D. Sequence of the lktD gene from Actinobacillus actinomycetemcomitans. Nucleic Acids Res. 1990 Sep 11;18(17):5292–5292. doi: 10.1093/nar/18.17.5292. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Haas B., Kraut J., Marks J., Zanker S. C., Castignetti D. Siderophore presence in sputa of cystic fibrosis patients. Infect Immun. 1991 Nov;59(11):3997–4000. doi: 10.1128/iai.59.11.3997-4000.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Hancock R. E., Nikaido H. Outer membranes of gram-negative bacteria. XIX. Isolation from Pseudomonas aeruginosa PAO1 and use in reconstitution and definition of the permeability barrier. J Bacteriol. 1978 Oct;136(1):381–390. doi: 10.1128/jb.136.1.381-390.1978. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Hancock R. E., Poole K., Benz R. Outer membrane protein P of Pseudomonas aeruginosa: regulation by phosphate deficiency and formation of small anion-specific channels in lipid bilayer membranes. J Bacteriol. 1982 May;150(2):730–738. doi: 10.1128/jb.150.2.730-738.1982. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Hancock R. E., Siehnel R., Martin N. Outer membrane proteins of Pseudomonas. Mol Microbiol. 1990 Jul;4(7):1069–1075. doi: 10.1111/j.1365-2958.1990.tb00680.x. [DOI] [PubMed] [Google Scholar]
  23. Hirai K., Aoyama H., Suzue S., Irikura T., Iyobe S., Mitsuhashi S. Isolation and characterization of norfloxacin-resistant mutants of Escherichia coli K-12. Antimicrob Agents Chemother. 1986 Aug;30(2):248–253. doi: 10.1128/aac.30.2.248. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Hirai K., Suzue S., Irikura T., Iyobe S., Mitsuhashi S. Mutations producing resistance to norfloxacin in Pseudomonas aeruginosa. Antimicrob Agents Chemother. 1987 Apr;31(4):582–586. doi: 10.1128/aac.31.4.582. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Hooper D. C., Wolfson J. S., Ng E. Y., Swartz M. N. Mechanisms of action of and resistance to ciprofloxacin. Am J Med. 1987 Apr 27;82(4A):12–20. [PubMed] [Google Scholar]
  26. Hooper D. C., Wolfson J. S., Souza K. S., Ng E. Y., McHugh G. L., Swartz M. N. Mechanisms of quinolone resistance in Escherichia coli: characterization of nfxB and cfxB, two mutant resistance loci decreasing norfloxacin accumulation. Antimicrob Agents Chemother. 1989 Mar;33(3):283–290. doi: 10.1128/aac.33.3.283. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Huyer M., Page W. J. Ferric reductase activity in Azotobacter vinelandii and its inhibition by Zn2+. J Bacteriol. 1989 Jul;171(7):4031–4037. doi: 10.1128/jb.171.7.4031-4037.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Huyer M., Page W. J. Zn Increases Siderophore Production in Azotobacter vinelandii. Appl Environ Microbiol. 1988 Nov;54(11):2625–2631. doi: 10.1128/aem.54.11.2625-2631.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Jacoby G. H., Young K. D. Cell cycle-independent lysis of Escherichia coli by cefsulodin, an inhibitor of penicillin-binding proteins 1a and 1b. J Bacteriol. 1991 Jan;173(1):1–5. doi: 10.1128/jb.173.1.1-5.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Kaatz G. W., Seo S. M. Mechanism of ciprofloxacin resistance in Pseudomonas aeruginosa. J Infect Dis. 1988 Sep;158(3):537–541. doi: 10.1093/infdis/158.3.537. [DOI] [PubMed] [Google Scholar]
  31. Klein J. R., Henrich B., Plapp R. Molecular analysis and nucleotide sequence of the envCD operon of Escherichia coli. Mol Gen Genet. 1991 Nov;230(1-2):230–240. doi: 10.1007/BF00290673. [DOI] [PubMed] [Google Scholar]
  32. Legakis N. J., Tzouvelekis L. S., Makris A., Kotsifaki H. Outer membrane alterations in multiresistant mutants of Pseudomonas aeruginosa selected by ciprofloxacin. Antimicrob Agents Chemother. 1989 Jan;33(1):124–127. doi: 10.1128/aac.33.1.124. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Liesegang H., Lemke K., Siddiqui R. A., Schlegel H. G. Characterization of the inducible nickel and cobalt resistance determinant cnr from pMOL28 of Alcaligenes eutrophus CH34. J Bacteriol. 1993 Feb;175(3):767–778. doi: 10.1128/jb.175.3.767-778.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Lugtenberg B., Meijers J., Peters R., van der Hoek P., van Alphen L. Electrophoretic resolution of the "major outer membrane protein" of Escherichia coli K12 into four bands. FEBS Lett. 1975 Oct 15;58(1):254–258. doi: 10.1016/0014-5793(75)80272-9. [DOI] [PubMed] [Google Scholar]
  35. Masuda N., Ohya S. Cross-resistance to meropenem, cephems, and quinolones in Pseudomonas aeruginosa. Antimicrob Agents Chemother. 1992 Sep;36(9):1847–1851. doi: 10.1128/aac.36.9.1847. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Michéa-Hamzehpour M., Auckenthaler R., Regamey P., Pechère J. C. Resistance occurring after fluoroquinolone therapy of experimental Pseudomonas aeruginosa peritonitis. Antimicrob Agents Chemother. 1987 Nov;31(11):1803–1808. doi: 10.1128/aac.31.11.1803. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Needleman S. B., Wunsch C. D. A general method applicable to the search for similarities in the amino acid sequence of two proteins. J Mol Biol. 1970 Mar;48(3):443–453. doi: 10.1016/0022-2836(70)90057-4. [DOI] [PubMed] [Google Scholar]
  38. Nies D. H., Nies A., Chu L., Silver S. Expression and nucleotide sequence of a plasmid-determined divalent cation efflux system from Alcaligenes eutrophus. Proc Natl Acad Sci U S A. 1989 Oct;86(19):7351–7355. doi: 10.1073/pnas.86.19.7351. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Nikaido H., Rosenberg E. Y. Cir and Fiu proteins in the outer membrane of Escherichia coli catalyze transport of monomeric catechols: study with beta-lactam antibiotics containing catechol and analogous groups. J Bacteriol. 1990 Mar;172(3):1361–1367. doi: 10.1128/jb.172.3.1361-1367.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Okii M., Iyobe S., Mitsuhashi S. Mapping of the gene specifying aminoglycoside 3'-phosphotransferase II on the Pseudomonas aeruginosa chromosome. J Bacteriol. 1983 Aug;155(2):643–649. doi: 10.1128/jb.155.2.643-649.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Piddock L. J., Hall M. C., Bellido F., Bains M., Hancock R. E. A pleiotropic, posttherapy, enoxacin-resistant mutant of Pseudomonas aeruginosa. Antimicrob Agents Chemother. 1992 May;36(5):1057–1061. doi: 10.1128/aac.36.5.1057. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Piddock L. J., Wijnands W. J., Wise R. Quinolone/ureidopenicillin cross-resistance. Lancet. 1987 Oct 17;2(8564):907–907. doi: 10.1016/s0140-6736(87)91387-0. [DOI] [PubMed] [Google Scholar]
  43. Poole K., Neshat S., Heinrichs D. Pyoverdine-mediated iron transport in Pseudomonas aeruginosa: involvement of a high-molecular-mass outer membrane protein. FEMS Microbiol Lett. 1991 Feb;62(1):1–5. [PubMed] [Google Scholar]
  44. Poole K., Schiebel E., Braun V. Molecular characterization of the hemolysin determinant of Serratia marcescens. J Bacteriol. 1988 Jul;170(7):3177–3188. doi: 10.1128/jb.170.7.3177-3188.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. Poole K., Young L., Neshat S. Enterobactin-mediated iron transport in Pseudomonas aeruginosa. J Bacteriol. 1990 Dec;172(12):6991–6996. doi: 10.1128/jb.172.12.6991-6996.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  46. Rella M., Haas D. Resistance of Pseudomonas aeruginosa PAO to nalidixic acid and low levels of beta-lactam antibiotics: mapping of chromosomal genes. Antimicrob Agents Chemother. 1982 Aug;22(2):242–249. doi: 10.1128/aac.22.2.242. [DOI] [PMC free article] [PubMed] [Google Scholar]
  47. 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]
  48. Russel M., Model P. Replacement of the fip gene of Escherichia coli by an inactive gene cloned on a plasmid. J Bacteriol. 1984 Sep;159(3):1034–1039. doi: 10.1128/jb.159.3.1034-1039.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  49. Sanders C. C., Sanders W. E., Jr, Goering R. V., Werner V. Selection of multiple antibiotic resistance by quinolones, beta-lactams, and aminoglycosides with special reference to cross-resistance between unrelated drug classes. Antimicrob Agents Chemother. 1984 Dec;26(6):797–801. doi: 10.1128/aac.26.6.797. [DOI] [PMC free article] [PubMed] [Google Scholar]
  50. Schnaitman C. A. Outer membrane proteins of Escherichia coli. 3. Evidence that the major protein of Escherichia coli O111 outer membrane consists of four distinct polypeptide species. J Bacteriol. 1974 May;118(2):442–453. doi: 10.1128/jb.118.2.442-453.1974. [DOI] [PMC free article] [PubMed] [Google Scholar]
  51. Schultz A. M., Oroszlan S. Murine leukemia virus gag polyproteins: the peptide chain unique to Pr80 is located at the amino terminus. Virology. 1978 Dec;91(2):481–486. doi: 10.1016/0042-6822(78)90395-1. [DOI] [PubMed] [Google Scholar]
  52. Surin B. P., Watson J. M., Hamilton W. D., Economou A., Downie J. A. Molecular characterization of the nodulation gene, nodT, from two biovars of Rhizobium leguminosarum. Mol Microbiol. 1990 Feb;4(2):245–252. doi: 10.1111/j.1365-2958.1990.tb00591.x. [DOI] [PubMed] [Google Scholar]
  53. Tabor S., Richardson C. C. A bacteriophage T7 RNA polymerase/promoter system for controlled exclusive expression of specific genes. Proc Natl Acad Sci U S A. 1985 Feb;82(4):1074–1078. doi: 10.1073/pnas.82.4.1074. [DOI] [PMC free article] [PubMed] [Google Scholar]
  54. Utsumi R., Yagi T., Katayama S., Katsuragi K., Tachibana K., Toyoda H., Ouchi S., Obata K., Shibano Y., Noda M. Molecular cloning and characterization of the fusaric acid-resistance gene from Pseudomonas cepacia. Agric Biol Chem. 1991 Jul;55(7):1913–1918. [PubMed] [Google Scholar]
  55. Vázquez M., Santana O., Quinto C. The NodL and NodJ proteins from Rhizobium and Bradyrhizobium strains are similar to capsular polysaccharide secretion proteins from gram-negative bacteria. Mol Microbiol. 1993 Apr;8(2):369–377. doi: 10.1111/j.1365-2958.1993.tb01580.x. [DOI] [PubMed] [Google Scholar]
  56. White J. R., Yeowell H. N. Iron enhances the bactericidal action of streptonigrin. Biochem Biophys Res Commun. 1982 May 31;106(2):407–411. doi: 10.1016/0006-291x(82)91125-1. [DOI] [PubMed] [Google Scholar]
  57. Wu H. C., Tokunaga M. Biogenesis of lipoproteins in bacteria. Curr Top Microbiol Immunol. 1986;125:127–157. doi: 10.1007/978-3-642-71251-7_9. [DOI] [PubMed] [Google Scholar]
  58. Yeowell H. N., White J. R. Iron requirement in the bactericidal mechanism of streptonigrin. Antimicrob Agents Chemother. 1982 Dec;22(6):961–968. doi: 10.1128/aac.22.6.961. [DOI] [PMC free article] [PubMed] [Google Scholar]

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

RESOURCES