Skip to main content
PMC home page
Antimicrobial Agents and Chemotherapy logoLink to Antimicrobial Agents and Chemotherapy
. 1991 Jun;35(6):1053–1059. doi: 10.1128/aac.35.6.1053

Mechanisms of clinical resistance to fluoroquinolones in Enterococcus faecalis.

N Nakanishi 1, S Yoshida 1, H Wakebe 1, M Inoue 1, S Mitsuhashi 1
PMCID: PMC284285  PMID: 1656852

Abstract

About 10% of 100 clinical isolates of Enterococcus faecalis were resistant to greater than or equal to 25 micrograms of norfloxacin, ofloxacin, ciprofloxacin, and temafloxacin per ml. In this study, the DNA gyrase of E. faecalis was purified from a fluoroquinolone-susceptible strain (ATCC 19433) and two resistant isolates, MS16968 and MS16996. Strains MS16968 and MS16996 were 64- to 128-fold and 16- to 32-fold less susceptible, respectively, to fluoroquinolones than was ATCC 19433; MICs of nonquinolone antibacterial agents for these strains were almost equal. The DNA gyrase from ATCC 19433 had two subunits, designated A and B, with properties similar to those of DNA gyrase from other gram-positive bacteria such as Bacillus subtilis and Micrococcus luteus. Inhibition of the supercoiling activity of the enzyme from ATCC 19433 by the fluoroquinolones correlated with their antibacterial activities. In contrast, preparations of DNA gyrase from MS16968 and MS16996 were at least 30-fold less sensitive to inhibition of supercoiling by the fluoroquinolones than the gyrase from ATCC 19433 was. Experiments that combined heterologous gyrase subunits showed that the A subunit from either of the resistant isolates conferred resistance to fluoroquinolones. These findings indicate that an alteration in the gyrase A subunit is the major contributor to fluoroquinolone resistance in E. faecalis clinical isolates. A difference in drug uptake may also contribute to the level of fluoroquinolone resistance in these isolates.

Full text

PDF
1057

Images in this article

1055Image
on p.1055
1056Image
on p.1056

Selected References

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

  1. Aoyama H., Sato K., Fujii T., Fujimaki K., Inoue M., Mitsuhashi S. Purification of Citrobacter freundii DNA gyrase and inhibition by quinolones. Antimicrob Agents Chemother. 1988 Jan;32(1):104–109. doi: 10.1128/aac.32.1.104. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Bradford M. M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem. 1976 May 7;72:248–254. doi: 10.1006/abio.1976.9999. [DOI] [PubMed] [Google Scholar]
  3. 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]
  4. Chin N. X., Neu H. C. Ciprofloxacin, a quinolone carboxylic acid compound active against aerobic and anaerobic bacteria. Antimicrob Agents Chemother. 1984 Mar;25(3):319–326. doi: 10.1128/aac.25.3.319. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Chow R. T., Dougherty T. J., Fraimow H. S., Bellin E. Y., Miller M. H. Association between early inhibition of DNA synthesis and the MICs and MBCs of carboxyquinolone antimicrobial agents for wild-type and mutant [gyrA nfxB(ompF) acrA] Escherichia coli K-12. Antimicrob Agents Chemother. 1988 Aug;32(8):1113–1118. doi: 10.1128/aac.32.8.1113. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. 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]
  7. Domagala J. M., Hanna L. D., Heifetz C. L., Hutt M. P., Mich T. F., Sanchez J. P., Solomon M. New structure-activity relationships of the quinolone antibacterials using the target enzyme. The development and application of a DNA gyrase assay. J Med Chem. 1986 Mar;29(3):394–404. doi: 10.1021/jm00153a015. [DOI] [PubMed] [Google Scholar]
  8. Drlica K. Biology of bacterial deoxyribonucleic acid topoisomerases. Microbiol Rev. 1984 Dec;48(4):273–289. doi: 10.1128/mr.48.4.273-289.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Fu K. P., Grace M. E., McCloud S. J., Gregory F. J., Hung P. P. Discrepancy between the antibacterial activities and the inhibitory effects on Micrococcus luteus DNA gyrase of 13 quinolones. Chemotherapy. 1986;32(6):494–498. doi: 10.1159/000238458. [DOI] [PubMed] [Google Scholar]
  10. Gellert M., Fisher L. M., O'Dea M. H. DNA gyrase: purification and catalytic properties of a fragment of gyrase B protein. Proc Natl Acad Sci U S A. 1979 Dec;76(12):6289–6293. doi: 10.1073/pnas.76.12.6289. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Gellert M., Mizuuchi K., O'Dea M. H., Itoh T., Tomizawa J. I. Nalidixic acid resistance: a second genetic character involved in DNA gyrase activity. Proc Natl Acad Sci U S A. 1977 Nov;74(11):4772–4776. doi: 10.1073/pnas.74.11.4772. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Gellert M., Mizuuchi K., O'Dea M. H., Nash H. A. DNA gyrase: an enzyme that introduces superhelical turns into DNA. Proc Natl Acad Sci U S A. 1976 Nov;73(11):3872–3876. doi: 10.1073/pnas.73.11.3872. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Greenwood D. Antibiotic resistance in enterococci. J Antimicrob Chemother. 1989 Nov;24(5):631–635. doi: 10.1093/jac/24.5.631. [DOI] [PubMed] [Google Scholar]
  14. Hirai K., Aoyama H., Irikura T., Iyobe S., Mitsuhashi S. Differences in susceptibility to quinolones of outer membrane mutants of Salmonella typhimurium and Escherichia coli. Antimicrob Agents Chemother. 1986 Mar;29(3):535–538. doi: 10.1128/aac.29.3.535. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. 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]
  16. Hooper D. C., Wolfson J. S., McHugh G. L., Winters M. B., Swartz M. N. Effects of novobiocin, coumermycin A1, clorobiocin, and their analogs on Escherichia coli DNA gyrase and bacterial growth. Antimicrob Agents Chemother. 1982 Oct;22(4):662–671. doi: 10.1128/aac.22.4.662. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Hooper D. C., Wolfson J. S. Mode of action of the quinolone antimicrobial agents. Rev Infect Dis. 1988 Jan-Feb;10 (Suppl 1):S14–S21. doi: 10.1093/clinids/10.supplement_1.s14. [DOI] [PubMed] [Google Scholar]
  18. 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]
  19. 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]
  20. Hooper D. C., Wolfson J. S. The fluoroquinolones: pharmacology, clinical uses, and toxicities in humans. Antimicrob Agents Chemother. 1985 Nov;28(5):716–721. doi: 10.1128/aac.28.5.716. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Hopewell R., Oram M., Briesewitz R., Fisher L. M. DNA cloning and organization of the Staphylococcus aureus gyrA and gyrB genes: close homology among gyrase proteins and implications for 4-quinolone action and resistance. J Bacteriol. 1990 Jun;172(6):3481–3484. doi: 10.1128/jb.172.6.3481-3484.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Imamura M., Shibamura S., Hayakawa I., Osada Y. Inhibition of DNA gyrase by optically active ofloxacin. Antimicrob Agents Chemother. 1987 Feb;31(2):325–327. doi: 10.1128/aac.31.2.325. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. King A., Phillips I. The comparative in-vitro activity of eight newer quinolones and nalidixic acid. J Antimicrob Chemother. 1986 Nov;18 (Suppl 500):1–20. doi: 10.1093/jac/18.supplement_d.1. [DOI] [PubMed] [Google Scholar]
  24. Kojima T., Inoue M., Mitsuhashi S. In vitro activity of AT-4140 against quinolone- and methicillin-resistant Staphylococcus aureus. Antimicrob Agents Chemother. 1990 Jun;34(6):1123–1127. doi: 10.1128/aac.34.6.1123. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Liu L. F., Wang J. C. Micrococcus luteus DNA gyrase: active components and a model for its supercoiling of DNA. Proc Natl Acad Sci U S A. 1978 May;75(5):2098–2102. doi: 10.1073/pnas.75.5.2098. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Masecar B. L., Celesk R. A., Robillard N. J. Analysis of acquired ciprofloxacin resistance in a clinical strain of Pseudomonas aeruginosa. Antimicrob Agents Chemother. 1990 Feb;34(2):281–286. doi: 10.1128/aac.34.2.281. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Mizuuchi K., O'Dea M. H., Gellert M. DNA gyrase: subunit structure and ATPase activity of the purified enzyme. Proc Natl Acad Sci U S A. 1978 Dec;75(12):5960–5963. doi: 10.1073/pnas.75.12.5960. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Muranaka K., Greenwood D. The response of Streptococcus faecalis to ciprofloxacin, norfloxacin and enoxacin. J Antimicrob Chemother. 1988 May;21(5):545–554. doi: 10.1093/jac/21.5.545. [DOI] [PubMed] [Google Scholar]
  29. Nakanishi N., Inoue M., Inoue K., Yamaguchi T., Mitsuhashi S. In vitro activity of temafloxacin hydrochloride (TA-167 or A-62254), a new fluorinated 4-quinolone. Chemotherapy. 1990;36(5):345–355. doi: 10.1159/000238787. [DOI] [PubMed] [Google Scholar]
  30. Neu H. C. Bacterial resistance to fluoroquinolones. Rev Infect Dis. 1988 Jan-Feb;10 (Suppl 1):S57–S63. doi: 10.1093/clinids/10.supplement_1.s57. [DOI] [PubMed] [Google Scholar]
  31. Orr E., Staudenbauer W. L. Bacillus subtilis DNA gyrase: purification of subunits and reconstitution of supercoiling activity. J Bacteriol. 1982 Jul;151(1):524–527. doi: 10.1128/jb.151.1.524-527.1982. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Piddock L. J., Wise R. The selection and frequency of streptococci with decreased susceptibility to ofloxacin compared with other quinolones. J Antimicrob Chemother. 1988 Sep;22 (Suppl 100):45–51. doi: 10.1093/jac/22.supplement_c.45. [DOI] [PubMed] [Google Scholar]
  33. 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]
  34. 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]
  35. Sato K., Inoue Y., Fujii T., Aoyama H., Inoue M., Mitsuhashi S. Purification and properties of DNA gyrase from a fluoroquinolone-resistant strain of Escherichia coli. Antimicrob Agents Chemother. 1986 Nov;30(5):777–780. doi: 10.1128/aac.30.5.777. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Staudenbauer W. L., Orr E. DNA gyrase: affinity chromatography on novobiocin-Sepharose and catalytic properties. Nucleic Acids Res. 1981 Aug 11;9(15):3589–3603. doi: 10.1093/nar/9.15.3589. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Sugino A., Bott K. F. Bacillus subtilis deoxyribonucleic acid gyrase. J Bacteriol. 1980 Mar;141(3):1331–1339. doi: 10.1128/jb.141.3.1331-1339.1980. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Sutcliffe J. A., Gootz T. D., Barrett J. F. Biochemical characteristics and physiological significance of major DNA topoisomerases. Antimicrob Agents Chemother. 1989 Dec;33(12):2027–2033. doi: 10.1128/aac.33.12.2027. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Takahata M., Nishino T. DNA gyrase of Staphylococcus aureus and inhibitory effect of quinolones on its activity. Antimicrob Agents Chemother. 1988 Aug;32(8):1192–1195. doi: 10.1128/aac.32.8.1192. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Watanabe M., Kotera Y., Yosue K., Inoue M., Mitsuhashi S. In vitro emergence of quinolone-resistant mutants of Escherichia coli, Enterobacter cloacae, and Serratia marcescens. Antimicrob Agents Chemother. 1990 Jan;34(1):173–175. doi: 10.1128/aac.34.1.173. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Wolfson J. S., Hooper D. C. The fluoroquinolones: structures, mechanisms of action and resistance, and spectra of activity in vitro. Antimicrob Agents Chemother. 1985 Oct;28(4):581–586. doi: 10.1128/aac.28.4.581. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from Antimicrobial Agents and Chemotherapy are provided here courtesy of American Society for Microbiology (ASM)

RESOURCES