Skip to main content
NCBI home page
Antimicrobial Agents and Chemotherapy logoLink to Antimicrobial Agents and Chemotherapy
. 1995 Aug;39(8):1667–1670. doi: 10.1128/aac.39.8.1667

Detection of gyrA and gyrB mutations in quinolone-resistant clinical isolates of Escherichia coli by single-strand conformational polymorphism analysis and determination of levels of resistance conferred by two different single gyrA mutations.

S Ouabdesselam 1, D C Hooper 1, J Tankovic 1, C J Soussy 1
PMCID: PMC162804  PMID: 7486897

Abstract

Twelve quinolone-resistant clinical isolates of Escherichia coli (nalidixic acid MICs, 64 to 512 micrograms/ml; norfloxacin MICs, 0.25 to 8 micrograms/ml) were transformed with plasmid pJSW101 carrying the gyrA+ gene and with plasmid pJB11 carrying the gyrB+ gene to examine the proportion of gyrA and gyrB mutations. Transformation with pJSW101 resulted in complementation (nalidixic acid MICs, 4 to 32 micrograms/ml; norfloxacin MICs, 0.06 to 0.25 micrograms/ml). In contrast, no change in MICs were observed after transformation with pJB11. A 418-bp fragment of gyrA from the 12 strains was amplified by PCR. Direct DNA sequencing of that fragment identified the causes of quinolone resistance in eight strains as a single point mutation leading to a substitution of the serine at position 83 (Ser-83) to Leu and in four strains as a single point mutation leading to a substitution of Asp-87 to Gly. Exchange of the fragment from one of these strains with that of gyrA+ and transformation of resistance with the hybrid gyrA plasmid indicated the contribution of Gly-87 to resistance and the stabilities of mutants containing GyrA (Gly-87). Thus, gyrA gene mutations are probably encountered more often than gyrB gene mutations in clinical isolates of E. coli. In addition, the substitution of Asp-87 to Gly can be encountered in such strains. On the basis of the level of resistance found in the fragment exchange experiment, the quinolone resistance attributable to Gly-87 appears to be comparable to that attributable to Leu-83. The levels of resistance found in the clinical isolates shown to have a Gly-87 mutation (nalidixic acid MICs, 64 to 512 micrograms/ml; norfloxacin MICs, 0.5 to 4 micrograms/ml) suggest that the Gly-87 mutation causes resistance at the level of the nalidixic acid MIC (64 micrograms/ml) or the norfloxacin MIC (0.5 micrograms/ml or less) and that the additional increments in resistance seen in the other strains with higher levels of resistance may be attributable to additional mutations. The single-strand conformational polymorphism analysis with PCR products readily detected te Leu-83 and Gly-87 mutations.

Full Text

The Full Text of this article is available as a PDF (303.3 KB).

Selected References

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

  1. Ariza R. R., Cohen S. P., Bachhawat N., Levy S. B., Demple B. Repressor mutations in the marRAB operon that activate oxidative stress genes and multiple antibiotic resistance in Escherichia coli. J Bacteriol. 1994 Jan;176(1):143–148. doi: 10.1128/jb.176.1.143-148.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. 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]
  3. Cullen M. E., Wyke A. W., Kuroda R., Fisher L. M. Cloning and characterization of a DNA gyrase A gene from Escherichia coli that confers clinical resistance to 4-quinolones. Antimicrob Agents Chemother. 1989 Jun;33(6):886–894. doi: 10.1128/aac.33.6.886. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Dower W. J., Miller J. F., Ragsdale C. W. High efficiency transformation of E. coli by high voltage electroporation. Nucleic Acids Res. 1988 Jul 11;16(13):6127–6145. doi: 10.1093/nar/16.13.6127. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Fisher L. M., Lawrence J. M., Josty I. C., Hopewell R., Margerrison E. E., Cullen M. E. Ciprofloxacin and the fluoroquinolones. New concepts on the mechanism of action and resistance. Am J Med. 1989 Nov 30;87(5A):2S–8S. doi: 10.1016/0002-9343(89)90010-7. [DOI] [PubMed] [Google Scholar]
  6. Heisig P., Schedletzky H., Falkenstein-Paul H. Mutations in the gyrA gene of a highly fluoroquinolone-resistant clinical isolate of Escherichia coli. Antimicrob Agents Chemother. 1993 Apr;37(4):696–701. doi: 10.1128/aac.37.4.696. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Hooper D. C., Wolfson J. S., Bozza M. A., Ng E. Y. Genetics and regulation of outer membrane protein expression by quinolone resistance loci nfxB, nfxC, and cfxB. Antimicrob Agents Chemother. 1992 May;36(5):1151–1154. doi: 10.1128/aac.36.5.1151. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. 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]
  9. 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]
  10. 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]
  11. Oram M., Fisher L. M. 4-Quinolone resistance mutations in the DNA gyrase of Escherichia coli clinical isolates identified by using the polymerase chain reaction. Antimicrob Agents Chemother. 1991 Feb;35(2):387–389. doi: 10.1128/aac.35.2.387. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Orita M., Suzuki Y., Sekiya T., Hayashi K. Rapid and sensitive detection of point mutations and DNA polymorphisms using the polymerase chain reaction. Genomics. 1989 Nov;5(4):874–879. doi: 10.1016/0888-7543(89)90129-8. [DOI] [PubMed] [Google Scholar]
  13. Piddock L. J., Wise R. Mechanisms of resistance to quinolones and clinical perspectives. J Antimicrob Chemother. 1989 Apr;23(4):475–480. doi: 10.1093/jac/23.4.475. [DOI] [PubMed] [Google Scholar]
  14. Revel V., Cambau E., Jarlier V., Sougakoff W. Characterization of mutations in Mycobacterium smegmatis involved in resistance to fluoroquinolones. Antimicrob Agents Chemother. 1994 Sep;38(9):1991–1996. doi: 10.1128/aac.38.9.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Sanger F., Nicklen S., Coulson A. R. DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci U S A. 1977 Dec;74(12):5463–5467. doi: 10.1073/pnas.74.12.5463. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Soussy C. J., Wolfson J. S., Ng E. Y., Hooper D. C. Limitations of plasmid complementation test for determination of quinolone resistance due to changes in the gyrase A protein and identification of conditional quinolone resistance locus. Antimicrob Agents Chemother. 1993 Dec;37(12):2588–2592. doi: 10.1128/aac.37.12.2588. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Swanberg S. L., Wang J. C. Cloning and sequencing of the Escherichia coli gyrA gene coding for the A subunit of DNA gyrase. J Mol Biol. 1987 Oct 20;197(4):729–736. doi: 10.1016/0022-2836(87)90479-7. [DOI] [PubMed] [Google Scholar]
  18. Telenti A., Imboden P., Marchesi F., Schmidheini T., Bodmer T. Direct, automated detection of rifampin-resistant Mycobacterium tuberculosis by polymerase chain reaction and single-strand conformation polymorphism analysis. Antimicrob Agents Chemother. 1993 Oct;37(10):2054–2058. doi: 10.1128/aac.37.10.2054. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Tokue Y., Sugano K., Saito D., Noda T., Ohkura H., Shimosato Y., Sekiya T. Detection of novel mutations in the gyrA gene of Staphylococcus aureus by nonradioisotopic single-strand conformation polymorphism analysis and direct DNA sequencing. Antimicrob Agents Chemother. 1994 Mar;38(3):428–431. doi: 10.1128/aac.38.3.428. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Vila J., Ruiz J., Marco F., Barcelo A., Goñi P., Giralt E., Jimenez de Anta T. Association between double mutation in gyrA gene of ciprofloxacin-resistant clinical isolates of Escherichia coli and MICs. Antimicrob Agents Chemother. 1994 Oct;38(10):2477–2479. doi: 10.1128/aac.38.10.2477. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Wolfson J. S., Hooper D. C. Bacterial resistance to quinolones: mechanisms and clinical importance. Rev Infect Dis. 1989 Jul-Aug;11 (Suppl 5):S960–S968. doi: 10.1093/clinids/11.supplement_5.s960. [DOI] [PubMed] [Google Scholar]
  22. Yamagishi J., Furutani Y., Inoue S., Ohue T., Nakamura S., Shimizu M. New nalidixic acid resistance mutations related to deoxyribonucleic acid gyrase activity. J Bacteriol. 1981 Nov;148(2):450–458. doi: 10.1128/jb.148.2.450-458.1981. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Yamagishi J., Yoshida H., Yamayoshi M., Nakamura S. Nalidixic acid-resistant mutations of the gyrB gene of Escherichia coli. Mol Gen Genet. 1986 Sep;204(3):367–373. doi: 10.1007/BF00331012. [DOI] [PubMed] [Google Scholar]
  24. Yoshida H., Kojima T., Yamagishi J., Nakamura S. Quinolone-resistant mutations of the gyrA gene of Escherichia coli. Mol Gen Genet. 1988 Jan;211(1):1–7. doi: 10.1007/BF00338386. [DOI] [PubMed] [Google Scholar]
  25. Yoshida H., Nakamura M., Bogaki M., Nakamura S. Proportion of DNA gyrase mutants among quinolone-resistant strains of Pseudomonas aeruginosa. Antimicrob Agents Chemother. 1990 Jun;34(6):1273–1275. doi: 10.1128/aac.34.6.1273. [DOI] [PMC free article] [PubMed] [Google Scholar]

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

RESOURCES