Skip to main content
NCBI home page
Sexually Transmitted Infections logoLink to Sexually Transmitted Infections
. 1998 Feb;74(1):59–62. doi: 10.1136/sti.74.1.59

Analysis of quinolone resistance mechanisms in Neisseria gonorrhoeae isolates in vitro

M Tanaka, S Sakuma, K Takahashi, T Nagahuzi, T Saika, I Kobayashi, J Kumazawa
PMCID: PMC1758082  PMID: 9634306

Abstract

BACKGROUND AND OBJECTIVES: Gonococcal fluoroquinolone resistance is now a significant problem in Japan. We generated gonococcal mutants resistant to norfloxacin in vitro from norfloxacin sensitive isolates and analysed the contribution of three known mechanisms of quinolone resistance in Neisseria gonorrhoeae. MATERIALS AND METHODS: Three clinical isolates of N gonorrhoeae susceptible to norfloxacin were exposed to increasing concentrations of norfloxacin. To identify mutations in the gyrA and parC genes of the gonococcal mutants, the quinolone resistance determining regions of the gyrA and parC genes were polymerase chain reaction (PCR) amplified and the PCR products were directly sequenced. Norfloxacin accumulation in the gonococcal cells was also measured. RESULTS: The MICs of norfloxacin for three variants containing a single GyrA mutation were 16-fold higher than that for their parent isolates. A variant showing reduced norfloxacin accumulation in the cells, without mutations in the GyrA or ParC proteins, was also less sensitive to norfloxacin, with a 16-fold increase in the MIC, compared with the parent strain. The MIC of norfloxacin for a variant which contained a single GyrA mutation with reduced norfloxacin accumulation in the cells was 128-fold higher than for the parent strain. A variant containing mutations in both GyrA and ParC proteins with reduced accumulation of norfloxacin in the cells showed a 256-fold increase in the norfloxacin MIC compared with the parent strain. There was no variant containing a ParC mutation without the simultaneous presence of a GyrA mutation. CONCLUSIONS: The results from this study suggest that not only a mutation in the gyrA gene but also reduced drug accumulation in cells contributes to the development of fluoroquinolone a mutation in the gyrA gene contributes to a high level of fluoroquinolone resistance in gonococci with decreases in accumulation in cells having an additional but lesser effect. 




Full Text

The Full Text of this article is available as a PDF (78.2 KB).

Selected References

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

  1. Belland R. J., Morrison S. G., Ison C., Huang W. M. Neisseria gonorrhoeae acquires mutations in analogous regions of gyrA and parC in fluoroquinolone-resistant isolates. Mol Microbiol. 1994 Oct;14(2):371–380. doi: 10.1111/j.1365-2958.1994.tb01297.x. [DOI] [PubMed] [Google Scholar]
  2. Bogaerts J., Tello W. M., Akingeneye J., Mukantabana V., Van Dyck E., Piot P. Effectiveness of norfloxacin and ofloxacin for treatment of gonorrhoea and decrease of in vitro susceptibility to quinolones over time in Rwanda. Genitourin Med. 1993 Jun;69(3):196–200. doi: 10.1136/sti.69.3.196. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Clendennen T. E., 3rd, Hames C. S., Kees E. S., Price F. C., Rueppel W. J., Andrada A. B., Espinosa G. E., Kabrerra G., Wignall F. S. In vitro antibiotic susceptibilities of Neisseria gonorrhoeae isolates in the Philippines. Antimicrob Agents Chemother. 1992 Feb;36(2):277–282. doi: 10.1128/aac.36.2.277. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. 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]
  5. 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]
  6. Gransden W. R., Warren C. A., Phillips I., Hodges M., Barlow D. Decreased susceptibility of Neisseria gonorrhoeae to ciprofloxacin. Lancet. 1990 Jan 6;335(8680):51–51. doi: 10.1016/0140-6736(90)90177-7. [DOI] [PubMed] [Google Scholar]
  7. Heisig P., Tschorny R. Characterization of fluoroquinolone-resistant mutants of escherichia coli selected in vitro. Antimicrob Agents Chemother. 1994 Jun;38(6):1284–1291. doi: 10.1128/aac.38.6.1284. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. 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]
  9. 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]
  10. 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]
  11. Jephcott A. E., Turner A. Ciprofloxacin resistance in gonococci. Lancet. 1990 Jan 20;335(8682):165–165. doi: 10.1016/0140-6736(90)90035-4. [DOI] [PubMed] [Google Scholar]
  12. Kaatz G. W., Seo S. M., Ruble C. A. Efflux-mediated fluoroquinolone resistance in Staphylococcus aureus. Antimicrob Agents Chemother. 1993 May;37(5):1086–1094. doi: 10.1128/aac.37.5.1086. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Kam K. M., Lo K. K., Ho N. K., Cheung M. M. Rapid decline in penicillinase-producing Neisseria gonorrhoeae in Hong Kong associated with emerging 4-fluoroquinolone resistance. Genitourin Med. 1995 Jun;71(3):141–144. doi: 10.1136/sti.71.3.141. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Knapp J. S., Ohye R., Neal S. W., Parekh M. C., Higa H., Rice R. J. Emerging in vitro resistance to quinolones in penicillinase-producing Neisseria gonorrhoeae strains in Hawaii. Antimicrob Agents Chemother. 1994 Sep;38(9):2200–2203. doi: 10.1128/aac.38.9.2200. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Knapp J. S., Washington J. A., Doyle L. J., Neal S. W., Parekh M. C., Rice R. J. Persistence of Neisseria gonorrhoeae strains with decreased susceptibilities to ciprofloxacin and ofloxacin in Cleveland, Ohio, from 1992 through 1993. Antimicrob Agents Chemother. 1994 Sep;38(9):2194–2196. doi: 10.1128/aac.38.9.2194. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Messing J. New M13 vectors for cloning. Methods Enzymol. 1983;101:20–78. doi: 10.1016/0076-6879(83)01005-8. [DOI] [PubMed] [Google Scholar]
  17. Sawai T., Hiruma R., Kawana N., Kaneko M., Taniyasu F., Inami A. Outer membrane permeation of beta-lactam antibiotics in Escherichia coli, Proteus mirabilis, and Enterobacter cloacae. Antimicrob Agents Chemother. 1982 Oct;22(4):585–592. doi: 10.1128/aac.22.4.585. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Tanaka M., Fukuda H., Hirai K., Hosaka M., Matsumoto T., Kumazawa J. Reduced uptake and accumulation of norfloxacin in resistant strains of Neisseria gonorrhoeae isolated in Japan. Genitourin Med. 1994 Aug;70(4):253–255. doi: 10.1136/sti.70.4.253. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Tanaka M., Kumazawa J., Matsumoto T., Kobayashi I. High prevalence of Neisseria gonorrhoeae strains with reduced susceptibility to fluoroquinolones in Japan. Genitourin Med. 1994 Apr;70(2):90–93. doi: 10.1136/sti.70.2.90. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Tanaka M., Matsumoto T., Kobayashi I., Uchino U., Kumazawa J. Emergence of in vitro resistance to fluoroquinolones in Neisseria gonorrhoeae isolated in Japan. Antimicrob Agents Chemother. 1995 Oct;39(10):2367–2370. doi: 10.1128/aac.39.10.2367. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Tanaka M., Otsuki M., Nishino T., Kobayashi I., Matsumoto T., Kumazawa J. Mutation in DNA gyrase of norfloxacin-resistant clinical isolates of Neisseria gonorrhoeae. Genitourin Med. 1996 Aug;72(4):295–297. doi: 10.1136/sti.72.4.295. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Yoshida H., Nakamura M., Bogaki M., Ito H., Kojima T., Hattori H., Nakamura S. Mechanism of action of quinolones against Escherichia coli DNA gyrase. Antimicrob Agents Chemother. 1993 Apr;37(4):839–845. doi: 10.1128/aac.37.4.839. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from Sexually Transmitted Infections are provided here courtesy of BMJ Publishing Group

RESOURCES