Journal of Antimicrobial Chemotherapy (2001) 48, 545-548
© 2001 The British Society for Antimicrobial Chemotherapy
Brief report |
Frequency of selection of fluoroquinolone-resistant mutants of Neisseria gonorrhoeae exposed to gemifloxacin and four other quinolones
a Institut Clínic d'Infeccions i Immunologia, IDIBAPS, Hospital Clínic, Villarroel 170, 08036 Barcelona; b GlaxoSmithKline, Madrid, Spain
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and methodsResults and discussionAcknowledgementsReferences |
|---|
We studied the frequency of mutation of clinical isolates of Neisseria gonorrhoeae (two nalidixic acid susceptible and two nalidixic acid resistant), and the stability of the mutants obtained, in the presence of three different concentrations of five fluoroquinolones. The frequency of mutation was low for all the quinolones. Only one N. gonorrhoeae mutant, obtained with trovafloxacin at 4 x MIC presented a stable increase in the MIC of this quinolone, not attributable to novel mutation(s), both in the gyrA and parC genes, although not showing any increase in the MIC of the other quinolones tested. In summary, gemifloxacin was the only quinolone tested for which resistant mutants were not obtained.
|
Introduction |
|---|
|
TopAbstractIntroductionMaterials and methodsResults and discussionAcknowledgementsReferences |
|---|
Quinolones have been employed to treat infections due to Neisseria gonorrhoeae, particularly for penicillin-resistant strains.1 However, during recent years an increase in the number of clinical isolates of N. gonorrhoeae with a decreased susceptibility to these antibacterial agents has been reported.1,2 Moreover, development of resistance during treatment with fluoroquinolones and therapeutic failure have been described.1,2
In vitro studies to establish the potential of different quinolones to select resistant mutants have been conducted on various microorganisms.3–6 In a series of 10 Gramnegative and Gram-positive microorganisms, not including N. gonorrhoeae, it has been shown5 that gemifloxacin has a low level of selection of resistant mutants, which is even lower than that of ciprofloxacin or trovafloxacin. Gemifloxacin is a novel fluoroquinolone that possesses enhanced activity against both Gram-negative and Gram-positive microorganisms.7 Previous studies have shown that it has excellent activity against N. gonorrhoeae.7,8 However, the role of this quinolone in the selection of resistant mutants of N. gonorrhoeae remains unknown.
The ability of antibacterial agents to select resistant mutants is a critical factor in their therapeutic usefulness. An antibacterial agent with high in vitro capacity to select resistant mutants may, potentially, act in a similar way during treatment, especially if the treatment is prolonged and subinhibitory concentrations are achieved in the infected tissue.9 Thus, evaluating the role of a new antibacterial agent in the development of resistant strains compared with other antibacterial agents may indicate the potential risk of developing resistance during therapy.
The aim of this study was to determine the frequency at which fluoroquinolone-resistant mutants were selected from two nalidixic acid susceptible and two nalidixic acid- resistant strains of N. gonorrhoeae exposed to gemifloxacin and four other fluoroquinolones.
|
Materials and methods |
|---|
|
TopAbstractIntroductionMaterials and methodsResults and discussionAcknowledgementsReferences |
|---|
Microorganisms
Four clinical isolates of N. gonorrhoeae analysed previously were used for the study.8 Two were resistant to nalidixic acid by virtue of a single mutation in codon 91 of the gyrA gene (Ser
Leu and SerPhe, respectively), whereas the other two were susceptible to nalidixic acid with no detectable alteration in either the gyrA or parC gene.
Antimicrobial susceptibility testing
The MICs of gemifloxacin (SmithKline Beecham Pharmaceuticals, Harlow, UK), levofloxacin (Hoechst Marion Rousell, Romainville, France), ciprofloxacin, moxifloxacin (Bayer, Leverkusen, Germany) and trovafloxacin (Pfizer Ltd, Sandwich, UK) were determined by an agar dilution method in accordance with the guidelines of the NCCLS.10 Escherichia coli ATCC 25922, N. gonorrhoeae ATCC 49226 and Staphylococcus aureus ATCC 29213 were used as control organisms.
Determination of spontaneous single-step resistance rates
The determination of spontaneous single-step resistance rates was performed as described previously,4 with the modification that the isolates were grown on GC-agar and then resuspended in saline solution before inoculation on to GC-agar containing the selected antibacterial agent at 2 x, 4 x and 8 x MIC. The plates were read at 24 and 48 h. The frequency of spontaneous resistance selected by each fluoroquinolone was calculated as the ratio of the number of bacteria growing divided by the number of bacteria in the original inoculum. The strains that did not appear on the plates were given a frequency of resistance reciprocal to that of the inoculum. The experiments were repeated three times.
Stability of the resistance
To establish the resistance stability of mutants, a representative mutant colony from each concentration of antibacterial agent was subcultured onto plates without antibacterial agent prior to MIC determination.
Analysis of the mutations present in the gyrA and parC genes
A fragment of the gyrA and parC genes containing the so-called ‘quinolone resistance-determining region’ was amplified using previously described primers and conditions.2,8
|
Results and discussion |
|---|
|
TopAbstractIntroductionMaterials and methodsResults and discussionAcknowledgementsReferences |
|---|
The susceptibility and genetic characteristics of the wild-type quinolone-susceptible strains and strains having a previous mutation in the gyrA gene used in this study are shown in Table 1
. Mutants able to grow at 2 x, 4 x or 8 x MIC were obtained with all quinolones tested, except gemifloxacin (Table 2). This finding is similar to those previously reported,5 which indicated that gemifloxacin selected resistant mutants at a lower frequency in Gram-negative and Gram-positive microorganisms, not including N. gonorrhoeae, compared with ciprofloxacin and trovafloxacin. In order to establish the frequency of mutation, all the colonies that grew on antibiotic-containing agar were considered as mutants, regardless of their MIC stability. The frequency of mutation with the quinolones analysed was low. Only when strain 97-692 was grown on agar containing trovafloxacin at 2 x MIC, and when strain 3174 was grown on ciprofloxacin at 2 x and 4 x MIC was the frequency of mutation in the order of 10–7. No differences were found when the plates were read at 24 and 48 h.
|
|
Only one strain (3174) selected at 4 x MIC of trovafloxacin presented a stable increase in its MIC from 0.06 to 0.25 mg/L of this antibacterial agent alone, i.e. did not present alterations in the MICs of the other quinolones studied. This phenomenon indicates that strain 3174 presents a particular system able to increase the MIC of trovafloxacin, but which does not affect gemifloxacin or the other quinolones studied. When the presence of alterations in the quinolone targets of this mutant was studied no new mutations were found in either the gyrA or parC gene, other than the previously described mutations in the parental strain. The other mutants selected did not present stable increases in the MICs of tested quinolones. The fact that strains able to grow in a medium with a concentration of an antibacterial agent above its MIC do not present a stable increase in the MIC of this antibacterial agent may be due to a temporary induction of some mechanism(s) of quinolone resistance, such as overexpression of some efflux pumps, in the presence of the fluoroquinolone, which reverts when the strain is grown in the absence of the antibiotic. If this were the case, the accumulation of fluoroquinolone would be lower but would favour the development of higher resistance.
In two different models (Gram-positive and Gramnegative) Drugeon et al.3 and Tavio et al.6 reported a higher frequency of mutation presented by strains carrying mutations in their quinolone targets. This phenomenon was not seen in our strains, which may be attributable to the high activity of the quinolones against N. gonorrhoeae.1,7,8 Despite this low frequency of mutation, the increase in resistance levels to quinolones among clinical isolates of N. gonorrhoeae is continuously rising.
The low level of selection by gemifloxacin of quinolone resistance in N. gonorrhoeae, together with its good activity against clinical isolates already carrying a substitution in GyrA,8 indicates that this quinolone may have an important role to play in the treatment of gonococcal infections.
|
Acknowledgements |
|---|
|
TopAbstractIntroductionMaterials and methodsResults and discussionAcknowledgementsReferences |
|---|
We thank Cristina Garcia de la Marìa for her useful suggestions and discussions. This work was supported by grants FIS00/0632 from Fondo de Investigaciones Sanitarias and from SmithKline Beecham, Harlow, UK.
|
Notes |
|---|
* Corresponding author. Tel: +34-93-227-5522; Fax: +34-93-227-5454; E-mail: vila{at}medicina.ub.es
|
References |
|---|
|
TopAbstractIntroductionMaterials and methodsResults and discussionAcknowledgementsReferences |
|---|
1 . Knapp, J. S., Fox, K. K., Trees, D. L. & Whittington, W. L. (1997). Fluoroquinolone resistance in Neisseria gonorrhoeae. Emerging Infectious Diseases 3, 33–9.[Web of Science][Medline]
2 . Vila, J., Olmos, L., Ballesteros, J., Vazquez, J. A., Giménez, M. J., Marco, F. et al. (1997). Development of in-vivo resistance after quinolone treatment of gonococcal urethritis. Journal of Antimicrobial Chemotherapy 39, 841.
3
.
Drugeon, H. B., Juvin, M. E. & Bryskier, A. (1999). Relative potential for selection of fluoroquinolone-resistant Streptococcus pneumoniae strains by levofloxacin: comparison with ciprofloxacin, sparfloxacin and ofloxacin. Journal of Antimicrobial Chemotherapy 43, Suppl. C, 55–9.
4
.
Evans, M. E. & Titlow, W. B. (1998). Levofloxacin selects fluoroquinolone-resistant methicillin-resistant Staphylococcus aureus less frequently than ciprofloxacin. Journal of Antimicrobial Chemotherapy 41, 285–8.
5 . Lowe, M. N. & Lamb, H. M. (2000). Gemifloxacin. Drugs 59, 1137–48.[Web of Science][Medline]
6
.
Tavio, M. M., Vila, J., Ruiz, J., Ruiz, J., Martin-Sanchez, A. M. & Jiménez de Anta, M. T. (1999). Mechanisms involved in the development of resistance to fluoroquinolones in Escherichia coli isolates. Journal of Antimicrobial Chemotherapy 44, 735–42.
7
.
Wise, R. & Andrews, J. M. (1999). The in-vitro activity and tentative breakpoint of gemifloxacin, a new fluoroquinolone. Journal of Antimicrobial Chemotherapy 44, 679–88.
8 . Ruiz, J., Marco, F., Sierra, J. M., Garcia, E., Aguilar, L., Mensa, J. et al. (2000). In vitro activity of gemifloxacin against clinical isolates of Neisseria gonorrhoeae with and without mutations in the gyrA gene. Revista Española de Quimioterapia 13, Suppl. 2, 6.
9
.
Thomas, J. K., Forrest, A., Bhavnani, S. M., Hyatt, J. M., Cheng, A., Ballow, C. H. et al. (1998). Pharmacodynamic evaluation of factors associated with the development of bacterial resistance in acutely ill patients during therapy. Antimicrobial Agents and Chemotherapy 42, 521–7.
10 . National Committee for Clinical Laboratory Standards. (2000). Methods for Dilution Antimicrobial Susceptibility Tests for Bacteria that Grow Aerobically—Fifth Edition: Approved Standard M7-A5. NCCLS, Wayne, PA.
Received 23 February 2001; returned 31 May 2001; revised 12 July 2001; accepted 30 July 2001
CiteULike 
Connotea 
Del.icio.us What's this?
This article has been cited by other articles:
|
|
 |
|
  G. P. Allen and C. D. Hankins Evaluation of the mutant selection window for fluoroquinolones against Neisseria gonorrhoeae J. Antimicrob. Chemother., August 1, 2009; 64(2): 359 - 363. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. Ruiz, L. Mensa, M. J. Pons, J. Vila, and J. Gascon Development of Escherichia coli rifaximin-resistant mutants: frequency of selection and stability J. Antimicrob. Chemother., May 1, 2008; 61(5): 1016 - 1019. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||