1 Molecular Genetics Group, Department of Biochemistry and Immunology, St Georges Hospital Medical School, University of London, Cranmer Terrace, London SW17 0RE; 2 Anti-Infective Marketing Group, Pharmaceutical Division, Bayer plc, Strawberry Hill, Newbury RG14 1JA, UK
Keywords: quinolones, dual activity, Streptococcus pneumoniae, gyrase, topoisomerase IV
Sir,
A recent article by Smith et al.1 reviewed studies on the target preferences of quinolones for DNA gyrase and/or topoisomerase IV in Streptococcus pneumoniae. Previous genetic work from our laboratory has indicated that quinolones can act in vivo through gyrase, through topoisomerase IV or through both enzymes (dual activity).2,3 In particular, clinafloxacin, gemifloxacin and other new quinolones such as moxifloxacin exhibit dual activity based on the minimal effect of either gyrA or parC mutations on resistance, whereas strains with mutations in both gyrA and parC exhibit high-level resistance to these agents.35 Smith et al.1 discuss our results including the well-known observation that for some quinolones there is a discrepancy between quinolone targets determined genetically and those suggested by in vitro enzyme inhibition.6,7 The authors go on to assume that the genetic and enzyme results should concur and on that basis claim that only clinafloxacin and sitafloxacin have proven dual activity in S. pneumoniae. We wish to emphasize that because genetic and enzyme approaches measure completely different parameters, they need not agree and therefore should not be conflated. Genetic data are the most reliable indicator of quinolone target preference(s) in vivo.
Several lines of evidence now demonstrate that differences in the results from genetic and enzymic studies of quinolones in S. pneumoniae are genuine and, contrary to Smith et al.,1 do not arise from methodological deficiencies (reviewed in Pan et al.8). For example, we have shown unequivocally that sparfloxacin and ciprofloxacin act, respectively, through gyrase and through topoisomerase IV in S. pneumoniae, yet they behave identically in enzyme inhibition assays in vitro, with each preferentially inhibiting topoisomerase IV.8 In addressing such findings, we could discount alternative explanations rehearsed by Smith et al.,1 e.g. undetected topoisomerase IV mutations lying outside the known quinolone resistance-determining regions.8 Rather, the discrepancy with genetic results arises from the inability of enzyme data to reflect the totality of quinolone action in vivo. Thus, quinolones exert their antibacterial effects by forming a drugenzymeDNA ternary complexthe cleavable complexwhich is converted into a lethal lesion by cellular processes such as collisions with replication forks and subsequent processing.6,8 IC50 data for enzyme inhibition cited by Smith et al.1 simply provide a measure of drugtopoisomerase binding affinities in vitro. Given the mechanism of quinolone action, we have argued that quinolone-induced DNA breakage assays indicating cleavable complex formation are more relevant than IC50 data.6 Unfortunately, even these assays do not reproduce the exact in vivo conditions relevant to complex formation, e.g. chromosomal supercoiling, salt and ATP, nor do they take into account the different intracellular expression levels of gyrase and topoisomerase IV. However, the key limitation of in vitro enzyme assays is that they relate to only one step of a multistep killing pathway: they cannot reflect the contribution of cellular processing and repair of drug-induced lesions, processes that may also be sensitive to quinolone structure. Clearly, IC50 data are not appropriate in determining in vivo target preferences. It is genetic data that are necessary in assigning preferential killing pathways and in ascertaining dual activity in vivo. Moreover, the genetic targets are the important factors clinically.
Based on the lack of effect of gyrA or parC resistance mutations, previous studies have identified gemifloxacin, gatifloxacin and moxifloxacin as well as clinafloxacin as having dual activity in S. pneumoniae.35 In further support of this idea for topoisomerases reconstituted with S. pneumoniae S81F GyrA and S79F ParC subunits, cleavable complex formation was reduced 16- to 64-fold for gemifloxacin, gatifloxacin and moxifloxacin, yet the proteins conferred little or no resistance to these agents when expressed in S. pneumoniae.5 The largely silent phenotype in vivo can be explained if these quinolones act substantially through gyrase and topoisomerase IV. Moreover, several studies have reported difficulty in selecting resistant topoisomerase mutants of S. pneumoniae by challenge with gatifloxacin, gemifloxacin, moxifloxacin and clinafloxacin, presumably because of the need for two target mutations for emergence of resistance.3
Finally, Smith et al.1 conclude that clinically available, safe and effective dual-activity fluoroquinolones have yet to be created. This statement is clearly incorrect. Both gatifloxacin and moxifloxacin act substantially through both gyrase and topoisomerase IV in S. pneumoniae and are widely used clinically. Intelligent use of such agents and proper understanding of the scientific basis underlying their antibacterial action will be important in combating the emergence of clinical resistance.
Footnotes
* Corresponding author. Tel: +44-20-8725-5782; Fax: +44-20-8725-2992; E-mail: lfisher{at}sghms.ac.uk
References
1
.
Smith, H. J., Nichol, K. A., Hoban, D. J. & Zhanel, G. G. (2002). Dual activity of fluoroquinolones against Streptococcus pneumoniae: the facts behind the claims. Journal of Antimicrobial Chemotherapy 49, 8935.
2 . Pan, X.-S. & Fisher, L. M. (1997). Targeting of DNA gyrase in Streptococcus pneumoniae by sparfloxacin: selective targeting of gyrase or topoisomerase IV by quinolones. Antimicrobial Agents and Chemotherapy 41, 4714.[Abstract]
3
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Pan, X.-S. & Fisher, L. M. (1998). DNA gyrase and topoisomerase IV are dual targets of clinafloxacin action in Streptococcus pneumoniae. Antimicrobial Agents and Chemotherapy 42, 28106.
4
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Heaton, V. J., Ambler, J. E. & Fisher L. M. (2000). Potent antipneumococcal activity of gemifloxacin is associated with dual targeting of gyrase and topoisomerase IV, an in vivo target preference for gyrase, and enhanced stabilization of cleavable complexes in vitro. Antimicrobial Agents and Chemotherapy 44, 31127.
5
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Yague, G., Morris, J. E., Pan, X.-S., Gould, K. A. & Fisher, L. M. (2002). Cleavable complex formation by wild-type and quinolone-resistant Streptococcus pneumoniae type II topoisomerases mediated by gemifloxacin and other fluoroquinolones. Antimicrobial Agents and Chemotherapy 46, 4139.
6
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Pan, X.-S. & Fisher, L. M. (1999). Streptococcus pneumoniae DNA gyrase and topoisomerase IV: overexpression, purification and differential inhibition by fluoroquinolones. Antimicrobial Agents and Chemotherapy 43, 112936.
7
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Morrissey, I. & George, J. (1999). Activities of fluoroquinolones against Streptococcus pneumoniae type II topoisomerases purified as recombinant proteins. Antimicrobial Agents and Chemotherapy 43, 257985.
8
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Pan, X.-S., Yague, G. & Fisher, L. M. (2001). Quinolone resistance mutations in Streptococcus pneumoniae GyrA and ParC proteins: mechanistic insights into quinolone action from enzymatic analysis, intracellular levels, and phenotypes of wild-type and mutant proteins. Antimicrobial Agents and Chemotherapy 45, 31407.