Department of Medical Microbiology, Faculties of 1Medicine and 3Pharmacy, University of Manitoba, Manitoba; Departments of 2Clinical Microbiology and 4Medicine, Health Sciences Centre, Winnipeg, Manitoba, Canada
Recently, there have been several reports of the dual activity of some fluoroquinolones used to treat infections probably caused by Streptococcus pneumoniae.13 Furthermore, there have been numerous declarations as to the clinical advantages of using dual-activity agents, including minimizing the development of resistance. These reports have been inconsistent, with fluoroquinolones being reported as having dual activity in some reports but not in others, and thus remain controversial.1,37 In order to clarify the reasons behind the inconsistencies, we have summarized the flaws of the methods by which the current data on the dual activity of fluoroquinolones against S. pneumoniae have been collected, and the conclusions that have been drawn. We offer suggestions of methods that may be used to clarify these reports of dual activity in the future, as well as a critical analysis of whether any truly dual-activity fluoroquinolones have yet been created.
Fluoroquinolones function by inhibiting two enzymes essential for prokaryotic cellular replication: DNA gyrase and topoisomerase IV.1,2,4,5,812 DNA gyrase, an A2B2 tetramer encoded by gyrA and gyrB, removes positive superhelical twists ahead of the replication fork and catalyses negative supercoiling.1,2,4,9,10,12 Topoisomerase IV, a C2E2 tetramer encoded by parC and parE, aids in chromosome partitioning by decatenating sister chromatids.1,2,4,9,10,12 Fluoroquinolone activity relies upon the formation of a ternary complex involving the fluoroquinolone, DNA gyrase or topoisomerase IV and the DNA on which the enzyme is bound.1,2,8 Collision of the replication fork with one of these ternary complexes leads to the release of a lethal double-stranded DNA break by an as yet undetermined mechanism.1,2,8
Resistance to fluoroquinolones in S. pneumoniae can be attributed to chromosomal mutations and/or efflux.5,8,11,12 Amino acid substitutions most commonly associated with resistance occur in the quinolone resistance-determining region (QRDR) of ParC and/or GyrA.1,8,12 QRDR mutations in gyrB and/or parE are rarely functionally associated with fluoroquinolone resistance in S. pneumoniae. Active efflux in S. pneumoniae is mediated by the secondary multidrug transporter PmrA.8
The majority of fluoroquinolones are reported to target preferentially either DNA gyrase or topoisomerase IV, although all fluoroquinolones can bind both enzymes to varying degrees.2,5,1013 In order to limit the emergence of resistance, it has been the aspiration of drug discovery programmes to identify fluoroquinolones that possess dual activity. Dual-acting fluoroquinolones demonstrate comparable activity against both DNA gyrase and topoisomerase IV.1,3,10 An organism would have to generate point mutations in both DNA gyrase and topoisomerase IV in order to become resistant to such a fluoroquinolone, as single point mutations in one target alone would not yield clinically relevant resistance, i.e. organisms whose MICs increased beyond breakpoint levels.9,11 As double mutations are a rare genetic event (they occur at a frequency of 1014 for fluoroquinolones in S. pneumoniae),14 the preferential use of fluoroquinolones with dual activity could limit the incidence of fluoroquinolone resistance in S. pneumoniae.
Target specificities of fluoroquinolones have been assessed by two methods: genetic and enzymic studies.1,9 Genetic studies identify the QRDR mutations acquired during the selection of clinical or laboratory-created (step-wise selected) fluoroquinolone-resistant mutants.1,2,5,8,9,15 Those agents for which mutations first appear in gyrA are reported to target preferentially DNA gyrase.13,15 Likewise, those agents correlated with mutations in parC have topoisomerase IV designated as their preferred in vivo target.13,15 Laboratory-created ciprofloxacin-, levofloxacin- and gemifloxacin-resistant mutants selected QRDR substitutions in parC, whereas mutations were first observed in gyrA in moxifloxacin- and gatifloxacin-resistant mutants (H. J. Smith, H. Walters, D. J. Hoban and G. G. Zhanel, unpublished results). Fluoroquinolones that interact equally with both parC and gyrA, and that do not demonstrate increased MICs until S. pneumoniae has acquired mutations in both DNA gyrase and topoisomerase IV, are considered to have dual activity. Enzymic studies evaluate the activities of fluoroquinolones against purified DNA gyrase and topoisomerase IV in vitro.9 These results are commonly reported as IC50 values. IC50 studies determine the fluoroquinolone concentration that is required to inhibit DNA gyrase- or topoisomerase IV-mediated supercoiling by 50%.5 Topoisomerase IV inhibition is noted by a 50% reduction of the decatenation activity.2,4,9,15 The inhibition of DNA gyrase is measured by a 50% reduction in supercoiling.2,4,9,15 A smaller IC50 value implies greater target affinity. Fluoroquinolones with dual activity will have very similar IC50 values for both DNA gyrase and topoisomerase IV.
There are various references to dual activity amongst the newer fluoroquinolones used to treat infections caused by S. pneumoniae.13 Unfortunately, the genetic and enzymic results do not correlate in S. pneumoniae as they do for Escherichia coli.9 A comparison of the enzymic results for fluoroquinolones is complicated by the use of various protocols by different research groups. In order to compare these results, IC50 ratios of DNA gyrase to topoisomerase IV are listed in Table 1 for various fluoroquinolones. For example, if the DNA gyrase IC50 for a fluoroquinolone is 40 and the topoisomerase IV IC50 is 5, the IC50 ratio of DNA gyrase to topoisomerase IV for that fluoroquinolone is 40:5 (8). A ratio near 1 indicates that the fluoroquinolone has similar activity against both DNA gyrase and topoisomerase IV (i.e. dual activity).6 The target specificities, as determined by genetic studies, are listed in Table 2. Discrepancies between the results of these two methods are evident. Enzymic studies identify all the fluoroquinolones as preferentially selecting topoisomerase IV except clinafloxacin and sitafloxacin, which are reported to be dual acting.2,4,6,9,15 Conversely, the genetic results show a target preference of DNA gyrase for gatifloxacin, gemifloxacin, moxifloxacin and sparfloxacin.13,5,810,15 Ciprofloxacin, levofloxacin, norfloxacin, pefloxacin and trovafloxacin are reported as preferentially selecting topoisomerase IV.13,5,8,10,15 Clinafloxacin, sitafloxacin and, in some cases, gemifloxacin, show dual activity.13,5,6,8,9,11,15 As shown with gemifloxacin, the reports generated from genetic and enzymic studies do not concur, as gemifloxacin is reported as a dual-activity agent or as binding preferentially to DNA gyrase or topoisomerase IV in different publications.1,4 The dual activity of clinafloxacin and sitafloxacin is indicated by both studies; however, the results for the other fluoroquinolones are varied.
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Some insight into the discrepancies between enzymic and genetic studies may be provided by the recent suggestion that mutations causing resistance exist outside the reported QRDRs of gyrA and parC in S. pneumoniae.2,4,8,9 The QRDRs commonly analysed in S. pneumoniae were based on those determined for E. coli.9,10 This may explain why the genetic and enzymic results agree in E. coli but not in S. pneumoniae. A larger QRDR may need to be elucidated for S. pneumoniae. If mutations mediating resistance lie outside the currently evaluated QRDR, true first-step mutations may have been overlooked in genetic studies,2,4,8,9 and mutated DNA gyrase or topoisomerase IV may have been used inadvertently in enzymic studies. Future analyses using the complete QRDR of S. pneumoniae in genetic and enzymic studies may then yield concurring results. The assessment of target specificity would be greatly simplified.
Although various fluoroquinolones have been claimed to possess dual activity against S. pneumoniae and thus to be less likely to select for fluoroquinolone resistance than non-dual-activity agents, presently genetic and enzymic studies suggest that only clinafloxacin and sitafloxacin are truly dually active. Insufficient data are currently available to conclude that gemifloxacin is a dual-activity agent. Whether the preferential use of dual-activity fluoroquinolones will limit the development of resistance in S. pneumoniae is unclear. What is clear is that clinically available, safe and effective dual-activity fluoroquinolones have yet to be created.
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References |
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2
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Pan, X. & Fisher, L. M. (1999). Streptococcus pneumoniae DNA gyrase and topoisomerase IV: overexpression, purification, and differential inhibition by fluoroquinolones. Antimicrobial Agents and Chemotherapy 43, 112936.
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Fukuda, H., Kishii, R., Takei, M. & Hosaka, M. (2001). Contributions of the 8-methoxy group of gatifloxacin to resistance selectivity, target preference, and antibacterial activity against Streptococcus pneumoniae. Antimicrobial Agents and Chemotherapy 45, 164953.
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Morrissey, I. & George, J. T. (2000). Purification of pneumococcal type II topoisomerases and inhibition by gemifloxacin and other quinolones. Journal of Antimicrobial Chemotherapy 45, Suppl. S1, 1016.
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Pestova, E., Millichap, J. J., Noskin, G. A. & Peterson, L. R. (2000). Intracellular targets of moxifloxacin: a comparison with other fluoroquinolones. Journal of Antimicrobial Chemotherapy 45, 58390.
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Onodera, Y., Uchida, Y., Tanaka, M. & Sato, K. (1999). Dual inhibitory activity of sitafloxacin (DU-6859a) against DNA gyrase and topoisomerase IV of Streptococcus pneumoniae. Journal of Antimicrobial Chemotherapy 44, 5336.
7 . Yamada, H., Hisada, H., Mitsuyama, M., Takahata, M., Todo, Y., Minami, S. et al. (2000). BMS-284756 (T-3811ME), a des-F(6)-quinolone: selectivity between bacterial and human type II DNA toposiomerases. In Program and Abstracts of the Fortieth Interscience Conference on Antimicrobial Agents and Chemotherapy, Toronto, Canada, 2000. Abstract 753, p. 82. American Society for Microbiology, Washington, DC.
8 . Bush, K. & Goldschmidt, R. (2000). Effectiveness of fluoroquinolones against Gram-positive bacteria. Current Opinion in Investigational Drugs 1, 2230.[Medline]
9
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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.
10 . Pan, X. & 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]
11 . Sanders, C. C. (2001). Mechanisms responsible for cross-resistance and dichotomous resistance among the quinolones. Clinical Infectious Diseases 32, Suppl. 1, S18.[ISI][Medline]
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Varon, E., Janoir, C., Kitzis, M. & Gutmann, L. (1999). ParC and GyrA may be interchangeable initial targets of some fluoroquinolones in Streptococcus pneumoniae. Antimicrobial Agents and Chemotherapy 43, 3026.
13 . Zhanel, G. G., Ennis, K., Vercaigne, L., Gin, A. S., Embil, J., Smith, H. et al. (2002). A critical review of the fluoroquinolones: Focus on respiratory infections. Drugs 62, 1359.[ISI][Medline]
14
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Blondeau, J. M., Xilin, A., Hansen, G. & Drlica, K. (2001). Mutant prevention concentrations of fluoroquinolones for clinical isolates of Streptococcus pneumoniae. Antimicrobial Agents and Chemotherapy 45, 4338.
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Alovero, F. L., Pan, X., Morris, J. E., Manzo, R. H. & Fisher, L. M. (2000). Engineering the specificity of antibacterial fluoroquinolones: benzenesulfonamide modifications at C-7 of ciprofloxacin change its primary target in Streptococcus pneumoniae from topoisomerase IV to gyrase. Antimicrobial Agents and Chemotherapy 44, 3205.