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2The fluoroquinolones are broad-spectrum antibacterial agents that are becoming increasingly popular as bacterial resistance erodes the effectiveness of other agents (fluoroquinolone sales accounted for 18% of the antibacterial market in 2006) (41). One of the attractive features of the quinolones is their ability to kill bacteria rapidly, an ability that differs widely among the various derivatives. For example, quinolones differ in rate and extent of killing, in the need for aerobic metabolism to kill cells, and in the effect of protein synthesis inhibitors on quinolone lethality. Understanding the mechanisms underlying these differences could lead to new ways for identifying the most bactericidal quinolone derivatives.Before describing the types of damage caused by the quinolones, it is useful to define lethal activity. Operationally, it is the ability of drug treatment to reduce the number of viable cells, usually measured as CFU on drug-free agar after treatment. This assay is distinct from measurements that detect inhibition of growth (e.g., MIC), since with the latter bacteria are exposed to drug throughout the measurement. The distinction between killing and blocking growth is important because it allows susceptibility determinations to be related to particular biological processes. For example, inhibition of growth is typically reversed by the removal of drug, while cell death is not. Thus, biochemical events associated with blocking growth should be readily reversible, while those responsible for cell death should be difficult to reverse. Reversibility can be used to distinguish among quinolone derivatives and assign functions to particular aspects of drug structure. Moreover, protective functions, such as repair and stress responses, can be distinguished by whether their absence affects inhibition of growth, killing, or both.The intracellular targets of the quinolones are two DNA topoisomerases: gyrase and topoisomerase IV. Gyrase tends to be the primary target in gram-negative bacteria, while topoisomerase IV is preferentially inhibited by most quinolones in gram-positive organisms (28). Both enzymes use a doublestrand DNA passage mechanism, and it is likely that quinolone biochemistry is similar for both. However, physiological differences between the enzymes exist, some of which may bear on quinolone lethality.In the present minireview we consider cell death through a two-part "poison" hypothesis in which the quinolones form reversible drug-topoisomerase-DNA complexes that subsequently lead to several types of irreversible (lethal) damage. Other consequences of quinolone treatment, such as depletion of gyrase and topoisomerase IV activity, are probably less immediate (42). To provide a framework for considering quinolone lethality, we begin by briefly describing the drug-topoisomerase-DNA complexes. Readers interested in a more comprehensive discussion of quinolones are referred to a previously published work (28). QUINOLONE-TOPOISOMERASE-DNA COMPLEXESAs a normal part of their reaction mechanism, g...

The quinolones trap DNA gyrase and DNA topoisomerase IV on DNA as complexes in which the DNA is broken but constrained by protein. Early studies suggested that drug binding occurs largely along helix-4 of the GyrA (gyrase) and ParC (topoisomerase IV) proteins. However, recent X-ray crystallography shows drug intercalating between the -1 and +1 nucleotides of cut DNA, with only one end of the drug extending to helix-4. These two models may reflect distinct structural steps in complex formation. A consequence of drug-enzyme-DNA complex formation is reversible inhibition of DNA replication; cell death arises from subsequent events in which bacterial chromosomes are fragmented through two poorly understood pathways. In one pathway, chromosome fragmentation stimulates excessive accumulation of highly toxic reactive oxygen species that are responsible for cell death. Quinolone resistance arises stepwise through selective amplification of mutants when drug concentrations are above the MIC and below the MPC, as observed with static agar plate assays, dynamic in vitro systems, and experimental infection of rabbits. The gap between MIC and MPC can be narrowed by compound design that should restrict the emergence of resistance. Resistance is likely to become increasingly important, since three types of plasmid-borne resistance have been reported.

The mutant selection window hypothesis postulates that, for each antimicrobial-pathogen combination, an antimicrobial concentration range exists in which selective amplification of single-step, drug-resistant mutants occurs. This hypothesis suggests an antimutant dosing strategy that is keyed to the upper boundary of the selection window: the mutant prevention concentration. Correlations are described between the mutant prevention concentration-a static parameter that is measured with agar plates-and fluctuating drug concentrations that restrict mutant amplification in vitro and in animals. When drug resistance is acquired stepwise, the mutant selection window increases, making the suppression of each successive mutant increasingly more difficult. For agents that kill drug-resistant mutants in a drug concentration-dependent manner, the use of the area under the 24-h time-drug concentration curve value divided by the value of the mutant prevention concentration is suggested as an index for designing antimutant dosing regimens. The need for such regimens is emphasized by a clinical example in which acquisition of drug resistance occurs concurrently with eradication of susceptible bacterial cells. These data support using the mutant selection window to optimize antimicrobial dosing regimens.Antimicrobial resistance is a complex problem that is likely to require attention at many levels [1]. Issues concerning dosing are addressed by the mutant selection window hypothesis [2,3]. This hypothesis maintains that drug-resistant mutant subpopulations present prior to initiation of antimicrobial treatment are enriched and amplified during therapy when antimicrobial concentrations fall within a specific range (the mutant selection window). The upper boundary of the mutant selection window is the MIC of the least drugsusceptible mutant subpopulation, a value called the mutant prevention concentration (MPC) [4]. The lower boundary of the mutant selection window is the lowest concentration that exerts selective pressure, often approximated by the minimal concentration that inhibits colony formation by 99% (MIC 99 ). Two principles emerge from the hypothesis. First, traditional dos-

Studies with fluoroquinolones have led to a general method for restricting the selection of antibiotic-resistant mutants. The strategy is based on the use of antibiotic concentrations that require cells to obtain 2 concurrent resistance mutations for growth. That concentration has been called the "mutant prevention concentration" (MPC) because no resistant colony is recovered even when >10(10) cells are plated. Resistant mutants are selected exclusively within a concentration range (mutant selection window) that extends from the point where growth inhibition begins, approximated by the minimal inhibitory concentration, up to the MPC. The dimensions of the mutant selection window can be reduced in a variety of ways, including adjustment of antibiotic structure and dosage regimens. The window can be closed to prevent mutant selection through combination therapy with > or =2 antimicrobial agents if their normalized pharmacokinetic profiles superimpose at concentrations that inhibit growth. Application of these principles could drastically restrict the selection of drug-resistant pathogens.