Mechanisms of Action and Resistance of Older and Newer Fluoroquinolones
- Division of Infectious Diseases, Massachusetts General Hospital, Harvard Medical School, Boston, Massachusetts
- Reprints or correspondence: Dr. David C. Hooper, Division of Infectious Diseases, Massachusetts General Hospital, 55 Fruit St., Boston, MA 02114-2696 (dhooper{at}partners.org).
Abstract
The fluoroquinolones interact with 2 bacterial targets, the related enzymes DNA gyrase and topoisomerase IV, both of which are involved in DNA replication. Quinolones form complexes of these enzymes with DNA, complexes that block movement of the DNA-replication fork and thereby inhibit DNA replication. Many older quinolones differ in their relative activities against gyrase and topoisomerase IV in a bacterial cell, having greater potency against gyrase than against topoisomerase IV in many gram-negative bacteria and greater potency against topoisomerase IV than against gyrase in many gram-positive bacteria. Several newer quinolones appear to have more closely balanced activity against these enzymes. Resistance to fluoroquinolones occurs as a result of mutational amino acid substitutions in the subunits of the more sensitive (or primary-target) enzyme within the cell. If, however, both enzymes are similarly susceptible to a fluoroquinolone, then the level of resistance caused by a primary-target mutation may be low and may be limited by the sensitivity of the secondary target. Fluoroquinolones also differ in the extent to which common bacterial multidrug efflux pumps affect their activity, with some compounds being unaffected by resistance mechanisms because of overexpression of such pumps. Newer fluoroquinolone interaction with dual targets and avoidance of efflux-resistance mechanisms may each contribute to the lower frequencies of selection of resistant mutants in the laboratory.
A clearer understanding of the mechanisms of fluoroquinolone action and resistance has evolved with the development of newer agents in this class. Older quinolones, such as ciprofloxacin and ofloxacin, were followed by new agents, such as levofloxacin, sparfloxacin, grepafloxacin, and trovafloxacin. Additional newer agents, such as gatifloxacin and moxifloxacin, are being developed. Currently, fluoroquinolones are known to have 2 enzyme targets, DNA gyrase and topoisomerase IV, in the bacterial cell; both of these targets are essential for bacterial DNA replication [1]. DNA gyrase is a tetramer composed of 2 GyrA and 2 GyrB subunits. Topoisomerase IV is similarly structured and is composed of 2 ParC and 2 ParE subunits, which are also known as GrlA and GrlB, respectively, in Staphylococcus aureus [2]. ParC is homologous to GyrA, and ParE is homologous to GyrB.
DNA gyrase is the only bacterial enzyme that introduces negative superhelical twists into DNA [3]. Negatively supertwisted DNA is important for initiation of DNA replication. DNA gyrase also facilitates DNA replication by removing positive superhelical twists that accumulate ahead of the replication fork or as a result of the transcription of certain genes [3, 4]. Topoisomerase IV acts in the terminal stages of DNA replication, allowing for the decatenation (separation) of interlinked daughter chromosomes so that segregation into daughter cells can occur [5, 6]. Fluoroquinolones inhibit these enzymes by stabilizing either the DNA-DNA gyrase complex [1] or the DNA-topoisomerase IV complex. The stabilized DNA-DNA gyrase complex blocks movement of the replication fork, causing formerly reversible DNA-enzyme complexes to become irreversible [7]. Damage to DNA and the generation of DNA-strand breaks then trigger a set of events, as yet poorly defined, that follow the rapid inhibition of DNA synthesis and result in eventual cell death [1, 4].
Targets of Quinolone Action and Resistanceby Alterations in Targets
Although all fluoroquinolones are active, to some extent, against both DNA gyrase and topoisomerase IV, they differ in their relative activities against these enzymes. In earlier studies with Escherichia coli, DNA gyrase was found to be a primary target of quinolone activity [8–10]; the activity of quinolones against topoisomerase IV appeared to be somewhat limited, however, and topoisomerase IV was shown to be a secondary quinolone target [11, 12]. Later, the results of studies done with S. aureus indicated that, in contrast to E. coli, topoisomerase IV was the primary quinolone target in this species [2, 13–15]. In addition, the results of studies done with both E. coli and S. aureus demonstrated that different levels of quinolone resistance occurred, depending on whether alterations were made in the primary target, the secondary target, or both. Mutations in the primary target precede those in the secondary target, in a stepwise selection for resistance; mutations in both targets produce higher levels of resistance.
These genetic relationships have generally been understood on the basis of the relative sensitivities of DNA gyrase and topoisomerase IV within a given organism. Results from studies done with E. coli and S. aureus are illustrated by the work of Blanche et al. [16], who investigated the inhibitory activities of several fluoroquinolones (including ciprofloxacin, sparfloxacin, norfloxacin, and ofloxacin) against both purified DNA gyrase and purified topoisomerase IV. With E. coli, the concentration of fluoroquinolone that was needed to inhibit DNA gyrase activity by 50% (IC50) ranged from 0.5 to 1.5 µg/mL (table 1). In contrast, the IC50 that was needed to inhibit topoisomerase IV activity was much higher, ranging from 2 to 12 µg/mL. In S. aureus, topoisomerase IV was more sensitive to fluoroquinolones than was DNA gyrase; IC50 values for topoisomerase IV ranged from 4 to 10 µg/mL, whereas IC50 values for DNA gyrase were higher, ranging from 12 to >100 µg/mL. For both organisms, the MIC was closer to the IC50 of the more susceptible enzyme, suggesting that the fluoroquinolone MIC is determined by activity against the primary (more susceptible) target. Thus, the primary enzyme target, as determined by genetic testing, correlated with the more susceptible of the 2 enzymes in a given organism.
Table 1
Inhibitory activities of quinolones against gyrase, topoisomerase IV, and whole cells of Escherchia coli and Staphylococcus aureus.
The fluoroquinolone sensitivities of both enzymes are relevant to the development of resistance. A single mutational event in the more sensitive primary target can result in an increase in the MIC of the drug. If the altered primary-target enzyme remains more sensitive to fluoroquinolone than does the secondary target, it continues to determine the MIC. However, if the altered primary target becomes less sensitive than the secondary target, MIC is determined by the inhibitory activity of the fluoroquinolone against the secondary target. Theoretically, therefore, if the original sensitivities of both DNA gyrase and topoisomerase IV are the same, no single mutational alteration in either enzyme would result in an increase in MIC. Rather, resistance would require concurrent alteration in both enzymes. Properties of this type have been reported for the newer quinolones clinafloxacin [17] and sitafloxacin [18].
The activities of a newer fluoroquinolone (gatifloxacin) and an older one (ciprofloxacin) against parent and mutant strains of S. aureus were compared by Ince et al. [19]. Against the parent strain, gatifloxacin had a lower MIC than did ciprofloxacin (0.125–0.25 µg/mL versus 0.25–0.5 µg/mL, respectively; table 2). When the 2 fluoroquinolones were tested against a gyrA mutant strain, no change in the MIC of either compound was observed. However, a 4- to 8-fold increase in the ciprofloxacin MIC and a 2- to 4-fold increase in the gatifloxacin MIC occurred in strains with mutations in either the grlA or the grlB gene. These results support those of previous studies that reported topoisomerase IV to be the primary quinolone target of ciprofloxacin in S. aureus [15].
Table 2
The effects of DNA gyrase and topoisomerase IV mutations in Staphylococcus aureus on the MICs of Cpfx and Gtfx.
When gatifloxacin and ciprofloxacin were tested against a strain that had both grlA and gyrA mutations, an even higher increase in MIC was observed (table 2) [19]. The MIC of ciprofloxacin for the doubly mutant strain was 16–32 µg/mL, which is up to 128 times greater than its MIC for the parent strain. Against the doubly mutant strain, the activity of gatifloxacin was better than that of ciprofloxacin, since the gatifloxacin MIC remained relatively low at 4 µg/mL.
In general, the relationships between DNA gyrase and topoisomerase IV as primary or secondary targets have followed the pattern found with E. coli and S. aureus in which DNA gyrase is the primary fluoroquinolone target for gram-negative bacteria and topoisomerase IV is the primary target for gram-positive bacteria [20]. However, some interesting exceptions to this pattern have been seen with some fluoroquinolones that are active against Streptococcus pneumoniae [21–23]. In a study by Fukuda and Hiramatsu [22], parC mutations resulted in higher MICs of trovafloxacin, levofloxacin, ciprofloxacin, and norfloxacin, indicating that topoisomerase IV may be the primary target for these fluoroquinolones. In contrast, changes in gyrA resulted in higher MICs of gatifloxacin and sparfloxacin. The results of genetic tests indicated that DNA gyrase is a primary target of these drugs. Surprisingly, however, purified topoisomerase IV was more susceptible to sparfloxacin than was DNA gyrase [18, 21]. Thus, other as-yet-undefined factors, in addition to relative drug sensitivity, may determine whether certain drugs primarily target DNA gyrase or topoisomerase IV.
Efflux Mechanisms of Resistance
Another important factor in the determination of fluoroquinolone resistance is the expression or overexpression of energy-dependent efflux pumps that can actively remove antibacterial agents from the cell. Currently, almost all bacteria are thought to have such efflux pumps, many of which are multidrug pumps that recognize a number of different antibacterial classes and other compounds as potential substrates. Because overexpression of these pumps in a number of bacterial species may result in multidrug resistance, an antibacterial agent that is not affected by these efflux mechanisms may have certain advantages, such as a lower likelihood of selecting resistant mutants.
The NorA efflux pump has been shown to contribute to antibacterial resistance in S. aureus [24–27]. The effect of increased NorA expression on gatifloxacin and ciprofloxacin activity was recently investigated by Ince et al. [19]. When overexpression of the NorA efflux pump was effected through a single mutation of the promoter region of the norA gene, the MIC of ciprofloxacin increased 2- to 4-fold (from 0.25 to 0.5–1.0 µg/mL); the increase in gatifloxacin MIC was 2-fold (from 0.125 to 0.25 µg/mL) (table 3). This same mutation was shown to cause an 8-fold increase in the MIC of norfloxacin [25]. In other laboratory-constructed strains that overexpress NorA, the MICs of sparfloxacin and gatifloxacin were less affected than those of ciprofloxacin and norfloxacin [28]. As discussed earlier, both ciprofloxacin and gatifloxacin had decreased activity against strains with the double mutation grlA, gyrA (MICs of 16–32 µg/mL and 4.0 µg/mL, respectively). Against a strain of S. aureus that had grlA and gyrA mutations and a mutation causing overexpression of NorA, the ciprofloxacin MIC was 32 µg/mL and the gatifloxacin MIC was 4.0 µg/mL [19]. These data suggest that increasing the expression of efflux pumps, such as NorA in S. aureus, may have only a limited effect on the activity of some fluoroquinolones, particularly sparfloxacin and gatifloxacin.
Table 3
Effects of increased expression of NorA efflux pumpof Staphylococcus aureus on activity of Cpfx and Gtfx.
The effects of efflux pumps on fluoroquinolone activity in S. pneumoniae were reported by Piddock et al. [29]. With fluoroquinolone-selected mutant strains of S. pneumoniae exhibiting characteristics of efflux pump overexpression (cross-resistance to unrelated antibiotic agents and reduction of ciprofloxacin MIC when reserpine, an efflux pump inhibitor, was added), newer fluoroquinolones, including gatifloxacin, sparfloxacin, and trovafloxacin, had only slightly higher MICs (or no change, in the case of levofloxacin) than they did with the parent strain (table 4). More recently, the gene encoding an efflux pump, PmrA, which is related to NorA, was identified in S. pneumoniae [30], and its inactivation resulted in increased susceptibility to norfloxacin (8-fold) and ciprofloxacin (4-fold) but had no effect on 2 newer quinolones (sparfloxacin or moxifloxacin), a finding that suggests that the latter 2 drugs were poor substrates for PmrA. The hydrophobicity of a fluoroquinolone has been associated with its activity being little affected by expression of the NorA efflux pump [31], but other structural features of quinolones may also contribute to the extent to which efflux pumps affect quinolone activity [32].
Table 4
Susceptibility of efflux mutant strains of Streptococcus pneumoniae to fluoroquinolones.
Selection of Resistance Mutations by Newer Fluoroquinolones
Newer fluoroquinolones act against both DNA gyrase and topoisomerase IV, and the activity of many are less affected by dual mutations than are older fluoroquinolones. In addition, these agents appear to be only mildly affected by the overexpression of some efflux pumps, such as NorA and PmrA. How relevant such findings are for the selection of resistance has been tested in both S. aureus and S. pneumoniae. In the study by Ince et al. [19], mutant strains could be readily selected by plating them on agar containing twice the MIC of either gatifloxacin or ciprofloxacin (table 5). However, at an MIC 4 times that of gatifloxacin, mutant strains were selected at a frequency of 2.8 × 10−9, and, at an MIC 8 times that of gatifloxacin, no mutants could be selected, for a frequency of <4.5 × 10−11 to <2.8 × 10−9. In comparison, selection of resistant strains continued to be observed when bacteria were plated on agar containing 8 times the MIC of ciprofloxacin.
Table 5
Frequency of selection of resistant mutants of Staphylococcus aureus ISP794 plated on agar containing Cpfx or Gtfx.
In the study by Fukuda and Hiramatsu [22], the frequency with which various fluoroquinolones selected mutant strains of S. pneumoniae was measured and compared. Of the tested fluoroquinolones, gatifloxacin was the least likely to select resistant strains (table 6). At 2 times its MIC for S. pneumoniae, gatifloxacin selected mutants at an undetectable level (<1.1 × 10−9). In comparison, higher concentrations of either levofloxacin or ciprofloxacin (4 times MIC) and either trovafloxacin or norfloxacin (8 times MIC) were necessary to reach an undetectable level of selection of mutants. Sparfloxacin was the most likely to select mutants, requiring plating on 32 times its MIC before no mutants were selected.
Table 6
Frequency of selection of resistant mutants of Streptococcus pneumoniae IID553 plated on agar containing fluoroquinolones.
Conclusion
The continued development and testing of newer fluoroquinolones have provided a better understanding of the mechanisms of action and resistance in this class. Currently, 2 different mechanisms are considered to be major determinants of fluoroquinolone resistance. First, mutations resulting in changes in the primary enzyme target may result in bacterial resistance, although the magnitude of this increase may differ among fluoroquinolones. An even higher level of resistance may result if a stepwise mutation occurs in both the primary and secondary targets. Second, overexpression of multidrug efflux pumps may also contribute to fluoroquinolone resistance in a wide variety of bacterial organisms.
The effects of altered enzyme targets and overexpressed efflux systems have already been studied with many of the newer fluoroquinolones. Results from such studies indicate that bacterial enzyme targets may be more susceptible to the newer fluoroquinolones. These data are supported by studies demonstrating a lower likelihood of selecting resistant strains with such antibacterial agents as gatifloxacin, levofloxacin, clinafloxacin, and sitafloxacin.
As the testing of newer fluoroquinolones continues, a number of topics will require additional study and definition. Such topics include the effect of fluoroquinolone structure on the mechanisms of action and resistance, the relevance of pharmacodynamic modeling in determining dosing and predicting efficacy, and the actual clinical efficacy of fluoroquinolones for various indications in different patient populations. In addition, it is hoped that future inquiries into the mechanisms of action and resistance will translate into the continued development of agents that are less likely to contribute to the increasing worldwide prevalence of antibacterial resistance.
- © 2000 by the Infectious Diseases Society of America
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