JAC Advance Access originally published online on September 26, 2006
Journal of Antimicrobial Chemotherapy 2006 58(6):1283-1286; doi:10.1093/jac/dkl388
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Bactericidal activity and target preference of a piperazinyl-cross-linked ciprofloxacin dimer with Staphylococcus aureus and Escherichia coli
1 Public Health Research Institute, 225 Warren Street Newark, NJ 07103, USA 2 Division of Medicinal and Natural Products Chemistry, The University of Iowa, Iowa City IA 52242, USA
*Corresponding author. Tel: +1-973-854-3360; Fax: +1-973-854-3101; E-mail: drlica{at}phri.org
Received 15 May 2006; returned 12 July 2006; revised 10 August 2006; accepted 4 September 2006
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Abstract |
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Background: Previous work showed that piperazinyl-cross-linked ciprofloxacin dimer exhibits good bacteriostatic activity with Streptococcus pneumoniae and Staphylococcus aureus; lethal activity was not measured. Subsequently, the dimer failed to kill Mycobacterium smegmatis but blocked growth. Whether the compound is lethal with non-mycobacterial species is not known.
Methods: Bacteriostatic and bactericidal activities were measured with wild-type cells and topoisomerase mutants of S. aureus and Escherichia coli for ciprofloxacin and a dimer of ciprofloxacin. Spontaneous resistance mutants were selected with S. aureus for both compounds, followed by target identification by nucleotide sequence determination of the quinolone-resistance-determining-region of gyrA (gyrase) and parC (topoisomerase IV).
Results: The dimer was lethal, in some cases exhibiting more activity than ciprofloxacin (particularly with wild-type cells and a parC mutant of S. aureus). Dimerization affected target preference with S. aureus but not with E. coli. Resistance mutations in either gyrA or parC of S. aureus raised the MIC of the dimer, but only a parC mutation raised the MIC of ciprofloxacin. With S. aureus, the dimer selected spontaneous resistant gyrA mutants, whereas ciprofloxacin selected a parC mutant. With E. coli, a gyrA, but not a parC, mutation raised the MIC of both compounds.
Conclusion: The dimer readily killed S. aureus and E. coli, representative Gram-positive and Gram-negative bacteria. In both cases the preferred target was DNA gyrase. The switch in target preference may be responsible for the greater lethality of the dimer seen with S. aureus.
Keywords: fluoroquinolones , gyrase , topoisomerase IV
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The fluoroquinolones trap DNA gyrase and DNA topoisomerase IV on bacterial chromosomes, forming complexes of drug, enzyme and broken DNA. These ternary complexes block DNA replication and transcription, thereby inhibiting bacterial growth. Cell death is proposed to occur subsequently when DNA breaks are released from the ternary complexes.1 The existence of such a two-step process is consistent with some quinolones having similar MICs but different abilities to kill cells,2 with some quinolones being only bacteriostatic,3 and with some quinolones blocking DNA synthesis under conditions in which cells are not killed.1 A distinction between bacteriostatic and bactericidal activities means that low MIC values cannot be assumed to correlate with excellent lethal activity.
In an attempt to improve the activity of fluoroquinolones, dimer molecules were constructed in which the C-7 piperazinyl rings of two drug molecules were joined.4,5 When tested with Streptococcus pneumoniae and Staphylococcus aureus, fluoroquinolone dimers exhibited greater bacteriostatic activity than the parent monomers,5,6 but lethal activity was not measured. We later found that Mycobacterium smegmatis is not killed by the dimer even though growth is blocked.3 Since lethal activity is a central aspect of quinolone efficacy, knowing whether dimers can be lethal with both wild-type cells and resistant mutants of pathogenic species is important to their refinement as antimicrobials.
This work examined a piperazinyl-linked ciprofloxacin dimer for lethal activity with S. aureus and Escherichia coli. Both organisms were readily killed by the dimer. Determination of MICs for resistant mutants and selection of spontaneous, single-step resistant mutants revealed that, as reported with S. pneumoniae,4 the target of the dimer in S. aureus is primarily gyrase (topoisomerase IV is the primary target of monomeric ciprofloxacin).7 Genetic experiments have led to the expectation that resistant mutants are less likely to be recovered when gyrase is the target.8 Knowing how to alter target preference and understanding the subsequent effect on killing pathways may help guide quinolone development.
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Bacterial strains, fluoroquinolones and susceptibility testing
S. aureus and E. coli K-12 strains were grown as described previously.9 The ciprofloxacin dimer 7,7'-[(2E)-2-butene-1,4-diyldi-4,1-piperazinediyl]bis[1-cyclopropyl-6-fluoro-1,4-dihydro-4-oxo-3-quinoline carboxylic acid]4,5 was dissolved in DMSO at 1 mg/mL. Ciprofloxacin hydrochloride salt (Bayer Corp., West Haven, CT, USA) was dissolved in sterile water at 10 mg/mL.
MIC was the minimal concentration that inhibited visible growth following broth dilution and overnight incubation. Starting cultures contained about 104–105 cells; quinolone concentrations increased linearly by no more than 50% per increment. To measure lethal action, exponentially growing cells were divided into 1 mL aliquots to which various concentrations of fluoroquinolone were added. After incubation for 0.5 h (E. coli) or 3 h (S. aureus) with shaking, cells were diluted in cold 0.9% NaCl and applied to drug-free agar. cfu were determined following overnight incubation at 37°C. Viable counts after various treatments were compared with those from a control sample taken at the time of fluoroquinolone addition. LD99 was the drug concentration that caused a 100-fold reduction of bacterial survival.
Selection of resistant mutants
Single-step, spontaneous mutants were obtained from three independent overnight cultures of wild-type S. aureus (strain ISP794). Various amounts of culture (1–3 x 109 cells) were applied to GL agar plates containing ciprofloxacin or its dimer at 1.5, 2, 3 and 4x MIC. After 48–72 h of incubation, colonies were recovered and streaked on drug-free agar. Mutant MIC was determined, and the nucleotide sequence of the gyrA and parC (grlA) quinolone-resistance-determining-regions (QRDRs) was determined as described previously.10
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Susceptibility of S. aureus to ciprofloxacin dimer
The dimer was lethal with exponentially growing S. aureus (Figure 1a, circles). Thus the dimer behaves differently with S. aureus than with M. smegmatis, which is not killed by the dimer even at 6 mg/L or 20x MIC [ref. 3; confirmed in the present study (data not shown)]. The dimer was 
3-fold more lethal than ciprofloxacin (wild-type LD99 = 0.5 and 1.7 mg/L, for the dimer and ciprofloxacin, respectively; on a molar basis this difference would be roughly 2-fold greater). When bacteriostatic activity (MIC) was measured, the dimer was 6- to 7-fold more active than ciprofloxacin with wild-type S. aureus (strain ISP794, Table 1), confirming previous work.4,5
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Activity with gyrase and topoisomerase IV resistance mutants of S. aureus
Topoisomerase IV is the primary target for ciprofloxacin,7 which was confirmed by a gyrA resistance mutant having an MIC similar to that for wild-type cells and a parC mutant having an MIC 6-fold higher (Table 1). In contrast, the dimer MIC was 4.5-fold higher for the gyrA mutant than for wild-type cells, consistent with a shift to gyrase as a target. The parC mutant MIC was 2- to 3-fold higher (Table 1), indicating that bacteriostatic, dimer-containing complexes form with both enzymes. For both compounds, mutations in both gyrA and parC increased the MIC more than 10-fold above that seen when only one gene was mutated (not shown). Thus complexes between dimer and either wild-type enzyme inhibit growth; ciprofloxacin inhibits growth largely through complexes formed with topoisomerase IV.
When lethality was measured for the dimer, little difference was observed at concentrations below 0.5 mg/L for wild-type, parC mutant and gyrA mutant strains (Figure 1a). At high concentrations the gyrA mutant was less susceptible (Figure 1a, squares). Loss of lethal activity at high concentration, which is observed with many quinolones, is currently unexplained. Ciprofloxacin lethality was markedly reduced by the parC mutation and unaffected by the gyrA mutation (Figure 1b). Thus dual targeting with a small preference for gyrase is involved in dimer lethality while ciprofloxacin lethality is mediated primarily by topoisomerase IV.
Selection of resistant mutants of S. aureus
Since mutations in either gyrA or parC raised the MIC of the dimer, we examined resistant mutants selected with the dimer as an independent indicator of target preference. Mutants were readily selected when more than 109 cells were applied to agar plates containing the dimer or ciprofloxacin at 1.5-, 2- and 3-fold MIC. Mutants selected with the dimer showed a 2- to 4-fold increase in dimer MIC but no change in ciprofloxacin MIC mutants selected with ciprofloxacin had a 4-fold increase in ciprofloxacin MIC but no change in dimer MIC (not shown). DNA sequence determination revealed that five of six mutants obtained with the dimer harboured a GyrA Ser-84 to Leu substitution; one contained a Ser-84 to Ala substitution. No variation in the QRDR of ParC was observed with any dimer-resistant mutant. A single-step mutant selected with ciprofloxacin had an Ala-116 to Glu substitution in ParC and no change in the QRDR of GyrA, as expected from previous work11 (substitutions of Ser-80 and Glu-84 tend to be more common than Ala-116 substitutions). These data are consistent with the dimer and ciprofloxacin preferentially targeting gyrase and topoisomerase IV, respectively.
Activity of ciprofloxacin dimer with E. coli
The dimer killed E. coli (Figure 1c). However, the dimer was roughly 9-fold less active than ciprofloxacin (Figure 1c and d; wild-type LD99 = 0.69 and 0.076 mg/L, for the dimer and ciprofloxacin, respectively). Both compounds killed the parC mutant; neither was very effective with the gyrA mutant (Figure 1c and d). The dimer was also less effective than ciprofloxacin at blocking growth of wild-type E. coli (Table 1). Introduction of a parC resistance allele (strain KD1373, Table 1) had no effect on MIC; however, a gyrA resistance mutant (strain KD2750) was considerably less susceptible to both compounds (Table 1). Thus gyrase is the primary target for both quinolones with E. coli, unlike the situation with S. aureus.
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The results described above demonstrate that a dimer of ciprofloxacin is bactericidal with both Gram-positive and Gram-negative bacteria. With S. aureus, the dimer exhibited greater bacteriostatic and bactericidal activity than ciprofloxacin; the opposite was seen with E. coli (Table 1 and Figure 1). The absence of dimer lethality with M. smegmatis3 probably reflects a unique interaction between dimer and mycobacterial gyrase, since other fluoroquinolones readily kill mycobacteria.3
With S. aureus, both gyrA and parC mutations increased the MIC of the dimer (Table 1); single-step GyrA variants having either Leu or Ala at codon 84 were readily selected with the ciprofloxacin dimer. In contrast, ciprofloxacin MIC was raised only by the parC mutant, and a single-step, spontaneous ParC variant was selected with ciprofloxacin. Thus dimerization of ciprofloxacin changes the target preference from topoisomerase IV to gyrase. A shift towards gyrase as a target has been observed with S. pneumoniae for a fluoroquinolone dimer6 and a derivative of ciprofloxacin having a large C-7 ring structure.12 One ciprofloxacin moiety of the dimer may behave as a large C-7 ring system for the other ciprofloxacin moiety. A target shift was not observed with E. coli: both compounds blocked growth primarily by targeting gyrase (Table 1).
In summary, bactericidal activity is readily observed for a dimer of ciprofloxacin with S. aureus and E. coli but not with M. smegmatis, even though all three organisms have gyrase as the primary target. With S. aureus the dimer was more lethal than ciprofloxacin, perhaps because it preferentially targets gyrase rather than topoisomerase IV. Understanding target effects may lead to new ways to make quinolones more lethal.
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None to declare for the present work.
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Acknowledgements |
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We thank Marila Gennaro and Richard Pine for critical comments on the manuscript. The work was supported by NIH grants AI35257 and AIO63431 (K. D.) and University of Iowa Biological Sciences Funding Program (R. K.).
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1 Chen C-R, Malik M, Snyder M, et al. (1996) DNA gyrase and topoisomerase IV on the bacterial chromosome: quinolone-induced DNA cleavage. J Mol Biol 258:627–37.[CrossRef][Web of Science][Medline]
2 Morrissey I and George J. (2000) Bactericial activity of gemifloxacin and other quinolones against Streptococcus pneumoniae. J Antimicrob Chemother 45:Suppl S1, 107–10.[Abstract]
3
Malik M, Lu T, Zhao X, et al. (2005) Lethality of quinolones against Mycobacterium smegmatis in the presence or absence of chloramphenicol. Antimicrob Agents Chemother 49:2008–14.
4 Kerns R, Rybak M, Kaatz G, et al. (2003) Structural features of piperazinyl-linked ciprofloxacin dimers required for activity against drug-resistant strains of Staphylococcus aureus. Bioorg Med Chem Lett 13:2109–12.[CrossRef][Medline]
5 Kerns RJ, Rybak MJ, Kaatz GW, et al. (2003) Piperazinyl-linked fluoroquinolone dimers possessing potent antibacterial activity against drug-resistant strains of Staphylococcus aureus. Bioorg Med Chem Lett 13:1745–9.[CrossRef][Medline]
6
Gould K, Pan X, Kerns R, et al. (2004) Ciprofloxacin dimers target gyrase in Streptococcus pneumoniae. Antimicrob Agents Chemother 48:2108–15.
7 Ferrero L, Cameron B, Crouzet J. (1995) Analysis of gyrA and grlA mutations in stepwise-selected ciprofloxacin-resistant mutants of Staphylococcus aureus. Antimicrob Agents Chemother 39:1554–8.[Abstract]
8
Fukuda H, Kishii R, Takei M, et al. (2001) Contributions of the 8-methoxy group of gatifloxacin to resistance selectivity, target preference, and antibacterial activity against Streptococcus pneumoniae. Antimicrob Agents Chemother 45:1649–53.
9 Zhao X and Drlica K. (2002) Restricting the selection of antibiotic-resistant mutants: measurement and potential uses of the mutant selection window. J Inf Dis 185:561–5.[CrossRef][Web of Science][Medline]
10
Lu T, Zhao X, Li X, et al. (2001) Enhancement of fluoroquinolone activity by C-8 halogen and methoxy moieties: action against a gyrase resistance mutant of Mycobacterium smegmatis and a gyrase-topoisomerase IV double mutant of Staphylococcus aureus. Antimicrob Agents Chemother 45:2703–9.
11 Ng EY, Trucksis M, Hooper DC. (1996) Quinolone resistance mutations in topoisomerase IV: relationship to the flqA locus and genetic evidence that topoisomerase IV is the primary target and DNA gyrase is the secondary target of fluoroquinolones in Staphylococcus aureus. Antimicrob Agents Chemother 40:1881–8.[Abstract]
12 Alovero F, Pan X-S, Morris J, et al. (1999) 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. Antimicrob Agents Chemother 44:320–5.
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