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JAC Advance Access originally published online on April 4, 2008
Journal of Antimicrobial Chemotherapy 2008 62(1):98-104; doi:10.1093/jac/dkn136
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© The Author 2008. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved. For Permissions, please e-mail: journals.permissions@oxfordjournals.org

Original research

Dual-targeting properties of the 3-aminopyrrolidyl quinolones, DC-159a and sitafloxacin, against DNA gyrase and topoisomerase IV: contribution to reducing in vitro emergence of quinolone-resistant Streptococcus pneumoniae

Ryo Okumura1,2,*, Tsuyoshi Hirata1, Yoshikuni Onodera1, Kazuki Hoshino1, Tsuyoshi Otani1 and Tomoko Yamamoto2

1 Biological Research Laboratories IV, Daiichi Sankyo Co., Ltd, 1-16-13 Kitakasai, Edogawa-ku, Tokyo 134-8630, Japan 2 Department of Microbiology and Molecular Genetics, Graduate School of Pharmaceutical Sciences, Chiba University, Chiba 263-8522, Japan

* Corresponding author. Tel: +81-3-3680-0151; Fax: +81-3-5696-4264; E-mail: okumura.ryo.x7{at}daiichisankyo.co.jp

Received 13 November 2007; returned 24 January 2008; revised 3 March 2008; accepted 10 March 2008


   Abstract
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Objectives: DC-159a (a novel quinolone) and sitafloxacin (DU-6859a) are structurally related quinolones, bearing a 3-aminopyrrolidyl substitution. We investigated the relationship between the target preferences of these 3-aminopyrrolidyl quinolones, in vitro potencies and emergence of quinolone-resistant mutants in Streptococcus pneumoniae, compared with other quinolones.

Methods: MICs, resistance frequencies and mutant prevention concentrations (MPCs) were determined using quinolone-susceptible strains and first-step parC mutant strains of S. pneumoniae. Target preferences were tested by the following two methods: antibacterial activities against gyrA or parC mutants and in vitro enzyme assays for the determination of 50% inhibition (IC50) values.

Results: DC-159a and sitafloxacin exhibited potent antibacterial activities, low frequencies of mutant selection, low MPCs and narrow mutant selection windows against both quinolone-susceptible strains and first-step parC mutants of S. pneumoniae, compared with gatifloxacin, moxifloxacin and other quinolones tested. DC-159a and sitafloxacin showed relatively low MIC ratios against single gyrA or parC mutants relative to the wild-type strain and low IC50 ratios against DNA gyrase and topoisomerase IV.

Conclusions: DC-159a and sitafloxacin demonstrated a more balanced dual-targeting activity than gatifloxacin, moxifloxacin and other quinolones tested. In addition, DC-159a and sitafloxacin have a lower propensity for selecting first- and second-step resistant mutants.

Keywords: quinolones , dual-activity , MPC


   Introduction
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 Introduction
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Streptococcus pneumoniae is the most important pathogen in community-acquired respiratory tract infections, resulting in significant levels of morbidity and mortality.1 There is growing concern about emerging quinolone-resistant S. pneumoniae, although the prevalence of quinolone resistance remains low in the USA, Canada and Japan.2–4 However, in the other parts of the world, a higher prevalence of quinolone resistance has been reported. These countries include Northern Ireland (15.2%),5 Hong Kong (13.3%)6 and Spain (7.1%).7 Thus, some clinicians have cautioned against inadequate dose or duration of quinolone therapy, which may lead to the shortening of the life cycle of this class of agents due to the emergence of resistance.8

The quinolones target bacterial type II topoisomerases, DNA gyrase and topoisomerase IV (TopoIV), which play important roles in DNA replication, chromosome segregation and DNA compaction.9,10 DNA gyrase is composed of two GyrA and two GyrB subunits, and TopoIV is composed of two ParC and two ParE subunits. Although mutations occur in these genes and/or efflux pumps are induced in some organisms, the activity of most quinolones, except ciprofloxacin and norfloxacin, is less affected by the efflux pumps in S. pneumoniae.11,12 Spontaneous point mutations most commonly occur in the quinolone-resistance-determining regions (QRDRs) of gyrA (DNA gyrase) and/or parC (TopoIV). Recently, some researchers have reported a high prevalence of the first-step parC mutant in S. pneumoniae. Lim et al.13 presented data that 48 of the 82 isolates (59%) having levofloxacin MICs of 2 mg/L, classified as susceptible according to the CLSI criteria, had a first-step mutation in parC. In addition, Davies et al.14 reported that 10 of the 14 strains (71%) having levofloxacin MICs of 2 mg/L had a parC mutation. With the increasing prevalence of first-step mutants, attention should be paid to the risk of treatment failure with quinolones and the emergence of S. pneumoniae isolates fully resistant to quinolones, harbouring a pre-existing parC mutation.15

Dealing with concerns about the prevalence of mutants, we believe that an important property for the next generation of quinolones, such as DC-159a and sitafloxacin, is to be more effective in preventing the development of first- and second-step quinolone resistance. Thus, in order to explore this, we estimated the mutant prevention concentration (MPC), a value which is thought to reflect the concentration of drug required to suppress the selection of drug-resistant mutants.16

DC-159a {(+)-7-[(7S)-7-amino-7-methyl-5-azaspiro (2.4) heptan-5-yl]-6-fluoro-1-[(1R, 2S)-2-fluoro-1-cyclopropyl]-1, 4-dihydro-8-methoxy-4-oxo-3-quinolinecarboxylic acid} is a novel 8-methoxy fluoroquinolone with extended activity against Gram-positive pathogens, particularly streptococci and staphylococci from community-acquired infections.17 In addition, it exhibits activity against Gram-negative pathogens similar to that of levofloxacin. Sitafloxacin {DU-6859a; (–)-7-[(7S)-7-amino-5-azaspiro (2.4) heptan-5-yl]-8-chloro-6-fluoro-1-[(1R, 2S)-2-fluoro-1-cyclopropyl]-1, 4-dihydro-4-oxo-3-quinolinecarboxylic acid}, which was recently approved in Japan, also has enhanced broad-spectrum antibacterial activity against Gram-positive and Gram-negative aerobes and anaerobes.18 Sitafloxacin is particularly active against S. pneumoniae, including quinolone-resistant isolates, compared with other quinolones.19

The objective of the present study was to investigate the relationship between the emergence of resistant mutants and the balanced inhibitory activity of quinolones against both target enzymes. Thus, we determined the antibacterial activities of DC-159a, sitafloxacin and other quinolones against genetically defined mutants of S. pneumoniae; further, the frequency of selection of resistant mutants and the MPC against quinolone-susceptible isolates with no mutations in the QRDRs and isolates containing a parC mutation were also investigated. Furthermore, we determined the inhibitory activities of DC-159a and sitafloxacin against DNA gyrase and TopoIV from S. pneumoniae in comparison with those of other quinolones.


   Materials and methods
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 Abstract
 Introduction
 Materials and methods
Results
 Discussion
 Funding
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Antimicrobial agents

DC-159a, sitafloxacin, levofloxacin and the other quinolones tested were synthesized at Daiichi Sankyo Co. Ltd, Tokyo, Japan.

Medium

For the determination of the frequency of mutation and MPC, starter cultures were spread on heart infusion agar containing 5% sheep-defibrinated blood (Eiken Chemical Co., Ltd, Tokyo, Japan); thereafter, they were grown in brain heart infusion broth (BHI; Becton Dickinson, Sparks, MD, USA) supplemented with 0.5% yeast extract (BHIY). Spontaneous first-step S. pneumoniae mutants were obtained on BHI agar containing 10% horse-defibrinated blood supplemented with various concentrations of each quinolone.

Bacterial strains and antimicrobial susceptibility testing

Two quinolone-susceptible strains of S. pneumoniae (EG00093 and EG00218), which did not harbour mutations in the QRDRs of the gyrA, gyrB, parC and parE genes, and two first-step parC mutants (60 and 1026523) were used for mutant selection experiments. In 2002, EG00093 and EG00218 were selected as representative strains of quinolone-susceptible S. pneumoniae from a surveillance collection in Japan. Strain 60 harbouring a single QRDR mutation in parC (Ser-79->Tyr) and isolate SP39 with a gyrA mutation (Ser-81->Phe) are both mutants of ATCC 49619. Strain 1026523 is a clinical isolate harbouring a combination of mutations within the QRDRs of parC (Ser-79->Phe) and parE (Ile-460->Val), which was obtained from the GLOBAL surveillance study in 2003 (Focus Bio-Inova Inc., Herndon, VA, USA). The determination of MICs was performed at least in duplicate, according to a standard agar dilution method.20

Determination of mutant selection frequency and MPC

The determination of mutant selection frequency and MPC experiments were performed concurrently. The method for measuring the mutation frequency and MPC was a modification of that described previously.21 Each isolate was grown for 12 h on heart infusion agar containing 5% sheep blood at 35°C. Several colonies were then suspended in sterile PBS (Takara Bio Inc., Shiga, Japan) at a turbidity equivalent to that of a 0.5 McFarland standard (1 x 108 cfu/mL). The suspension (20 mL) was divided equally into four flasks each containing 500 mL of fresh BHIY broth (total volume of 2 L) and incubated for an additional 6 h without shaking. Cultures were then concentrated 10- to 30-fold by centrifugation (5000 g for 30 min at 20°C) to yield a concentration of 1010–1011 cfu/mL. Aliquots of 100 µL of the bacterial suspension (containing 109–1010 cfu) were spread onto BHI agar plates containing 10% horse-defibrinated blood with multiples of the MICs for each quinolone. The plates containing quinolones were incubated at 35°C for 72 h, and the antibiotic-free plates were incubated at 35°C for 16–24 h. The frequency of mutant selection was calculated as the ratio of the number of resistant colonies at 72 h to the number of cfu plated. The MPC of each quinolone was determined as the lowest concentration that prevented the growth of resistant colonies when more than 1010 bacteria were spread on agar plates and incubated for 72 h at 35°C.

PCR amplification of QRDRs and DNA sequence analysis

The presence of mutations in the QRDRs of the gyrA, gyrB, parC and parE genes was investigated by PCR. The primer sequences used to amplify the gyrA QRDR were as follows: SPGA3, 5'-GTCAATCTGACAAAGGAGATGAAGG-3' (position 25 to 49) and SPGA6, 5'-CAATCTCTGTACGAGAACGTAGGAC-3' (position 715 to 739). For the amplification of the gyrB QRDR, SPGB3: 5'-TTACCAATCGCCTCTTCAGTGAAGC-3' (position 1070 to 1094) and SPGB4: 5'-CTTCCAACCTTGACACCATAGATTGG-3' (position 1621 to 1646) were used; for the amplification of the parC QRDR, SPPC1: 5'-GGCTTTGTATCTTATGTCTAACATTC-3' (position –14 to 27) and SPPC6: 5'-AAACTGCAGCATCTATGACCTCAGC-3' (position 548 to 573) and for the amplification of the parE QRDR, SPPE3: 5'-AGTTGTGGATGGAATAGTGGCT-3' (position 1061 to 1084) and SPPE4: 5'-GGACATCTTGTAAGAGGTGGGAG-3' (position 1615 to 1638). Amplification was performed in a total volume of 50 µL containing 1 µL of template DNA, 4 µL of each deoxynucleoside triphosphate (2.5 mM each), 5 µL of 10x Ex Taq DNA polymerase buffer, 1 µL of each primer (10 pmol/µL) and 0.25 µL of Ex Taq DNA polymerase (5 U/µL, Takara Bio Inc.). Amplification conditions were 94°C for 2 min, 35 cycles of 98°C for 20 s, 62°C for 2 min and 72°C for 3 min, with a final extension of 3 min at 68°C. DNA sequencing of PCR products was carried out with an ABI PRISM 3100 Avant Genetic Analyzer (Applied Biosystems, Foster City, CA, USA) in accordance with the manufacturer's instructions.

Determination of inhibitory activities against DNA gyrase and topoisomerase IV from S. pneumoniae

DNA gyrase supercoiling and TopoIV decatenation assays were carried out as described previously22,23 with minor modifications. For the DNA gyrase supercoiling assay, the reaction mixture contained 40 mM Tris–HCl (pH 7.5), 30 mM KCl, 5 mM MgCl2, 1 mM spermidine, 4 mM ATP, 1 mM dithiothreitol (DTT) and 20 µg of BSA per mL. One unit of each purified GyrA and GyrB was incubated with 0.1 µg of relaxed pBR322 plasmid DNA (Boehringer Mannheim, Mannheim, Germany) for 60 min at 37°C.

For the TopoIV decatenation assay, the reaction mixture contained 40 mM Tris–HCl (pH 7.5), 20 mM KCl, 5 mM MgCl2, 0.5 mM ATP, 1 mM DTT and 50 µg of BSA per mL. The decatenation activity was performed using 1 U of each purified ParC and ParE as well as 0.4 µg of catenated kinetoplast DNA (k-DNA, TopoGen, Inc., Columbus, OH, USA) for 60 min at 37°C.

IC50 values were obtained from the experiments performed in duplicate with five different concentrations of each agent and calculated by the linear regression analysis using EXSAS 7.10 (ARMSYSTEX Co., Ltd, Osaka, Japan).


   Results
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Antibacterial activities of DC-159a and sitafloxacin against gyrA or parC mutants of S. pneumoniae

To investigate the target preference of DC-159a and sitafloxacin, we first determined the MICs of DC-159a and sitafloxacin against a wild-type strain, first-step gyrA (Ser-81->Phe) mutant and parC (Ser-79->Phe) mutant of S. pneumoniae and compared these MICs with those of other quinolones tested (Table 1). Sitafloxacin was found to be the most active against wild-type S. pneumoniae, followed by clinafloxacin and DC-159a. DC-159a and sitafloxacin were 2- to 16-fold and 8- to 64-fold more active against the wild-type strain, respectively, than sparfloxacin, gatifloxacin, levofloxacin, ciprofloxacin and norfloxacin, respectively. In contrast, DC-159a showed comparable activity to moxifloxacin, but it was 2-fold less active than clinafloxacin. DC-159a, sitafloxacin and clinafloxacin showed relatively high activities with an MIC of 0.5 mg/L or below against the SP39 mutant, which harbours a single gyrA mutation (Ser-81->Phe). In the case of 1026523 bearing a single parC mutation (Ser-79->Phe), DC-159a, sitafloxacin and clinafloxacin showed relatively high activities with an MIC of 0.12 mg/L or below.


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  		Table 1. Antibacterial activities of DC-159a, sitafloxacin and other quinolones against gyrA or parC mutants of S. pneumoniaea

 
The increases in the MICs of quinolones tested against either the gyrA or parC mutant relative to the wild-type strain were also determined (Table 1). The MIC of DC-159a for the gyrA mutant increased 4-fold when compared with the wild-type strain. In contrast, the parC mutation in 1026523 had no effect on the MIC of DC-159a. These results suggested that the primary target for DC-159a in S. pneumoniae is DNA gyrase. In contrast, the MICs of sparfloxacin and moxifloxacin against the gyrA mutant increased 16-fold; however, the increase was only 2-fold against the parC mutant when compared with that against the wild-type strain.

Frequencies of mutant selection and MPCs of DC-159a and sitafloxacin against quinolone-susceptible strains of S. pneumoniae

Mutation frequencies and MPCs of DC-159a, sitafloxacin and other quinolones were determined using two quinolone-susceptible strains of S. pneumoniae (Table 2). There was little difference between the results from the two isolates. Among the quinolones tested, sitafloxacin at its MIC enabled selection of resistant mutants at the lowest frequency and yielded no resistant mutants at twice its MIC. At its MIC, DC-159a showed a lower mutation frequency than gatifloxacin, moxifloxacin or ciprofloxacin. Moxifloxacin and ciprofloxacin exhibited high mutation frequencies against both isolates at twice their MICs. The frequencies of resistance to gatifloxacin and levofloxacin were below the detectable levels at twice their MICs; this was in agreement with previous reports.24,25 No mutant was detected at four times the MIC of any quinolone tested.


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  		Table 2. Frequency of selection of resistant mutants and MPCs against quinolone-susceptible strains of S. pneumoniae

 
The MPCs of DC-159a and sitafloxacin against quinolone-susceptible strains of S. pneumoniae were determined (Table 2). Consistent with the result indicating that sitafloxacin had a higher activity and a lower mutation frequency than the other quinolones, the MPC of sitafloxacin (0.06 mg/L) was the lowest among the quinolones tested. The MPC of DC-159a (0.25–0.5 mg/L) was 4- to 8-fold higher than that of sitafloxacin, but the same or lower than that of gatifloxacin or moxifloxacin (0.5 mg/L). In contrast, higher concentrations of levofloxacin and ciprofloxacin were required to prevent the emergence of mutants with MPCs of 2 and 4 mg/L, respectively.

Frequencies of mutant selection and MPCs of DC-159a and sitafloxacin against first-step parC mutants of S. pneumoniae

The determination of mutation frequencies and MPCs with DC-159a, sitafloxacin and other quinolones was also investigated in two isolates with first-step parC mutations (Table 3). As a consequence, DC-159a and sitafloxacin had the lowest frequencies at eight times their MICs, which was approximately 1 log unit lower than the frequencies of the other quinolones. Moreover, no resistant mutant was selected with DC-159a and sitafloxacin at 16 times their respective MICs, whereas gatifloxacin, moxifloxacin, levofloxacin and ciprofloxacin failed to select a mutant at up to 32–64 times their respective MICs. Therefore, the MPCs for DC-159a and sitafloxacin were 4- to 64-fold and 8- to 128-fold lower than those of moxifloxacin, gatifloxacin, levofloxacin or ciprofloxacin, respectively.


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  		Table 3. Frequency of selection of resistant mutants and MPCs against first-step parC mutants of S. pneumoniae

 
Genetic characterization of DC-159a-selected first-step mutants

To understand the resistance mechanism and to confirm the primary target of DC-159a, we characterized several mutants of S. pneumoniae ATCC 49619 and a clinical isolate with no mutation within QRDRs selected using DC-159a at its MIC or twice its MIC. The obtained mutants exhibited a 2- or 4-fold increase with DC-159a at its MIC when compared with the parent strains. The sequence analysis revealed that all DC-159a mutants had a single-step mutation in gyrA, which was a commonly observed mutation (Ser-81->Phe or Tyr), and no mutation in the QRDRs of gyrB, parC and parE was observed, strongly suggesting that the primary target for DC-159a was DNA gyrase (data not shown).

Inhibitory activities of DC-159a and sitafloxacin against DNA gyrase and topoisomerase IV from S. pneumoniae

To assess the target specificities of quinolones quantitatively, DNA gyrase supercoiling assays and TopoIV decatenation assays were performed for the determination of IC50 values. Against DNA gyrase and TopoIV, the IC50s of DC-159a and sitafloxacin were 13.3, 4.38 and 6.78, 3.12 mg/L, respectively (Table 4). Thus, sitafloxacin showed the lowest IC50 for both targets among the quinolones tested. Against S. pneumoniae DNA gyrase, DC-159a demonstrated more potent inhibitory activity when compared with the other quinolones, with the exception of sitafloxacin. The IC50 ratio of DNA gyrase and TopoIV is often used as a measure of target specificity of the quinolone. If the ratio is close to 1, the quinolone exhibits similar activity against both enzymes, i.e. dual activity.26 Based on this theory, the IC50 ratio for sitafloxacin was found to be 1.40 in this study, suggesting that sitafloxacin was a dual-acting quinolone as reported previously.22,26,27 In addition, DC-159a had similar target preference against DNA gyrase and TopoIV with an IC50 ratio of 1.97 (Table 4). Consequently, DC-159a and sitafloxacin demonstrated more balanced activities against both DNA gyrase and TopoIV than gatifloxacin, moxifloxacin and the other quinolones tested.


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  		Table 4. Inhibitory activities of DC-159a, sitafloxacin and other quinolones against DNA gyrase and topoisomerase IV from S. pneumoniae

 

   Discussion
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Quinolone antibacterial agents have been recommended for use in the treatment of respiratory tract infections, particularly pneumonia caused by multidrug-resistant S. pneumoniae. Although S. pneumoniae harbouring a first-step parC mutation is often found in clinical settings, this mutation is often undetected because isolates with first-step parC mutation are reported as susceptible using standard susceptibility testing. This is alarming because isolates with first-step parC mutations are the progenitors for fully quinolone-resistant strains of S. pneumoniae, which acquire additional mutations in gyrA. Therefore, we sought to examine whether our quinolones, DC-159a and sitafloxacin, had advantageous features in preventing the selection of quinolone-resistant S. pneumoniae.

Our results indicate that the MPCs of DC-159a and sitafloxacin are lower than those of moxifloxacin, gatifloxacin, levofloxacin and ciprofloxacin (Tables 2 and 3). Moreover, DC-159a and sitafloxacin exhibited lower mutant frequencies than moxifloxacin and gatifloxacin at comparable concentrations and displayed much lower mutant frequencies than levofloxacin and ciprofloxacin (Tables 2 and 3). The MPCs of moxifloxacin and levofloxacin were similar to previously reported results.20,28 Another concept related to MPC is the mutant selection window (MSW), which is the antibiotic concentration range between the MIC and the MPC.29 It is hypothesized that next-step mutants are selectively amplified in the MSW, and hence the assessment of MSW may predict the relative abilities of quinolone to prevent mutant selection. In this study, the MSWs of DC-159a and sitafloxacin were 2-fold smaller than those of other quinolones tested against first-step parC mutants.

Some researchers have also reported that dual-targeting quinolones have lower mutation frequencies or MPCs with S. pneumoniae30 and Staphylococcus aureus.31,32 In the present study, DC-159a and sitafloxacin demonstrated relatively low MIC ratios against a single gyrA or parC mutant relative to the wild-type strains. Furthermore, DC-159a and sitafloxacin showed well-balanced inhibitory activities against purified DNA gyrase and TopoIV in contrast to gatifloxacin, moxifloxacin, levofloxacin and ciprofloxacin (Table 4).

DC-159a and sitafloxacin have the same fluoroquinolone structure with a 3-aminopyrrolidyl substituent at the C-7 position; this substituent is also present in clinafloxacin. Both clinafloxacin and sitafloxacin contain a chloride atom at the C-8 position, and higher antibacterial activity against both quinolone-susceptible and quinolone-resistant strains of S. pneumoniae can be attributed to this chloride atom.19,33 The structure of DC-159a differs from that of sitafloxacin and clinafloxacin by the presence of a methoxy substituent at the C-8 position. Although previous research has indicated that quinolones possessing a methoxy substituent at the C-8 position are better able to prevent the development of resistance than other C-8 moieties and confer a dual-targeting of DNA gyrase and TopoIV,28,31 our study suggests that DC-159a evidently exhibited different characteristics in this respect when compared with the same C-8 methoxy quinolones, gatifloxacin and moxifloxacin. As far as we can determine, DC-159a (also classified as 8-methoxy quinolone) and sitafloxacin are likely to have a lower propensity for resistance development and have a more balanced dual activity than gatifloxacin and moxifloxacin.

We also investigated the target specificities of DC-159a, sitafloxacin and other quinolones (Figure 1). Although the MIC of DC-159a did not change against a first-step parC mutant, the MIC of DC-159a was affected to a greater extent by a first-step gyrA mutant (4-fold). In contrast, the MIC of sitafloxacin was affected by mutations in both gyrA (4-fold) and parC (2-fold). The observation of an increased ratio of MIC for sparfloxacin and moxifloxacin was in agreement with the results reported previously.34 On the basis of the determination of first-step mutants or mutants with reduced susceptibility, each quinolone appears to have a preferential target of either DNA gyrase or TopoIV.26,34 We found that DC-159a selected first-step gyrA mutants, suggesting that the primary target for DC-159a in S. pneumoniae is DNA gyrase.


Figure 1
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  		Figure 1. Effect of the QRDR mutations in S. pneumoniae on the antibacterial activities of the quinolones tested. Grey bars represent the fold increase in MIC against SP39, a gyrA mutant (Ser-81->Phe) in comparison with ATCC 49619. Likewise, black bars show fold MICs against 1026523, having a parC mutation (Ser-79->Phe), relative to ATCC 49619. SPX, sparfloxacin; MXF, moxifloxacin; GAT, gatifloxacin; CLX, clinafloxacin; SIT, sitafloxacin; LVX, levofloxacin; CIP, ciprofloxacin; NOR, norfloxacin.

 
The mutation at position 460 in parE in 1026523 was not considered to be responsible for resistance to quinolones. However, Kawamura-Sato et al.35 demonstrated that this mutation alone was involved in the low-level resistance to norfloxacin and ciprofloxacin.

In conclusion, the balanced dual-targeting activity of DC-159a and sitafloxacin against DNA gyrase and TopoIV might provide a greater potential to minimize the development of quinolone resistance. DC-159a and sitafloxacin could be potential therapeutic options for pneumococcal pneumonia caused by quinolone-susceptible and less quinolone-susceptible isolates.


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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Funding
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 References
 
This study was supported by Daiichi Sankyo Co., Ltd.


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R. O., T. H., Y. O., K. H. and T. O. are employees of Daiichi Sankyo Co., Ltd.


   Acknowledgements
 
This work was presented in part at the Forty-sixth Interscience Conference on Antimicrobial Agents and Chemotherapy, San Francisco, CA, 2006 (Abstract F1-0478). We would like to thank Dr Kenichi Sato for the helpful discussions and Dr Ian Morrissey for reviewing the manuscript prior to submission. We thank Shinichiro Yamachika and Hitomi Awaya for their scientific support and technical assistance.


   References
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1 Centers for Disease Control and Prevention (CDC). Assessment of susceptibility testing practices for Streptococcus pneumoniae—United States, February 2000. MMWR Morb Mortal Wkly Rep (2002) 51:392–4.[Medline]

2 Draghi DC, Jones ME, Sahm DF, et al. Geographically-based evaluation of multidrug resistance trends among Streptococcus pneumoniae in the USA: findings of the FAST surveillance initiative (2003–2004). Int J Antimicrob Agents (2006) 28:525–31.[CrossRef][Web of Science][Medline]

3 Powis J, McGeer A, Green K, et al. In vitro antimicrobial susceptibilities of Streptococcus pneumoniae clinical isolates obtained in Canada in 2002. Antimicrob Agents Chemother (2004) 48:3305–11.[Abstract/Free Full Text]

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5 Goldsmith CE, Moore JE, Murphy PG, et al. Increased incidence of ciprofloxacin resistance in penicillin-resistant pneumococci in Northern Ireland. J Antimicrob Chemother (1998) 41:420–1.[Free Full Text]

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7 Perez-Trallero E, Fernandez-Mazarrasa C, Garcia-Rey C, et al. Antimicrobial susceptibilities of 1,684 Streptococcus pneumoniae and 2,039 Streptococcus pyogenes isolates and their ecological relationships: results of a 1-year (1998–1999) multicenter surveillance study in Spain. Antimicrob Agents Chemother (2001) 45:3334–40.[Abstract/Free Full Text]

8 Weiss K, Tillotson GS. Fluoroquinolones for respiratory infection: too valuable to overuse (and too valuable to misuse!). Chest (2002) 122:1102–3.[CrossRef][Web of Science][Medline]

9 Khodursky AB, Peter BJ, Schmid MB, et al. Analysis of topoisomerase function in bacterial replication fork movement: use of DNA microarrays. Proc Natl Acad Sci USA (2000) 97:9419–24.[Abstract/Free Full Text]

10 Levine C, Hiasa H, Marians KJ. DNA gyrase and topoisomerase IV: biochemical activities, physiological roles during chromosome replication, and drug sensitivities. Biochim Biophys Acta (1998) 1400:29–43.[Medline]

11 Piddock LJ, Johnson MM. Accumulation of 10 fluoroquinolones by wild-type or efflux mutant Streptococcus pneumoniae. Antimicrob Agents Chemother (2002) 46:813–20.[Abstract/Free Full Text]

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N. German, M. Malik, J. D. Rosen, K. Drlica, and R. J. Kerns
Use of Gyrase Resistance Mutants To Guide Selection of 8-Methoxy-Quinazoline-2,4-Diones
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