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

Transcription

1 Journal of Antimicrobial Chemotherapy (2008) 62, doi: /jac/dkn136 Advance Access publication 4 April 2008 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 Okumura 1,2 *, Tsuyoshi Hirata 1, Yoshikuni Onodera 1, Kazuki Hoshino 1, Tsuyoshi Otani 1 and Tomoko Yamamoto 2 1 Biological Research Laboratories IV, Daiichi Sankyo Co., Ltd, Kitakasai, Edogawa-ku, Tokyo , Japan; 2 Department of Microbiology and Molecular Genetics, Graduate School of Pharmaceutical Sciences, Chiba University, Chiba , Japan Received 13 November 2007; returned 24 January 2008; revised 3 March 2008; accepted 10 March 2008 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 quinoloneresistant 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 (IC 50 ) 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 IC 50 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 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... *Corresponding author. Tel: þ ; Fax: þ ; okumura.ryo.x7@daiichisankyo.co.jp # The Author Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved. For Permissions, please journals.permissions@oxfordjournals.org

2 3-Aminopyrrolidyl quinolones against S. pneumoniae 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 f(þ)-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 acidg 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 fdu-6859a; (2)-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 acidg, 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 quinolonesusceptible 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 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 ) 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 Strain 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 358C. 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 ( 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 208C) to yield a concentration of cfu/ml. Aliquots of 100 ml of the bacterial suspension (containing 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 358C for 72 h, and the antibiotic-free plates were incubated at 358C for 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 bacteria were spread on agar plates and incubated for 72 h at 358C. 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 0 -GTCAATCTGACAAAGGAGATGAAGG-3 0 (position 25 to 49) and SPGA6, 5 0 -CAATCTCTGTACGAGAACGTAGGAC-3 0 (position 715 to 739). For the amplification of the gyrb QRDR, SPGB3: 5 0 -TTACCAATCGCCTCTTCAGTGAAGC-3 0 (position 1070 to 1094) and SPGB4: 5 0 -CTTCCAACCTTGACACCATAGATTGG-3 0 (position 1621 to 1646) were used; for the amplification of the parc QRDR, SPPC1: 5 0 -GGCTTTGTATCTTATGTCTAACATTC-3 0 (position 214 to 27) and SPPC6: 5 0 -AAACTGCAGCATCTATGAC 99

3 Okumura et al. CTCAGC-3 0 (position 548 to 573) and for the amplification of the pare QRDR, SPPE3: 5 0 -AGTTGTGGATGGAATAGTGGCT-3 0 (position 1061 to 1084) and SPPE4: 5 0 -GGACATCTTGTAA GAGGTGGGAG-3 0 (position 1615 to 1638). Amplification was performed in a total volume of 50 ml containing 1 ml of template DNA, 4 ml of each deoxynucleoside triphosphate (2.5 mm each), 5 ml of10 Ex Taq DNA polymerase buffer, 1 ml of each primer (10 pmol/ml) and 0.25 ml ofextaq DNA polymerase (5 U/mL, Takara Bio Inc.). Amplification conditions were 948C for 2 min, 35 cycles of 988C for 20 s, 628C for 2 min and 728C for 3 min, with a final extension of 3 min at 688C. 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 previously 22,23 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 MgCl 2, 1 mm spermidine, 4 mm ATP, 1 mm dithiothreitol (DTT) and 20 mg of BSA per ml. One unit of each purified GyrA and GyrB was incubated with 0.1 mg of relaxed pbr322 plasmid DNA (Boehringer Mannheim, Mannheim, Germany) for 60 min at 378C. For the TopoIV decatenation assay, the reaction mixture contained 40 mm Tris HCl (ph 7.5), 20 mm KCl, 5 mm MgCl 2, 0.5 mm ATP, 1 mm DTT and 50 mg of BSA per ml. The decatenation activity was performed using 1 U of each purified ParC and ParE as well as 0.4 mg of catenated kinetoplast DNA (k-dna, TopoGen, Inc., Columbus, OH, USA) for 60 min at 378C. IC 50 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 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 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. Table 1. Antibacterial activities of DC-159a, sitafloxacin and other quinolones against gyra or parc mutants of S. pneumoniae a Quinolone MIC (mg/l) b ATCC SP DC-159a (4) 0.12 (1) Sitafloxacin (4) 0.06 (2) Clinafloxacin (4) 0.12 (2) Levofloxacin 1 2 (2) 2 (2) Ciprofloxacin 1 2 (2) 4 (4) Gatifloxacin (8) 0.25 (1) Sparfloxacin (16) 0.5 (2) Moxifloxacin (16) 0.25 (2) Norfloxacin 2 4 (2) 32 (16) a ATCC 49619, wild-type; SP39, gyra mutant (gyra: Ser-81!Phe); , parc mutant ( parc: Ser-79!Phe). b The values in parentheses represent the fold increase in the MIC relative to that of the wild-type strain. 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 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 quinolonesusceptible 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. The MPCs of DC-159a and sitafloxacin against quinolonesusceptible 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 ( mg/l) was 4- to 8-fold higher than that of sitafloxacin, but the same or lower than that of gatifloxacin or moxifloxacin 100

4 3-Aminopyrrolidyl quinolones against S. pneumoniae Table 2. Frequency of selection of resistant mutants and MPCs against quinolone-susceptible strains of S. pneumoniae Frequency of selection of resistant mutants Strain Quinolone MIC (mg/l) 1 MIC 2 MIC 4 MIC MPC (mg/l) EG DC-159a , , sitafloxacin , , gatifloxacin , , moxifloxacin , levofloxacin , , ciprofloxacin , EG DC-159a , sitafloxacin , , gatifloxacin , , moxifloxacin , levofloxacin , , ciprofloxacin , Results are derived from representative values of at least two independent experiments. (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 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. 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 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 Table 3. Frequency of selection of resistant mutants and MPCs against first-step parc mutants of S. pneumoniae Mutant Quinolone MIC (mg/l) Frequency of selection of resistant mutants 2 MIC 4 MIC 8 MIC 16 MIC 32 MIC 64 MIC MPC (mg/l) 60 DC-159a , , , sitafloxacin , , , gatifloxacin , , moxifloxacin , , levofloxacin , , ciprofloxacin , , DC-159a , , , sitafloxacin , , , gatifloxacin 0.25 N.D , moxifloxacin , , levofloxacin , , ciprofloxacin , , ND, not determined. Results are derived from representative values of at least two independent experiments. 101

5 Okumura et al. Table 4. Inhibitory activities of DC-159a, sitafloxacin and other quinolones against DNA gyrase and topoisomerase IV from S. pneumoniae Quinolone 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 IC 50 values. Against DNA gyrase and TopoIV, the IC 50 s 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 IC 50 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 IC 50 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 IC 50 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 IC 50 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. Discussion DNA gyrase IC 50 (mg/l) a TopoIV Ratio b DC-159a Sitafloxacin Gatifloxacin Levofloxacin Moxifloxacin Ciprofloxacin a The IC 50 s for DNA gyrase supercoiling activity and TopoIV decatenation activity. b Determined by dividing IC 50 for DNA gyrase by IC 50 for topoisomerase IV. 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 quinoloneresistant 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. pneumoniae 30 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 wildtype 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 102

6 3-Aminopyrrolidyl quinolones against S. pneumoniae 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 Likewise, black bars show fold MICs against , having a parc mutation (Ser-79!Phe), relative to ATCC SPX, sparfloxacin; MXF, moxifloxacin; GAT, gatifloxacin; CLX, clinafloxacin; SIT, sitafloxacin; LVX, levofloxacin; CIP, ciprofloxacin; NOR, norfloxacin. 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. The mutation at position 460 in pare in 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. 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. Funding This study was supported by Daiichi Sankyo Co., Ltd. Transparency declarations R. O., T. H., Y. O., K. H. and T. O. are employees of Daiichi Sankyo Co., Ltd. References 1. Centers for Disease Control and Prevention (CDC). Assessment of susceptibility testing practices for Streptococcus pneumoniae United States, February MMWR Morb Mortal Wkly Rep 2002; 51: 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 ( ). Int J Antimicrob Agents 2006; 28: Powis J, McGeer A, Green K et al. In vitro antimicrobial susceptibilities of Streptococcus pneumoniae clinical isolates obtained in Canada in Antimicrob Agents Chemother 2004; 48: Yamaguchi K, Ohno A; Levofloxacin Surveillance Group. Investigation of the susceptibility trends in Japan to fluoroquinolones and other antimicrobial agents in a nationwide collection of clinical isolates: a longitudinal analysis from 1994 to Diagn Microbiol Infect Dis 2005; 52: 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: Ho PL, Yung RW, Tsang DN et al. Increasing resistance of Streptococcus pneumoniae to fluoroquinolones: results of a Hong Kong multicentre study in J Antimicrob Chemother 2001; 48: 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 ( ) multicenter surveillance study in Spain. Antimicrob Agents Chemother 2001; 45: Weiss K, Tillotson GS. Fluoroquinolones for respiratory infection: too valuable to overuse (and too valuable to misuse!). Chest 2002; 122: 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: 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: Piddock LJ, Johnson MM. Accumulation of 10 fluoroquinolones by wild-type or efflux mutant Streptococcus pneumoniae. Antimicrob Agents Chemother 2002; 46: Zhanel GG, Walkty A, Nichol K et al. Molecular characterization of fluoroquinolone resistant Streptococcus pneumoniae clinical isolates obtained from across Canada. Diagn Microbiol Infect Dis 2003; 45: Lim S, Bast D, McGeer A et al. Antimicrobial susceptibility breakpoints and first-step parc mutations in Streptococcus pneumoniae: redefining fluoroquinolone resistance. Emerg Infect Dis 2003; 9: Davies TA, Evangelista A, Pfleger S et al. Prevalence of single mutations in topoisomerase type II genes among levofloxacinsusceptible clinical strains of Streptococcus pneumoniae isolated in the United States in 1992 to 1996 and 1999 to Antimicrob Agents Chemother 2002; 46: Kays MB, Zhanel GG, Reimann MA et al. Selection of a gyra mutation and treatment failure with gatifloxacin in a patient with Streptococcus pneumoniae with a preexisting parc mutation. Pharmacotherapy 2007; 27: Dong Y, Zhao X, Domagala J et al. Effect of fluoroquinolone concentration on selection of resistant mutants of Mycobacterium bovis BCG and Staphylococcus aureus. Antimicrob Agents Chemother 1999; 43: Hoshino K, Inoue K, Murakami Y et al. In vitro and in vivo antibacterial activities of DC-159a, a new fluoroquinolone. Antimicrob Agents Chemother 2008; 52: Sato K, Hoshino K, Tanaka M et al. Antimicrobial activity of DU-6859, a new potent fluoroquinolone, against clinical isolates. Antimicrob Agents Chemother 1992; 36:

7 Okumura et al. 19. Touyama M, Higa F, Nakasone C et al. In vitro activity of sitafloxacin against clinical strains of Streptococcus pneumoniae with defined amino acid substitutions in QRDRs of gyrase A and topoisomerase IV. J Antimicrob Chemother 2006; 58: National Committee for Clinical Laboratory Standards. Methods for Dilution Antimicrobial Susceptibility Tests for Bacteria That Grow Aerobically Sixth Edition: Approved Standard M7-A6. NCCLS, Wayne, PA, USA, Li X, Zhao X, Drlica K. Selection of Streptococcus pneumoniae mutants having reduced susceptibility to moxifloxacin and levofloxacin. Antimicrob Agents Chemother 2002; 46: Onodera Y, Uchida Y, Tanaka M et al. Dual inhibitory activity of sitafloxacin (DU-6859a) against DNA gyrase and topoisomerase IV of Streptococcus pneumoniae. J Antimicrob Chemother 1999; 44: Tanaka M, Sato K, Kimura Y et al. Inhibition by quinolones of DNA gyrase from Staphylococcus aureus. Antimicrob Agents Chemother 1991; 35: Fukuda H, Hiramatsu K. Primary targets of fluoroquinolones in Streptococcus pneumoniae. Antimicrob Agents Chemother 1999; 43: Drugeon HB, Juvin ME, Bryskier A. Relative potential for selection of fluoroquinolone-resistant Streptococcus pneumoniae strains by levofloxacin: comparison with ciprofloxacin, sparfloxacin and ofloxacin. J Antimicrob Chemother 1999; 43 Suppl C: Smith HJ, Nichol KA, Hoban DJ et al. Dual activity of fluoroquinolones against Streptococcus pneumoniae: the facts behind the claims. J Antimicrob Chemother 2002; 49: Morrissey I, George J. Activities of fluoroquinolones against Streptococcus pneumoniae type II topoisomerases purified as recombinant proteins. Antimicrob Agents Chemother 1999; 43: Allen GP, Kaatz GW, Rybak MJ. Activities of mutant prevention concentration-targeted moxifloxacin and levofloxacin against Streptococcus pneumoniae in an in vitro pharmacodynamic model. Antimicrob Agents Chemother 2003; 47: Zhao X, Drlica K. Restricting the selection of antibiotic-resistant mutants: a general strategy derived from fluoroquinolone studies. Clin Infect Dis 2001; 33 Suppl 3: S Kishii R, Takei M, Fukuda H et al. Contribution of the 8-methoxy group to the activity of gatifloxacin against type II topoisomerases of Streptococcus pneumoniae. Antimicrob Agents Chemother 2003; 47: Strahilevitz J, Hooper DC. Dual targeting of topoisomerase IV and gyrase to reduce mutant selection: direct testing of the paradigm by using WCK-1734, a new fluoroquinolone, and ciprofloxacin. Antimicrob Agents Chemother 2005; 49: Ince D, Zhang X, Silver LC et al. Dual targeting of DNA gyrase and topoisomerase IV: target interactions of garenoxacin (BMS , T-3811ME), a new desfluoroquinolone. Antimicrob Agents Chemother 2002; 46: Jorgensen JH, Weigel LM, Ferraro MJ et al. Activities of newer fluoroquinolones against Streptococcus pneumoniae clinical isolates including those with mutations in the gyra, parc, and pare loci. Antimicrob Agents Chemother 1999; 43: Pestova E, Millichap JJ, Noskin GA et al. Intracellular targets of moxifloxacin: a comparison with other fluoroquinolones. J Antimicrob Chemother 2000; 45: Kawamura-Sato K, Hasegawa T, Torii K et al. Prevalence of Ile-460-Val/ParE substitution in clinical Streptococcus pneumoniae isolates that were less susceptible to fluoroquinolones. Curr Microbiol 2005; 51: