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9（ 9 ）

Rapid bactericidal activity of sitafloxacin against
Streptococcus pneumoniae
HIROKO KANDA1), KAZUE INOUE2), RYO OKUMURA1) and
KAZUKI HOSHINO1)
1

Biological Research Laboratories, R&D Division,
Daiichi Sankyo Co., Ltd., Tokyo
2
Vaccine Business Strategy Department, Vaccine Business Intelligence
Division, Daiichi Sankyo Co., Ltd., Tokyo(Received for publication November 7, 2012)

The initial bactericidal activity of quinolones against Streptococcus pneumoniae at
the concentration equivalent to their respective peak serum concentration (Cmax) andfree drug fraction of Cmax (fCmax) were investigated. The bactericidal activity ofsitaÀoxacin (STFX), levoÀoxacin (LVFX), moxiÀoxacin (MFLX), and garenoxacin(GRNX) were compared by determining the actual killing of bacteria at Cmax andfCmax for 1 and 2 hours based on the Japanese maximum dose per administration(100, 500, 400, and 400 mg, respectively). Against 4 quinolone-susceptible clinicalisolates (wild-type), STFX with Cmax and fCmax exhibited the most rapid bactericidal
activity resulting in an average reduction of 侒3.0 log10 colony forming units (CFU)/
mL in 1 hour. STFX with Cmax and fCmax also showed the most rapid and potent
bactericidal activity against 9 clinical isolates with single par (C/E) mutation,
resulting in 侒3.0 log10 CFU/mL average reduction in viable cells in 1 hour. STFX
showed a statistically signi¿cant advantage in initial bactericidal activity over otherquinolones for single mutants (P＜0.001). The propensity that the difference in theinitial bactericidal activity between STFX and other quinolones was higher in single
mutants than wild-type strains, was con¿rmed using S. pneumoniae ATCC49619(wild-type) and its laboratory single parC mutant. As a result, STFX showed asimilar rapid and potent initial bactericidal activity against both strains, while initialbactericidal activity for other quinolones was signi¿cantly reduced in the single
mutant (P＜0.05). In conclusion, STFX has the most rapid and potent initialbactericidal activity against wild-type and single mutants of S. pneumoniae and its
bactericidal activity is not affected by the presence of a single par mutationcompared to LVFX, MFLX, and GRNX.

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Introduction
Streptococcus pneumoniae is one of the most signi¿cant pathogens of community-acquired
respiratory tract infections and accounts for two-thirds of mortality of those patients requiring
hospital treatment1). Due to increasing levels of resistance in S. pneumoniae to commonly used
antimicrobials, such as penicillins and macrolides, Àuoroquinolone with improved antibacterial
activity against S. pneumoniae are being recommended and used for the treatment of patientswith community acquired pneumonia（CAP）caused by multidrug resistant strains2). Emerging
quinolone-resistant S. pneumoniae is also a growing concern because high rates of levoÀoxacin(LVFX) used by geographic region were reported to be associated with increased quinolone-resistant pneumococci3), however, current global resistance rates remain low4〜6).
In pneumococci, resistance to quinolones is primarily mediated via mutations in the quinolone resistance-determining region (QRDR) of target enzymes7). Even in the LVFX-susceptible
clinical isolates, 6–25% of strains with LVFX MIC at 1 ȝg/mL and 30–71% of strains with LVFX
MIC at 2 ȝg/mL were reported to possess a single mutation in QRDR8〜10). The frequency of selection of resistant mutants were higher in single QRDR mutants than wild-type strains11〜13). It isnecessary to use an appropriate quinolone and regimen to eradicate bacteria and to minimise thedevelopment of resistance.
SitaÀoxacin (STFX) is a Àuoroquinolone with enhanced broad-spectrum antibacterial activity against Gram-positive and Gram-negative aerobes and anaerobes14). STFX was con¿rmed topossess a potent antimicrobial activity against S. pneumoniae, including quinolone-resistant isolates, compared with other quinolones15).
For the evaluation of the bactericidal activities of quinolones, many studies were conductedusing the concentration based on the minimum inhibitory concentration (MIC). In the case of
STFX, the MIC based study showed potent bactericidal activity of STFX against S.
pneumoniae16), however, the concentration should be examined considering the clinical setting.
Since the pharmacodynamics of quinolones are concentration-dependent17,18), some investigators reported the antibactericidal activity using pharmacokinetics/pharmacodynamics parameters of the therapeutic ef¿cacy of quinolones, AUC/MIC, peak serum concentration (Cmax)/MIC
and Cmax19〜22). In this study, we investigated the initial bactericidal activities of STFX and otherquinolones against wild-type and single QRDR mutants of S. pneumoniae based on their Cmax andfree drug fraction of Cmax (fCmax), which might have an effect on the initial bactericidal activitiesof quinolones in the clinical setting.

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11（ 11 ）Table 1. Amino acid substitutions in the quinolone resistance-determining regions of ParC and
ParE and susceptibility to quinolones of used Streptococcus pneumoniae strains.

Materials and Methods
Antimicrobial agents
STFX and LVFX were synthesized at Daiichi Sankyo Propharma Co., Ltd., Tokyo, Japan.
MoxiÀoxacin (MFLX) was purchased from Sequoia Research Products Ltd., Pangbourne, UK
and LKT Laboratories, Inc., Minnesota, USA. Garenoxacin mesilate (GRNX) was synthesized at
Daiichi Sankyo Co., Ltd., Tokyo, Japan. The determination of MICs was performed by the broth
dilution method according to the Clinical and Laboratory Standards Institute guidelines23).
Microorganisms
The bacterial strains used in this study are shown in Table 1. Four quinolone-susceptible
clinical isolates of S. pneumoniae (GE00175, GE01045, GE01085, and 05712), which did notharbour mutations in the QRDR of the gyrA, gyrB, parC and parE genes, and 9 clinical isolates(GE00215, GE01064, 05101, 05667, 05696, 05298, 05674, 05706, and 1026523) harbouring asingle mutation relating to quinolone-resistance in the QRDR of parC or parE of topoisomelase12（ 12 ）THE JAPANESE JOURNAL OF ANTIBIOTICS

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IV24,25) isolated from Japan or UK were used in this study. Strains ATCC49619 and strain 60,
which is a mutant of ATCC49619 harbouring a single QRDR mutation in parC (Ser79Tyr)13),
were also used in this study. The bacterium were stored at −80°C before experiments.
Time-kill study
The bacterial strains were inoculated on heart infusion agar (HIA; Eiken Chemical Co., Ltd.,Tokyo, Japan) containing 5% sheep-de¿brinated blood and incubated overnight at 35°C. Several
fresh colonies on the plate were suspended in brain heart infusion broth (BHI; Becton, Dickinson
and Company (BD), NJ, USA) supplemented with 0.5% yeast extract (BD) (BHIY) and incubated at 37°C with shaking, to obtain the culture containing approximately 106 colony forming
units (CFU)/mL of bacteria. The initial bactericidal activities of STFX, LVFX, MFLX, and
GRNX were examined with exposure of the culture to the concentration equivalent to theirrespective Cmax at the Japanese maximum dose per administration (100, 500, 400, and 400 mg,
respectively) and their fCmax at 37°C for 2 hours. Cmax of the quinolones was as follows: STFX
1.0 ȝg/mL26), LVFX 8.0 ȝg/mL27), MFLX 4.1 ȝg/mL28), and GRNX 7.2 ȝg/mL29). fCmax of the quinolones which was calculated using their respective protein binding ratio by the ultra¿ltration
method26〜29) was as follows: STFX 0.5 ȝg/mL, LVFX 5.6 ȝg/mL, MFLX 2.05 ȝg/mL, and GRNX
1.44 ȝg/mL. Samples were taken for viable count determinations at 0, 1, and 2 hours after addition of the drug. Each sample was then diluted appropriately and spread on HIA plates. Antibacterial agents carryover was minimized by serial saline dilution. Saline dilution reduced antibacterial
agents concentrations around the MIC for study organisms. The viable cell counts (log10 CFU/mL) were determined based on the numbers of colonies grown on the plates after incubation at
35°C overnight combined with dilution factors. The detection limit was 1×102 CFU/mL.
Analysis of time-kill data
Initial bactericidal activities were represented as the change in viable cell counts before andafter treatment with quinolones. This was shown as the mean±SD of the results of quinolonesusceptible isolates or mutant strains, calculated by Microsoft Excel 2003.
Statistical analysis
The statistical signi¿cance between the STFX-treated group and other quinolone-treatedgroups was evaluated using Tukeyʼs comparison test. The statistical signi¿cance between S.
pneumoniae ATCC49619 and 60 treated with the same quinolone was evaluated using Studentʼst-test. These analyses were performed by SAS System Release 8.2 (SAS Institute Inc., NC,
USA).

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13（ 13 ）Results
Antimicrobial activity
The antimicrobial activity of STFX and other quinolones to S. pneumoniae strains used areshown in Table 1. The MICs of STFX, LVFX, MFLX, and GRNX against wild-type strains ofS. pneumoniae were 0.03–0.06, 1, 0.12–0.25, and 0.03–0.06 ȝg/mL, respectively. The MICs of
STFX, LVFX, MFLX, and GRNX against single mutants of S. pneumoniae were 0.03–0.06, 1–2,
0.12–0.5, and 0.03–0.12 ȝg/mL, respectively.
Bactericidal activity against wild-type and single QRDR mutant of S. pneumoniae
The changes in mean viable cell counts of 4 wild-type strains of S. pneumoniae after treatment with quinolones at Cmax and fCmax for 1 and 2 hours are shown in Fig. 1 and Fig. 2, respectively. STFX at Cmax and fCmax exhibited the most potent initial bactericidal activity resulting in
3.75 and 3.77 log10 CFU/mL reduction in viable counts at 1 hour and 4.46 and 4.13 log10 CFU/mL
reduction in viable counts at 2 hours, respectively. Especially, STFX at fCmax exhibited signi¿cantly rapid bactericidal activity compared to other quinolones tested (1 hour: P＜0.001 vs.
LVFX and GRNX, P＜0.01 vs. MFLX, 2 hours: P＜0.05 vs. MFLX and GRNX).
The changes in mean viable cell counts of 9 single mutants of S. pneumoniae after treatment
with quinolones at Cmax or fCmax for 1 and 2 hours are shown in Fig. 3 or Fig. 4, respectively.
STFX at Cmax and fCmax exhibited the most potent initial bactericidal activity resulting in 3.75 and3.26 log10 CFU/mL reduction in viable counts at 1 hour and 3.96 and 3.85 log10 CFU/mL reduction
in viable counts at 2 hours, respectively. STFX showed a statistically signi¿cant advantage in initial bactericidal activity over other quinolones tested for single mutants at any condition (P＜0.001).
The changes in the mean viable cell counts after treatment with STFX at Cmax for 1 and 2hours were 0.87–1.53 and 0.75–1.27 log10 CFU/mL, respectively in the wild-type strains and
2.14–3.05 and 1.27–2.37 log10 CFU/mL, respectively in the single mutants. The changes in themean viable cell counts after treatment with STFX at fCmax for 1 and 2 hours were 1.35–2.22 and
0.97–1.25 log10 CFU/mL, respectively in the wild-type strains and 1.98–2.56 and 1.73–2.39 log10
CFU/mL, respectively in the single mutants. This means that the STFX showed a more rapid bactericidal activity in the single mutants than wild type strains.
Bactericidal activity against wild-type and its laboratory single parC mutant of S.
pneumoniae
The initial bactericidal activity of wild-type S. pneumoniae ATCC49619 (parent strain) andstrain 60, which is a single parC mutant of ATCC49619, was investigated. The mean changes in
viable cell counts with 3 times of individual experiments after treatment of two strains with quinolones at Cmax or fCmax for 1 and 2 hours are shown in Fig. 5 or Fig. 6, respectively. STFX at14（ 14 ）THE JAPANESE JOURNAL OF ANTIBIOTICS

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Fig. 1. Change in viable cell count of wild-type Streptococcus pneumoniae after (A) 1 hour and(B) 2 hour treatment with quinolones at Cmax in humans.

Each bar represents the mean±S.D. of the change in viable cell count of
4 strains of wild-type S. pneumoniae. The statistical signi¿cance between
the STFX-treated group and other quinolone-treated groups wasevaluated using Tukeyʼs comparison test.

Cmax exhibited the most potent early bactericidal activity against wild-type and a single parC mutant, resulting in 2.92 and 3.00 log10 CFU/mL reduction in viable counts at 1 hour, respectively,
and 3.70 and 3.71 log10 CFU/mL reduction in viable counts at 2 hours, respectively. No signi¿cant effect on the initial bactericidal activity of STFX by a single parC mutation was con¿rmed.

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15（ 15 ）Fig. 2. Change in viable cell count of wild-type Streptococcus pneumoniae after (A) 1 hour and(B) 2 hour treatment with quinolones at fCmax in humans.

Each bar represents the mean±S.D. of the change in viable cell count of 4 strains of wildtype S. pneumoniae. The statistical signi¿cance between the STFX-treated group and otherquinolone-treated groups was evaluated using Tukeyʼs comparison test.

On the contrary, in the case of other quinolones tested, signi¿cant reduction of bactericidal activity was con¿rmed in a single parC mutant compared to wild-type (P＜0.01 vs. LVFX at 1 and 2hours, P＜0.05 vs. MFLX and GRNX at 1 and 2 hours). STFX at fCmax exhibited the most potent
early bactericidal activity against wild-type and a single parC mutant resulting in 2.80 and2.41 log10 CFU/mL reduction in viable counts at 1 hour, respectively and 3.55 and 3.24 log10
CFU/mL reduction in viable counts at 2 hours, respectively. No signi¿cant effect on the initial16（ 16 ）THE JAPANESE JOURNAL OF ANTIBIOTICS

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Fig. 3. Change in viable cell count of Streptococcus pneumoniae with a single mutation after(A) 1 hour and (B) 2 hour treatment with quinolones at Cmax in humans.

Each bar represents the mean±S.D. of the change in viable cell count of 9
strains of S. pneumoniae with a single mutation. The statistical signi¿cancebetween the STFX-treated group and other quinolone-treated groups wasevaluated using Tukeyʼs comparison test.

bactericidal activity of STFX by the substitution of single amino acid was con¿rmed. In contrast,in the case of other quinolones tested, signi¿cant reductions of bactericidal activities were con¿rmed in the single mutant compared to wild-type strain (P＜0.01 vs. LVFX at 1 and 2 hours,
MFLX at 2 hours, GRNX at 1 hour, P＜0.05 vs. MFLX at 1 hour and GRNX at 2 hours).

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17（ 17 ）Fig. 4. Change in viable cell count of Streptococcus pneumoniae with a single mutation after(A) 1 hour and (B) 2 hour treatment with quinolones at fCmax in humans.

Each bar represents the mean±S.D. of the change in viable cell count of 9
strains of S. pneumoniae with a single mutation. The statistical signi¿cancebetween the STFX-treated group and other quinolone-treated groups wasevaluated using Tukeyʼs comparison test.

Discussion
The in vitro study described here was performed with the aim of determining the initial bactericidal activities of STFX, LVFX, MFLX, and GRNX against wild-type strains and single18（ 18 ）THE JAPANESE JOURNAL OF ANTIBIOTICS

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Fig. 5. Change in viable cell count of Streptococcus pneumoniae ATCC49619 and 60 after(A) 1 hour and (B) 2 hour treatment with quinolones at Cmax in humans.

Each bar represents the mean±S.D. of the change in viable cell count of 3 times
of individual experiments. The statistical signi¿cance between ATCC49619 and60 treated with the same quinolone was evaluated using Studentʼs t-test.

QRDR mutants of S. pneumoniae at the concentration equivalent to their Cmax and fCmax in 2
hours to predict the initial bactericidal activities of quinolones in the clinical setting. We revealed
that STFX not only at Cmax but also fCmax exhibited distinguished potent and rapid bactericidal
activity against S. pneumoniae, compared to LVFX, MFLX, and GRNX. The advantage of theFeb. 2013

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19（ 19 ）Fig. 6. Change in viable cell count of Streptococcus pneumoniae ATCC49619 and 60 after(A) 1 hour and (B) 2 hour treatment with quinolones at fCmax in humans.

Each bar represents the mean±S.D. of the change in viable cell count of 3 times
of individual experiments. The statistical signi¿cance between ATCC49619 and60 treated with the same quinolone was evaluated using Studentʼs t-test.

bactericidal activity of STFX over other quinolones tested stood out in single QRDR mutants.
Among the other quinolones tested, GRNX was con¿rmed to possess an almost equivalent antibacterial activity to STFX against S. pneumoniae and the Cmax and fCmax of GRNX were about 7
times and 3 times higher than those of STFX, respectively. However, the initial antibactericidal

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Feb. 2013activity of GRNX at Cmax and fCmax were lower than those of STFX at Cmax and fCmax. YOKOTA etal. demonstrated a similar experiment except for the concentration of STFX. They used Cmax andfCmax for the dosage of STFX 50 mg, which was half of our study and reported a different resultthat the antibactericidal activity of STFX 50 mg at Cmax and fCmax was lower than GRNX21). Since
the protein binding ratio of STFX is 50%, fCmax of STFX 100 mg is almost equal to Cmax of STFX
50 mg. However, the initial bactericidal activity of fCmax of STFX 100 mg was com¿rmed to behigher than that of Cmax of GRNX 400 mg in our study. This difference is considered to be attributable to the difference of bacterial strains, medium, and bacterial counts at the beginning of drug
treatment between the two studies. On the contrary, in terms of LVFX and MFLX, SCHAFER, J. et
al. reported that MFLX initially displayed faster killing than LVFX against a parC mutant with a
Ser-79Phe mutation19). It was con¿rmed in the report by ANDEREGG, T. R. et al. that GRNX and
LVFX showed slow initial bactericidal activity against wild-type and QRDR mutant strains ofS. pneumoniae at clinically achievable serum concentration22), where these data support ourstudy.
STFX was con¿rmed to possess a more potent and balanced dual-target inhibition against
wild-type and single mutated DNA gyrase and topoisomerase IV of S. pneumoniae than otherquinolones30,31). Moreover, OKUMURA et al. reported that the rapid bactericidal activity of STFX
was due to its ability to induce autolysis mediated by LytA32), which is resposible for autolysis in
S. pneumoniae33). We speculate that these features are part of the reasons why STFX showed
rapid and potent bactericidal activity against wild-type and single QRDR mutant strains of S.
pneumoniae.
In conclusion, STFX at around the Cmax of the clinical dose (100 mg) showed the most potent
and rapid antibactericidal activity against S. pneumoniae regardless of existence or non-existenceof single QRDR mutation. These results suggest that STFX may show potent and rapid bactericidal activity in the treatment of infectious disease caused by S. pneumoniae.
Acknowledgment
We gratefully acknowledge the technical assistance of MIHO TANABE.

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23（ 23 ）SitaÀoxacin の Streptococcus pneumoniae に対する迅速な殺菌効果
神田裕子 1）・井上和恵 2）・奥村

亮 1）・星野一樹 1）

1）

第一三共株式会社研究開発本部生物医学研究所

2）

第一三共株式会社ワクチン事業本部ワクチン事業部

キノロン系抗菌薬の Streptococcus pneumoniae に対する初期殺菌効果を，最高血漿中濃度（Cmax）及びフリー体 Cmax（fCmax）で検討した。すなわち，SitaÀoxacin（STFX），

LevoÀoxacin（LVFX），MoxiÀoxacin（MFLX），及び Garenoxacin（GRNX）を健康
成 人 に 国 内における 1 回あたり最高用量（それぞれ 100 mg, 500 mg, 400 mg, 及び400 mg）を投与した際の Cmax 及び fCmax を作用させ，薬剤作用 1 時間後及び 2 時間後に お け る 殺 菌 効 果 を 比 較 検 討 し た。そ の 結 果， STFX は 野 生 型 の 臨 床 分 離 S.

pneumoniae 菌株，計 4 株に対し，Cmax, fCmax いずれの濃度を作用した場合も，薬剤作用 1 時間以内に平均生菌数を 3 log colony forming units（CFU）/mL 以上減少させ，最も 高 い 初 期 殺 菌 効 果 を 示 し た。 STFX は， par （C/E ）に 1 ヵ 所 変 異 を 有 す る S.
，計 9 株に対しても，最も高い初期殺菌効果を示し，
pneumoniae（一変異型 S. pneumoniae）
薬剤作用 1 時間以内に平均生菌数を 3 log CFU/mL 以上減少させた。STFX の一変異型 S.

pneumoniae に対する初期殺菌効果は，対照キノロン系抗菌薬と比較して有意に高い。STFX と対照キノロン系抗菌薬との殺菌効果の差が，野生型ものであった（P＜0.001）
より一変異型 S. pneumoniae で 大 き く な る 傾 向 を 確 認 す る た め に， S. pneumoniae

ATCC49619（野生型）と本株から実験的に作製された parC 一変異株を用いて同様の検討を実施した。その結果，STFX は両株に対して同等の高い初期殺菌効果を示したが，対照キノロン系抗菌薬では，一変異型 S. pneumoniae に対する初期殺菌効果が顕著に減少した（P＜0.05）。
以上の結果より，STFX は野生型及び一変異型 S. pneumoniae に対して最も高い初期殺菌効果を示すことが明らかとなった。さらに，STFX の殺菌効果は LVFX，

MFLX，及び GRNX と比較して，標的酵素の一変異の影響を受けにくいことが明らかになった。