International Journal of Antimicrobial Agents 44 (2014) 443–449

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In vitro activity of WQ-3810, a novel fluoroquinolone, against multidrug-resistant and fluoroquinolone-resistant pathogens Daichi Kazamori ∗ , Hiroshi Aoi, Kaori Sugimoto, Taichi Ueshima, Hirotaka Amano, Kenji Itoh, Yasuhiro Kuramoto, Akira Yazaki Drug Discovery Laboratory, Wakunaga Pharmaceutical Co., Ltd., 1624 Shimokotachi, Koda-cho, Akitakata-shi, Hiroshima 739-1195, Japan

a r t i c l e

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Article history: Received 30 June 2014 Accepted 21 July 2014 Keywords: WQ-3810 Fluoroquinolones Antimicrobial resistance

a b s t r a c t The aim of this study was to examine the in vitro antibacterial activity of WQ-3810, a new fluoroquinolone, against clinically relevant pathogens such as Acinetobacter baumannii, Escherichia coli and Streptococcus pneumoniae, including multidrug-resistant (MDR) and fluoroquinolone-resistant (FQR) isolates, compared with those of ciprofloxacin, levofloxacin, moxifloxacin and gemifloxacin. WQ-3810 demonstrated the most potent activity against the antimicrobial-resistant pathogens tested. Against A. baumannii, including MDR isolates, the potency of WQ-3810 [minimum inhibitory concentration for 90% of the organisms (MIC90 ) = 1 mg/L] was more than eight-fold higher than that of ciprofloxacin (64 mg/L) and levofloxacin (8 mg/L). Against E. coli and S. pneumoniae, including FQR isolates, WQ-3810 (MIC90 = 4 mg/L and 0.06 mg/L, respectively) was also more active than ciprofloxacin (64 mg/L and 2 mg/L) and levofloxacin (32 mg/L and 2 mg/L). Furthermore, WQ-3810 was the most potent among the fluoroquinolones tested against meticillin-resistant Staphylococcus aureus (MRSA) and Neisseria gonorrhoeae, including FQR isolates. In particular, WQ-3810 demonstrated highly potent activity against FQR isolates of A. baumannii, E. coli and S. pneumoniae with amino acid mutation(s) in the quinolone resistance-determining region of DNA gyrase and/or topoisomerase IV, which are the target enzymes of fluoroquinolones. An enzyme inhibition study performed using FQR E. coli DNA gyrase suggested that the potent antibacterial activity of WQ-3810 against drug-resistant isolates partly results from the strong inhibition of the target enzymes. In conclusion, this study demonstrated that WQ-3810 exhibits extremely potent antibacterial activity over the existing fluoroquinolones, particularly against MDR and FQR pathogens. © 2014 Elsevier B.V. and the International Society of Chemotherapy. All rights reserved.

1. Introduction Fluoroquinolones belong to one of the most prescribed classes of antibiotics for the treatment of many types of infections owing to their broad spectrum and potent antibacterial activity as well as desirable pharmacokinetics and a good safety profile. Since the discovery of norfloxacin as the first fluoroquinolone, which is structurally characterised by a C6 fluorine atom in the quinolone ring that results in improved potency and spectrum of activity, intensive efforts have been focused on the development of improved successors of norfloxacin [1]. Consequently, newly developed fluoroquinolones, such as ciprofloxacin, levofloxacin and moxifloxacin, have been successfully used in the therapy of various infections, including respiratory tract, urinary tract and intra-abdominal infections. However, excessive use of fluoroquinolones has led to the

∗ Corresponding author. Tel.: +81 826 45 2331; fax: +81 826 45 4351. E-mail address: kazamori [email protected] (D. Kazamori).

emergence of fluoroquinolone-resistant (FQR) bacteria. The prevalence and spread of FQR bacteria have been reported globally in a variety of important human pathogens, including Escherichia coli, Streptococcus pneumoniae and meticillin-resistant Staphylococcus aureus (MRSA), and have thus become major public health concerns over the past years [2,3]. In addition, a high rate of FQR Neisseria gonorrhoeae isolated among patients with sexually transmitted infections has been the focus of much attention [4]. The mechanism of bacterial resistance to fluoroquinolones primarily involves amino acid substitution(s) in the target enzymes, DNA gyrase and topoisomerase IV, through mutations in the quinolone resistancedetermining region (QRDR) of the gyrA and parC genes [5–7]. Overexpression of drug efflux pumps in the bacterial cell membrane is also associated with fluoroquinolone resistance [8–10]. In addition, a plasmid-mediated resistance mechanism, namely the production of Qnr protein capable of protecting DNA gyrase from fluoroquinolones, has been proposed [11]. Recently, the emergence and spread of multidrug-resistant (MDR) pathogens, defined as pathogens that are resistant to at

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lowest concentration at which bacterial growth was invisible. The susceptibility rate was determined according to the breakpoint criteria established by the Clinical and Laboratory Standards Institute (CLSI) [17].

2.4. DNA sequence of the quinolone resistance-determining region in quinolone-resistant isolates

Fig. 1. Structure of the fluoroquinolone WQ-3810.

least three antibiotic classes, have been a growing threat for infection therapy. For example, it has been reported that Acinetobacter baumannii, which causes severe nosocomial opportunistic infections, including hospital-acquired pneumonia, ventilatorassociated pneumonia and sepsis, has developed resistance to several major classes of antibiotics [12]. In addition, infectious diseases caused by MDR pathogens have been associated with a higher mortality rate and longer hospital stay because of the lack of therapeutically effective drugs [13–15]. As a result of increasing bacterial resistance, the management of serious bacterial infections with currently available agents has become challenging [13–15]. In this context, the development of new agents with effective activity to cope with the emerging resistant bacteria is strongly desired. WQ-3810 (Fig. 1) is a newly developed fluoroquinolone with unique substituents at the N1 and C7 positions of the quinolone ring for the treatment of antibiotic-resistant infections. In this study, the in vitro antibacterial activity of WQ-3810 was evaluated against clinical pathogens, including MDR and FQR isolates, compared with those of several currently available fluoroquinolones. In addition, an enzyme assay was also conducted to address whether the potent antibacterial activity of WQ-3810 is attributable to a strong inhibition of the target enzymes. 2. Materials and methods 2.1. Antibiotics WQ-3810 and ciprofloxacin were synthesised by Wakunaga Pharmaceutical Co., Ltd. (Osaka, Japan). Levofloxacin was purchased from Sigma-Aldrich (St Louis, MO). Gemifloxacin and moxifloxacin were purchased from Genesoft Pharmaceuticals (San Francisco, CA) and Bayer Yakuhin (Osaka, Japan), respectively, as tablets with stated potency. 2.2. Bacterial isolates Clinical isolates of Gram-positive and Gram-negative bacteria were collected from hospitals and research laboratories of universities in Japan from 1995 to 2010. 2.3. Minimum inhibitory concentration (MIC) determination The MIC was determined by the agar dilution method according to the standard method described by the Japanese Society of Chemotherapy [16]. A bacterial culture (1–3 × 104 CFU) was inoculated onto appropriate agar plates containing each test drug and the plates were then incubated at 37 ◦ C for 24 h. Mueller–Hinton agar (MHA) (Becton Dickinson, Franklin Lakes, NJ) supplemented with 5% (v/v) sheep defibrinated whole blood (Nippon Bio-Supply Center, Tokyo, Japan), ABCM agar (Eiken Chemical Co. Ltd., Tokyo, Japan) and MHA were used for S. pneumoniae, N. gonorrhoeae and other pathogens, respectively. The MIC was determined as the

The gyrA and parC genes of A. baumannii, E. coli and S. pneumoniae were individually cloned. Also, the gyrA and grlA genes of MRSA were individually cloned. These genes were amplified by PCR using a GeneAmp® PCR System 9700 (Applied Biosystems, Foster City, CA). DNA sequencing was performed using a BigDye® Terminator v.3.1 Cycle Sequencing Kit (Applied Biosystems) with an ABI Prism® 3130xI DNA Sequencer (Applied Biosystems).

2.5. Cloning, protein expression and purification of fluoroquinolone-resistant E. coli DNA gyrase E. coli gyrA and gyrB genes were amplified by PCR of genomic DNA isolated from a clinical FQR isolate using gyrA-specific primers (F, 5 -AGGATCCTAGCGACCTTGCGAGAGAAATTACACC-3 ; R, 5 - AGCGGCCGCTTCTTCTTCTGGCTCGTCGTCAACG-3 ) or gyrBspecific primers (F, 5 -AGGATCCTTCGAATTCTTATGACTCCTCCAG3 ; R, 5 - AGCGGCCGCAATATCGATATTCGCCGCTTTCAG-3 ). These genes were inserted into the T7 promoter-based expression vector pET48b containing a His-tag sequence (Novagen, Madison, WI) and were individually transformed into E. coli DH5␣ competent cells (Toyobo, Osaka, Japan). Two expression plasmids containing the gyrA or gyrB gene were individually transformed into recombination-deficient E. coli OrigamiTM 2(DE3)pLysS (Novagen). Protein expression was induced by incubation of the transformed cells with 0.4 mM isopropyl ␤-d-1-thiogalactopyranoside (IPTG) at 22 ◦ C. After centrifugation for 15 min at 7000 × g, each cell pellet was re-suspended in 75 mM Tris–HCl (pH 8.0), 0.1% Triton X-100 and Halt protease inhibitor (Thermo Fisher Scientific, Waltham, MA) and was lysed with LysonaseTM Bioprocessing Reagent (Novagen). After centrifugation for 20 min at 20 000 × g, the supernatant was filtered through a 0.45-␮m Millex-HV membrane (Millipore, Billerica, MA). The filtrate was applied to a HiTrap Q Sepharose Column (GE Healthcare, Pollards Wood, UK) and the bound protein was eluted with 75 mM Tris–HCl (pH 8.0) and 300 mM NaCl. Each eluent containing His-tagged GyrA or GyrB was applied to a HisTrap HP column (GE Healthcare) and was eluted with 20 mM phosphate buffer (pH 7.5) containing 150 mM NaCl and 50–120 mM imidazole. The protein fraction was dialysed in 20 mM Tris–HCl (pH 7.5) and 200 mM KCl at 4 ◦ C for 16 h and was then treated with HRV-3C protease (Novagen) and thrombin (GE Healthcare) to cleave the 5 -His-tag and 3 -unrelated peptides, respectively. GyrA was purified by affinity chromatography using a HisTrap HP column (GE Healthcare). GyrB was purified by gel filtration (Superdex 75; GE Healthcare). The solutions containing each purified protein were dialysed in 20 mM Tris–HCl (pH 7.5), 4 mM dithiothreitol (DTT), 0.2 mM ethylene diamine tetra-acetic acid (EDTA) and 200 mM KCl at 4 ◦ C for 16 h and were concentrated by centrifugal filtration using Ultrafree-15 Biomax-3 membrane centrifugal filter units (Millipore). The concentrated GyrA and GyrB were diluted two times with 80% glycerol and were stored at −80 ◦ C until use. The protein concentration of the GyrA and GyrB solutions was determined using the BCA Protein Assay Reagent (Bio-Rad, Hercules, CA). GyrA and GyrB were mixed on ice for 20 min to reconstitute the complex prior to use.

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2.6. DNA gyrase supercoiling and topoisomerase IV decatenation assays The inhibitory activities of the test drugs for E. coli DNA gyrase and topoisomerase IV were determined by supercoiling and decatenation assays, respectively [18,19]. In the DNA gyrase supercoiling assay, reaction mixtures containing 35 mM Tris–HCl (pH 7.5), 24 mM KCl, 4 mM MgCl2 , 2 mM DTT, 1.8 mM spermidine, 1 mM ATP, 6.5% glycerol, 0.1 mg/mL albumin, 500 ng of relaxed pBR322 (Inspiralis, Norwich, UK), DNA gyrase [0.04 U of commercially available E. coli gyrase (New England Biolabs, Ipswich, MA) or 1 ␮g of recombinant FQR E. coli gyrase] and a series of concentrations of the test drugs were incubated at 37 ◦ C for 15 min. In the topoisomerase IV decatenation assay, reaction mixtures containing 40 mM HEPES–KOH (pH 7.6), 100 mM potassium glutamate, 10 mM magnesium acetate, 10 mM DTT, 1 mM ATP, 0.1 mg/mL albumin, 100 ng of kinetoplast DNA (Inspiralis), 0.03 U of commercially available E. coli topoisomerase IV (Inspiralis) and a series of concentrations of the test drugs were incubated at 37 ◦ C for 10 min. Both assays were terminated by addition of loading buffer containing 300 mM Tris–HCl (pH 8.0), 0.3 mM EDTA, 0.5 mg/mL bromophenol blue/xylene cyanol and 40% sucrose, followed by electrophoretic analysis on a 1% agarose gel. The supercoiling activity of DNA gyrase and the decatenation activity of topoisomerase IV were quantified from the intensity of the supercoiled

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and decatenated DNA bands, respectively, using ImageQuantTM 4000 and ImageQuantTM TL (GE Healthcare). The half maximal inhibitory concentration (IC50 ) was calculated by non-linear curve fitting of the enzyme activity inhibition using KyPlot v.5.0 (Keyence, Osaka, Japan). 3. Results The in vitro activity of WQ-3810 against various clinical pathogens, including MDR and FQR isolates, was compared with those of comparator fluoroquinolones (ciprofloxacin, levofloxacin, moxifloxacin and gemifloxacin) (Table 1). WQ-3810 showed the most potent activity against clinical isolates of A. baumannii, including MDR isolates, with a MIC90 value (MIC for 90% of the organisms) of 1 mg/L, which is 64-, eight-, eight- and two-fold lower than those of ciprofloxacin (MIC90 = 64 mg/L), levofloxacin (8 mg/L), moxifloxacin (8 mg/L) and gemifloxacin (2 mg/L), respectively. The susceptibility rates for these clinical isolates of A. baumannii, as determined by the CLSI criteria, were 45% and 59% for ciprofloxacin and levofloxacin, respectively. Against six MDR isolates of A. baumannii (with MICs of ≥4, ≥16 and ≥64 mg/L for ciprofloxacin, imipenem/cilastatin and amikacin), WQ-3810 also demonstrated prominent activity, with all of the isolates being inhibited at 0.12–0.25 mg/L, whereas the MICs of ciprofloxacin (32–64 mg/L), levofloxacin, moxifloxacin and gemifloxacin (2–4 mg/L) were significantly higher than that of WQ3810.

Table 1 Antibacterial activity of WQ-3810 and comparator fluoroquinolones against clinical pathogens, including multidrug-resistant and fluoroquinolone-resistant isolates. Organism (no. of isolates)/antimicrobial agent

MIC50 Acinetobacter baumannii (22) WQ-3810 Ciprofloxacin Levofloxacin Moxifloxacin Gemifloxacin Escherichia coli (98) WQ-3810 Ciprofloxacin Levofloxacin Moxifloxacin Gemifloxacin Streptococcus pneumoniae (74) WQ-3810 Ciprofloxacin Levofloxacin Moxifloxacin Gemifloxacin HA-MRSA (25) WQ-3810 Ciprofloxacin Levofloxacin Moxifloxacin Gemifloxacin CA-MRSA (11) WQ-3810 Ciprofloxacin Levofloxacin Moxifloxacin Gemifloxacin Neisseria gonorrhoeae (12) WQ-3810 Ciprofloxacin Levofloxacin Moxifloxacin Gemifloxacin

Susceptibility rate (%)a

MIC (mg/L) MIC90

Range

S

R

0.12 16 2 2 0.25

1 64 8 8 2

0.06–2 0.12–64 0.06–32 0.03–16 0.008–16

– 45 59 – –

– 55 14 – –

2 32 16 16 16

4 64 32 32 32

0.015–64 0.008–128 0.03–128 0.03–128 0.008 to >128

– 49 49 – –

– 51 49 – –

0.015–1 0.5–32 0.5–8 0.06–4 0.008–1

– – 95 99 –

– – 1 1 –

0.06–16 16 to >128 4 to >128 0.5–128 1 to >128

– 0 0 24 28

– 100 100 76 52

0.03 1 1 0.12 0.03 2 >128 32 8 8

0.06 2 2 0.25 0.12 4 >128 >128 16 64

0.015 0.5 0.25 0.06 0.03

0.06 16 4 1 1

0.008–0.5 0.25–16 0.12–8 0.03–2 0.008–1

– 73 73 73 100

– 27 27 9 0

0.008 0.12 0.12 0.03 0.015

0.25 32 16 8 4

128 >128 >128

16 32 32 54 >128 68

4.0 6.7 8.0 8.0 38 11

4.0 16 8.0 13 91 16

CIP, ciprofloxacin; LEV, levofloxacin; MOX, moxifloxacin; GEM, gemifloxacin.

comparator fluoroquinolones. The MIC90 of WQ-3810 (0.25 mg/L) was 128-, 64- and 32-fold lower than those of ciprofloxacin (32 mg/mL), levofloxacin (16 mg/mL) and moxifloxacin (8 mg/L), respectively. 4. Discussion In this study, we demonstrated the potent activity of WQ-3810 against clinically troublesome pathogens, including MDR and FQR isolates, by comparing its activity with those of comparator fluoroquinolones. WQ-3810 showed the most potent activity against A. baumannii, including MDR isolates, among the fluoroquinolones tested. A. baumannii is one of the major problematic pathogens because it causes nosocomial pneumonia, such as hospital-acquired pneumonia and ventilator-associated pneumonia. A significant increase in nosocomial pneumonia caused by this pathogen was recently reported [20] and this increase is associated with an increase in A. baumannii strains resistant to various types of antibiotics, including fluoroquinolones [3]. A recent surveillance study reported that the prevalence of resistant A. baumannii was 96.7% for ciprofloxacin and >60% for most commercially available antibiotics, including ␤-lactams and aminoglycosides [12]. We examined the antibacterial activity of several classes of antibiotics against clinical isolates of A. baumannii, some of which were highly resistant to multiple classes of antibiotics. Notably, WQ-3810 had remarkable activity even against the MDR isolates, for which the MICs of ciprofloxacin, imipenem/cilastatin, amikacin and WQ-3810 were 16–32, 32, 64–128 and 0.12–0.25 mg/L, respectively. WQ-3810 was also the most active against S. pneumoniae, which also is recognised as the leading pathogen for pneumonia,

particularly that acquired in community settings. The emergence of QRSP has been reported in recent years, although the global incidence of QRSP has been documented to be low (ca. 1%) [2,21]. QRSP has become a serious problem in specific regions, e.g. the rate of fluoroquinolone resistance among S. pneumoniae is comparatively high (>10%) in Hong Kong [21]. Against one QRSP isolate, WQ-3810 exhibited superior activity to levofloxacin and moxifloxacin; the MICs of WQ-3810, levofloxacin and moxifloxacin were 1, 8 and 4 mg/L, respectively. WQ-3810 also showed the most potent activity against FQR E. coli. E. coli is a typical bacterium that is primarily involved in urinary tract infection, and an increasing trend in E. coli strains resistant to fluoroquinolones has been reported for decades [22–24]. In particular, the increasing prevalence of ESBL-producing FQR E. coli resistant not only to fluoroquinolones but also to ␤lactams limits the therapeutic options using antibacterial agents [2]. This study demonstrated that WQ-3810 was also active against E. coli isolates with concurrent resistance to fluoroquinolones (MIC ≥ 4 mg/mL for ciprofloxacin) and ␤-lactams (MIC ≥ 16 mg/mL for ceftazidime); the MICs of WQ-3810 (1–4 mg/L) were more than 8-fold lower than those of the comparator fluoroquinolones. These results indicated that WQ-3810 can exert potent antibacterial activity against clinically problematic bacteria, even against those that are resistant to the existing fluoroquinolones. The prevalence of drug-resistant pathogens varies among countries, regions and hospitals but continues to steadily increase [3,14,21]. Therefore, it is important to investigate the activity of antibiotics against pathogens isolated in multiple regions. Deane et al. [25,26] presented the in vitro activity of WQ-3810 (WQ-3810 was named KPI-10 in the presentations) against a large panel of clinical Gram-positive and Gram-negative bacteria collected in the USA. Their studies demonstrated that the MIC90 values

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of WQ-3810 were 2, 0.03 and 2 mg/L against 199 isolates of Acinetobacter spp., 198 isolates of S. pneumoniae and 198 isolates of E. coli, respectively. These results are consistent with the results of the current study using clinical isolates of bacteria collected from several Japanese institutions, suggesting that WQ-3810 is likely to exhibit comparable antibacterial activity against clinically isolated pathogens regardless of their geographic region. In this study, the activity of WQ-3810 and comparator fluoroquinolones was investigated against isolates of clinically important pathogens with mutations in the target enzymes for fluoroquinolones. DNA gyrase and topoisomerase IV are known to be the primary targets of fluoroquinolone antibiotics and consist of two subunits (GyrA and GyrB, and ParC and ParE, respectively) [5,6]. Therefore, substitutions of key amino acids in either or both of the two enzymes confer fluoroquinolone resistance through the prevention of the interaction between each enzyme and the fluoroquinolones [7]. Indeed, it has been reported that the resistance level to the existing fluoroquinolones develops depending on the number of amino acid substitutions in these enzymes [27]. The representative fluoroquinolones used for the treatment of Gram-negative infections, namely ciprofloxacin and levofloxacin, were not active against A. baumannii and E. coli with the various types of mutations in the QRDR of GyrA and ParC (MIC range, 35–64 mg/L and 8.0–49 mg/L, respectively, for ciprofloxacin, and 3.2–11 mg/L and 8.0–17 mg/L, respectively, for levofloxacin). In contrast, WQ-3810 exhibited potent activity against these mutated isolates, with MICs ranging from 0.27–1.4 mg/L and 1.0–4.0 mg/L, respectively (Table 2). In addition, WQ-3810 inhibited the enzyme activities of DNA gyrase and topoisomerase IV from wild-type E. coli with comparable potency to ciprofloxacin and levofloxacin, whereas WQ-3810 exhibited four- to nine-fold more potent inhibition than ciprofloxacin and levofloxacin against an E. coli DNA gyrase with amino acid mutations in the QRDR of GyrA (Table 3). These results indicated that WQ-3810 exhibits potent antibacterial activity against FQR pathogens, in part due to its highly potent inhibition of the target enzymes. Kuramoto et al. reported the structure–activity relationship of fluoroquinolones with a 5-amino-2,4-difluorophenyl group at the N1 position on the quinolone ring [28]. In their report, they demonstrated that the specific combination of substituents at the N1, C7 and C8 positions of the quinolone ring gave extremely potent antibacterial activity, speculating that the distorted orientation of the N1 phenyl ring out of the quinolone ring owing to steric repulsion between the N1 phenyl ring and the C8 chloride (or bromide, methyl) atom results in enhanced antibacterial activity [28]. WQ-3810 has a similar combination of substituents at the N1, C7 and C8 positions: 5-amino-2,4-difluoropyridyl, 3-isopropylaminoazetizine-1-yl and methyl, respectively. Taken together, these results suggested that WQ-3810 would exert potent antibacterial activity against FQR pathogens through strong inhibition of the mutated target enzymes owing to its unique conformational features distinguished by the N1, C7 and C8 substituents of the quinolone ring. WQ-3810 may interact with some sites of the target enzymes that are different from those with which the conventional fluoroquinolones interact. It is critical to confirm whether the potent in vitro activity of WQ-3810 can be translated to efficacy. Regarding in vivo efficacy, it has been reported that WQ-3810 demonstrates potent therapeutic efficacy in systemic and local infection models caused by MDR and FQR pathogens [29]. A phase 1 study using an oral formulation of WQ-3810 (named KPI-10 in the study) at dosages of 100–1000 mg demonstrated that WQ-3810 was quickly absorbed and showed dose-linear pharmacokinetics with large area under the plasma concentration–time curve (AUC) values (8.3–64 mg h/L), a long elimination half-life (7.5–8.7 h) and moderate urinary excretion (38–60% of the dose) [30].

In conclusion, we demonstrated that WQ-3810 has highly potent activity against clinically relevant MDR and FQR pathogens, which may be due to its strong inhibition of the target enzymes with mutations conferring resistance to the conventional fluoroquinolones. Based on these results, WQ-3810 is a promising new agent with potent in vitro activity against important human pathogens, including bacteria that are resistant to the currently available fluoroquinolones. Acknowledgments The authors are grateful to Keiichi Hiramatsu, PhD, at Juntendo University (Tokyo, Japan) and Yoshihito Otsuka, PhD, at Kameda Medical Center (Chiba, Japan) for supplying bacterial isolates. Funding: This research was funded by Wakunaga Pharmaceutical Co., Ltd. (Osaka, Japan). Competing interests: All authors are current or former employees of Wakunaga Pharmaceutical Co., Ltd. (Osaka, Japan). Ethical approval: Not required. References [1] Koga H, Itoh A, Murayama S, Suzue S, Irikura T. Structure–activity relationships of antibacterial 6,7- and 7,8-disubstituted 1-alkyl-1,4-dihydro4-oxoquinolone-3-carboxylic acids. J Med Chem 1980;23:1358–63. [2] Zhanel GG, DeCorby M, Nichol KA, Wierzbowski A, Baudry PJ, Karlowsky JA, et al. Antimicrobial susceptibility of 3931 organisms isolated from intensive care units in Canada: Canadian National Intensive Care Unit Study, 2005/2006. Diagn Microbiol Infect Dis 2008;62:67–80. [3] Lockhart SR, Abramson MA, Beekmann SE, Gallagher G, Riedel S, Diekema DJ, et al. Antimicrobial resistance among Gram-negative bacilli causing infections in intensive care unit patients in the United States between 1993 and 2004. J Clin Microbiol 2007;45:3352–9. [4] Donegan EA, Wirawan DN, Muliawan P, Schachter J, Moncada J, Parekh M, et al. Fluoroquinolone-resistant Neisseria gonorrhoeae in Bali, Indonesia: 2004. Sex Transm Dis 2006;33:625–9. [5] Drlica K, Zhao X. DNA gyrase, topoisomerase IV, and the 4-quinolones. Microbiol Mol Biol Rev 1997;61:377–92. [6] Hooper DC. Bacterial topoisomerases, anti-topoisomerases, and antitopoisomerase resistance. Clin Infect Dis 1998;27:S54–63. [7] Hooper DC. Emerging mechanisms of fluoroquinolone resistance. Emerg Infect Dis 2001;7:337–41. [8] Fukuda H, Hori S, Hiramatsu K. Antibacterial activity of gatifloxacin (AM-1155, CG5501, BMS-206584), a newly developed fluoroquinolone, against sequentially acquired quinolone-resistant mutants and the norA transformant of Staphylococcus aureus. Antimicrob Agents Chemother 1998;42:1917–22. [9] Masuda N, Sakagawa E, Ohya S, Gotoh N, Tsujimoto H, Nishino T. Substrate specificities of MexAB–OprM, MexCD–OprJ, and MexXY–OprM efflux pumps in Pseudomonas aeruginosa. Antimicrob Agents Chemother 2000;44:3322–7. [10] Su XZ, Chen J, Mizushima T, Kuroda T, Tsuchiya T. AbeM, an H+ -coupled Acinetobacter baumannii multidrug efflux pump belonging to the MATE family of transporters. Antimicrob Agents Chemother 2005;49:4362–4. [11] Robicsek A, Jacoby GA, Hooper DC. The worldwide emergence of plasmidmediated quinolone resistance. Lancet Infect Dis 2006;6:629–40. [12] Yau W, Owen RJ, Poudyal A, Bell JM, Turnidge JD, Yu HH, et al. Colistin hetero-resistance in multidrug-resistant Acinetobacter baumannii clinical isolates from the Western Pacific region in the SENTRY antimicrobial surveillance programme. J Infect 2009;58:138–44. [13] Dent LL, Marshall DR, Pratap S, Hulette RB. Multidrug resistant Acinetobacter baumannii: a descriptive study in a city hospital. BMC Infect Dis 2010;10:196. [14] Morata L, Cobos-Trigueros N, Martinez JA, Soriano A, Almela M, Marco F, et al. Influence of multidrug resistance and appropriate empirical therapy on the 30day mortality rate of Pseudomonas aeruginosa bacteremia. Antimicrob Agents Chemother 2012;56:4833–7. [15] Tabah A, Koulenti D, Laupland K, Misset B, Valles J, Bruzzi de Carvalho F, et al. Characteristics and determinants of outcome of hospital-acquired bloodstream infections in intensive care units: the EUROBACT International Cohort Study. Intensive Care Med 2012;38:1930–45. [16] Nagayama A, Yamaguchi K, Watanabe K, Tanaka M, Kobayashi I, Nagasawa Z. Final report from the Committee on Antimicrobial Susceptibility Testing, Japanese Society of Chemotherapy, on the agar dilution method (2007). J Infect Chemother 2008;14:383–92. [17] Clinical and Laboratory Standards Institute. Performance standards for antimicrobial susceptibility testing; twenty-third informational supplement. Document M100-S23. Wayne, PA: CLSI; 2013. [18] Akasaka T, Kurosaka S, Uchida Y, Tanaka M, Sato K, Hayakawa I. Antibacterial activities and inhibitory effects of sitafloxacin (DU-6859a) and its optical isomers against type II topoisomerases. Antimicrob Agents Chemother 1998;42:1284–7.

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