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In vitro and in vivo antibacterial activities of garenoxacin against group G Streptococcus dysgalactiae subsp. equisimilis
International Journal of Antimicrobial Agents 38 (2011) 226–230
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In vitro and in vivo antibacterial activities of garenoxacin against group G Streptococcus dysgalactiae subsp. equisimilis Masahiro Takahata ∗ , Yoko Sugiura, Yuko Shiokawa, Naoko Futakuchi, Yoshiko Fukuda, Nobuhiko Nomura, Junichi Mitsuyama Research Laboratories, Toyama Chemical Co. Ltd., 4-1, Shimookui 2-chome, Toyama 930-8508, Japan
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Article history: Received 19 January 2011 Accepted 20 April 2011 Keywords: Streptococcus dysgalactiae subsp. equisimilis Garenoxacin In vitro activity In vivo activity
a b s t r a c t In this study, garenoxacin showed potent in vitro activity against clinical isolates of group G Streptococcus dysgalactiae subsp. equisimilis [minimum inhibitory concentration for 90% of the organisms (MIC90 ) = 0.125 g/mL] and was superior to levofloxacin (MIC90 = 1 g/mL) and moxifloxacin (MIC90 = 0.25 g/mL). In experimental pneumonia caused by group G S. dysgalactiae subsp. equisimilis in mice, the effective dose for 50% survival (ED50 ) of garenoxacin following single oral administration was 1.87 mg/kg, >10.7-fold and 4.6-fold less than the ED50 values of levofloxacin (>20 mg/kg) and moxifloxacin (8.54 mg/kg), respectively. The area under the free serum concentration–time curve from 0–24 h (fAUC0–24 )/MIC ratio of garenoxacin in serum following oral administration of 20 mg/kg was 73.2, which was 8.7–11.4-fold and 1.4-fold greater than that of levofloxacin (6.44–8.46) and moxifloxacin (51.4), respectively. These results suggest that garenoxacin has potential for the treatment of infectious diseases caused by S. dysgalactiae subsp. equisimilis. © 2011 Elsevier B.V. and the International Society of Chemotherapy. All rights reserved.
1. Introduction Streptococcus dysgalactiae subsp. equisimilis can be classified as follows on the basis of a combination of phenotypic and genotypic characteristics: Lancefield group C ␣-haemolytic; Lancefield group C -haemolytic; Lancefield group G -haemolytic; and Lancefield group L -haemolytic [1]. Streptococcus dysgalactiae subsp. equisimilis may exist amongst the normal flora of the skin, oropharynx, and gastrointestinal and genitourinary tracts [2]. Group C and G streptococci are thought to show low pathogenicity. However, in 1996 Vandamme et al. [3] proposed that S. dysgalactiae subsp. equisimilis is a clinical pathogen. Recently it was reported that Lancefield group G S. dysgalactiae subsp. equisimilis is responsible for 5–8% of human streptococcal infections, including suppurative diseases, bacteraemia, wound infections, otitis media, purulent pharyngitis, pneumonia, empyema and streptococcal toxic shock syndrome [1,4–8]. The M protein encoded by the emm gene is the major virulence determinant of Streptococcus pyogenes, one of the group A streptococci (GAS), and contributes considerably to the invasive capability of GAS by mediating antiphagocytic, adherence and internalisation processes [9]. Group G S. dysgalactiae subsp. equisimilis express homologues of the M proteins of S. pyogenes [10,11]
∗ Corresponding author. Tel.: +81 76 431 8306; fax: +81 76 431 8208. E-mail address: MASAHIRO [email protected] (M. Takahata).
and it is reported that the M proteins of group G S. dysgalactiae subsp. equisimilis may also play an important role in pathogenesis [5]. Penicillins have usually been used for the treatment of infections caused by group C and G streptococci; however, some reports present examples of failed penicillin treatment. The reason for this failure is that group C and G streptococci may survive inside pharyngeal cells or phagocytes, particularly in the carrier state [12]. Macrolides and tetracyclines have also been used for the treatment of various infectious diseases caused by streptococci; however, a rapid increase in the frequency of macrolide- and/or tetracyclineresistant group C and G streptococci was reported during the 1990s [13]. Furthermore, recent studies have shown the continuing prevalence of macrolide [14] and tetracycline resistance [15]; therefore, chemotherapy with new antibacterial agents for infectious diseases caused by group C and G streptococci is required. Garenoxacin, a des-fluoro(6) quinolone, exhibits more potent activity against Streptococcus pneumoniae, a major respiratory pathogen, than other fluoroquinolones such as levofloxacin and moxifloxacin [16]. In addition, following oral administration to humans, garenoxacin shows more favourable pharmacokinetics than other developed quinolones [17]; therefore, garenoxacin is expected to show good clinical efficacy against various infectious diseases caused by streptococci, including macrolide- and tetracycline-resistant strains. The present study investigated the in vitro antibacterial activity of garenoxacin against clinical isolates of group G S. dysgalactiae
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M. Takahata et al. / International Journal of Antimicrobial Agents 38 (2011) 226–230
subsp. equisimilis, in which the emm gene type and macrolide and tetracycline resistance genes were analysed. In addition, because it has been reported that pneumonia often develops to bacteraemia, which has frequently been seen in infections caused by this bacterial species, the in vivo efficacy of garenoxacin against experimental pneumonia induced by group G S. dysgalactiae subsp. equisimilis in mice was evaluated and was compared with levofloxacin and moxifloxacin.
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MIC determination and dilution of antibacterial agents was cationadjusted Mueller–Hinton broth (Nippon Becton, Dickinson & Co., Tokyo, Japan) supplemented with 5% lysed horse blood (Nihon Biotest Laboratories Inc., Tokyo, Japan). A 5 L bacterial suspension was inoculated into 96-well microplates containing 95 L of antibiotic solution [final inoculum size ca. 105 colony-forming units (CFU)/mL]. The lowest drug concentration that prevented visible growth after 22 h of incubation at 35 ◦ C was defined as the MIC.
2. Materials and methods 2.2. In vivo efficacy 2.1. In vitro activity 2.1.1. Bacterial strains For minimum inhibitory concentration (MIC) determination, 42 clinical isolates of group G S. dysgalactiae subsp. equisimilis isolated in Japan during the period 2005–2008 were used. Isolates were identified on the basis of phenotypic characteristics determined by the VITEK® Gram-Positive Identification card (Sysmex bioMérieux Co. Ltd., Tokyo, Japan) and the streptococcal grouping kit UNIBLUE (Kanto Chemical Co. Inc., Tokyo, Japan). The sources of the isolates were as follows: sputum, 9; pus, 9; pharyngeal exudates, 8; vaginal exudates, 2; other exudates, 3; urine, 3; and other, 8. Group G S. dysgalactiae subsp. equisimilis strain D-4691 was used to induce experimental pneumonia. 2.1.2. Typing of the M protein gene of clinical isolates The M protein type of the 42 clinical isolates of group G S. dysgalactiae subsp. equisimilis was determined on the basis of the M protein gene (emm) amplified by polymerase chain reaction (PCR) using oligonucleotide primers reported previously [18]. The first 240 bases of the sequence were used to query the US Centers for Disease Control and Prevention (CDC) streptococcal emm sequence database An (http://www.cdc.gov/ncidod/biotech/strep/strepindex.htm). emm gene type was defined as one that was 95% identical with regard to the first 160 bases of the sequence. 2.1.3. Detection of drug resistance genes of clinical isolates Five drug resistance genes (macrolide resistance genes ermA, ermB and mefA and tetracycline resistance genes tetM and tetO) were detected by PCR with the respective oligonucleotide primers as reported previously [15,19–22]. 2.1.4. Antibacterial agents The following antibacterial agents were used: garenoxacin (garenoxacin methanesulfonate monohydrate); levofloxacin; moxifloxacin; azithromycin; clarithromycin; tetracycline; cefcapene; and benzylpenicillin. Garenoxacin was synthesised at Research Laboratories, Toyama Chemical Co. Ltd. (Tokyo, Japan). Moxifloxacin and cefcapene were extracted from commercially available tablets (Shionogi & Co. Ltd., Osaka, Japan); the purity of each of these two agents was >99.8% as measured by high-performance liquid chromatography (HPLC). Levofloxacin was purchased from Chem-Impex International Inc. (Wood Dale, IL). Azithromycin and clarithromycin were purchased from LKT Laboratories Inc. (Saint Paul, MN). Tetracycline and benzylpenicillin were purchased from Sigma-Aldrich Japan (Tokyo, Japan) and Meiji Seika Kaisha Ltd. (Tokyo, Japan), respectively. 2.1.5. Minimum inhibitory concentration determination Precultures for MIC determination were performed in brain–heart infusion broth (Eiken Chemical Co. Ltd., Tokyo, Japan) by incubation at 35 ◦ C for 18 h. MICs were determined using a microdilution method recommended by the Clinical and Laboratory Standards Institute (CLSI) [23]. The medium used for
2.2.1. Laboratory animals Four-week-old male Institute of Cancer Research (ICR) mice were purchased from Japan SLC Inc. (Shizuoka, Japan) and were used after acclimation for 3 days. All murine experiments were performed according to the Laboratory Animal Use Management Regulations at Toyama Chemical Co. Ltd. (9 November 2005). 2.2.2. Experimental murine model of pneumococcal pneumonia Streptococcus dysgalactiae subsp. equisimilis D-4691 (emm gene type stG652.0) was used for experimental pneumonia studies to determine the therapeutic effects of the quinolones. Following intraperitoneal administration of cyclophosphamide (Shionogi & Co. Ltd.) (200 mg/kg and 100 mg/kg at 4 days and 1 day before infection, respectively), experimental pneumonia was induced in mice. Mice were anaesthetised by intramuscular injection of a solution containing a mixture of ketamine hydrochloride (Ketalar; DAIICHI SANKYO Co. Ltd., Tokyo, Japan), xylazine hydrochloride (Ceractal; Bayer Medical Co. Ltd., Tokyo, Japan) and sterile physiological saline (2:1:3) at 0.1 mL per mouse via the femoral region. Pulmonary infection was induced by intranasal injection of 40 L of bacterial suspension (ca. 105 CFU per mouse). On the day of infection, mice were randomly allocated to each group (N = 10). 2.2.3. Administration of antimicrobial agents Garenoxacin, levofloxacin and moxifloxacin were suspended in 0.5% (w/v) methyl cellulose 400 (Wako Pure Chemical Industries Ltd., Osaka, Japan) and were orally administered once at 2 h after infection (N = 10). 2.2.4. Evaluation of the therapeutic effects of antimicrobial agents From the number of surviving mice 7 days after antibiotic administration, the 50% effective dose (ED50 ) was calculated by the probit method (SAS release 8.2; SAS Institute Japan Ltd., Tokyo, Japan). 2.3. Determination of drug concentrations in serum and lung tissue Drug concentrations in serum and lung tissue following oral administration of 20 mg/kg garenoxacin, levofloxacin or moxifloxacin (N = 4 in each group) were determined by HPLC. The dose of 20 mg/kg was decided on the basis of the HPLC assay limit. At 0.25, 0.5, 1, 2, 4 and 6 h after administration of each quinolone, mice were sacrificed by exsanguination from the inferior vena cava under ether anaesthesia at each sampling time. Blood collected from the inferior vena cava was used to measure drug concentrations. Blood was kept in Sepaclen-A-5 (Eiken Kizai Co. Ltd., Tokyo, Japan) at room temperature for >30 min and serum was then obtained by centrifugation. Lung tissue was also removed at each sampling time and was homogenised in a four-fold volume of phosphate-buffered saline (pH 7.0, 1/15 mol/L). Homogenised tissue was centrifuged and the supernatant and serum were frozen at −40 ◦ C until HPLC assay. For the HPLC assay, an equal volume of methanol was added to each sample, which was then mixed and
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Table 1 Antibacterial activities of garenoxacin and other antimicrobial agents against 42 clinical isolates of group G Streptococcus dysgalactiae subsp. equisimilis. Antibacterial agent
Garenoxacin Levofloxacin Moxifloxacin Azithromycin Clarithromycin Tetracycline Cefcapene Benzylpenicillin
MIC (g/mL) Range
MIC50
MIC90
0.0313–0.125 0.5–2.0 0.0625–0.5 0.0625 to >128 0.0156 to >128 0.25–64 0.0078–0.0313 0.0078–0.01
0.0625 0.5 0.125 0.125 0.0313 2 0.0156 0.0156
0.125 1 0.25 4 2 32 0.0156 0.0156
MIC, minimum inhibitory concentration; MIC50/90 , MIC for 50% and 90% of the organisms, respectively.
centrifuged. The supernatant was used to measure the concentration of each quinolone by HPLC (LC series; Shimadzu Corporation, Kyoto, Japan). The maximum concentration of drug in serum (Cmax ) and the time to reach Cmax (tmax ) were determined directly from the actual mean drug concentrations in serum. The area under the serum concentration–time curve from 0–24 h (AUC0–24 ) and the half-life (t1/2 ) were calculated from the mean drug concentrations in serum, which were determined by non-compartmental analysis with WinNonlin Professional software v5.0.1 (Pharsight Corporation, Sunnyvale, CA). Moreover, the free (f) AUC0–24 in serum was calculated on the basis of the protein-binding percent of each quinolone in mice (garenoxacin 78%, levofloxacin 12–33% and moxifloxacin 31%) [24–26].
Table 2 Therapeutic effects of garenoxacin and other antimicrobial agents on experimental pneumonia induced in micea by Streptococcus dysgalactiae subsp. equisimilis D-4691. Antibacterial agent
MIC (g/mL)
ED50 (mg/kg) (95% CI)
Garenoxacin Levofloxacin Moxifloxacin
0.0313 0.5 0.0625
1.87 (0.990–2.73) >20 8.54 (4.59–13.6)
MIC, minimum inhibitory concentration; ED50 , effective dose for 50% survival; CI, confidence interval. a Animals, 5-week-old (at the time of infection) male ICR mice, 10 animals per group; infection, transnasal infection, 3.2 × 105 colony-forming units per mouse.
3.2. Minimum inhibitory concentrations of antibacterial agents Garenoxacin showed the most potent activity [MIC for 90% of the organisms (MIC90 ) = 0.125 g/mL] amongst the tested quinolones against the 42 clinical isolates of group G S. dysgalactiae subsp. equisimilis and was superior to levofloxacin (MIC90 = 1 g/mL) and moxifloxacin (MIC90 = 0.25 g/mL) (Table 1). The MIC90 of garenoxacin was 16-fold and 32-fold lower than that of clarithromycin (MIC90 = 2 g/mL) and azithromycin (MIC90 = 4 g/mL), respectively, and 256-fold lower than that of tetracycline (MIC90 = 32 g/mL). The MIC90 values of benzylpenicillin and cefcapene were both 0.0156 g/mL, the lowest amongst all the antibiotics tested. Although no strain with resistance to quinolones was found, a few strains highly resistant to macrolides and tetracycline were detected.
3.3. Therapeutic effect 3. Results
The ED50 of garenoxacin in the experimental pneumonia model was 1.87 mg/kg, which was >10.7-fold and 4.6-fold less than that of levofloxacin (>20 mg/kg) and moxifloxacin (8.54 mg/kg), respectively (Table 2).
3.1. Typing of the emm gene and detection of drug resistance genes The 42 clinical isolates of group G S. dysgalactiae subsp. equisimilis used for MIC determination were divided into >15 emm gene types, of which stG485.0 was the most common (8 strains), followed by stG245.0 (7 strains), stG652.0 (4 strains), stG10.0 (4 strains) and stG6.1 (3 strains). The isolates had a high frequency of the tetM gene (15/42 strains); isolates of the emm gene types stG485.0, stG245.0 and stG10.0 showed a high frequency of the tetM gene (6/8 strains, 5/7 strains and 4/4 strains, respectively). The macrolide resistance genes ermA, ermB and mefA were detected in two, one and three strains of various emm gene types, respectively.
3.4. Pharmacokinetic/pharmacodynamic parameters The fAUC0–24 of garenoxacin in serum following oral administration of 20 mg/kg in the experimental pneumonia model was 2.29 g h/mL. This value was slightly lower than those of levofloxacin (3.22–4.23 g h/mL) and moxifloxacin (3.21 g h/mL), respectively (Table 3); however, the fAUC0–24 /MIC ratio of garenoxacin in serum was 73.2, which was 8.7–11.4-fold and 1.4-fold greater than that of levofloxacin (6.44–8.46) and moxifloxacin (51.4), respectively (Table 3).
Table 3 Pharmacokinetic/pharmacodynamic parameters of garenoxacin and other antimicrobial agents following oral administration of 20 mg/kg to mice with experimental pneumoniaa induced by Streptococcus dysgalactiae subsp. equisimilis D-4691. Antibacterial agent Serum Garenoxacin Levofloxacin Moxifloxacin Lung tissue Garenoxacin Levofloxacin Moxifloxacin
Cmax (g/mL or g/g)
tmax (h)
t1/2 (h)
Total AUC0–24 [fAUC0–24 b ] (g h/mL or g h/g)
Total AUC0–24 /MICc [fAUC0–24 /MICb , c ]
1.97 1.00 0.97
0.25 0.25 0.25
5.02 4.32 6.36
10.4 [2.29] 4.81 [3.22–4.23] 4.65 [3.21]
332 [73.2] 9.62 [6.44–8.46] 74.4 [51.4]
2.81 1.95 3.12
0.25 0.25 0.25
4.86 3.77 4.47
14.1 7.39 12.0
452 14.8 191
Cmax , maximum drug concentration; tmax , time to reach Cmax ; t1/2 , half-life; AUC0–24 , area under the concentration–time curve from 0–24 h; f, free; MIC, minimum inhibitory concentration. a Animals, 5-week-old (at the time of infection) male ICR mice were intraperitoneally administered cyclophosphamide 200 mg/kg and 100 mg/kg at 4 days and 1 day before infection, respectively; inoculum, 2.7 × 105 colony-forming units per mouse; dose, 20 mg/kg orally administered at 2 h after infection. b Protein-binding percent: garenoxacin, 78%; levofloxacin, 12–33%; and moxifloxacin, 31% [24–26]. c MIC against strain D-4691: garenoxacin, 0.0313 g/mL; levofloxacin, 0.5 g/mL; and moxifloxacin, 0.0625 g/mL.
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4. Discussion Group G streptococci (GGS) were first identified by Lancefield and Hare [27]. Recently, group G S. dysgalactiae subsp. equisimilis has been reported as a causative organism of infections of the respiratory tract, skin and soft tissues, endocarditis, bacteraemia and meningitis [28,29]. Cases of pneumonia and empyema caused by group G S. dysgalactiae subsp. equisimilis have recently been reported in Japan [2,28,30] and Denmark [8]. In the present study, the antibacterial activity of garenoxacin was investigated against clinical isolates of group G S. dysgalactiae subsp. equisimilis in which the emm gene and drug resistance genes were studied. In addition, the efficacy of quinolones against experimental pneumonia induced in mice by group G S. dysgalactiae subsp. equisimilis was evaluated. The M protein, a key virulence factor encoded by the emm gene, has also been used to differentiate streptococcal strains such as GAS. The sequence-based typing system developed for GAS, which depends on the hypervariable region of the emm gene, was successfully applied to GGS and group C streptococci (GCS) [31]. Furthermore, it is considered that the M protein plays an important role in the pathogenesis of GGS and GCS infections. Most of the strains in the present study were isolated from sputum, pus and pharyngeal exudates, as previously reported [31]. Forty-two isolates of group G S. dysgalactiae subsp. equisimilis were divided into >15 emm gene types, of which stG485.0 was the most common (19.0%), followed by stG245.0 (16.7%). Although a large number of strains of emm gene type stG10.0 were isolated in previous studies [31,32], a comparable number of strains of emm gene type stG485.0 were also isolated. The isolates of emm gene type stG485.0 and stG245.0 had a high frequency of the tetM gene (73.3%); however, only a few strains possessed macrolide resistance genes and there was no quinolone-resistant strain. Garenoxacin showed 2-fold and 8-fold more potent antibacterial activity against the clinical isolates of group G S. dysgalactiae subsp. equisimilis than moxifloxacin and levofloxacin, respectively. MICs of benzylpenicillin against all strains tested were ≤0.01 g/mL, and no resistant strains were identified. Zaoutis et al. [29] also reported that the majority of GCS strains demonstrated in vitro susceptibility to penicillins; however, penicillin hardly shows antibacterial activity against intracellular bacterial parasites [33]. It is reported that S. dysgalactiae subsp. equisimilis is also both an extracellular and intracellular pathogen and it is considered that this organism may survive inside phagocytes, particularly in the carrier state [34]; therefore, when penicillin proves ineffective, antibiotics such as macrolides and quinolones should be administered. Regarding in vivo efficacy, the ED50 of garenoxacin in mice with experimental pneumonia was lower than that of moxifloxacin and levofloxacin. The fAUC0–24 /MIC ratio of garenoxacin in serum was 8.7–11.4-fold and 1.4-fold greater than that of levofloxacin and moxifloxacin, respectively. It was considered that the potent antibacterial activity and favourable pharmacokinetic profile of garenoxacin reflected its excellent therapeutic effect on experimental pneumonia caused by GGS. Garenoxacin is a des-fluoro(6) quinolone with potent antibacterial activity against various streptococci, including their various drug-resistant strains [16,35]. It also exhibits a favourable pharmacokinetic profile in humans, with good penetration into bronchial mucosa and alveolar macrophages [17,36]. In previous studies, the fAUC of garenoxacin in human plasma following single oral administration [400 mg once daily (q.d.), protein binding (PB) = 75%] was 14.6 g h/mL [17,37]. It was therefore considered that the fAUC/MIC90 (MIC90 of garenoxacin for S. dysgalactiae subsp. equisimilis of 0.125 g/mL) ratio was 116.8, much greater than those of levofloxacin and moxifloxacin
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administered at the clinical dosage (levofloxacin 500 mg q.d., PB = 35%, fAUC = 30.9 g h/mL, MIC90 = 1 g/mL, fAUC/MIC90 = 30.9; moxifloxacin 400 mg q.d., PB = 45%, fAUC = 16.4 g h/mL, MIC90 = 0.25 g/mL, fAUC/MIC90 = 65.6) [26,38–40]. Because the fAUC/MIC ratio is one of the most important predictors of the clinical efficacy of fluoroquinolones, a favourable clinical effect of garenoxacin is expected in the treatment of streptococci against which garenoxacin possesses potent activity. In conclusion, it was considered that garenoxacin is a valuable quinolone for the treatment of infectious diseases caused by group G S. dysgalactiae subsp. equisimilis. Funding: No funding sources. Competing interests: None declared. Ethical approval: All murine experiments were performed according to the Laboratory Animal Use Management Regulations at Toyama Chemical Co. Ltd. (9 November 2005). References [1] Woo PC, Fung AM, Lau SK, Wong SS, Yuen KY. Group G -hemolytic streptococcal bacteremia characterized by 16S ribosomal RNA gene sequencing. J Clin Microbiol 2001;39:3147–55. [2] Takahashi T, Ubukata K, Watanabe H. Invasive infection caused by Streptococcus dysgalactiae subsp. equisimilis: characteristics of strains and clinical features. J Infect Chemother 2011;17:1–10. [3] Vandamme P, Pot B, Falsen E, Kersters K, Devriese LA. Taxonomic study of Lancefield streptococcal groups C, G, and L (Streptococcus dysgalactiae) and proposal of S. dysgalactiae subsp. equisimilis subsp. nov. Int J Syst Bacteriol 1996;46:774–81. [4] Ueno K, Kawayama T, Edakuni N, Koga T, Aizawa H. A case of thoracic empyema with gas formation associated with Streptococcus dysgalactiae subsp. equisimilis [in Japanese]. Kansenshogaku Zasshi 2006;80:527–30. [5] Hashikawa S, Iinuma Y, Furushita M, Ohkura T, Nada T, Torii K, et al. Characterization of group C and G streptococcal strains that cause streptococcal toxic shock syndrome. J Clin Microbiol 2004;42:186–92. [6] Horii T, Izumida S, Takeuchi K, Tada T, Ishikawa J, Tsuboi K. Acute peritonitis and salpingitis associated with streptococcal toxic shock syndrome caused by Lancefield group G ␣-haemolytic Streptococcus dysgalactiae subsp. equisimilis. J Med Microbiol 2006;55:953–6. [7] Lopardo HA, Vidal P, Sparo M, Jeric P, Centron D, Facklam RR, et al. Sixmonth multicenter study on invasive infections due to Streptococcus pyogenes and Streptococcus dysgalactiae subsp. equisimilis in Argentina. J Clin Microbiol 2005;43:802–7. [8] Ekelund K, Skinhøj P, Madsen J, Konradsen HB. Invasive group A, B, C and G streptococcal infections in Denmark 1999–2002: epidemiological and clinical aspects. Clin Microbiol Infect 2005;11:569–76. [9] Cunningham MW. Pathogenesis of group A streptococcal infections. Clin Microbiol Rev 2000;13:470–511. [10] Collins CM, Kimura A, Bisno AL. Group G streptococcal M protein exhibits structural features analogous to those of class I M protein of group A streptococci. Infect Immun 1992;60:3689–96. [11] Schnitzler N, Podbielski A, Baumgarten G, Mignon M, Kaufhold A. M or M-like protein gene polymorphisms in human group G streptococci. J Clin Microbiol 1995;33:356–63. [12] Savini V, Catavitello C, Talia M, Manna A, Pompetti F, Di Bonaventura G, et al. -Lactam failure in treatment of two group G Streptococcus dysgalactiae subsp. equisimilis pharyngitis patients. J Clin Microbiol 2008;46:814–6. [13] Kataja J, Seppälä H, Skurnik M, Sarkkinen H, Huovinen P. Different erythromycin resistance mechanisms in group C and group G streptococci. Antimicrob Agents Chemother 1998;42:1493–4. [14] Merino Díaz L, Torres Sánchez MJ, Aznar Martín J. Prevalence and mechanisms of erythromycin and clindamycin resistance in clinical isolates of -haemolytic streptococci of Lancefield groups A, B, C and G in Seville, Spain. Clin Microbiol Infect 2008;14:85–7. [15] Jeric PE, Lopardo H, Vidal P, Arduino S, Fernandez A, Orman BE, et al. Multicenter study on spreading of the tet(M) gene in tetracycline-resistant Streptococcus group G and C isolates in Argentina. Antimicrob Agents Chemother 2002;46:239–41. [16] Takahata M, Mitsuyama J, Yamashiro Y, Yonezawa M, Araki H, Todo Y, et al. In vitro and in vivo antimicrobial activities of T-3811ME, a novel des-F(6)quinolone. Antimicrob Agents Chemother 1999;43:1077–84. [17] Gajjar DA, Bello A, Ge Z, Christopher L, Grasela DM. Multiple-dose safety and pharmacokinetics of oral garenoxacin in healthy subjects. Antimicrob Agents Chemother 2003;47:2256–63. [18] Whatmore AM, Kehoe MA. Horizontal gene transfer in the evolution of group A streptococcal emm-like genes: gene mosaics and variation in Vir regulons. Mol Microbiol 1994;11:363–74. [19] Seppälä H, Skurnik M, Soini H, Roberts MC, Huovinen P. A novel erythromycin resistance methylase gene (ermTR) in Streptococcus pyogenes. Antimicrob Agents Chemother 1998;42:257–62.
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[31] Sunaoshi KS, Aburahashi H, Kobayashi R, Yamamoto Y, Okuzumi K, Yoshida A, et al. emm typing by genetic identification of Streptococcus dysgalactiae subsp. equisimilis and susceptibility to oral antibiotics [in Japanese]. Kansenshogaku Zasshi 2006;80:488–95. [32] Pinho MD, Melo-Cristino J, Ramirez M. Clonal relationships between invasive and noninvasive Lancefield group C and G streptococci and emm-specific differences in invasiveness. J Clin Microbiol 2006;44:841–6. [33] Neeman R, Keller N, Barzilai A, Korenman Z, Sela S. Prevalence of internalisation-associated gene, prtF1, among persisting group-A streptococcus strains isolated from asymptomatic carriers. Lancet 1998;352: 1974–7. [34] Zaoutis T, Attia M, Gross R, Klein J. The role of group C and group G streptococci in acute pharyngitis in children. Clin Microbiol Infect 2004;10:37– 40. [35] Jones RN, Biedenbach DJ. Comparative activity of garenoxacin (BMS 284756), a novel desfluoroquinolone, tested against 8,331 isolates from communityacquired respiratory tract infections: North American results from the SENTRY Antimicrobial Surveillance Program (1999–2001). Diagn Microbiol Infect Dis 2003;45:273–8. [36] Andrews J, Honeybourne D, Jevons G, Boyce M, Wise R, Bello A, et al. Concentrations of garenoxacin in plasma, bronchial mucosa, alveolar macrophages and epithelial lining fluid following a single oral 600 mg dose in healthy adult subjects. J Antimicrob Chemother 2003;51:727– 30. [37] Bello A, Hollenbaugh D, Gajjar D, Christopher L, Grasela D. Abstracts of the 41st Interscience Conference on Antimicrobial Agents and Chemotherapy (ICAAC). Washington, DC: ASM Press; 2001 [Abstract A-45]. [38] Chien SC, Rogge MC, Gisclon LG, Curtin C, Wong F, Natarajan J, et al. Pharmacokinetic profile of levofloxacin following once-daily 500-milligram oral or intravenous doses. Antimicrob Agents Chemother 1997;41:2256– 60. [39] Zeitlinger MA, Dehghanyar P, Mayer BX, Schenk BS, Neckel U, Heinz G, et al. Relevance of soft-tissue penetration by levofloxacin for target site bacterial killing in patients with sepsis. Antimicrob Agents Chemother 2003;47: 3548–53. [40] Stass H, Kubitza D. Pharmacokinetics and elimination of moxifloxacin after oral and intravenous administration in man. J Antimicrob Chemother 1999;43(Suppl. B):83–90.
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International Journal of Antimicrobial Agents journal homepage: http://www.elsevier.com/locate/ijantimicag
In vitro and in vivo antibacterial activities of garenoxacin against group G Streptococcus dysgalactiae subsp. equisimilis Masahiro Takahata ∗ , Yoko Sugiura, Yuko Shiokawa, Naoko Futakuchi, Yoshiko Fukuda, Nobuhiko Nomura, Junichi Mitsuyama Research Laboratories, Toyama Chemical Co. Ltd., 4-1, Shimookui 2-chome, Toyama 930-8508, Japan
a r t i c l e
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Article history: Received 19 January 2011 Accepted 20 April 2011 Keywords: Streptococcus dysgalactiae subsp. equisimilis Garenoxacin In vitro activity In vivo activity
a b s t r a c t In this study, garenoxacin showed potent in vitro activity against clinical isolates of group G Streptococcus dysgalactiae subsp. equisimilis [minimum inhibitory concentration for 90% of the organisms (MIC90 ) = 0.125 g/mL] and was superior to levofloxacin (MIC90 = 1 g/mL) and moxifloxacin (MIC90 = 0.25 g/mL). In experimental pneumonia caused by group G S. dysgalactiae subsp. equisimilis in mice, the effective dose for 50% survival (ED50 ) of garenoxacin following single oral administration was 1.87 mg/kg, >10.7-fold and 4.6-fold less than the ED50 values of levofloxacin (>20 mg/kg) and moxifloxacin (8.54 mg/kg), respectively. The area under the free serum concentration–time curve from 0–24 h (fAUC0–24 )/MIC ratio of garenoxacin in serum following oral administration of 20 mg/kg was 73.2, which was 8.7–11.4-fold and 1.4-fold greater than that of levofloxacin (6.44–8.46) and moxifloxacin (51.4), respectively. These results suggest that garenoxacin has potential for the treatment of infectious diseases caused by S. dysgalactiae subsp. equisimilis. © 2011 Elsevier B.V. and the International Society of Chemotherapy. All rights reserved.
1. Introduction Streptococcus dysgalactiae subsp. equisimilis can be classified as follows on the basis of a combination of phenotypic and genotypic characteristics: Lancefield group C ␣-haemolytic; Lancefield group C -haemolytic; Lancefield group G -haemolytic; and Lancefield group L -haemolytic [1]. Streptococcus dysgalactiae subsp. equisimilis may exist amongst the normal flora of the skin, oropharynx, and gastrointestinal and genitourinary tracts [2]. Group C and G streptococci are thought to show low pathogenicity. However, in 1996 Vandamme et al. [3] proposed that S. dysgalactiae subsp. equisimilis is a clinical pathogen. Recently it was reported that Lancefield group G S. dysgalactiae subsp. equisimilis is responsible for 5–8% of human streptococcal infections, including suppurative diseases, bacteraemia, wound infections, otitis media, purulent pharyngitis, pneumonia, empyema and streptococcal toxic shock syndrome [1,4–8]. The M protein encoded by the emm gene is the major virulence determinant of Streptococcus pyogenes, one of the group A streptococci (GAS), and contributes considerably to the invasive capability of GAS by mediating antiphagocytic, adherence and internalisation processes [9]. Group G S. dysgalactiae subsp. equisimilis express homologues of the M proteins of S. pyogenes [10,11]
∗ Corresponding author. Tel.: +81 76 431 8306; fax: +81 76 431 8208. E-mail address: MASAHIRO [email protected] (M. Takahata).
and it is reported that the M proteins of group G S. dysgalactiae subsp. equisimilis may also play an important role in pathogenesis [5]. Penicillins have usually been used for the treatment of infections caused by group C and G streptococci; however, some reports present examples of failed penicillin treatment. The reason for this failure is that group C and G streptococci may survive inside pharyngeal cells or phagocytes, particularly in the carrier state [12]. Macrolides and tetracyclines have also been used for the treatment of various infectious diseases caused by streptococci; however, a rapid increase in the frequency of macrolide- and/or tetracyclineresistant group C and G streptococci was reported during the 1990s [13]. Furthermore, recent studies have shown the continuing prevalence of macrolide [14] and tetracycline resistance [15]; therefore, chemotherapy with new antibacterial agents for infectious diseases caused by group C and G streptococci is required. Garenoxacin, a des-fluoro(6) quinolone, exhibits more potent activity against Streptococcus pneumoniae, a major respiratory pathogen, than other fluoroquinolones such as levofloxacin and moxifloxacin [16]. In addition, following oral administration to humans, garenoxacin shows more favourable pharmacokinetics than other developed quinolones [17]; therefore, garenoxacin is expected to show good clinical efficacy against various infectious diseases caused by streptococci, including macrolide- and tetracycline-resistant strains. The present study investigated the in vitro antibacterial activity of garenoxacin against clinical isolates of group G S. dysgalactiae
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M. Takahata et al. / International Journal of Antimicrobial Agents 38 (2011) 226–230
subsp. equisimilis, in which the emm gene type and macrolide and tetracycline resistance genes were analysed. In addition, because it has been reported that pneumonia often develops to bacteraemia, which has frequently been seen in infections caused by this bacterial species, the in vivo efficacy of garenoxacin against experimental pneumonia induced by group G S. dysgalactiae subsp. equisimilis in mice was evaluated and was compared with levofloxacin and moxifloxacin.
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MIC determination and dilution of antibacterial agents was cationadjusted Mueller–Hinton broth (Nippon Becton, Dickinson & Co., Tokyo, Japan) supplemented with 5% lysed horse blood (Nihon Biotest Laboratories Inc., Tokyo, Japan). A 5 L bacterial suspension was inoculated into 96-well microplates containing 95 L of antibiotic solution [final inoculum size ca. 105 colony-forming units (CFU)/mL]. The lowest drug concentration that prevented visible growth after 22 h of incubation at 35 ◦ C was defined as the MIC.
2. Materials and methods 2.2. In vivo efficacy 2.1. In vitro activity 2.1.1. Bacterial strains For minimum inhibitory concentration (MIC) determination, 42 clinical isolates of group G S. dysgalactiae subsp. equisimilis isolated in Japan during the period 2005–2008 were used. Isolates were identified on the basis of phenotypic characteristics determined by the VITEK® Gram-Positive Identification card (Sysmex bioMérieux Co. Ltd., Tokyo, Japan) and the streptococcal grouping kit UNIBLUE (Kanto Chemical Co. Inc., Tokyo, Japan). The sources of the isolates were as follows: sputum, 9; pus, 9; pharyngeal exudates, 8; vaginal exudates, 2; other exudates, 3; urine, 3; and other, 8. Group G S. dysgalactiae subsp. equisimilis strain D-4691 was used to induce experimental pneumonia. 2.1.2. Typing of the M protein gene of clinical isolates The M protein type of the 42 clinical isolates of group G S. dysgalactiae subsp. equisimilis was determined on the basis of the M protein gene (emm) amplified by polymerase chain reaction (PCR) using oligonucleotide primers reported previously [18]. The first 240 bases of the sequence were used to query the US Centers for Disease Control and Prevention (CDC) streptococcal emm sequence database An (http://www.cdc.gov/ncidod/biotech/strep/strepindex.htm). emm gene type was defined as one that was 95% identical with regard to the first 160 bases of the sequence. 2.1.3. Detection of drug resistance genes of clinical isolates Five drug resistance genes (macrolide resistance genes ermA, ermB and mefA and tetracycline resistance genes tetM and tetO) were detected by PCR with the respective oligonucleotide primers as reported previously [15,19–22]. 2.1.4. Antibacterial agents The following antibacterial agents were used: garenoxacin (garenoxacin methanesulfonate monohydrate); levofloxacin; moxifloxacin; azithromycin; clarithromycin; tetracycline; cefcapene; and benzylpenicillin. Garenoxacin was synthesised at Research Laboratories, Toyama Chemical Co. Ltd. (Tokyo, Japan). Moxifloxacin and cefcapene were extracted from commercially available tablets (Shionogi & Co. Ltd., Osaka, Japan); the purity of each of these two agents was >99.8% as measured by high-performance liquid chromatography (HPLC). Levofloxacin was purchased from Chem-Impex International Inc. (Wood Dale, IL). Azithromycin and clarithromycin were purchased from LKT Laboratories Inc. (Saint Paul, MN). Tetracycline and benzylpenicillin were purchased from Sigma-Aldrich Japan (Tokyo, Japan) and Meiji Seika Kaisha Ltd. (Tokyo, Japan), respectively. 2.1.5. Minimum inhibitory concentration determination Precultures for MIC determination were performed in brain–heart infusion broth (Eiken Chemical Co. Ltd., Tokyo, Japan) by incubation at 35 ◦ C for 18 h. MICs were determined using a microdilution method recommended by the Clinical and Laboratory Standards Institute (CLSI) [23]. The medium used for
2.2.1. Laboratory animals Four-week-old male Institute of Cancer Research (ICR) mice were purchased from Japan SLC Inc. (Shizuoka, Japan) and were used after acclimation for 3 days. All murine experiments were performed according to the Laboratory Animal Use Management Regulations at Toyama Chemical Co. Ltd. (9 November 2005). 2.2.2. Experimental murine model of pneumococcal pneumonia Streptococcus dysgalactiae subsp. equisimilis D-4691 (emm gene type stG652.0) was used for experimental pneumonia studies to determine the therapeutic effects of the quinolones. Following intraperitoneal administration of cyclophosphamide (Shionogi & Co. Ltd.) (200 mg/kg and 100 mg/kg at 4 days and 1 day before infection, respectively), experimental pneumonia was induced in mice. Mice were anaesthetised by intramuscular injection of a solution containing a mixture of ketamine hydrochloride (Ketalar; DAIICHI SANKYO Co. Ltd., Tokyo, Japan), xylazine hydrochloride (Ceractal; Bayer Medical Co. Ltd., Tokyo, Japan) and sterile physiological saline (2:1:3) at 0.1 mL per mouse via the femoral region. Pulmonary infection was induced by intranasal injection of 40 L of bacterial suspension (ca. 105 CFU per mouse). On the day of infection, mice were randomly allocated to each group (N = 10). 2.2.3. Administration of antimicrobial agents Garenoxacin, levofloxacin and moxifloxacin were suspended in 0.5% (w/v) methyl cellulose 400 (Wako Pure Chemical Industries Ltd., Osaka, Japan) and were orally administered once at 2 h after infection (N = 10). 2.2.4. Evaluation of the therapeutic effects of antimicrobial agents From the number of surviving mice 7 days after antibiotic administration, the 50% effective dose (ED50 ) was calculated by the probit method (SAS release 8.2; SAS Institute Japan Ltd., Tokyo, Japan). 2.3. Determination of drug concentrations in serum and lung tissue Drug concentrations in serum and lung tissue following oral administration of 20 mg/kg garenoxacin, levofloxacin or moxifloxacin (N = 4 in each group) were determined by HPLC. The dose of 20 mg/kg was decided on the basis of the HPLC assay limit. At 0.25, 0.5, 1, 2, 4 and 6 h after administration of each quinolone, mice were sacrificed by exsanguination from the inferior vena cava under ether anaesthesia at each sampling time. Blood collected from the inferior vena cava was used to measure drug concentrations. Blood was kept in Sepaclen-A-5 (Eiken Kizai Co. Ltd., Tokyo, Japan) at room temperature for >30 min and serum was then obtained by centrifugation. Lung tissue was also removed at each sampling time and was homogenised in a four-fold volume of phosphate-buffered saline (pH 7.0, 1/15 mol/L). Homogenised tissue was centrifuged and the supernatant and serum were frozen at −40 ◦ C until HPLC assay. For the HPLC assay, an equal volume of methanol was added to each sample, which was then mixed and
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Table 1 Antibacterial activities of garenoxacin and other antimicrobial agents against 42 clinical isolates of group G Streptococcus dysgalactiae subsp. equisimilis. Antibacterial agent
Garenoxacin Levofloxacin Moxifloxacin Azithromycin Clarithromycin Tetracycline Cefcapene Benzylpenicillin
MIC (g/mL) Range
MIC50
MIC90
0.0313–0.125 0.5–2.0 0.0625–0.5 0.0625 to >128 0.0156 to >128 0.25–64 0.0078–0.0313 0.0078–0.01
0.0625 0.5 0.125 0.125 0.0313 2 0.0156 0.0156
0.125 1 0.25 4 2 32 0.0156 0.0156
MIC, minimum inhibitory concentration; MIC50/90 , MIC for 50% and 90% of the organisms, respectively.
centrifuged. The supernatant was used to measure the concentration of each quinolone by HPLC (LC series; Shimadzu Corporation, Kyoto, Japan). The maximum concentration of drug in serum (Cmax ) and the time to reach Cmax (tmax ) were determined directly from the actual mean drug concentrations in serum. The area under the serum concentration–time curve from 0–24 h (AUC0–24 ) and the half-life (t1/2 ) were calculated from the mean drug concentrations in serum, which were determined by non-compartmental analysis with WinNonlin Professional software v5.0.1 (Pharsight Corporation, Sunnyvale, CA). Moreover, the free (f) AUC0–24 in serum was calculated on the basis of the protein-binding percent of each quinolone in mice (garenoxacin 78%, levofloxacin 12–33% and moxifloxacin 31%) [24–26].
Table 2 Therapeutic effects of garenoxacin and other antimicrobial agents on experimental pneumonia induced in micea by Streptococcus dysgalactiae subsp. equisimilis D-4691. Antibacterial agent
MIC (g/mL)
ED50 (mg/kg) (95% CI)
Garenoxacin Levofloxacin Moxifloxacin
0.0313 0.5 0.0625
1.87 (0.990–2.73) >20 8.54 (4.59–13.6)
MIC, minimum inhibitory concentration; ED50 , effective dose for 50% survival; CI, confidence interval. a Animals, 5-week-old (at the time of infection) male ICR mice, 10 animals per group; infection, transnasal infection, 3.2 × 105 colony-forming units per mouse.
3.2. Minimum inhibitory concentrations of antibacterial agents Garenoxacin showed the most potent activity [MIC for 90% of the organisms (MIC90 ) = 0.125 g/mL] amongst the tested quinolones against the 42 clinical isolates of group G S. dysgalactiae subsp. equisimilis and was superior to levofloxacin (MIC90 = 1 g/mL) and moxifloxacin (MIC90 = 0.25 g/mL) (Table 1). The MIC90 of garenoxacin was 16-fold and 32-fold lower than that of clarithromycin (MIC90 = 2 g/mL) and azithromycin (MIC90 = 4 g/mL), respectively, and 256-fold lower than that of tetracycline (MIC90 = 32 g/mL). The MIC90 values of benzylpenicillin and cefcapene were both 0.0156 g/mL, the lowest amongst all the antibiotics tested. Although no strain with resistance to quinolones was found, a few strains highly resistant to macrolides and tetracycline were detected.
3.3. Therapeutic effect 3. Results
The ED50 of garenoxacin in the experimental pneumonia model was 1.87 mg/kg, which was >10.7-fold and 4.6-fold less than that of levofloxacin (>20 mg/kg) and moxifloxacin (8.54 mg/kg), respectively (Table 2).
3.1. Typing of the emm gene and detection of drug resistance genes The 42 clinical isolates of group G S. dysgalactiae subsp. equisimilis used for MIC determination were divided into >15 emm gene types, of which stG485.0 was the most common (8 strains), followed by stG245.0 (7 strains), stG652.0 (4 strains), stG10.0 (4 strains) and stG6.1 (3 strains). The isolates had a high frequency of the tetM gene (15/42 strains); isolates of the emm gene types stG485.0, stG245.0 and stG10.0 showed a high frequency of the tetM gene (6/8 strains, 5/7 strains and 4/4 strains, respectively). The macrolide resistance genes ermA, ermB and mefA were detected in two, one and three strains of various emm gene types, respectively.
3.4. Pharmacokinetic/pharmacodynamic parameters The fAUC0–24 of garenoxacin in serum following oral administration of 20 mg/kg in the experimental pneumonia model was 2.29 g h/mL. This value was slightly lower than those of levofloxacin (3.22–4.23 g h/mL) and moxifloxacin (3.21 g h/mL), respectively (Table 3); however, the fAUC0–24 /MIC ratio of garenoxacin in serum was 73.2, which was 8.7–11.4-fold and 1.4-fold greater than that of levofloxacin (6.44–8.46) and moxifloxacin (51.4), respectively (Table 3).
Table 3 Pharmacokinetic/pharmacodynamic parameters of garenoxacin and other antimicrobial agents following oral administration of 20 mg/kg to mice with experimental pneumoniaa induced by Streptococcus dysgalactiae subsp. equisimilis D-4691. Antibacterial agent Serum Garenoxacin Levofloxacin Moxifloxacin Lung tissue Garenoxacin Levofloxacin Moxifloxacin
Cmax (g/mL or g/g)
tmax (h)
t1/2 (h)
Total AUC0–24 [fAUC0–24 b ] (g h/mL or g h/g)
Total AUC0–24 /MICc [fAUC0–24 /MICb , c ]
1.97 1.00 0.97
0.25 0.25 0.25
5.02 4.32 6.36
10.4 [2.29] 4.81 [3.22–4.23] 4.65 [3.21]
332 [73.2] 9.62 [6.44–8.46] 74.4 [51.4]
2.81 1.95 3.12
0.25 0.25 0.25
4.86 3.77 4.47
14.1 7.39 12.0
452 14.8 191
Cmax , maximum drug concentration; tmax , time to reach Cmax ; t1/2 , half-life; AUC0–24 , area under the concentration–time curve from 0–24 h; f, free; MIC, minimum inhibitory concentration. a Animals, 5-week-old (at the time of infection) male ICR mice were intraperitoneally administered cyclophosphamide 200 mg/kg and 100 mg/kg at 4 days and 1 day before infection, respectively; inoculum, 2.7 × 105 colony-forming units per mouse; dose, 20 mg/kg orally administered at 2 h after infection. b Protein-binding percent: garenoxacin, 78%; levofloxacin, 12–33%; and moxifloxacin, 31% [24–26]. c MIC against strain D-4691: garenoxacin, 0.0313 g/mL; levofloxacin, 0.5 g/mL; and moxifloxacin, 0.0625 g/mL.
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4. Discussion Group G streptococci (GGS) were first identified by Lancefield and Hare [27]. Recently, group G S. dysgalactiae subsp. equisimilis has been reported as a causative organism of infections of the respiratory tract, skin and soft tissues, endocarditis, bacteraemia and meningitis [28,29]. Cases of pneumonia and empyema caused by group G S. dysgalactiae subsp. equisimilis have recently been reported in Japan [2,28,30] and Denmark [8]. In the present study, the antibacterial activity of garenoxacin was investigated against clinical isolates of group G S. dysgalactiae subsp. equisimilis in which the emm gene and drug resistance genes were studied. In addition, the efficacy of quinolones against experimental pneumonia induced in mice by group G S. dysgalactiae subsp. equisimilis was evaluated. The M protein, a key virulence factor encoded by the emm gene, has also been used to differentiate streptococcal strains such as GAS. The sequence-based typing system developed for GAS, which depends on the hypervariable region of the emm gene, was successfully applied to GGS and group C streptococci (GCS) [31]. Furthermore, it is considered that the M protein plays an important role in the pathogenesis of GGS and GCS infections. Most of the strains in the present study were isolated from sputum, pus and pharyngeal exudates, as previously reported [31]. Forty-two isolates of group G S. dysgalactiae subsp. equisimilis were divided into >15 emm gene types, of which stG485.0 was the most common (19.0%), followed by stG245.0 (16.7%). Although a large number of strains of emm gene type stG10.0 were isolated in previous studies [31,32], a comparable number of strains of emm gene type stG485.0 were also isolated. The isolates of emm gene type stG485.0 and stG245.0 had a high frequency of the tetM gene (73.3%); however, only a few strains possessed macrolide resistance genes and there was no quinolone-resistant strain. Garenoxacin showed 2-fold and 8-fold more potent antibacterial activity against the clinical isolates of group G S. dysgalactiae subsp. equisimilis than moxifloxacin and levofloxacin, respectively. MICs of benzylpenicillin against all strains tested were ≤0.01 g/mL, and no resistant strains were identified. Zaoutis et al. [29] also reported that the majority of GCS strains demonstrated in vitro susceptibility to penicillins; however, penicillin hardly shows antibacterial activity against intracellular bacterial parasites [33]. It is reported that S. dysgalactiae subsp. equisimilis is also both an extracellular and intracellular pathogen and it is considered that this organism may survive inside phagocytes, particularly in the carrier state [34]; therefore, when penicillin proves ineffective, antibiotics such as macrolides and quinolones should be administered. Regarding in vivo efficacy, the ED50 of garenoxacin in mice with experimental pneumonia was lower than that of moxifloxacin and levofloxacin. The fAUC0–24 /MIC ratio of garenoxacin in serum was 8.7–11.4-fold and 1.4-fold greater than that of levofloxacin and moxifloxacin, respectively. It was considered that the potent antibacterial activity and favourable pharmacokinetic profile of garenoxacin reflected its excellent therapeutic effect on experimental pneumonia caused by GGS. Garenoxacin is a des-fluoro(6) quinolone with potent antibacterial activity against various streptococci, including their various drug-resistant strains [16,35]. It also exhibits a favourable pharmacokinetic profile in humans, with good penetration into bronchial mucosa and alveolar macrophages [17,36]. In previous studies, the fAUC of garenoxacin in human plasma following single oral administration [400 mg once daily (q.d.), protein binding (PB) = 75%] was 14.6 g h/mL [17,37]. It was therefore considered that the fAUC/MIC90 (MIC90 of garenoxacin for S. dysgalactiae subsp. equisimilis of 0.125 g/mL) ratio was 116.8, much greater than those of levofloxacin and moxifloxacin
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administered at the clinical dosage (levofloxacin 500 mg q.d., PB = 35%, fAUC = 30.9 g h/mL, MIC90 = 1 g/mL, fAUC/MIC90 = 30.9; moxifloxacin 400 mg q.d., PB = 45%, fAUC = 16.4 g h/mL, MIC90 = 0.25 g/mL, fAUC/MIC90 = 65.6) [26,38–40]. Because the fAUC/MIC ratio is one of the most important predictors of the clinical efficacy of fluoroquinolones, a favourable clinical effect of garenoxacin is expected in the treatment of streptococci against which garenoxacin possesses potent activity. In conclusion, it was considered that garenoxacin is a valuable quinolone for the treatment of infectious diseases caused by group G S. dysgalactiae subsp. equisimilis. Funding: No funding sources. Competing interests: None declared. Ethical approval: All murine experiments were performed according to the Laboratory Animal Use Management Regulations at Toyama Chemical Co. Ltd. (9 November 2005). References [1] Woo PC, Fung AM, Lau SK, Wong SS, Yuen KY. Group G -hemolytic streptococcal bacteremia characterized by 16S ribosomal RNA gene sequencing. J Clin Microbiol 2001;39:3147–55. [2] Takahashi T, Ubukata K, Watanabe H. Invasive infection caused by Streptococcus dysgalactiae subsp. equisimilis: characteristics of strains and clinical features. J Infect Chemother 2011;17:1–10. [3] Vandamme P, Pot B, Falsen E, Kersters K, Devriese LA. Taxonomic study of Lancefield streptococcal groups C, G, and L (Streptococcus dysgalactiae) and proposal of S. dysgalactiae subsp. equisimilis subsp. nov. Int J Syst Bacteriol 1996;46:774–81. [4] Ueno K, Kawayama T, Edakuni N, Koga T, Aizawa H. A case of thoracic empyema with gas formation associated with Streptococcus dysgalactiae subsp. equisimilis [in Japanese]. Kansenshogaku Zasshi 2006;80:527–30. [5] Hashikawa S, Iinuma Y, Furushita M, Ohkura T, Nada T, Torii K, et al. Characterization of group C and G streptococcal strains that cause streptococcal toxic shock syndrome. J Clin Microbiol 2004;42:186–92. [6] Horii T, Izumida S, Takeuchi K, Tada T, Ishikawa J, Tsuboi K. Acute peritonitis and salpingitis associated with streptococcal toxic shock syndrome caused by Lancefield group G ␣-haemolytic Streptococcus dysgalactiae subsp. equisimilis. J Med Microbiol 2006;55:953–6. [7] Lopardo HA, Vidal P, Sparo M, Jeric P, Centron D, Facklam RR, et al. Sixmonth multicenter study on invasive infections due to Streptococcus pyogenes and Streptococcus dysgalactiae subsp. equisimilis in Argentina. J Clin Microbiol 2005;43:802–7. [8] Ekelund K, Skinhøj P, Madsen J, Konradsen HB. Invasive group A, B, C and G streptococcal infections in Denmark 1999–2002: epidemiological and clinical aspects. Clin Microbiol Infect 2005;11:569–76. [9] Cunningham MW. Pathogenesis of group A streptococcal infections. Clin Microbiol Rev 2000;13:470–511. [10] Collins CM, Kimura A, Bisno AL. Group G streptococcal M protein exhibits structural features analogous to those of class I M protein of group A streptococci. Infect Immun 1992;60:3689–96. [11] Schnitzler N, Podbielski A, Baumgarten G, Mignon M, Kaufhold A. M or M-like protein gene polymorphisms in human group G streptococci. J Clin Microbiol 1995;33:356–63. [12] Savini V, Catavitello C, Talia M, Manna A, Pompetti F, Di Bonaventura G, et al. -Lactam failure in treatment of two group G Streptococcus dysgalactiae subsp. equisimilis pharyngitis patients. J Clin Microbiol 2008;46:814–6. [13] Kataja J, Seppälä H, Skurnik M, Sarkkinen H, Huovinen P. Different erythromycin resistance mechanisms in group C and group G streptococci. Antimicrob Agents Chemother 1998;42:1493–4. [14] Merino Díaz L, Torres Sánchez MJ, Aznar Martín J. Prevalence and mechanisms of erythromycin and clindamycin resistance in clinical isolates of -haemolytic streptococci of Lancefield groups A, B, C and G in Seville, Spain. Clin Microbiol Infect 2008;14:85–7. [15] Jeric PE, Lopardo H, Vidal P, Arduino S, Fernandez A, Orman BE, et al. Multicenter study on spreading of the tet(M) gene in tetracycline-resistant Streptococcus group G and C isolates in Argentina. Antimicrob Agents Chemother 2002;46:239–41. [16] Takahata M, Mitsuyama J, Yamashiro Y, Yonezawa M, Araki H, Todo Y, et al. In vitro and in vivo antimicrobial activities of T-3811ME, a novel des-F(6)quinolone. Antimicrob Agents Chemother 1999;43:1077–84. [17] Gajjar DA, Bello A, Ge Z, Christopher L, Grasela DM. Multiple-dose safety and pharmacokinetics of oral garenoxacin in healthy subjects. Antimicrob Agents Chemother 2003;47:2256–63. [18] Whatmore AM, Kehoe MA. Horizontal gene transfer in the evolution of group A streptococcal emm-like genes: gene mosaics and variation in Vir regulons. Mol Microbiol 1994;11:363–74. [19] Seppälä H, Skurnik M, Soini H, Roberts MC, Huovinen P. A novel erythromycin resistance methylase gene (ermTR) in Streptococcus pyogenes. Antimicrob Agents Chemother 1998;42:257–62.
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