Abstract
Objectives: The in vitro activity of S-013420, a novel bicyclolide, was investigated.
Abstract
Methods: All test strains for this study were isolated from Japanese medical facilities. MICs were determined by the microbroth dilution method or agar dilution method according to the CLSI guidelines. In time -kill kinetics, viable cells were measured at 1, 2.5, 4 and 6 h after exposure to antimicrobials. The frequencies of single-step resistance acquisition at 4× MIC and 8× MIC were determined using 10 7 cfu of bacterial cells. The MICs of S-013420 for atypical pathogens such as erythromycin-susceptible Mycoplasma pneumoniae and Chlamydophila pneumoniae were 0.00049 -0.001 and 0.0039 mg/L, respectively, although the activity of S-013420, as well as other macrolide agents, against erythromycin-resistant M. pneumoniae was significantly weak. S-013420 caused a 3 log 10 reduction in viable cells against all test strains of S. pneumoniae and H. influenzae. Acquisition of resistance to S-013420 was not observed for three strains of S. pneumoniae.
Abstract
Conclusions: S-013420 shows potent in vitro activity against respiratory tract pathogens. Against streptococci, including erythromycin-resistant strains, S-013420 demonstrated the most potent in vitro activity among the antimicrobials tested.
Introduction
Macrolide antimicrobials, such as clarithromycin and azithromycin, have played a key role in the treatment of community-acquired respiratory tract infections (RTIs). However, erythromycin-resistant Streptococcus pneumoniae (ERSP) is one of the major problems in clinical practice.
Introduction
1,2 ERSP accounted for 40% of S. pneumoniae in the USA, 10% in Canada, 58% in France and 80%-90% in Asia. 3, 4 Two major mechanisms of macrolide resistance have been reported. One is ribosomal modification mediated by erythromycin ribosome methylation (erm) genes and the other is alteration of antibiotic accumulation due to macrolide efflux (mef) genes. 5 Most mef(A) and erm(B) genes of S. pneumoniae have been reported to lead to low and high levels of resistance to erythromycin, respectively. 2, 5, 6 Macrolide resistance is occasionally ascribed to mutations in 23S rRNA or ribosomal proteins such as L4 and L22 in clinical isolates. 3 Telithromycin, a ketolide antimicrobial agent, demonstrates in vitro activity against RTI pathogens including erythromycin-resistant streptococci. 2, 4, 5 S-013420 (EDP-420) is a bicyclolide (bridged bicyclic macrolide) that is characterized as a 6,11-bridged oxime macrolide discovered by Enanta Pharmaceuticals, Inc. (Figure 1 ). 7 S-013420 has shown good activity against respiratory tract pathogens including ERSP and good distribution into the lungs in healthy Japanese subjects. 7, 8 The purpose of this study was to evaluate the in vitro activity of S-013420 against clinical isolates obtained in Japan.
Materials and methods

Antimicrobial agents
S-013420 and telithromycin were chemically synthesized by Shionogi & Co., Ltd (Osaka, Japan). Clarithromycin, azithromycin and erythromycin # The Author 2010. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved. For Permissions, please e-mail: mcmahonshannon@example.org J Antimicrob Chemother 2010; 65: 1433 -1440 doi:10.1093/jac/dkq147 Advance Access publication 14 May 2010 were purchased from commercial sources. Levofloxacin was kindly supplied by Daiichi Sankyo Co., Ltd (Tokyo, Japan).
Bacterial isolates
Most bacterial isolates for MIC determination were clinical isolates collected at various Japanese medical facilities in 2004.
Bacterial isolates
9,10 S. pneumoniae ATCC 49619 was purchased from the ATCC. Clinical isolates of Mycoplasma pneumoniae and Chlamydophila pneumoniae were gifts from Kitasato University and Kurume University, respectively. The timekill kinetic study or single-step mutant selection study used six strains (SR26102, SR26100, SR26132, SR23965, SR23996 and SR26116) of S. pneumoniae and one strain (SR24169) of Haemophilus influenzae (collected in 2002). The mef(A) and erm(B) genes in S. pneumoniae were detected by PCR using a penicillin-resistant S. pneumoniae detection kit (Wakunaga Pharmaceutical, Osaka, Japan). The mef(A), erm(B) and erm(TR) genes in Streptococcus pyogenes were detected by PCR as described previously.
11,12

MIC determination
MICs for aerobic bacteria were determined by the microbroth dilution method according to the CLSI guidelines 13 with cation-adjusted Mueller-Hinton broth (CAMHB; Becton Dickinson, Sparks, MD, USA). CAMHB supplemented with 15 mg/L of b-NAD (Sigma-Aldrich, St Louis, MO, USA), 15 mg/L of haematin (Sigma) and 0.5% yeast extract (Gibco, Invitrogen Japan K.K., Tokyo, Japan) was used for H. influenzae and CAMHB supplemented with 5% lysed horse blood (CAMHB+LHB; Nippon Biotest Laboratories Inc., Japan) was used for S. pneumoniae. MICs for Neisseria gonorrhoeae were determined by the agar dilution method with GC medium (Becton Dickinson) containing growth supplement after incubation at 358C for 18 h in the presence of 5% CO 2 .
MIC determination
14 MICs for anaerobic bacteria were determined by the agar dilution method with Wilkins -Chalgren medium (Becton Dickinson) after incubation under anaerobic conditions at 358C after 18 h.
MIC determination
14 MICs for M. pneumoniae were determined by a broth microdilution method. 15 PPLO (Difco Inc., Detroit, MI, USA) medium containing 20% inactivated horse serum (Gibco), 2.5% yeast extract, 0.5% glucose and 0.002% phenol red (Tokyo Kasei Co., Ltd, Tokyo, Japan) was used for the growth medium. Aliquots of 10 3 -10 5 cfu/mL were added to each well containing 100 mL of the medium containing the substance and were incubated at 378C in ambient air. The colour of the growth control well was observed daily, and MICs were determined 3 days after the colour changed from red to yellow in the growth control well. The MIC was defined as the minimum concentration of the substance that inhibited the growth of mycoplasmas. The MIC endpoint was the well in which the colour was the same of that in the uninoculated well. MICs for C. pneumoniae were determined by a modified method according to the standard method of the Japan Society of Chemotherapy. 16 Dulbecco's modified Eagle's Medium (Gibco) supplemented with 10% heat-inactivated fetal bovine serum (Gibco) and 1 mg/L cycloheximide (Nacalai Tesque Inc., Tokyo, Japan) was used for cultivation of HeLa 229. One millilitre of culture medium containing HeLa 229 (10 5 cells/mL) was dispensed into each well of plastic 24-well culture plates. These plates were then incubated in 5% CO 2 at 378C for 20 h. After confirming the growth of a confluent monolayer, each chlamydial strain (10 4 inclusion-forming units/ mL) was used to infect HeLa 229 cells by centrifugation adsorption at 500 g for 1 h at room temperature. After removal of the supernatant, the culture medium containing antibacterials was dispensed into each well. After incubation in 5% CO 2 at 358C for 70 h, the medium was removed from the well and HeLa 229 cells were fixed and stained for inclusions with the Chlamydia FA reagent Seiken (Denka Seiken, Tokyo, Japan). The MIC was defined as the lowest concentration at which no inclusions were found.
Time -kill kinetics
Exponentially growing bacterial suspensions containing 10 8 cfu/mL were prepared by mcmahonshannon@example.org. After incubation, the bacterial suspensions were diluted to 5×10 5 cfu/mL. The bacterial suspensions were incubated with shaking at 378C in the presence of various concentrations (ranging from 0.5× MIC to 8× MIC) of each antimicrobial. A portion of the bacterial suspension was collected at 0, 1, 2.5, 4 and 6 h after exposure to antimicrobials. The numbers of viable bacterial cells in the suspension were determined using brain heart infusion agar (BHIA; Becton Dickinson) with 5% defibrinated horse blood (DHB; Nippon Biotest) for S. pneumoniae and BHIA with 2.5% enrichment (Becton Dickinson) for H. influenzae. The detection limit of the number of viable bacterial cells was 100 cfu/mL.
Single-step resistance acquisition
Bacterial cell suspensions of up to 1×10 7 cfu in 100 mL aliquots were spread on MHB (Becton Dickinson)/5% DHB (Japan Lamb) plates containing each antimicrobial at 4× MIC and 8× MIC. After incubation at 358C in 5% CO 2 for 48 h, the number of colonies that appeared on each plate was determined. Representative colonies on each antimicrobial plate were picked up to see if the MICs had increased. If an elevation in MIC was observed, the rate that the colonies appeared per inoculum was determined as the frequency of single-step resistance acquisition.
Results

MIC determination
The MIC ranges, MIC 50 (the concentration at which the growth of 50% of the strains was inhibited) values and MIC 90 values of S-013420 and other antimicrobials for aerobic pathogens are shown in Table 1 . Whereas clarithromycin and azithromycin showed MIC 90 s of .64 mg/L, S-013420 showed an MIC 90 of 0.125 mg/L for S. pneumoniae, which was 2-and 8-fold lower than that of telithromycin and levofloxacin, respectively. Moreover, S-013420 also showed an MIC 90 Homma et al. Table 2 shows the MIC values of each antimicrobial for four strains of S. pyogenes carrying macrolide resistance genes. Against these strains, S-013420 displayed potent activity with MICs of ≤0.063-1 mg/L, which were significantly lower than those of clarithromycin and azithromycin. Telithromycin also showed weak activity against the erm(B)-possessing strain with an MIC of 32 mg/L, although it showed potent activity against the other three strains. Furthermore, the MIC of S-013420 for S. pneumoniae ATCC 49619 was 0.063 mg/L and was comparable to those of telithromycin (0.031 mg/L), clarithromycin (0.063 mg/L) and azithromycin (0.125 mg/L). S-013420 has potent antibacterial activity against streptococci including erythromycin-resistant strains, which is comparable to or better than that of telithromycin and better than that of clarithromycin and azithromycin. S-013420 showed an MIC 90 value of 0.25 mg/L for erythromycin-susceptible S. aureus, which was equal to that of telithromycin and lower than those of the other antimicrobials. Against coagulase-negative Staphylococcus spp., S-013420 showed an MIC 90 value of 0.25 mg/L, which was comparable to those of other macrolide agents and much lower than that of levofloxacin. But all tested macrolide agents including S-013420 showed weak activity with MIC 90 values of .64 mg/L for erythromycin-intermediate and resistant strains. The MIC 90 values of S-013420 were 8 mg/L for Enterococcus faecalis and 16 mg/L for Enterococcus faecium, which were equal to those of telithromycin and lower than those of the other antimicrobials. S-013420 showed an MIC 90 of 8 mg/L for H. influenzae, which was equal to that of clarithromycin and 4-to 8-fold higher than those of telithromycin and azithromycin, respectively. Against Moraxella catarrhalis and Neisseria gonorrhoeae, S-013420 showed an MIC 90 of 0.5 mg/L, which was comparable to those of telithromycin and clarithromycin.
MIC determination
The in vitro activity of S-013420 against anaerobic pathogens is shown in Table 3 . S-013420 showed an MIC 90 of 0.25 mg/L for Peptostreptococcus spp., which was 8-fold lower than that of telithromycin and was 128-fold or more lower than those of clarithromycin, azithromycin and levofloxacin. S-013420 also showed an MIC 90 of 0.25 mg/L for Prevotella spp., which was 32-to .512-fold lower than those of the other antimicrobials. Against Clostridium difficile and Bacteroides spp., S-013420 was not active with MIC 90 s of .128 and 64 mg/L, respectively.
MIC determination
The MICs of S-013420 for atypical pathogens such as M. pneumoniae and C. pneumoniae are shown in Table 4 . For M. pneumoniae, the MICs of S-013420 were 0.00049-0.001 mg/L for erythromycin-susceptible M. pneumoniae, which were 2-to 4-fold higher than those of telithromycin and azithromycin and 2-to 8-fold lower than those of clarithromycin and erythromycin. On the other hand, S-013420 showed weak activity against erythromycin-resistant strains with an MIC of 8-16 mg/L, which was comparable to those of telithromycin and azithromycin. Although there were only three test strains of C. pneumoniae, the MIC of S-013420 for C. pneumoniae was 0.0039 mg/L, which was lower than those of any of the other antimicrobials.
Bactericidal activity
The killing kinetics of S-013420, telithromycin and clarithromycin at various concentrations against three strains of S. pneumoniae including mef(A)-and erm(B)-positive strains and one strain of H. influenzae are shown in Figure 3 . For S. pneumoniae, S-013420 and telithromycin showed rapid bactericidal action against all test strains, causing 3 log 10 reductions in viable cells at 4× MIC within 4 h regardless of the resistance genotype. However, clarithromycin showed similar bactericidal action only against the mef(A)-positive strain, and much weaker bactericidal action against strain SR26102 (erythromycin-susceptible strain). For H. influenzae, S-013420 and telithromycin caused 3 log 10 reductions at 2× MIC within 4 h and clarithromycin caused a 3 log 10 reduction at 2× MIC within 6 h. Similar time -kill kinetics profiles were also observed against three other strains of P. anaerobius (9), P. asaccharolyticus (21), P. hydrogenalis (1), P. ivorii (1), P. magnus (6), P. micros (10) and P. vaginalis (4). b B. caccae (2), B. distasonis (8), B. fragilis (40), B. ovatus (1), B. thetaiotaomicron (13), B. uniformis (6) and B. vulgatus (6). c P. bivia (12), P. buccae (6), P. intermedia (10), P. loescheii (2), P. melaninogenica (7) and P. oralis (2).
Bactericidal activity
In vitro activity of S-013420 1437 JAC H. influenzae (data not shown). S-013420 caused rapid killing of S. pneumoniae and H. influenzae and its bactericidal activity was comparable to or more potent than that of telithromycin and clarithromycin.
Single-step resistance acquisition
To estimate the tendency towards resistance to S-013420 of S. pneumoniae, the frequency of single-step resistance acquisition was investigated ( Table 5 ). The acquisition of resistance to S-013420 from 10 7 cfu of bacterial cells was not observed for all of the three strains tested. The colonies of SR26116 for S-013420 at 4× MIC appeared at a rate of 5.7×10
Single-step resistance acquisition
24 . But the susceptibility of these colonies did not decrease (the same MIC value as the parent). The acquisition of resistance to telithromycin was observed for two strains. As for clarithromycin, resistance acquisition was observed for one mef(A)-positive strain (out of the two strains tested). These results suggested that the development of resistance to S-013420 occurred less frequently than to telithromycin and clarithromycin in some strains.
Discussion
Macrolides are used in the treatment of community-acquired RTIs around the world. However, many studies have shown an increase in macrolide resistance rates among streptococci.
Discussion
2,5,6 S-013420, a novel bicyclolide, has shown a good pharmacokinetic profile, safety and efficacy in clinical trials. 8, 17 The present study was conducted to characterize S-013420, which has a broad antibacterial spectrum against a variety of RTI pathogens including anaerobic and atypical pathogens. S-013420 also showed potent activity against macrolide-resistant streptococci including telithromycinresistant S. pyogenes.
Discussion
In S. pneumoniae, mef(A)-positive strains are of the macrolide resistance phenotype (M phenotype) and erm(B)-positive strains are of the macrolide-lincosamide -streptogramin B resistance phenotype (MLS B ). 6 Many surveillance studies have reported that telithromycin showed potent activity against erythromycinresistant S. pneumoniae including erm(B)-positive strains, 2, 5, 6, 12 Homma et al.
Discussion
due to the lack of ability to induce the erm(B) gene. 18, 19 However, there have been reports of the appearance of telithromycinresistant S. pneumoniae in clinical studies. 20, 21 Although resistance by erm(B) in S. pneumoniae has been shown to be an inducible phenotype, 22, 23 S. pneumoniae was shown to acquire resistance to telithromycin by constitutive erm(B) expression. 18, 24 On the other hand, telithromycin-resistant erm(B)-positive S. pyogenes appeared more frequently as Morosini et al. 11 reported that 11 out of 19 strains of erm(B)-positive S. pyogenes exhibited a wide telithromycin MIC range (4 -32 mg/L). In the present study, one telithromycin-resistant strain of S. pyogenes with an MIC of 32 mg/L was used, but no telithromycin-resistant S. pneumoniae were found among macrolide-resistant streptococci. S-013420 showed potent activity against all strains of macrolide-resistant streptococci including telithromycin-resistant S. pyogenes. The antibacterial activity of S-013420 against telithromycin-resistant S. pyogenes with an MIC of 1 mg/L was 32-fold more potent than that of telithromycin. Although the resistance mechanism of telithromycin-resistant S. pyogenes is still unclear, S-013420 might have antimicrobial activity against constitutive erm(B)-positive streptococci, greater than that of telithromycin. This might be because S-013420 has a ribosome binding site that differs from that of telithromycin. S-013420, as well as telithromycin, exhibits stronger binding to both domain IV and domain II in 23S rRNA. 25 -28 In Escherichia coli, susceptibility to S-013420 was barely affected by the U2609C mutation in domain IV, which affected the binding of telithromycin. It suggested that S-013420 became bound to a site in 23S rRNA that was not responsible for the binding of other macrolide antimicrobials. 27 The excellent activity of S-013420 against erm(B)-positive S. pyogenes might be due to the S-013420 specific binding mechanism to 23S rRNA. More investigations are needed to understand the mechanism of S-013420 against telithromycin-resistant streptococci.
Discussion
S-013420 showed potent activity against mef(A)-positive S. pneumoniae, although the IC 50 of S-013420 in the cell-free transcription-translation system was similar to that of erythromycin. 27 Decrease in susceptibility to erythromycin in M-phenotype streptococci was reported to be caused by an efflux mechanism. 29 These results suggested that S-013420 might have a low affinity for the mef efflux system.
Discussion
In conclusion, S-013420 showed good in vitro antibacterial activity, good bactericidal activity and low frequency of resistance acquisition against respiratory tract pathogens. S-013420 had particularly potent activity against streptococci including erythromycin-resistant strains, which was greater than the other antimicrobials. In clinical trials, the distribution of S-013420 in the human lung was greater than that of the macrolide previously reported 8 and S-013420 showed good therapeutic efficacy in the murine lung infection model caused by S. pneumoniae and H. influenzae (R. Nakamura, H. Matsuda, T. Sato, T. Yoshitomi and M. Tsuji, unpublished results). From these results, S-013420 shows promise as the first-choice drug for treatment of community-acquired RTIs.

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