Moxifloxacin

Antibiotic powders of daptomycin (Merck & Co. Inc., Kenilworth, NJ) and quinupristin/dalfopristin (AG Scientific, San Diego, CA) were dissolved in sterile water for injection and stored as stocks at −20 °C. Sterile liquid formulations of clindamycin (Pfizer, New York, NY), moxifloxacin (Bayer AG, Leverkusen, Germany), gentamicin (GPO, Bangkok, Thailand), and amikacin (Siam Bheasach, Bangkok, Thailand) were stored at 4 °C. Final antibiotic solutions were freshly prepared before use by diluting stock solutions with cell culture medium.

1599 and 1810 were synthesized as described (Figure S1).³ (link) Isoniazid, rifampicin, pyrazinamide, ethambutol, levofloxacin, kanamycin, amikacin, ethionamide, ofloxacin, cycloserine, rifapentine and clofazimine were purchased from Sigma-Aldrich. Linezolid was purchased from 21CEC. Moxifloxacin was obtained from Bayer. PA-824 (Pretomanid) was obtained from the Global Alliance for TB Drug Development. Bedaquiline (Sirturo) was provided by Johnson and Johnson. SQ109 was synthesized as described.⁹ (link)

The susceptibilities of the 284 toxigenic C. difficile clinical isolates to 15 antimicrobial agents were determined by the agar dilution method described by the Clinical and Laboratory Standards Institute (CLSI)²⁴ . The antimicrobial agents tested include cefotaxime, cefoperazone, ceftazidime (GlaxoSmithKline), ciprofloxacin, clindamycin, erythromycin, fusidic acid, levofloxacin, metronidazole (B. Braun Medical Industries), meropenem (Astra Zeneca), moxifloxacin (Bayer), piperacillin-tazobactam, rifampicin, tetracycline and vancomycin, all reagents were purchased from Sigma unless otherwise stated. C. difficile ATCC 700057 and Bacteroides fragilis ATCC 25285 were used as control strains for each run of agar dilution testing. The minimal inhibitory concentration (MIC) was defined as the lowest concentration of the drug that inhibits bacterial growth. The breakpoints for metronidazole, clindamycin, tetracycline, moxifloxacin, meropenem, piperacillin-tazobactam, cefotaxime and cefoperazone were determined with MIC criteria described by CLSI guidelines²⁴ . For vancomycin and fusidic acid, breakpoints recommended by the European Committee on Antimicrobial Susceptibility Testing (EUCAST) were used²⁵ . For erythromycin, ciprofloxacin, levofloxacin, and rifampicin, we adapted the breakpoints from Huang et al.^{26 (link)}. No breakpoint for ceftazidime was available at the time of this study.

The antimicrobials for susceptibility testing were azithromycin (Novatec, Cuba), erythromycin (Sigma, USA), ciprofloxacin (Sigma, USA), ofloxacin (Sigma, USA), levofloxacin (Novatec, Cuba), moxifloxacin (Bayer, Italy), tetracycline (Sigma, USA) and doxycycline (Bayer, Italy). All compounds were diluted and conserved according to the specifications in the CLSI-Guideline M43-A: “Methods for Antimicrobial Susceptibility Testing for Human Mycoplasmas” [15 ].

Antibiotics were supplied by their respective manufacturers as pure titrated powders (ciprofloxacin and moxifloxacin by Bayer Healthcare SAS, levofloxacin by Sanofi-Aventis, France). Five strains of each species were selected for time-kill studies, based on their different susceptibility patterns to the antibiotics (Table 2). For each strain, time kill studies were performed for the three molecules at concentrations equal to the theoretical plasma peak (ciprofloxacin = 4 μg/mL; levofloxacin = 10 μg/mL; moxifloxacin = 3 μg/mL), then at concentrations equal to one fold and two fold the MIC of the antibiotic used. Bacteria were initially cultured for 24 h at 37°C on Mueller-Hinton agar. These cultures were then considered as being on stationary growth-phase and used to prepare exponential growth phase at standard inoculum (10⁶ CFU/mL) in Mueller-Hinton broth (MHB, bioMérieux, France). The inoculum of 10⁶ CFU/ml was obtained by standardizing optical density at 550 nm to 0.125 followed by a 1:100 dilution. Final suspensions of bacteria were supplemented with ciprofloxacin, levofloxacin or moxifloxacin at different concentrations and cultured for 24 h at 37°C. Culture aliquots of 100 ml were removed at 2, 4, 6 and 24 h and plated on agar for colony counts.