Moxifloxacin

The MICs of standard antibiotics were determined using a resazurin assay as described previously (48 (link)). Antibiotics used were amoxicillin (Sigma-Aldrich; beta-lactam antibiotic), cefuroxime (Sigma-Aldrich; beta-lactam antibiotic), moxifloxacin (Sigma-Aldrich; fluoroquinolone), and vancomycin (Sigma-Aldrich; glycopeptide), four clinically relevant antibiotics that are either commonly used to treat S. pneumoniae infections (amoxicillin, cefuroxime, and moxifloxacin) or used as a last resort, mostly in a hospital environment (vancomycin) (82 – (link)84 (link)). Briefly, a 1:2 serial dilution of the antibiotic was made in triplicates in MHL in a 96-well plate with a final volume of 100 μL. Then, 100 μL of a bacterial suspension was added to each well, except negative-control wells, to a final concentration of 5 × 10⁵ CFU/mL in 200 μL. Positive-control wells contained 200 μL of bacterial suspension (5 × 10⁵ CFU/mL) without antibiotics, and negative-control wells contained 200 μL of MHL without antibiotics or bacteria. Plates were incubated at 37°C and 5% CO₂ for 20 h before 20 μL of a 0.005% resazurin solution was added. Plates were further incubated for 90 min and fluorescence was measured (λ_em = 590 nm; λ_ex = 520 nm) using a spectrophotometer (Promega; Discover).

The fluoroquinolones evaluated in this study were enoxacin (Alfa Aesar, Tewksbury, MA, USA), ciprofloxacin (Sigma-Aldrich, St. Louis, MO, USA), levofloxacin (Sigma-Aldrich, St. Louis, MO, USA), and moxifloxacin (Sigma-Aldrich, St. Louis, MO, USA) (Figure S1). The drugs were prepared as previously described [12 (link)]. Briefly, the ciprofloxacin, levofloxacin, and moxifloxacin were solubilized in millipure water to 1.5 mM. enoxacin was solubilized in ultrapure water to 1.5 mM with lactic acid (3 mM; Sigma-Aldrich, St. Louis, MO, USA) to improve the solubility. Following solubilization, all drugs were filter-sterilized with a 0.2 μm filter and diluted to 600 μM in the cell specific media before further 2-fold serial dilution in the cell specific media.

Ciprofloxacin, moxifloxacin, tobramycin and gentamycin were purchased from Merck. The PcrV antibody Mab 166 was purchased from Creative Biolabs (New York, U.S.A.). The pOPINF E. coli expression vector was purchased from Addgene. ExoU with a C-terminal 6xHistidine tag was cloned into pUCPT20 and transformed into PA103 where indicated. LIVE/DEAD assay reagents were purchased from Invitrogen. LDH assay reagents were purchased from Thermofisher.