Bead beater

Protein extraction from S. mutans cultures was carried out as previously described (22 (link)). Briefly, the culture was grown at 37°C to the late stationary phase (OD₆₀₀ ∼1.0 to 1.2). The culture was centrifuged at 8,000 rpm for 10 min at 4°C and resuspended in 700 µL of buffer A [25 mM HEPES-NaOH (pH 7.5), 300 mM NaCl, 5% glycerol). The resuspended cells were then lysed using a bead beater (MP Biomedicals) at 5 m/s for 30 s (five times). E. coli cultures were similarly grown, collected at their stationary phase, and lysed by sonication (intensity 5 for 2 min with 10 s on and 20 s off). The lysed cell culture was centrifugated, and the clarified supernatant was collected.

Intracellular iron levels were measured using the ferrozine-based colorimetric assay described previously (70 (link), 71 (link)). Briefly, exponentially grown cultures of Mtb strains (OD₆₀₀ ~ 0.8) were harvested by centrifugation and washed twice with ice-cold PBS. The cell pellets were resuspended in 1 ml of 50 mM NaOH and lysed using a bead beater (MP Biomedicals, Santa Ana, CA). HCl (10 mM, 300 μl) was added to the cell lysate samples (300 μl), followed by the addition of the Fe-detection reagent (6.5 mM ferrozine, 6.5 mM neocuproine, 1 M ascorbic acid, and 2.5 M ammonium acetate in water) (90 μl). This reaction mix was incubated for 30 min at 37°C, followed by reading the absorbance of the samples at 562 nm using a microplate reader (Versa-Max; Molecular Devices, San Jose, CA). The cellular free iron concentration was equated by plotting the absorbance values against a standard curve of FeCl₃ concentration gradient and readouts normalized to the protein content of the respective samples. Protein concentration was estimated using a Pierce bicinchoninic acid (BCA) protein assay kit (Thermo Fisher Scientific, Rockford, IL).

A total of 24 female C57BL/6J mice of 6 weeks were randomly assigned into four groups (n = 6 per group) in ventilated cages and had ad libitum access to feed and water. At the day of infection, the mice were anesthetized with 1.5% pentobarbital sodium before being infected with UEPC CFT073 or corresponding mutants as indicated (10⁸ CFU, 50 µL). The infected animals were divided into four groups and respectively received i) PBS (control, 100 µL), ii) AMK (7.5 mg kg⁻¹, 100 µL), iii) NIT (3.75 mg kg⁻¹, 100 µL), and iv) combination of AMK (3.75 mg kg⁻¹, 50 µL) with NIT (1.875 mg kg⁻¹, 50 µL) at 12 h post infection for once. At the 3 days post the infection, the mice were sacrificed after anesthesia and the bladders of tested animals were removed. The bladders were homogenized in cold PBS using a bead beater (MP Biomedicals, USA) and the homogenized mixtures were then subjected for bacteria enumeration by plating.