Sheep blood

AN11251 was provided by Calibr at Scripps Research (San Diego, CA, USA). All the other chemicals were purchased from Sigma (St Louis, MO, USA) without further purification. Rats were purchased from Charles River (Wilmington, NC, USA) and fasted overnight before dosing. Yeast Extract (OXOID, from Thermo Fisher (Waltham, MA, USA)), Sheep Blood (Yuanye Bio-Technology in Shanghai, China), Haemophilus Test Medium Base (HTM, HalingBio in Shanghai, China), Tryptic Soy Broth (TSA, BD (Franklin Lakes, NJ, USA)), Cation Adjusted Muller-Hinton broth (CAMHB, BD), 7H9 broth (BD), OADC (BD), glycerol (Sigma), Tyloxapol (Sigma), and Chocolate Agar (BD) were from commercial sources as indicated. Rifampicin (RIF, Sigma), Ciprofloxacin (CIP, MedChemExpress (South Brunswick, NJ, USA)), Alamar Blue^TM Cell Viability Reagent from Thermo Fisher, and other chemical reagents were purchased.

The hemolysis assay of thucin A1 was performed using a previously described method with some modifications [5 ]. The hemolytic activity of thucin A1 was tested by measuring the release of hemoglobin from a suspension of defibrillated sheep blood (Shanghai yuanye Biotechnology Co., Ltd., China) at an absorbance of 540 nm. Briefly, defibrillated sheep blood was centrifuged at 3000 rpm for 10 min and the sedimented cells were washed three times with PBS buffer (pH 7.0). Then, 30 μL of re-suspended red blood cells were added to a 96-well plate containing 70 μL of thucin A1 at different final concentrations (3.75, 7.5, 15, 30, 60 and 120 μM) and incubated for 1 h at 37 °C. PBS buffer and Triton X-100 (1%) were used as negative and positive controls, respectively. After incubation, the samples were centrifuged at 3,000 rpm for 5 min, and the release of hemoglobin from the supernatant was tested at a wavelength of 540 nm. Hemolysis activity was calculated using the formula: hemolysis (%) = [(Abs₅₄₀ of the bacteriocin treated sample)−(Abs₅₄₀ of buffer treated sample)]/[(Abs₅₄₀ of Triton X-100 treated sample)−(Abs₅₄₀ of buffer treated sample)] × 100. All experiments were performed in triplicate.

Clinical isolates including S. aureus, MRSA, Staphylococcus epidermidis, Enterococcus faecium, Enterococcus faecalis, Streptococcus pneumoniae and Streptococcus agalactiae were obtained from Zhongshan Hospital of Xiamen University and The Second Affiliated Hospital of Fujian Medical University. Each strain was grown from a single colony and cultured in Brain Heart Infusion (BHI) broth (Solarbio, Bejing, China) at 37°C with 220 rpm shaking overnight with the following exceptions: RN450 was cultured in Mueller-Hinton (MH) broth (OXOID, UK), and Muller- Hinton2 (MH2) broth (OXOID, UK) was used to culture RN450 persister cells. 1.5% agar (BD, USA) was added to broth media to obtain solid media. Sheep blood (10%) (Yuanye, Shanghai, China) was added to growth medium for S. pneumoniae and incubation was at 37°C in the presence 5% CO₂.