Extracellular Staphylococcus aureus USA 300 Persister Cell Awakening Factor
Description
This is the raw data for experiments that identified and partially characterized an extracellular factor that awakens Staphylococcus aureus USA300 persister cells called staphylococcal awakening factor (SAF).
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Preparation of Staphylococcal Awakening Factor (SAF) An O/N was diluted 1:1000 in 500 mL TSB in a 2 L Erlenmeyer flask and incubated at 37 °C with shaking (150 rpm; Environmental Incubator Shaker, New Brunswick Scientific, Edison, NJ, USA) for 16–18 h. The culture was transferred into two centrifuge bottles and centrifuged at 10,000 rpm for 10 min at 4 °C using a fixed-angle rotor (Sorvall X Pro Series; Thermo Scientific, Waltham, MA, USA). The supernatant was decanted, filter sterilized (0.2-μm filter) and placed on ice. The supernatant was tranferred into 3 kDa molecular weight cutoff (MWCO) filtration devices (Pierce 3k MWCO; Thermo Fisher, Rockford, IL, USA) and centrifuged in a swinging-bucket rotor at 4,000 x g and 4 °C until the retentate was reduced to one-tenth original volume. Filtrate samples were pooled and stored at –70 °C. The retentate was washed three times with ice-cold TSB-5 mM arginine. The clarified retentate was suspended in TSB-5 mM arginine at one-tenth the original volume, aliquoted, and stored at –70 °C. A tenfold-concentrated TSB (10x TSB; 300 mg/mL) was used as a negative control and was filter sterilization (0.2-μm filter), aliquoted, and stored at –70 °C. SAF Activity Assay SAF activity assay was developed. Fifty microliters of O/N S. aureus culture were inoculated into 50 mL TSB in a sterile 250 mL Erlenmeyer flask and incubated at 37 °C with shaking (150 rpm) until an OD600 of 0.5 (±0.05). Next, 9.5 mL was transferred into each of two 15 mL conical tubes. From each tube, 100 μL was removed, serially 10-fold diluted, and plated in triplicate on TSA plates to determine the initial CFU. The remaining cultures were treated with 125 μg/mL vancomycin and incubated at 37 °C with rotation (40 rpm) for 24 h. After antibiotic treatment, 100 μL was removed, serially diluted, and plated in triplicate on TSA plates to determine the CFU of persister cells. Retentate, filtrate, or 10x TSB were thawed, sterilized (0.2-μm syringe filter), and added to the culture tubes (0.5 mL per tube) followed by incubation at 37 °C with rotation (40 rpm) for 24 h. After 48 h of antibiotic treatment and 24 h of 10x TSB, filtrate or retentate exposure, 1.0 mL from each sample was transferred into 1.5 mL Eppendorf tubes. Antibiotic was removed by centrifugation at 14,500 rpm for 60 s, the supernatant was decanted, and the bacterial pellet was suspended in 1.0 mL TSB. Finally, 100 μL of each sample was serially diluted, plated in triplicate on TSA plates, and incubated as above. Percent survival was calculated: Percent Survival = (mean CFU/mL at 48 h / mean CFU/mL at 24 h) x 100.
Institutions
- A.T. Still UniversityMissouri, Kirksville