Lysostaphin

For 28 antibiotic-resistant S. aureus strains, each was cultured overnight on a Mueller-Hinton agar plate at 37°C. Then, a single bacterial colony was suspended in 1 ml sterile LB medium, and subsequently incubated at 37°C with vigorous shaking for 6 hours, followed by centrifugation at 13000 × g for 2 minutes. Next, the culture media was discarded, and the pellet was mixed with 100 μl of Tris-HCl (200 mM, pH 8.0) and 2μl of lysostaphin (1 mg/100 μl, Sangon, Shanghai, China). Finally, each sample was heated at 37°C for 1 hour, followed by 95°C for 15 minutes, and centrifuged at 13000 × g for 2 minutes. Supernatant containing genomic DNA was collected and used as template for the PCR assays.

S. aureus isolates with FA resistance were cultured on blood agar overnight at 35 °C. Then, three to four bacterial colonies were suspended in 150 μl sterile distilled water with lysostaphin (1 mg/mL) (Sangon, Shanghai, China) and incubated at 37 °C for an hour. Finally, DNA was extracted following the instructions of the Genomic DNA Extraction kit (Sangon, Shanghai, China), stored at −20 °C and prepared for PCR assays.

An assay of bacterial invasion was performed as previously described (Liang et al., 2006 (link); Sharma et al., 2013 (link); Mechler et al., 2015 (link)). A549 human lung epithelial cells were cultured in Dulbecco’s modified Eagle’s medium/F-12 medium supplemented with 10% fetal calf serum, streptomycin (100 μg/ml), and penicillin (100 μg/ml) in a 5% CO₂ incubator at 37°C. The cells were passaged and expanded every 2 days. At 1 day prior to infection, 2 × 10⁵ cells were seeded in 24-well plates with an antibiotic-free cell culture medium and incubated at 37°C in a 5% CO₂ incubator. S. aureus strains were grown for 4 and 12 h in TSB. One milliliter of the bacterial culture was washed with ice-cold saline and resuspended in 1 ml of cell culture medium. Approximately 2 × 10⁶ CFU/ml of S. aureus strains were seeded in a 24-well plate. The plates were centrifuged at 1,000 rpm for 5 min to synchronize infection and then incubated for 1 h at 37°C in a 5% CO₂ incubator. The culture medium was removed. Extracellular bacteria were treated with 100 μg/ml gentamicin and 20 μg/ml lysostaphin (Sangon Biotech) for 30 min. The monolayer cells were washed three times with PBS (pH 7.4) and incubated for an additional 1 h (invasion capacity) or 24 h (intracellular survival). Intracellular bacteria were counted for the CFU by lysis of the host cells with 0.01% Triton X-100.

Bacterial culture samples were centrifuged at 13,000 × g and 4°C for 10 min; resuspended in TE buffer (10 mM of Tris HCl and 1 mM of EDTA, pH 8.0) with lysostaphin (1 mg/ml, Sangon Biotech, Shanghai, China) and proteinase K (20 mg/ml, TaKaRa, Dalian, China); and incubated at 56°C for 1 h for cell wall lysis. Total RNA was extracted using the MiniBEST Universal RNA Extraction Kit (TaKaRa, Dalian, China) according to the manufacturer’s instructions.