1.2 MRSA cultivation

The standard strain of Staphylococcus aureus ATCC25923 used by the Institute was purchased from the bacterial library of the Chinese Academy of Sciences.50 MRSA and 50 KO sdrc (purchased from Beijing Abace Biotechnology Co. LTD ) stored at -80 ℃ each μ Add 15ml of soybean broth culture medium, incubate overnight at 37 ℃ at 200r/min, and detect the knockout efficiency of sdrc using RT-PCR. Take 5 out of MRSA and KO sdrc recovered overnight μ Add 5ml of soybean broth medium and culture on a constant temperature shaker. Measure the absorbance value of OD600 at 1h, 2h, 3h, 4h, 5h, 7h, 9h, 12h, 18h, and 24h according to: <i mtid=’44’>hello</i> 07 Calculate and plot bacterial growth curves.

1.3 ELISA experiment

Collect samples and perform enzyme-linked immunosorbent assay according to the manufacturer’s protocol, including α- HL, PSM, CRP, TNF- α、 IL-6.

1.4 Crystal violet staining

Remove the bacteria cultured overnight from the culture medium and add 500-1000 to it μ Place 4% paraformaldehyde at room temperature for 30-40 minutes, remove excess liquid, tilt the plate, dry at 37 ℃ for 1 hour, add crystal violet that can cover the bottom of the hole, stain at room temperature for 20 minutes, add PBS to wash 2-3 times, and absorb the remaining liquid to observe the ability of biofilm formation.

1.5 Observation of MRSA biofilm formation using scanning electron microscopy

MRSA and KO sdrc were cultured overnight at 37 ℃ in a 24 well plate and placed under a scanning electron microscope to observe bacterial morphology.

1.6 RT-PCR experiment

Extract total RNA from each group separately, then perform reverse transcription reaction, and then use 2 x Universal Blue SYBR Green qPCR Master Mix reagent kit for qPCR reaction. The reaction procedure is: pre denaturation at 95 ℃ for 1 minute; 95 ℃ denaturation for 20 seconds, 55 ℃ annealing for 20 seconds, 72 ℃ extension for 30 seconds; 40 cycles. The primer sequence is shown in Table 1:

Table 1 The sequences of the Primers