TNF-α and IL-1β ELISA kits were purchased from Biolegend (CA, USA). Tissue protein extract and BCA Protein Assay Kit were bought from Thermo (Thermo, MA, USA). Trypticase Soy Broth (TSB) and bacillus medium were purchased from Qingdao Haibo Biotechnology Co., Ltd (Qingdao, China). Crystal Violet Stain solution, 1% was acquired from Solarbio (Solarbio, Beijing, China). All the monoclonal antibodies, including β-actin, p65, IκB, the phosphorylation of p65, phosphorylation IκB, ZO-1, Occludin, and Claudin-3 were recruited from Cell Signaling Technology (Beverly, MA, USA). MPO kit was available from Nanjing Jiancheng Co., Ltd (Nanjing, China).
A total of 60 Balb/c mice (40 females and 20 males) aged 6-8 weeks were purchased from the Liaoning Changsheng Biotechnology Co. Ltd. The animal experiments were subject to approval by the Animal Ethics Committee of Jilin University (KT202103058). Females and males are mixed in miniature isolation cages in about a ratio of 2 to 1 after adapting to the environment with free food and water. The mastitis model was established complying with the experimental animal manual. This study is built on the Handbook on the Care and use of Experimental Animals published by the National Institutes of Health.
Bacteria and culture conditions
Bacillus were isolated from healthy and mastitis milk samples from dairy cows in Baicheng, Jilin Province), ChiFeng, Inner Mongolia, and Weifang, Shandong Province, China. A total of 145 strains of Bacillus bacteria were separated and purified by the specific culture of bacillus medium plate. S. aureus (ATCC 35556) was acquired from American Type Culture Collection. In the present study, S. aureus was inoculated for about 6 hours at the condition of 37 ℃ and 120 r/min, and the OD 600 was about 0.5 (concentration was approximately 108 CFU/mL). Meanwhile, B. subtilis bacteria were inoculated into the TSB broth medium by the same inoculation method, and then the OD 600 reached about 0.6 (concentration was approximately 108CFU/mL) after 4 hours of growth.
Preparation of cell‑free supernatant (CFS) from Bacillus culture and treatments
To prepare Bacillus CFS, Bacillus strains were cultured at 37 ℃ under shaking at 200 rpm overnight until the cultures reached an OD 600 of 0.6 ± 0.05. The CFS of bacterial culture was collected by centrifugation at 6000 g for 10 min, and then filtered through a 0.22 µm sterilizing-grade filter (Millipore, SLGV033RB, USA) to remove bacteria. To evaluate the effect of Bacillus CFS on S. aureus genes expression, overnight culture of S. aureus strains was collected by a centrifuge, washed with PBS, re-suspended at 108 CFU/mL in TSB/PBS (1:1 v/v, control) or TSB/B. subtilis CFS (1:1 v/v) and incubated in 6-well-plate at 37 ℃ for 3 h. Finally, bacteria were collected for RNA extraction and analysis of genes expression.
To determine the antibacterial effect of B. subtilis CFS on S. aureus, the Bacillus supernatant was obtained using the method described previously (18). Briefly, the supernatant of Bacillus bacteria was added into 96-well plates. 10 µL of S. aureus suspension (5 × 108 CFU/mL) from a fresh overnight culture was inoculated into 200 µL TBS (control), Bacillus CFS, TSB/ Bacillus CFS (1:1 v/v) or TSB/ Bacillus CFS (0.5:1.5 v/v) and incubated at 37 ℃ or 24 h. The growth of S. aureus was determined by monitoring OD 600 of the cell culture.
Biofilm formation and viability assay
To evaluate the effect of Bacillus CFS on S. aureus biofilm formation, 10 µL of S. aureus (5 × 108 CFU/mL) was added to 200 µL of TSB (control) or TSB/ Bacillus CFS (1:1 v/v) in each well on a 96-well plate and incubated at 37 ℃ for indicated time points without shaking. Next, the wells were washed three times with sterile PBS after the medium was removed. Finally, the plates were air-dried for 45 min and the adherent cells and matrix were stained with 1% crystal violet solution. To quantify the biofilm production, crystal violet was extracted by incubation in a solution (95% ethanol) at room temperature for 15 min, and absorbance was measured at 570 nm in a microplate reader.
RNA extraction and Quantitative real‑time PCR (QRT‑PCR)
Total RNA of S. aureus was extracted with a Bacterial RNA Extraction Kit (B518655-0050, Sangon Biotech, Shanghai, China) following the manufacturer’s instructions. RNA purity was verified using a NanoDrop spectrophotometer (ND-1000, Nanodrop, USA). RNA was reversely transcribed using the 5× Prime Script RT Master Mix (RR036A, Takara, Shiga, Japan) according to the manufacturer’s instructions. QRT-PCR was carried out using TB Green Premix Ex Taq II (RR820A, Takara, Shiga, Japan). Fold changes in level of chosing genes expression were determined using the 2−ΔΔCt method.
Animal treatment and mastitis model
Totally forty female Balb/c mice 5-7 days after delivery were randomly divided into four groups: control group, S. aureus group (1 × 108 CFU per 100 µL PBS), B. subtilis H28 group (1 × 108 CFU per 100 µL PBS), B. subtilis H28 (1 × 108 CFU per 100 µL PBS) + S. aureus (1 × 108 CFU per 100 µL PBS) group. For induction of mastitis, S. aureus (1 × 108 CFU per 50 µL PBS) was an injected into each mammary gland of the mice using 100 µL syringes with a 30 gauge blunt needle. In the B. subtilis H28 group, the mammary gland of mice received an injection dose of intramammary with B. subtilis H28 (1 × 108 CFU per 100 µL PBS), later 2 hours S. aureus was an injected into each mammary gland. The control group mice received an injection dose of intramammary with an equal volume of sterile PBS. Twenty-four hours later, the mice were sacrificed and the mammary gland tissues were harvested and stored at −80°C for subsequent detection.
24 hours after S. aureus infection, the mammary gland tissues of each group were collected and fixed in 4% paraformaldehyde for 48 h. The sample was inserted into paraffin and cut into 4 µm sections. After deparaffinization, the sections were stained with hematoxylin and eosin (H&E), and histological analysis was conducted under an optical microscope. The main histopathological indicators are through hyperemia (grade 0-3, ranging from normal to severe, including normal, mild, moderate, and severe) and neutrophil infiltration (grade 0-5, grade 0, from nothing to transmural) to evaluate.
MPO activity assay
MPO activity is a functional and activation marker of neutrophils. The mammary gland tissue was harvested and homogenized on ice with reaction buffer (weight/volume ratio 1:19). The detection method of MPO activity was carried out according to the manufacturer's instructions (Nanjing Jiancheng Institute of Bioengineering, Nanjing, China).
The inflammatory cytokine was established using an ELISA kit, according to the manufacturer's instructions. 10% tissue homogenate was prepared and centrifuged at 4°C, 12000 r/min, for 10 min. The lipid layer was removed, and the middle supernatant was collected for detection. Use a microplate reader to test the read absorbance of the sample at 450 nm and 570 nm.
Mammary S. aureus load assay
To assess the S. aureus burden in the mammary gland, mammary tissues were aseptically obtained in mice and homogenized in 1 mL of PBS. A 10-fold dilution of the tissue homogenate was plated in mannitol high salt agar plates. Bacterial colonies were counted and calculated following plate incubation at 37 ℃ for 18 h. Results of bacterial burden were expressed on a log10 scale.
Western Blotting Analysis
The BCA protein detection kit was utilized to detect the protein concentration in mammary gland tissues. The protein samples were separated by 10% SDS-PAGE, then protein samples were transferred to the PVDF membrane and blocked in 5% skimmed milk at room temperature for 2 h. Use primary antibodies β-actin (1:1000), phospho-IκB (1:1000), IκB (1:1000), NF-κB p65 (1:1000) ZO-1 (1:500) Occludin(1:1000) and Claudin-3 (1:1000)and phospho-NF-κB p65 (1:1000) in Incubate overnight at 4°C. The membrane was washed three times with TBST for 10 minutes each time and incubated with goat anti-rabbit secondary antibody for 2 h at room temperature. Finally, use of the ECL Plus Western blotting detection system to detect the cell membrane.
The paraffin-embedded glass slides were dewaxed in xylene and different concentrations of alcohol. The antigen is restored with 0.01 M citrate buffer. Incubate in 3% H2O2 and then in diluted goat serum. The sections were incubated with primary antibodies: Claudin 3 (1:500) at 4°C. After washing 3 times, the sections were incubated with HRP-labeled goats with anti-rabbit secondary antibody (1:500, ZS-Bio, Beijing, China) at 37°C for 15 min. This section was stained with DAB and observed under a microscope.
GraphPad Prism 5 (Manufacturer, La Jolla, CA, USA) was performed for statistical analysis. The data are presented as mean ± SEM. p < 0.05 indicated statistical significance by one-way ANOVA and Tukey's multiple comparisons test.