Bacterial strains and growth conditions
S. aureus was isolated from a clinic patient in Yunnan first people's hospital, China, it was used as host bacteria for isolate phages. The host strain and phage host range determination strains were grown aerobically on BHI plates or in BHI broth (Difco, Detroit, MI, USA) and incubated in 37 ℃. Soft top agar containing of 0.5% (w/w) agar in BHI broth for phage plaque confirmation and BHI agar plates containing of 1.8% (w/w) agar. All S. aureus strains were stored in − 80 ℃ in BHI broth (Difco, Detroit, MI, USA) containing of 20% (v/v) glycerol.
Phage isolation and purification
S. aureus targeting phages were isolated from pig slaughter house. The phages isolate method was modified as follows . Briefly, 10 g of pig farm trash can was mixed with 20 mL of sterile normal saline (0.9% NaCl) buffered in 50 mL sterile centrifuge tube and then shocked incubator of 200 rpm for 2 h in room temperature. Then, samples were centrifuged at 5000 × g for 15 min and filtered with 0.22 µm filter membrane. 10 mL of each filtering medium was added to 30 mL of BHI broth that containing of 1% (v/v) of overnight culture of the host strain and then incubated for 48 h. After that, Cultures were centrifuged for 8 000 × g, 15 min and the supernatant was filtered with 0.22 µm filter membrane. The filtrate was diluted 10 times in series and mixing with 5 mL of molten BHI soft agar that containing of S. aureus (2 × 108 cfu/mL), and immediately added to BHI plate. Overnight culture and plaque formation was observed. Single phage plaque was selected for phage purification and repeated for three times.
The pH, thermotolerance, MOI, growth curve and TEM of isolated phage
Optimum pH，MOI and thermotolerance
The phage WX stock was diluted to 1 × 108 pfu/mL with BHI broth. Take of 0.99 mL liquid buffer for pH of 3, 4, 5, 6, 7, 8, 9, 10 and 11 (50 mmol/L Citrate buffer, pH 3, pH 4, pH 5; 50 mmol/L phosphate buffer, pH 6, pH 7, pH 8; 50 mmol/L Tris-HCl buffer, pH 9; 50 mmol/L Sodium carbonate buffer, pH 10, pH 11) in 1.5 mL sterile centrifuge tube, added 0.01 mL of diluted phage WX for titer of 1 × 106 pfu/mL to each tube. Place at room temperature for 1 h then detection the titer of phage WX in different pH buffer. The experiments were repeated for three times. Thermotolerance detection were placed of 1 mL diluted phage WX in temperature controller of 4 ℃, 25 ℃, 37 ℃, 42 ℃, 50 ℃, 60 ℃ and 90 ℃ for 1 h, respectively. Multiplicity of infection (MOI) was the ratio of phages to host bacteria of initial infection. According to the MOI of 0.0001, 0.001, 0.01, 0.1 1, 10 and 100 added phage WX stocks and S. aureus culture, cultured in 37 ℃ for 8 h. The culture were centrifuged at 10 000 × g for 15 min in 4 ℃, then supernatant was filtered with 0.22 µm filter and the titer of phage increment solution were determined through double plate method, the experiment was repeated for three times. For growth curve measure, 1 × 108 pfu/mL of phage WX were added to BHI culture containing of 1/250 S. aureus seed culture according the optimum MOI and shacked culture in 37 ℃, intermittent sampling was used to determine the titer of phage.
Transmission electron microscopy
The morphology of the phage particles was observed by transmission electron microscopy (TEM). Briefly, each phage stock dilution (approximately 2 × 108 to 2 × 109 pfu/mL) was deposited on copper grids with carbon-coated Formvar films, stained with 2% uranyl-acetate (pH 4.0). Phage samples were imaged using a Philips EM 300 electron microscope, operated at 80 kV in Jiangnan university (Wuxi, China). Phage was classified and identified referring to the International Committee on Taxonomy of Viruses (Rodhain et al., 1995).
Phage genome DNA extraction, sequencing and bioinformatics analysis
Firstly, phage was purified from concentrated to a high titer stock with 10 kDa filter (about 109 to 1010). Purified phage was treated with RNase and DNase in 37 ℃ for 1 h. Then, Takara minibest viral RNA/DNA Extraction kit (Cat#9766) was carried out to obtain purified phage genome DNA. Restriction endonuclease EcorI, Hind III, Not I and Xhol I were used for phage genome digestion, respectively. Extracted phage genomic DNA was sequenced using a Illumina Hiseq (Sangon Biotech, China). The original sequencing data were evaluated by FastQC and assembled with SPAdes assembler software. The NCBI Blast compare with multiple databases of CDD, KOG, COG, NR, NT, PFAM, Swissprot and TrEMBL were used for function annotation information of gene protein sequence.
Phage lytic spectrum and antimicrobial susceptibility of S. aureus
The host range of the phage WX was determined by the spot test method . The reference strains ( All strains were isolated from clinical patients) were tested for susceptibility of phage WX. Generally, each of 200 uL reference strains (109 cfu/mL) was added to 5 mL of liquefied BHI soft agar (BHI broth with 0.5% (w/w) agar), and poured over to BHI 1.8% (w/w) agar plate. Three minutes later, single drops of phage suspension were added and incubated in 37 ℃ for 24 h. Antibiotic sensitivity of S. aureus strains were tested against seventeen antibiotics by minimal inhibitory concentration (MIC) method. The antimicrobials tested were Penicillin, Streptomycin, Kanamycin sulfate, Gentamicin, Ciprofloxacin, Levofloxacin, Rifampicin, Vancomycin, Erythromycin, Teicoplanin and Tetracycline.
The different effects of phage and antibiotics on biofilms
Make first-phase preparations, A 48 - well cell slide was placed into a 24 - well plate. Seed solution was inoculated into 100 mL BHI culture solution at the rate of 4‰. Inoculate 1 mL of bacterial solution into 24-well plate. One group, added phage WX, streptomycin, and mixtures of streptomycin and phage WX, respectively, nothing added as control (The addition amount of phage was MOI = 1, The final concentration of streptomycin was 10 µg/ml), Incubation (37 °C, 24 h). The other group, firstly, host culture for 12 hours, after that, added phage, streptomycin, and mixtures of streptomycin and phage, respectively, nothing added as control (The addition amount of phage was MOI = 1, the final concentration of streptomycin was 10 µg/ml), Incubation (37 °C, 12 h). The cfu of each sample was measured through plate counting method. Following, the recovered culture washed twice with PBS buffer; and fixed with 2.5% pre-cooling glutaraldehyde at room temperature for 3 h in dark place. Wash twice with PBS buffer, then dehydrated in an increasing ethyl alcohol gradient (15%, 30%, 50%, 70%, 100% v/v) for 10 min for each step. Afterward, dry overnight and gilt, the results obtained through scanning electron microscope with an accelerating voltage of 20 kV. S. aureus seed solution was inoculated in BHI for the proportion of 4‰ of overnight culture. Then 200 times dilution with BHI and added to 96-well plate (200 uL/hole), each sample has three multiple holes. One group, added phage, streptomycin, and mixtures of streptomycin and phage, respectively, nothing added as control (The addition amount of phage was MOI = 0.1, the final concentration of streptomycin was 10 µg/ml), incubation (37 °C, 24 h). The other group, firstly, host culture for 12 hours, after that, added phage, streptomycin, and mixtures of streptomycin and phage, respectively, nothing added as control (The addition amount of phage was MOI = 0.1, the final concentration of streptomycin was 10 µg/ml), incubation (37 °C, 12 h). The bacterial population density (OD600 nm) was measured using a ELIASA (Thermo Scientific, EUA) and discarded bacteria solution. The wells washed twice with PBS to remove unattached bacteria, repeated three times, added of 99% methanol and fix for 15 min, then discard methanol and dry at room temperature, following added 2% crystal violet and stain for 8 min. Rinse the culture plate with running water until the water is colorless. After drying, measured the absorption light at 570 nm wavelength with a microplate reader. The experiment was repeated for three times.