Bacterial strains and growth conditions
Coli was isolated from the patient in Yunnan first people's hospital in China, was used as host for isolation of phages. The host strain and phage host range determination strains were grown aerobically on LB plates or in LB broth (Difco, Detroit, MI, USA) and stirring incubated at 37 ℃ for 18 h. Soft top agar containing LB broth was prepared with 0.5% agar for phage plaque confirmation and LB agar plates were prepared with broth supplemented with 1.8% agar. All strains of E. coli stock cultures were stored at −80 ℃ in the LB broth (Difco, Detroit, MI, USA) containing 20% (v/v) glycerol.
Bacteriophage isolation and purification
coli targeting phages were isolated from laboratory and drug. The phages isolate method was modified as follows[33]. Briefly, 10 g of each sample was mixed with 20 mL sterile normal saline (0.9% NaCl) buffered in sterile 50 mL centrifuge tube and then shock for 2 h using incubator with 200 rpm at room temperature. Then, samples were centrifuged at 5000×g for 15 min and filtered using 0.22 um filter membrane. 10 mL of each filtering medium was added to 30 mL of LB broth containing the 1% of overnight culture of the host strain and then incubated for 48 h. After that, Cultures were centrifuged at 8 000×g for 15 min and the supernatant was filtered using 0.22 um filter membrane. The filtrate was diluted 10 times in series and mixing in 5 mL of molten 0.5% LB soft agar containing E. coli (2×108 cfu/mL), and immediately add to LB plate that containing 1.8 agar. Overnight culture and plaque formation was observed. Single phage plaque was selected for phage purification and repeat three times.
The thermotolerance, optimum pH, optimum MOI, growth curve and transmission electron microscopy (TEM) of isolated phages
Thermotolerance
Take 1 mL phage pure cultures of titer for 1 × 107 pfu/mL, respectively set in 4 ℃, 25 ℃, 37 ℃, 42 ℃, 50 ℃, 60 ℃ and 90 ℃ for 1 h. Following determination of phage titer at different temperature. The experiment was repeated three times.
Optimum pH
Take 0.99 mL buffer liquid with pH of 3, 4, 5, 6, 7, 8, 9, 10 and 11 (Citrate buffer 50 mmol/L, pH 3, pH 4, pH 5; phosphate buffer 50 mmol/L, pH 6, pH 7, pH 8; Tris-HCl buffer 50 mmol/L, pH 9; Sodium carbonate buffer 50 mmol/L, pH 10, pH 11) in 1.5 mL sterile centrifuge tube, add phage pure cultures 0.01 mL of titer for 1 × 108 pfu/mL to each tube. Place at room temperature for 1 hour then determination of phage titer at different pH. The experiment was repeated three times.
Optimum MOI
Multiplicity of infection (MOI) was the ratio of the number of phages added to the number of host bacteria at the time of initial infection. According to the MOI of 0.0001, 0.001, 0.01, 0.1 1, 10 and 100 add phage pure culture solution and host bacteria suspension, then transfer to LB medium and shock culture at 37 ℃ for 8 hours. The cultures were centrifuged at 10 000×g for 15min at 4 ℃, then the supernatant was filtered by 0.22 um filter to obtain the phage increment solution, finally the titer of phage increment solution was determined through double plate method. The experiment was repeated three times.
Growth curve
Added phage (1 × 107 pfu/mL) to LB culture That containing 1/250 seed fluid of host bacteria according the optimum MOI and strring culture at 37 ℃, intermittent sampling was used to determine the titer of the phage.
Transmission electron microscopy
The morphology of the phages 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 at the Jiangnan university (Wuxi, China). Phages were classified and identified refering to the International Committee on Taxonomy of Viruses[34].
Bacteriophage genomic DNA extraction, sequencing and bioinformatics analysis
Bacteriophage genomic extraction and restriction enzyme digestion
Firstly, phage was purified from concentrated to a high titer stock using 10 kd filter (about 109 to 1010). Purified phages were treated with RNase (1U) and DNase (1U) at 37 ℃ for 1 h. Then, Takara minibest viral RNA/DNA Extraction kit (Cat#9766) was
carried out to obtain purified phage genomic DNA. Restriction endonuclease EcorI, Hind III, Not I and Xhol I were used for phage genome digestion, respectively.
Sequencing and bioinformatics analysis
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.
Host-range determination and characteristic of host
Host-range determination
The host range of the phages Flora, T4 and WJ were determined by the spot test method[35]. The reference strains ( ten strains of E. coli from clinical patients, E. coli DH5α and E. coli BL21) were tested for susceptibility of phage Flora, T4 and WJ. Generally, each of 200 uL reference strains (109 cfu/mL) was added to 5 mL of liquefied LB soft agar (LB broth with 0.5% agar), and poured over the LB 1.8% agar plate. Three minutes later, single drops of phage suspension were added and incubated at 37 ℃ for 24 h.
Antimicrobial susceptibility testing
Antibiotic sensitivity testing
Antibiotic sensitivity of the Ten strains of E. coli from clinical patients, E. coli DH5α and E. coli BL21 were tested against seventeen antibiotics by minimal inhibitory concentration (MIC) method. The antimicrobials tested were Penicillin, Streptomycin, kanamycin sulfate, cefoxitin, ampicillin, ceftriaxone, gentamicin, ertapenem, aztreonam, amoxicillin, Ciprofloxacin, imipenem, levofloxacin, cefepime, macrodantin, amikacin.
The different effects of phages and antibiotics on biofilms
Scanning electron microscopy
Make first-phase preparations, A 48 - well cell slide was placed into a 24 - well plate. Seed solution was inoculated into 100 mL LB culture solution at the rate of 4‰. Inoculate 1 mL of bacterial solution into 24-well plate. One group, added phages, antibiotics, and mixtures of antibiotics and phages, respectively, nothing added as a control (the addition amount of phage was MOI=1, The final concentration of kanamycin sulfate was 10 ug/ml), Incubation (37 °C, 24 h). The other group, firstly culture for 12 hours, after that, added phages, antibiotics, and mixtures of antibiotics and phages, respectively, nothing added as a control (The addition amount of phage was MOI=1, the final concentration of kanamycin sulfate was 10 ug/ml), Incubation (37 °C, 12 h). The cfu of each sample was measured by 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 ethanolic gradient (15%, 30%, 50%, 70%, 100% v/v), for 10 min at each step. Afterward, dry overnight and gilt, the results obtained through scanning electron microscope with an accelerating voltage 20 kV .
Microplate reader detected the ability of biofilm formation
coli seed solution was inoculated in LB for the proportion 4‰ and overnight culture. Then 200 times dilution with LB and added to 96-well plate (200 ul/hole), each sample has three multiple holes. One group, added phages, antibiotics (kanamycin sulfate (10 ug/mL) ), and mixtures of antibiotics and phages, respectively, nothing added as a control (The addition amount of phage was MOI= 0.1, the final concentration of kanamycin sulfate was 10 um/ml), incubation (37 °C, 24 h). The other group, firstly culture for 12 hours, after that, added phages, antibiotics, and mixtures of antibiotics and phages, respectively, nothing added as a control (The addition amount of phage was MOI= 0.1, the final concentration of kanamycin sulfate was 10 ug/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 cells, repeated three times, added 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 three times.