Bacterial strains and culture conditions
Burkholderia cenocepacia K56-2 was maintained in Luria-Bertani (LB) broth (1% tryptone, 0.5% yeast extract, and 1% NaCl) with shaking (120 rpm) at 37°C or on LB + 1.5% agar plates at 37°C unless otherwise noted. The Burkholderia cepacia complex species B. stabilis 14294, B. ambifaria AMMD, B. dolosa AU0645, B. cenocepacia J2315, B. anthina AU1293, B. seminalis AU0475, B. metallica AU0553, B. diffusa AU1075, B. pseudomultivorans AU3207, B. arboris ES263A, B. contaminans HI3429, B. lata HI4002, B. multivorans 17616, B. vietnamiensis PL259, B. cepacia 25416, B. pyrrocinia BC011 were grown in LB broth or LB agar at 37°C.
Isolation of bacteriophages from raw sewage
Raw sewage obtained from the Newark, Ohio Treatment Plant (40 ml) was combined with 5 ml of an overnight culture of B. cenocepacia K56-2 and 5 ml of 10X LB broth then incubated at 37°C with shaking. After 24 h, the culture (10 ml) was centrifuged at 14,000 x g for 3 min, the supernatant was filter sterilized (Fisherbrand 0.45 μm nylon filter), and stored at 4°C.
For the plaque assay, the filtered sewage sample was serially diluted in phage buffer (PB) (10 mM Tris-HCl, pH 8.0; 10 mM MgCl2). Each dilution (100 µl) was transferred to overnight K56-2 culture grown in ½Luria-Bertani (½LB) broth (500 µl), vortexed briefly, and incubated for 5 min at 37°C. The mixtures were transferred to 0.75% soft agar (5 ml) and poured onto ½LB agar for overnight incubation at 22°C (i.e., room temperature), as this temperature allowed plaques to be more visible. Phage buffer and K56-2 each were added to top agar and plated onto ½LB agar as controls. All plates were in duplicate.
Amplification of bacteriophage
For amplification in broth cultures, plaques were extracted using a sterile toothpick, placed in 0.2 ml PB, vortexed briefly, and transferred to 25 ml of an overnight culture of K56-2 in ½LB and incubated overnight at 37°C with shaking. The mixture was centrifuged at 5000 x g for 5 min at 4°C, filter sterilized, and stored at 4°C.
To determine the appropriate amount of phage needed to achieve a web pattern for amplification using plate, the phage stock was serially diluted in PB then used in a plaque assay as described previously. The appropriate dilution that achieved webbing was added to an overnight culture of K56-2 grown in ½LB. The mixture was incubated for 5 min at 37ºC, added to top agar (5 ml) then poured onto ½LB agar plates. Plates were incubated at 37°C for 24 h. Phage buffer (5 ml) was added to each plate and stored at room temperature overnight. The fluid was aspirated from the plate, centrifuged at 5000 x g for 10 min at 4°C, filter sterilized (0.45 μm), and stored at 4°C.
Determination of bacteriophage host range
The host range of the bacteriophage was determined by spotting 10 µl of phage (6 × 109 PFU/ml) onto ½LB plate swabbed with an overnight culture of a B. cepacia complex species, Pseudomonas aeruginosa, or Staphylococcus aureus. Lytic activity was measured after overnight incubation at 30 and 37°C. Species that exhibited clearing in the spot were further examined with a plaque assay to confirm lysis from the phage.
Visualization of bacteriophage
Virion morphology was visualized with electron microscopy at The Ohio State University Campus Microscopy & Imaging Facility. Stock phage (1 × 108 PFU/ml) was centrifuged at 100,000 x g for 1 h. Phage pellets were suspended in 1 M ammonium acetate, negatively stained with 2% (w/v) potassium phosphotungstate solution (pH 7) and visualized with a transmission electron microscope.
Isolation of bacteriophage DNA
Phage stock (1 ml) was transferred into four separate 15 ml conical tubes. In each tube, 20 units (10 µl for 2,000 units/ml stock solution) of RNase-free DNase 1 (New England Biolabs, Ipswich Massachusetts) was added and incubated at room temperature for 15 min, then a Norgen Phage DNA Isolation Kit (Thorold, ON, Canada) was used to isolate the DNA. The concentration of DNA was measured using the Synergy LX Multi-Mode Reader (BioTek Instruments, Inc., Winooski, Vermont) nanodrop. Phage DNA libraries were prepared using the Illumina DNA prep kit and IDT 10 bp indices, and were sequenced on an Illumina NextSeq 2000 platform (San Diego, CA), producing 2 × 151 bp reads. The total 2,743,288 read pairs were demultiplexed, quality controlled and adapter trimmed with the Illumina bcl-convert (v3.9.3). The genome was assembled with SPAdes (v3.13.0). The average fold coverage was 15,727. All sequencing and assembly procedures were performed by the Microbial Genome Sequencing Center (Pittsburgh, PA).
One step growth curve analysis
The viral propagation characteristics of KP1 were determined following a modified protocol from Summer et al. . B. cenocepacia K56-2 was grown in LB broth to log phase at 37°C. The phage lysate (1 ml) was added to bacterial cells (1 ml) at a multiplicity of infection (MOI) of approximately 0.1. The cells were centrifuged at 14,000 x g for three minutes at room temperature. The supernatant was removed, and the cells were suspended in 25 ml LB broth. The infected culture was incubated with shaking at 37°C for the 90-minute duration. At time zero, a 1 ml aliquot was removed from the culture. A volume of 50 µl was serially diluted in 450 µl PB. A 100 µl volume from each dilution was added to soft agar previously inoculated with 100 µl uninfected log phase culture and poured onto LB agar. This process was repeated every ten minutes for ninety minutes. To quantify the unadsorbed phage, or free phage, 50 µl of chloroform was added to the remaining 1 ml aliquot to lyse the bacterial cells. A volume of 100 µl from the lysate was serially diluted and added to soft agar previously inoculated with 100 µl of uninfected culture. This process was repeated every ten minutes for ninety minutes. All LB agar plates were incubated at 37°C, and plaques were quantified after overnight growth.
To determine the infected bacterial cell concentration, a 50 µl aliquot of the infected culture was serially diluted in PB, plated onto LB agar, and incubated at 37°C. After 24 h, the CFU/ml was determined. The burst size was determined by dividing the total phage by the infected cells.
Phage rescue assay with duckweed plants
Duckweed plants were purchased from Carolina Biological Supply Company, Burlington, NC. For the sterilization of the plants and determination of the LD50, the procedures in Thomson and Dennis  were used as described. Phage rescue assays were performed using a modification of the Thomson and Dennis procedure . Each well of a 96-well plate was filled with 160 µl Schenk-Hildebrandt medium supplemented with 1% w/v sucrose (SHS), one sterilized duckweed plant, and 20 µl of a K56-2 culture corresponding to 100×LD50 in ½LB broth. The plates were covered with sterile foil then incubated at 30°C in a sterile bag for four hours to allow infection. Phage was added (20 µl of MOI = 1) and then incubated for 96 h. Controls included uninfected plants with and without phage and infected plants without phage. Plants were identified as “alive” when more than 10% of the plant remained green after 96 h, and plants that displayed > 90% loss of green pigmentation were considered dead . An ANOVA (P < .05) and post-hoc Tukey-Kramer HSD tests were used to analyze differences. The study of plant material complies with relevant institutional, national, and international guidelines and legislation.
Phage-antibiotic synergy assay
To test the efficacy of phage-antibiotic synergy in the Artificial Sputum Medium model (ASMDM) , an overnight culture of B. cenocepacia K56-2 (1 ml) grown in ½LB broth (OD600 = ~ 1.00, approximately 1 × 107 CFU/ml) was centrifuged at 20,000 x g for 2 min and the pellet was suspended in 1ml saline. The wash was repeated. Under a sterile hood, 500µL 60% ASMDM  was added to wells in a gas-permeable 24-well plate (Coy Laboratory Products, Inc., Grass Lake, MI) then 10µL of the bacterial suspension was added. Two different concentrations of trimethoprim, a high dose (8.8µL of 100 mg/ml) and a low dose (8.8µL of 1 mg/ml or 1/2 x MIC) in DMSO and/or 50 µL of 9.10 × 108 KH1 phage (MOI = 17) was added to the wells. The plate was incubated at 37°C with 5% CO2 for 72 h. At time 0 and every 24 h, cell viability (CFU/ml) was determined with a standard plate count; the samples were diluted in saline, plated in duplicate on LB agar, and incubated for 48 h at 37°C. Every 24 h, phage viability (PFU/ml) was determined with plaque assays for wells containing KP1. A repeated measures MANOVA with follow-up contrasts analyzed differences in CFU/ml and PFU/ml.
Minimum inhibitory concentration of antibiotics
B. cenocepacia K56-2 was grown overnight in Mueller Hinton Broth (MHB) then diluted to an OD600 of 0.1 in MHB. A volume of 50 µl bacterial cells (approximately 5 × 105 CFU/ml) was delivered to wells containing serially diluted antibiotic in a 96-well SpectraPlate (PerkinElmer, Waltham, MA). The plates were incubated at 37°C, and growth was examined after 24 h. The MIC was determined to be the lowest concentration with no visible growth .