All water samples collected through sterile practices. All experiments were done in triplicate. In graphical representations, average value was taken. All cultural media, chemicals and ATCC bacterial strains were purchased from Himedia Labs Pvt. Ltd., Mumbai, India.
Isolation of S. aureus and its phage from water samples
The process of isolation of S. aureus and screening of phage has been discussed in the supplementary file 1 because the full description here in this paper was beyond the limits. In this study we have isolated a phage from a river water sample against the host W1 which is a confirmed local field strain of S. aureus and is an MRSA confirmed through antimicrobial susceptibility testing (AST).
Host Range vb_Sau_ARW1
The ability of the phage to infect the other bacterial hosts. For this, the following environmental S. aureus isolates were taken: W1, W2, W3, W4, E. coli ATCC 23848, S. aureus ATCC 25923, and a lab strain of Enterobacter. 8 µL of 0.22 µm membrane filtered vb_Sau_ARW1 lysate was added on the host culture plates. If a clear zone appeared on a culture plate, it may indicate phage active against that host. Agar overlay was done to exclude the doubt of lysis due to lysozyme in the lysate. For this, vb_Sau_ARW1 lysate was incubated with isolates mentioned above and kept for overnight incubation at 37°C in a rotator incubator and agar overlay was done the next day. In case of ambiguity, such as a faint or turbid plaque, further analysis by picking the plaque and re-incubating it with the host, followed by re-enumeration by double agar overlay.
Partial purification of vb_Sau_ARW1
Purification of phage is done by picking a single plaque and re-enriching with the host for three consecutive times and precipitated by PEG-NaCl for downstream applications.
For partial purification and concentration of phages (Pickard 2009), ten plates (agar overlay plating method) with dense plaque count was taken, in each plate, 4ml Phage buffer (P buffer) (10Mm Tris-Cl, 10mM MgSO4, 68mM NaCl, 1 mM CaCl2 constituted in molecular grade water and made up the volume to 1L) was added and kept overnight at 4°C. After overnight incubation, the lysate was centrifuged at 6000 rpm for 15 min at 4°C. The filtrate was then filtered through a 0.22 µm membrane filter. In the filtrate RNase A (20mg/ml) and DNase (1mg/ml) were added such that the final concentration was 4µl/ml. This was kept at 37°C for 30 minutes and then kept at room temperature for an additional one hour. Further, for phage precipitation, an ice-cold solution of 40% PEG-NaCl was added (PEG-6000 and 3M NaCl) such that its volume is one-fourth of the total volume of the treated filtered lysate. This was kept at 4°C on ice for an hour and then it was centrifuged at 6000 rpm for 40 min at 4°C. The precipitate was reconstituted in 0.1ml of P buffer. This partially purified phage was kept at -80°C for the next phase of analysis as described below.
Transmission electron microscopy
TEM analysis was done at the Indian Institute of Technology, Roorkee, India. Sample prepared briefly as- 10 µl of the PEG precipitated phage and purified crude lysate was loaded on a copper grid and stained by 10 µl of uranyl acetate (Ackermann 2009). Imaging was done at magnification 35000X. (TEM model: Tecnai G2 20)
vb_Sau_ARW1genomic DNA isolation and sequencing
The partially purified phage is subjected to the phenol-chloroform DNA extraction and ethanol precipitation (Pickard 2009). The precipitate was reconstituted in molecular grade water and was stored at -20°C.
Quality of the genomic DNA was checked on 0.8% agarose gel (loaded 3 μl) for the single intact band. The gel was run at 110 V for 30 mins. 1 μl of each sample was used for determining concentration using Qubit® 2.0 Fluorometer.
Sequencing of the phage genomic DNA and analysis of high quality data (HQ) was done at the Xcelris Labs Pvt. Ltd., India. It was sequenced on the Illumina platform with read length 2X 150 PE and coverage of more than 10000. For de novo assembly of the phage, SPAdes version–3.1.0 was used. BLASTn and BLASTx (version 2.2.28) were done for similarity search for the sequenced phage genome against the non-redundant protein database.
For reference based approach, reference phage genomes were downloaded from EMBI-EBI https://www.ebi.ac.uk/genomes/phage.html. There were total of 2401 phage genomes from which 104 genomes were representing S. aureus. HQ data were mapped with the reference phage genomes using CLC genomic workbench.
Genes prediction was done through Prodigal version 2.6.3 (Hyatt et al 2010). The phage lifecycle prediction was done through PHACTS (McNair et al 2012), a web tool (https://edwards.sdsu.edu/PHACTS/). Presence or absence of tRNA genes was done through a web-based tool called tRNAscan-SE (http://Lowelab.usu.edu/tRNASCAN-SE/) (Lowe and Chan 2016). For identification of antimicrobial resistance genes ‘Resistance Gene Identifier’ (RGI), a web based tool was used (https://card.mcmaster.ca/analyze/rgi) [17] (Alcock et al 2020).
HQ data is submitted in SRA submissions (in NCBI), submission ID is shared in the result section.
Adsorption Rate
For the determination of adsorption rate Kropinski (2009), Kaur et al. (2012) were considered. 9 ml of exponentially growing S. aureus (W1) culture of optical density at 600 nm (OD600) 0.2 OD (108 CFU/ml) was taken in a 100ml flask, to this 1ml of pre-warmed phage suspension of titre 1*105 PFU/ml was added. At time zero, 100 µl aliquot was withdrawn and added to 900 µl fresh broth (kept in 2 ml Eppendorf tube), thereafter at regular interval of 1.5 min., this process was repeated till 10 min. These aliquots were centrifuged at 14000 rpm for 15 min., the supernatant was carefully withdrawn and mixed with fresh host broth culture for phage enumeration. The amount of phage adsorbed to the host cell was determined as a decrease in the titer of free phage. Refer to table 1. Adsorption rate (ka) was calculated with following mathematical formula ka = (2.3/Bt)*log(Po/P), here B is host concentration, t is time taken for decrease in the plaque counts from initial, Po to the final plaque count, P.
One step growth
For one step growth, methods by Ellis and Delbruck (1939), Hyman and Abedon (2009) were followed. 1 ml of phage at a MOI of 0.1 was added in 9 ml of exponentially growing S. aureus culture (similar to adsorption rate method) and allowed to adsorb completely on S. aureus for 8 min, this was centrifuged at 6000 rpm for 10 min. This pellet was suspended in 10ml broth. After every 5 minutes, 100µl of the aliquot was taken and added in 900µl broth and plated by agar overlay method. An average value of five independent experiments was taken.
Phage viability
Viability of vb_Sau_ARW1 for a period of one year was tested. Filtered phage stocks were kept in the P buffer. Their viability was tested at temperatures ranging from -20°C, 4°C, 25°C, 40°C, and 60°C. Survivability of phage was studied at different PH: in the 9 ml of P buffer of different PH was prepared; that is., PH of 2,4,6,8,10, and 12 to this 1 ml of phage suspension was added and kept at 37°C for overnight incubation. 100 µl of sample was taken from each tube and mixed with 100 µl of actively growing host, and agar-overlay was done on this mixture for phage enumeration.