Bacterial cultures
For this study reference culture of Salmonella enteritidis was procured from American Type Culture Collection (ATCC-13076). Tryptic Soy broth (HIMEDIA, Mumbai) media was used to grow this culture and maintained at 4oC.
Isolation – Double Layer Agar Overlay Technique
Raw sewage sample (40ml) was collected aseptically in a sterile container (HIMEDIA, Mumbai) from Hyderabad Municipal sewerage treatment plant. To this sample, 5 ml of 10X nutrient broth and 5 ml of exponential phase culture of S. enteritidis were added. Flask was incubated at 370C for 24 h in a shaker incubator. After incubation 10 ml of culture was centrifuged at 2000 rpm for 5 min. The supernatant was taken in a syringe fitted with a 0.22 µ filter. The filtrate was collected in a microcentrifuge tube and stored at 40C. The filtrate (100 µl) and exponential phase culture of Salmonella (100 µl) are added to 3 ml of molten agarose (0.7%). Mixed contents gently and poured this mixture onto the pre-warmed agar plate. The mixture was spread evenly on the surface of the plate. Plates were allowed to cool down. Plates were incubated in an inverted position at 370C for 24 h. Plates were observed for plaques after incubation. The total number of plaques forming units (PFU) per ml of sample was calculated by using the formula number of plaques divided by Volume plated and dilution factor.
Phage Purification
This process was carried out by picking a single plaque with a sterilized pipette tip followed by serial purifications and amplifications from the host Salmonella enteritidis (ATCC13076).
Phage Host Spectrum Study
To perform this experiment, a spot test assay and agar well diffusion assay was done. For spot test assay 1 ml of exponential phase culture of S. enteritidis was cultured on nutrient agar by spread plate method. Filtrated supernatant of phage (10 µl) was spotted on the agar and allowed for air drying. The plates were incubated at 370C for 24 h and observed for the clear zone (lytic spot) formation over the bacterial lawn. Agar diffusion assay was performed by making a well with cork borer in a nutrient agar pre-inoculated with Salmonella. Phage filtrate (10 µl) was added to the well and the plates are incubated at 370C for 24 h. After incubation, the plates were observed for the zone of inhibition.
Host Range Assay
Spot test assay was performed to determine the host range. Reference cultures of Salmonella (Salmonella abony-NCTC 6017, Salmonella arizonae-ATCC 13314, S. typhi-MTCC 733, Salmonella poona-NCTC 4840, S. enteritidis-ATCC 13076, Salmonella spp- MTCC 1162) were procured from the National Collection of Type Cultures (NCTC), Microbial type culture collection (MTCC), and American Type Culture Collection (ATCC) to conduct this experiment.
Phage Morphology
TEM (Transmission Electron Microscopy) was used to obtain images of isolated phage at an acceleration of 80KV. A small drop of phage filtrate was loaded on a carbon-coated copper mesh grid. Negative staining phage filtrate was done by using 1% phosphotungstic acid.
Phage DNA isolation
Isolation of phage DNA was done using a phage DNA isolation kit (NORGEN, CANADA). For DNA isolation, phage filtrate (1 ml) was taken in a microcentrifuge tube and treated with DNase (10 µl). Phage filtrate was obtained by plucking individual plaque and subsequently inoculated to nutrient broth containing Salmonella exponential culture. After overnight incubation (370C for 24 h in a shaker incubator) and centrifugation (2000 rpm for 5 min) followed by filtration, the filtrate was obtained. Incubate this at 750C for 45 min in a water bath. Lysate buffer (500 µl) was added to this and vigorously vortexed for 10 sec. Proteinase K (4 µl) was added and incubated at 550C for 15 min. Again this solution was incubated at 650C for 15 min. Isopropanol (320 µl) was added to the lysate and vortexed. Centrifugation was done in a collection tube for 1 min at 8000 RPM. The flow-through was discarded from the collection tube. Once again centrifugation step was repeated and added 400 µl of ethanol as wash solution and again centrifuged for 1 min at 8000 RPM. This particular step was repeated twice. Elution buffer (75 µl) was added and centrifuged for 1 min at 8000 RPM. Phage DNA concentration was measured using nanodrop. The purified DNA sample was used for genome sequencing.
Whole-genome Sequencing
Whole-genome sequencing of phage DNA was performed in the Miseq platform (Illumina, USA). The genomic DNA library was prepared using the NexteraXT library prep kit (Illumina, USA) method using NexteraXT index kit and standard protocol provided by the manufacturer. The concentration of the DNA library was quantified in Qubit (ThermoScientific, USA) and the average library size was measured in Bioanalyzer (Agilent Technologies, Germany). Paired-end sequencing of 2×251 cycles read length; the run was performed using Miseq V3 kit (Illumina, USA). Upon completion of the sequence run, raw data fastq files for forward and reverse reads were carried forward for computational analysis.
Genome assembly and analysis
The raw reads were processed for QC using Trimmomatic v0.36 (Bolger et al., 2014). The reads that passed QC were assembled de novo using Unicycler v0.4.8 (Wick RR et al., 2017) assembler specific for Illumina short-reads. The assembly quality was checked with Quality Assessment Tool for Genome Assemblies (QUAST) v5.0.2 (Gurevich A 2013). Annotation of the genome was carried out by Rapid Annotation Subsystems Technology (RAST) tk which uses a k-mer based search method against CoreSEED (Brettin T et al., 2015). If the K-mer search of FIGfam didnt yield anay results a BLAT and BLASTP search of non-redundant genus-specific protein databases for the organism's genus was employed at cutoff of e-value < = 1e-5 and a percent identity > = 50%. A Phylogenetic tree was built for the Capsid sequence using GTRGAMMA model of RAxML v8.2.4 with 1000 bootstrapping value (Stamatakis A 2014). A comapartive study was carried using NCBI Pairwise Sequence Comparison (PASC) tool (Bao Y et al., 2014). A circular genome map for the assembled genome was developed using CGView (Grant JR 2008). Using ViPTree a whole-genome phylogeny was built by comparing against reference viral genomes obtained from Virus-Host DB of dsDNA category (Nishimura Y et al., 2017). GenBank Accession Number for the deposited sequence is MZ326168.