Salmonella enterica serovar Paratyphi MTCC735 was used to isolate the phage. Other bacterial strains used in this study to test the lytic spectrum and productivity of phage infection are listed in Table 1. All the isolates were propagated in nutrient broth (NB) at 37°C overnight with vigorous agitation at 180 rpm and preserved as glycerol stocks at −80°C. Antibiotic sensitivity test of all the bacterial isolates was conducted according to the Kirby-Bauer disk diffusion methods against 25 antibiotics [(Himedia, United States): ampicillin (AMP 10 μg), piperacillin (PI 100 μg), amoxicillin + clavulanate (AMC 20/10 μg), amikacin (AK 30 μg), gentamycin (GEN 10 μg), tetracycline (TE 30 μg), chloramphenicol (C 30 μg), aztreonam (AT 30 μg), cefoxitin (CX 30 μg), ceftazidime (CAZ 30 μg), cefepime (CPM 30 μg), cefpodoxime (CPD 10 μg), imipenem (IPM 10 μg), meropenem (MRP 10 μg), kanamycin (K 30 μg), tobramycin (TOB 10 μg), azithromycin (AZM 30 μg), ofloxacin (OF 5 μg), nalidixic acid (NA 30 μg), norfloxacin (NX 10 μg), ciprofloxacin (CIP 5 μg), fosfomycin (FO 50 μg), sulfafurazole (SF 300 μg), cotrimoxazole (COT 25 μg), and colistin (CL 10 μg)] as per Clinical and Laboratory Standards Institute (CLSI) guidelines (CLSI, 2016). Based on their susceptibilities against tested antibiotics, bacteria were categorized as multidrug-resistant (MDR) when they showed non-susceptibility to at least one agent in three or more antimicrobial classes (Magiorakos et al., 2012).

Pond water samples collected from Hisar City, India (N 29° 7′, 36.264″, E 75° 47′, 20.364″), were used for the isolation of bacteriophage. Sample enrichment, phage isolation, and purification were carried out by following the methods described by Anand et al. (2018) using the Salmonella enterica serovar Paratyphi MTCC735 as a host. In brief, 40 mL of sample water was mixed with log-phase bacterial culture (5 mL), 5X NB (5 mL), and 5 mM of CaCl₂ (Calcium chloride) and incubated overnight at 37°C with shaking at 220 rpm. The phage-enriched lysate was centrifuged, and the supernatant was passed through a 0.22-μm pore-size polyvinylidene difluoride (PVDF) syringe filter (Millipore). Afterward, a total of 10 μL of the filtrate was spotted on the bacterial lawn prepared by pouring 3 mL of molten agar containing 300 μL of log-phase bacterial culture using the double-agar overlay (DLA) method, and plates were incubated at 37°C for 3–4 h under static conditions. The qualifying filtrate was used to purify phages by picking and resuspending a single, well-separated plaque. A single plaque was lifted from the agar plate and resuspended in 100 μL of saline magnesium (SM) buffer [NaCl: 5.8 g/L; MgSO4: 2.0 g/L; 1 M Tris (pH 7.5): 50 mL/L; and gelatin (2%): 5 mL/L], and four rounds of purifications were performed to obtain the purified and homogenous plaques. A large volume (50 mL) of log-phase host culture infected with purified phage was incubated overnight at 37°C with shaking at 220 rpm. Lysate obtained after centrifugation was precipitated with NaCl and polyethylene glycol (PEG) to obtain a high-titer phage suspension (Sambrook and Russell, 2006). The bacteriophage suspension was used for titer estimation and preserved as glycerol stocks at −80°C, followed by accession in the Bacteriophage Repository at the National Centre for Veterinary Type Culture, ICAR-NRCE, Hisar, Haryana.

The bactericidal activity of phage phiSalP219 against planktonic cells of S. enterica serovar Paratyphi MTCC 735 and S. enterica serovar Typhimurium VTCCBAA501 was assessed as described previously (Esmael et al., 2021) with some modifications. In brief, in a 96-well microtiter plate, phage phiSalP219 was mixed with MTCC 735 and VTCCBAA501 at different MOIs of 0.0001, 0.001, 0.01, 0.1, 1, 10, or 100, respectively. Positive control wells contained MTCC 735 and VTCCBAA501, while negative control wells contained nutrient broth only. The microtiter plate was incubated at 37°C in a microplate reader with mild agitation, and bacterial growth was monitored at an interval of 30 min for 6 h by measuring optical densities at 600 nm using a Multiskan GO Microplate Spectrophotometer, using SkanIt™ Software (Thermo Scientific, Waltham, MA, United States, ver. 1.01.12x).

Bacteriophage DNA was extracted using the protocol described previously by Anand et al. (2018), and the concentration was determined using a Qubit 4 Fluorometer and NanoDrop spectrophotometer. Whole-genome sequencing of bacteriophage was carried out using Illumina NovaSeq 6,000 platform (Illumina, San Diego, CA, USA) and Nanopore GridION X5 (Oxford Nanopore Technologies, Oxford, United Kingdom) technology. Sequencing libraries for Illumina were prepared according to the QIASeq FX DNA library preparation protocol using the QIASeq FX DNA kit. For nanopore sequencing library preparation, genomic DNA was end-primed by using the NEBnext ultra II end repair kit and cleaned up with AMPure beads. Native barcoding of end-primed samples was carried out with EXP-NBD104(ONT). Finally, the equimolar concentration of the barcode sample was ligated with a sequencing adaptor, purified with AMPure beads, and eluted in 15 μL of elution buffer. Raw reads obtained from Illumina and nanopore sequencing were de-multiplexed using Bcl2Fastq software v2.20¹ and guppy-v2.3.4 (Wick et al., 2019), respectively. Trimgalore-v0.4.0² tool and Commander v2.0.20³ were used in Illumina sequencing, and Porechop-v0.2.3⁴ and Nanofilt-v2.8.0⁵ were used in Nanopore sequencing for trimming the raw reads and to assess their quality. The long read obtained from Nanopore sequencing and the short read obtained from Illumina were assembled using Unicycler v0.4.8. A hybrid sequencing approach was carried out for de novo assembly. Detailed information on the raw reads statistics is given in Supplementary Table S1.

The assembled genome of bacteriophage was analyzed by RASTtk (Brettin et al., 2015) pipeline for predicting and annotating open reading frames (ORFs). Functional prediction and validation of the ORFs were performed using BLASTp on the NCBI non-redundant protein (nr) sequences database. Predicted protein sequences were analyzed against InterProScan (Paysan-Lafosse et al., 2023) and CDD (Lu et al., 2020) for conservative domain identification. Putative tRNAs were identified using tRNAscan-SE by using bacterial sequence source and default parameter (Lowe and Chan, 2016). ARG genes were screened by using CARD (Alcock et al., 2020), and CG View software was used for genomic map visualization (Grant et al., 2023). The complete genome of Salmonella phage phiSalP219 was submitted to the GenBank database, and its accession number is PP595732.

A phylogenetic analysis was conducted to investigate the relatedness of phage phiSalP219 with the known phages of the subfamily Vequintavirinae. Genome-BLAST Distance Phylogeny method in the Virus Classification and Tree Building Online Resource (VICTOR) was used for the whole-genome phylogenetic analysis and tree building (Meier-Kolthoff and Göker, 2017). The resulting intergenomic distances (including 100 pseudo-bootstrap replicates each) were used to infer a balanced minimum evolution tree with branch support via FASTME, including subtree pruning and regrafting postprocessing for the formula D0 (Farris, 1972). The tree was visualized using ggTree (Yu, 2020). For determining the phylogenetic positions and DNA packaging strategies, phage-conserved gene sequences (major capsid protein and terminase large subunits) were used. The phylogenetic tree of their amino acid sequence was constructed in MEGA X 10.2.6 software (Kumar et al., 2018) using the Clustal W alignment algorithm by neighbor joining method with 1,000 bootstrap replications (Thompson et al., 1994). Comparative genome alignment with four reference phages (Salmonella phages PVP-SE1, SSE-121, and NINP13076 and Escherichia phage 4MG) was performed using Mauve 2.4.0 (Darling et al., 2004).

To check the stability of Salmonella phage phiSalP219 in foods, phage stability experiments were conducted according to a previously described method (Guenther et al., 2012). In brief, packed chicken ham and salami were obtained from the local market, sliced into 1 g pieces using a surgical blade, and sterilized using UV radiation for 30 min. In total, 50 μL volume of phage suspension was added to reach a final titer of 1×10⁹ PFU/mL. Inoculated samples were incubated for 0, 6, 12, 24, and 48 h separately at 28°C. After each time interval, the respective sample was transferred into 5 mL of PBS and homogenized. Then, the phage titer was measured using the double-layer agar method and calculated as PFU/mL.

Packaged/processed chicken ham and salami were obtained from a local supermarket and then sliced aseptically in the laboratory. The food samples were cut into pieces (1 g) using a sterile scalpel blade and then sterilized by exposing them to 30 min of UV light. After UV exposure, we checked the presence of bacterial contamination in the food sample, before starting the experiment and found that UV exposure sterilized the food sample. Then, the food sections were placed in the center of sterile Petri dishes and inoculated with S. typhimurium VTCCBAA501 (As S. typhimurium is the leading agent of foodborne infection, therefore to find out the biocontrol capability to phage phiSalP219 in the control of S. typhimurium strain in the food sample, this strain of S. typhimurium was used) to a final viable count of 4.3 log10 CFU/g and was allowed to adsorb for 15–20 min. Phage suspension was then applied to the sample surface with an MOI of 10⁴. These samples were incubated at 4°C or 28°C for 48 h. During incubation, the samples were taken at 0, 6, 12, 24, and 48 h from each group and transferred to 5 mL of PBS buffer. They were homogenized with a sterile handheld homogenizer and vortexed. The proportions of recoverable bacteria among the control group and the experimental group were determined using the direct spread plate method by plating 100 μL of successive serial dilutions of the homogenized tissue and calculating the CFU/mL by counting the number of colonies.

For assessing the biofilm eradication capability of phage phiSalP219, all the bacterial strains as shown in Table 1 were tested for their biofilm-forming ability; out of these strains, VTCCBAA501 was chosen based on its good biofilm formation capacity. Overnight grown culture of VTCCBAA501 in Tryptic Soy Broth (TSB) was re-inoculated in a 12-well culture plate containing 2X TSB broth with 1% glucose. Borosilicate glass coverslips of size 18–22 mm were immersed in the wells containing inoculated bacterial culture and placed at 37°C in a static incubator for 72 h. After incubation, the media in the wells were removed, and the wells were washed twice with sterile PBS. Bacteriophage suspension (10¹¹ PFU/mL) at a volume to submerge the coverslips completely was added in the wells and placed at 37°C for 24 h under static conditions. Following the treatment, the coverslips were removed and washed with PBS twice. The control group was treated with SM buffer without any phage in it. Then, the coverslips were washed with PBS and placed in a 2.5% glutaraldehyde solution for 1 h at 4°C to fix the biofilm. Thereafter, biofilm was dehydrated by placing coverslips in the ethanol gradient [25, 50, 75, 90, and 100% (2X)] (Nikara et al., 2020). Dehydrated coverslips were affixed to a copper stub, sputtered with gold for 1 min, and then viewed at an accelerating voltage of 5–10 kV in a JSM-7610F Plus Scanning Electron Microscope, Jeol, Japan.

The data analyses of all the experiments were performed using GraphPad Prism 8.0.2 (GraphPad Software, La Jolla, CA, United States). All experiments were conducted in triplicate. The two-way ANOVA with Bonferroni’s multiple comparisons was used to determine the significance among groups at a significance level of p < 0.05.

The outcomes of the antibiotic sensitivity test revealed that 13 Salmonella isolates belonging to the serovar Typhimurium, Enteritidis, Paratyphi, and Gallinarum were MDR (Refer to Table 1), while 73.33% of the Salmonella isolates displayed resistance to either one or two antibiotics falling within the fluoroquinolone class of antimicrobial agents.

Salmonella phage phiSalP219 was isolated from the pond water sample using Salmonella enterica serovar Paratyphi MTCC735 as a host and was named Salmonella phage phiSalP219 according to Adriaenssens and Brister (2017). Phage phiSalP219 was preserved as glycerol stocks in the Bacteriophage Repository of the National Centre on Veterinary Type Culture–National Research Centre on Equine NCVTC-NRCE, Hisar (see Figure 1).

Salmonella phage phiSalP219 produced clear plaques with a surrounding halo. Phage head and diameter were measured by the software used for capturing TEM images only using the inbuilt scale. Phage dimensions are shown as the average of measurements taken from five phage particles of the same phage. TEM divulged that phage phiSalP219 belongs to the class Caudoviricetes, with an icosahedral head of 155.9 ± 7.2 diameter and a long tail of 365.6 ± 5.0.5 diameter (Figure 1). pH sensitivity analysis showed that phage phiSalP219 was stable without any significant change in their viable titer at pH values ranging from 6 to 10, while reductions in phage titer were detected at pH 2, pH 3, and pH 4 (Figure 2B). Similarly, the results obtained from the temperature stability assay demonstrated that phage remained stable at temperatures ranging from 25 to 45°C. Beyond 45°C up to 70°C, a gradual significant reduction in phage stability in terms of phage titer was observed (as shown in Figure 2A), while treatment at 80°C led to huge stability loss in phage titer (9.74 log reduction).

Host range evaluation by spot assay revealed that phage phiSalP219 was able to lyse 28/30 tested strains of Salmonella with varied strength of lysis (Table 1). Hence, it was characterized as a broad-host range phage, which was further supported by the results from the EOP study. In the EOP study, we found that phage phiSalP219 showed high productivity against 7/28, medium productivity against 12/28, low productivity against 3/28, and inefficient productivity against 6/28 tested strains of the Salmonella enterica.

The results from the one-step growth curve indicate that phage phiSalP219 required 65 min to complete its infection cycle with a latent period of 35 min, followed by a short rise period in the next 5 min, and the burst size was calculated to be 68 phage particles per infected bacterial cell (Figure 2C). To find out the best lytic efficiency of Salmonella phage phiSalP219 for carrying out the biocontrol of S. typhimurium (VTCCBAA501) in ready-to-use food samples and in the eradication of biofilm formed by S. typhimurium (VTCCBAA501), Salmonella phage phiSalP219 was tested against MTCC 735 and VTCCBAA501 at different Multiplicity of infection (MOIs), that is, 1,000, 100, 10, 1, 0.1, 0.01, 0.001, and 0.0001, and respective time-kill curves were obtained. In the case of MTCC 735, phage phiSalP219 inhibited bacterial growth up to MOI of 0.001, while in the case of S. typhimurium VTCCBAA501, phage phiSalP219 inhibited bacterial growth up to MOI of 1 (Figures 2D,E).

The detailed genomic information of Salmonella phage phiSalP219 is given in Supplementary Tables S2, S3. The genome size of the phage phiSalP219 was 146,994 bp, and the overall G + C content was 44.5%. Most CDS of phage phiSalP219 (218/250) began with ATG, followed by GTG (17/250) and TTG (15/250). Among termination codons, TAA (144/250) was the most common, followed by TAG (84/250) and TGA (22/250).

Bioinformatic analysis revealed that the genomes of phage phiSalP219 contained ~250 putative ORFs (including ~100 functional, ~ 19 uncharacterized, and ~ 131 hypothetical proteins) and 25 tRNAs. The predicted CDS were divided into five groups according to their functions: nucleotide metabolism, DNA replication and transcription, structural proteins, lysis proteins, and other proteins (Figure 3).

A BLAST search revealed that phage phiSalP219 showed >97% identity with Salmonella phages belonging to the subfamily Vequintavirinae (NCBI: txid1914851; Vequintavirinae, Caudoviricetes). To further investigate and visualize the genomic distance between Salmonella phage phiSalP219 and other Vequintavirinae phages, phylogeny was performed using whole-genome and phage-conserved gene sequences (major capsid protein and terminase large subunit). The whole-genome phylogeny constructed with the VICTOR server showed that Salmonella phage phiSalP219 was the new member of the Vequintavirinae subfamily, which was further supported by the results of the phylogenetic analysis of the major capsid protein and large terminase subunits (see Figures 4–7).

We observed that phage remained stable on the food matrices, and no significant change in phage concentrations was observed in the salami and chicken ham. Both the samples were inoculated with phage at 1×10⁹ PFU/mL final concentration, and after 48 h, we found 0.8×10⁸ PFU/mL concentration of phage in salami and 0.15×10⁸ PFU/mL of phage in chicken ham. We also found that Salmonella phage phiSalP219 had a significant antibacterial effect on S. enterica serovar Typhimurium VTCCBAA501 in different food matrices under different temperature conditions when applied at MOI = 10,000 (p < 0.05). For Salmonella-contaminated salami samples administered with phage phiSalP219 at an MOI of 10,000, the viable bacterial count was reduced by 0.661 log10 CFU/mL at 4°C and 3.191 log10 CFU/mL at 28°C compared to the control group after 48 h of incubation (Figures 8A,C), while for Salmonella-spiked chicken ham samples, phage phiSalP219 applied at an MOI of 10,000 reduced the viable Salmonella count by 0.529 log10 CFU/mL at 4°C and 2.046 log10 CFU/mL at 28°C relative to the control group after 48 h of incubation (Figures 8B,D). Hence, it is observed that the antibacterial effect of phage phiSalP219 was higher at 28°C than at 4°C.

The 72-h-old biofilms developed on the glass surface were used to demonstrate the process of phage-mediated eradication. The biofilms that formed on the coverslips have projections with many layers of different densities and heights. These come from the cell layers below (Figures 9A,B). Within the gelatinous extracellular polymeric substance (EPS) matrix, cells exhibited adhesion to both each other and the surfaces of the coverslip (Figures 9A–C). After the application of bacteriophage, the biofilm was clearly eliminated (as shown in Figures 9D–F), resulting in the presence of only cellular debris and the absence of any intact cellular structure.