Isolation, Identification, and In Vivo Anti-Infective Efficacy of Lytic Bacteriophage SEP1 Against Salmonella Paratyphi

Isolation, Identification, and In Vivo Anti-Infective Efficacy of Lytic Bacteriophage SEP1 Against Salmonella Paratyphi | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Isolation, Identification, and In Vivo Anti-Infective Efficacy of Lytic Bacteriophage SEP1 Against Salmonella Paratyphi Zhiyi Ge, Di Lian, Wei Zhao, Weiru Song, Shengyi Han, Chunyan Xu This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8201091/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Background The emergence of multidrug-resistant Salmonella Paratyphi has created a critical need for novel therapeutic options beyond conventional antibiotics. Bacteriophages, with their ability to selectively lyse bacterial hosts, offer a promising alternative. This study aimed to characterize the lytic phage SEP1 and evaluate its efficacy against S. Paratyphi infection. Methods SEP1 was isolated from poultry farm sewage in Lanzhou, China, using S. Paratyphi QH as the host. We performed transmission electron microscopy for morphological analysis, double-layer agar assays for host range, one-step growth curves for lifecycle characteristics, and stability tests under varying temperature and pH conditions. Genomic DNA was sequenced and analyzed. In vivo efficacy was assessed in a lethal murine model of S. Paratyphi infection, with monitoring of survival, body weight, bacterial loads in liver and spleen, histopathology of liver/spleen/intestine, and serum cytokine levels (IL-1β, IL-6, IL-10, IFN-γ). Results SEP1 was classified as a member of the Felixounavirus genus, featuring a double-stranded DNA genome of 85,703 bp with 39.00% GC content and 123 predicted open reading frames. No lysogenic or virulence genes were identified. The phage exhibited a host range infecting 31.25% of tested Salmonella strains, a latent period of 20 minutes, and a burst size of approximately 141 PFU per cell. It remained stable at temperatures between 10–50°C and pH values from 4 to 9. In the mice model, a single dose of SEP1 administered 30 minutes post-infection resulted in 100% survival, compared to 0% in the challenge control group (p < 0.001). Treatment prevented weight loss, reduced bacterial loads in organs (p < 0.01), mitigated tissue damage, and restored cytokine levels to near baseline. Salmonella Paratyphi Bacteriophage Biological Characteristics Genomics Phage therapy Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 1. Introduction Salmonella , a facultative anaerobic, Gram-negative rod-shaped bacterium of the Enterobacteriaceae family, represents a major global foodborne pathogen impacting both human and animal health [ 1 ]. Its remarkable adaptability facilitates persistent colonization in diverse agricultural environments—including soil, water, and animal feed—with the fecal-oral route serving as the primary mode of transmission [ 2 ]. While numerous serovars contribute to its global burden, S. Paratyphi is particularly notable for causing enteric fever, a systemic illness whose severity contrasts sharply with the typically self-limiting gastroenteritis associated with other foodborne strains [ 3 ]. Humans and domestic animals act as key reservoirs, and clinical outcomes range from asymptomatic carriage to life-threatening septicemia, largely influenced by host immunity and an array of sophisticated bacterial virulence mechanisms[ 4 , 5 ]. Although antibiotics remain the standard treatment for invasive salmonellosis, the increasing prevalence of multidrug-resistant strains and the emergence of persistent bacterial subpopulations have progressively compromised their efficacy [ 6 , 7 ],This reality underscores the growing urgency for developing alternative therapeutic approaches. In this context, bacteriophages (phages)—viruses that specifically infect and replicate within bacteria—have regained prominence as promising antimicrobial agents. Their life cycle can proceed through a lytic pathway, directly lysing the host bacterium, or a lysogenic one, facilitating genetic exchange. Phages offer several unique advantages for clinical and biotechnological applications: high specificity, an inherent capacity for self-amplification at infection sites, and minimal toxicity to the host [ 8 , 9 ]. Notably, Salmonella -targeting phages have demonstrated potential in several areas, from enabling rapid pathogen detection and enhancing food safety through biocontrol to serving as effective therapeutics against active infections [ 10 – 14 ]. Against this backdrop, the current study aims to contribute to the field by characterizing SEP1, a novel lytic phage targeting S. Paratyphi . We present its complete genome sequence alongside detailed morphological and biological analyses. Furthermore, we critically evaluate the therapeutic potential of SEP1 using a murine infection model, highlighting its viability as an alternative to conventional antibiotics. 2. Materials and Methods 2.1 Experimental Materials 2.1.1 Strains and Samples For this study, 32 Salmonella strains (including the one used for in vivo experiments) were obtained from the Engineering Laboratory of Tarim Animal Diseases Diagnosis and Control, Xinjiang Production & Construction Corps. Sewage samples used in the experiment were collected from various poultry farms in Lanzhou City (Sampling point coordinates: 102°36′-104°35′E, 35°34′-37°00′N). 2.1.2 Experimental Animals Female SPF BALB/c mice (6–8 weeks old, 16–22 g) were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. All animals were housed under standard laboratory conditions with ad libitum access to sterile water and a commercial rodent diet (Beijing Ke'ao Xieli Feed Co., Ltd.). After a two-day acclimatization period, experiments were conducted in accordance with guidelines approved by the Animal Ethics Committee of Tarim University (Approval No. 2023061), following the Guidelines for the Ethical Review of Laboratory Animal Welfare (GB/T 35892-2018). Note: This study only involved animal experiments and did not include human clinical trials; thus, Clinical trial number: not applicable. 2.2 Isolation, Purification, Biological Characterization and Genomic Analysis of Phage SEP1 2.2.1 Phage isolation and purification A bacteriophage was isolated from poultry farm sewage using S. Paratyphi QH as the host. Approximately 20 g of sewage was suspended in 100 mL of SM buffer (0.01% gelatin (w/v), 100 mM NaCl, 10 mM MgSO₄, 50 mM Tris-HCl, pH 7.5) and centrifuged at 8,000 ×g for 10 min. The supernatant was filtered through a 0.22 μm membrane. Then, 10 mL of filtrate was mixed with 10 mL of a S. Paratyphi QH culture (OD 600 ≈ 0.6) and incubated with shaking (180 r/min) at 37°C for 12 h. After centrifugation (8,000 ×g, 10 min), 100 µL of supernatant was adsorbed with 100 µL of fresh host culture (OD 600 ≈ 1.2) for 10 min at 37°C. Plaque isolation was performed using the double-layer agar method: 200 µL of the adsorption mixture was mixed with 0.7% LB soft agar and overlaid onto solid LB agar plates. After incubation at 37°C for 12 h, visible plaques were subjected to five successive rounds of purification. Phage concentration and purification followed Gavrić and Knežević [15], involving CsCl density gradient centrifugation (densities: 1.45, 1.50, 1.70 g/mL) and ultracentrifugation at 25,000 ×g for 4 h at 4°C. 2.2.2 Morphological observation A purified phage suspension (10 9 –10 10 PFU/mL) was applied to a 400-mesh carbon-coated copper grid for 10 min. After blotting excess liquid, the grid was negatively stained with 1% phosphotungstic acid for 2 min. Samples were air-dried and imaged using an HT7700 TEM (Hitachi, Japan) at 50 kV. 2.2.3 Host range The host range of SEP1 was determined against 31 Salmonella strains using the standard double-layer agar plaque assay. Lytic activity was qualitatively assessed and quantified using a predefined infection efficiency scale (Table 1). 2.2.4 One step growth curve Host cultures (OD 600 = 0.6) were infected with phage at an MOI of 0.01 and incubated at 37°C with shaking (180 r/min). Samples (100 μL) were collected at 20 min intervals, centrifuged (8,000 ×g, 10 min, 4°C), and the supernatants were titrated using the double-layer agar method. Experiments were performed in triplicate. 2.2.5 Optimal multiplicity of infection Phage serial dilutions were mixed with equal volumes of logarithmic-phase bacterial cultures to achieve MOIs of 0.0001 to 10. After adsorption (10 min, 37°C), mixtures were centrifuged (8,000 ×g, 10 min), pellets resuspended in 10 mL fresh LB broth, and cultured (180 r/min, 37°C) for 5 h. Cultures were centrifuged, supernatants filtered (0.22 μm), and phage titers determined. All experiments were conducted in triplicate. 2.2.6 Temperature and pH stability For thermal stability, phage suspensions were incubated at 10–70°C (10°C increments) for 1 h, and viable particles were quantified. For pH stability, 100 µL phage suspension was mixed with 900 µL SM buffer adjusted to pH 2–13, incubated at 37°C for 1 h, and titrated. All assays were performed in triplicate. 2.2.7 Phage genome extraction, sequencing, and analysis Phage genomic DNA was extracted using the Bacteriophage DNA Isolation Kit (Norgen Biotek, Canada). Sequencing libraries were prepared using the Illumina TruSeq Nano DNA LT Library Prep Kit. Raw reads were processed with Trimmomatic (v0.39) and assembled de novo using SPAdes (v3.12.0). ORFs were predicted and annotated via BLASTp. Antibiotic resistance and virulence genes were screened using ResFinder and VirulenceFinder databases. tRNA genes were predicted with tRNAscan-SE 2.0. A neighbor-joining phylogenetic tree was constructed in MEGA 7.0 [16]based on major capsid protein sequences. Intergenomic comparisons were performed with VIRIDIC [17], and genomic comparisons were visualized using Easyfig 2.2.5 [18] 2.3 In Vivo Experimental Design and Treatment 2.3.1 Preparation of challenge solution S. Paratyphi QH was grown in LB medium at 37°C with shaking (180 r/min) to late logarithmic phase (OD 600 ≈ 5.0 × 10 8 CFU/mL). Cells were harvested by centrifugation (8,000 ×g, 10 min, 4°C), resuspended in sterile PBS, and adjusted to 2.0 × 10 8 CFU/mL. 2.3.2 Grouping and processing BALB/c mice were randomly divided into four groups (n=12 per group): Challenge group: Intraperitoneal (i.p.) injection of 100 μL S. Paratyphi QH suspension (2.0 × 10 8 CFU/mice). After 30 min, 100 μL sterile PBS was administered i.p., repeated daily for three days. Phage treatment group: i.p. injection of bacterial suspension as above. After 30 min, 100 μL phage SEP1 suspension (5.0 × 10 10 PFU/mice, MOI=0.01) was given i.p., followed by an i.p. injection of the same dose daily for the next three days. PBS control group: i.p. injection of 100 μL sterile PBS daily for three days. Phage control group: i.p. injection of 100 μL phage SEP1 suspension (5.0 × 10 10 PFU/mL) daily for three days. After treatments concluded on day 3, a seven-day observation period followed. General health status was monitored daily. 2.4 Detection Indicators and Methods 2.4.1 Animal euthanasia method When it was necessary to collect tissue and blood samples (on days 1, 3, 5, and 7 post - infection), the mice were euthanized humanely following the ethical requirements of laboratory animal welfare. First, the mice were anesthetized with an intraperitoneal injection of pentobarbital sodium at a dosage of 50 mg/kg body weight. After confirming that the mice were completely unconscious (judged by the loss of corneal reflex and pain response), cervical dislocation was performed to ensure rapid and painless death. This euthanasia method was selected to minimize animal suffering and comply with the Guidelines for the Ethical Review of Laboratory Animal Welfare (GB/T 39760 − 2021). 2.4.2 Mice phenotypic monitoring Survival rates were recorded daily. Body weight was measured daily at 9:00 AM. Body condition scores (BCS) were assessed daily using a scale of 0 (dead) to 5 (healthy)(Table2) [19]. 2.4.3 Detection of bacterial loads in liver and spleen On days 1, 3, 5, and 7 post-infection, liver and spleen samples were aseptically collected from three mice per group. Tissues (0.5 g) were homogenized in 4.5 mL PBS. Serial dilutions were plated on LB agar, incubated at 37°C for 12 h, and CFU/g calculated. 2.4.4 Histopathological analysis Liver, spleen, and jejunum samples were fixed in 4% paraformaldehyde, dehydrated, embedded in paraffin, sectioned at 5 μm, and stained with H&E. Sections were examined under an Olympus BX53 microscope. Pathology scores were assigned as follows:[20] Liver: 0 (normal), 1 (mild edema), 2 (moderate necrosis, localized inflammation), 3 (severe necrosis, inflammatory foci, congestion). Spleen: 0 (normal), 1 (minimal apoptosis), 2 (moderate congestion, inflammation), 3 (severe necrosis, follicle loss, extensive inflammation). Gut: 0 (normal), 1 (mild villous edema), 2 (partial villous disruption, mild crypt damage), 3 (extensive villous loss, crypt necrosis, massive inflammation). 2.4.4 Peripheral blood cytokine levels Blood was collected on days 1, 3, 5, and 7. Plasma levels of IL-1β, IL-6, IL-10, and IFN-γ were quantified using ELISA kits according to the manufacturer’s instructions. 2.5 Statistical analysis Data are presented as mean ± standard deviation from three independent replicates. Statistical analyses were performed using GraphPad Prism 9.0: one-way ANOVA with LSD post-hoc test for multiple comparisons; Log-rank (Mantel-Cox) test for survival analysis; two-way ANOVA for phage properties under varying conditions. P < 0.05 was considered significant. 3. Results 3.1 Isolation and microscopy Using the double-layer agar method, we isolated a lytic bacteriophage from poultry farm sewage. After five purification rounds, SEP1 formed clear, circular plaques with halos (Fig. 1A). TEM revealed an icosahedral head (70 ± 1 nm) and a contractile tail (145 ± 2 nm) (Fig. 1B), morphologically typical of the Myoviridae family. 3.2 Biological Characteristics Host specificity profiling showed phage SEP1 exhibited lytic activity against a moderate range of Salmonella isolates, infecting 10 out of 32 tested strains (31.25%; Table 3). One-step growth analysis was performed at an MOI of 0.01, showing that SEP1 had a latent period of 20 minutes, underwent exponential phage production until reaching a plateau at 120 minutes, with a mean burst size of 141 PFU/infected cell (Fig. 2A). The optimal MOI for SEP1 was determined to be 0.01, at which a maximal phage titer of 6.9×10 7 PFU/mL was achieved after 4 hours of propagation in S. Paratyphi QH (Fig. 2B). SEP1 retained lytic activity across a certain temperature range (10–50°C) but was irreversibly inactivated at temperatures > 50°C (Fig. 2C) and showed a certain pH resilience, maintaining infectivity between pH 4 and 9 with complete inactivation only observed under extreme pH conditions (pH 10; Fig. 2D). 3.3 Genomic Characterization The complete genome sequence of phage SEP1 (GenBank accession: MW311372.1) is an 85,703 bp linear double-stranded DNA molecule containing 123 predicted coding sequences, of which 67 ORFs were functionally annotated through homology analysis (the remaining 56 encode hypothetical proteins). Functional annotation grouped these ORFs into three distinct modules: structural components (major capsid, portal, tail sheath, tail fiber proteins), DNA replication/repair machinery (DNA helicase, primase, polymerase), and a host lysis system (holin, endolysin, spanin proteins). Notably, the genome lacks lysogeny-associated genes (e.g., integrases, excisionases, repressors) but encodes eight tRNA genes (for Asp, Trp, Arg, Leu, Pro, Met, Tyr, Thr) (Fig. 3A, Table 4). Phylogenetic analysis showed SEP1 clusters most closely with Shigella phage Tf (GenBank: OR980947.1) and Escherichia phage garuso (GenBank: MN850566.1)—both members of the Felixounavirus genus. Escherichia phage garuso (isolated in Denmark in 2019) has an 85,798 bp genome encoding 126 protein-coding genes [ 21 ], while Shigella phage Tf has a similarly sized genome (89,550 bp) with 132 predicted genes—this shared phylogenetic lineage supports SEP1’s taxonomic placement within Felixounavirus (Fig. 3B). Comparative analysis of the major capsid protein (MCP) further confirmed this placement, showing high amino acid identity to Salmonella phage MBP4696116 (GenBank: OP515798.1; Fig. 3C). Using Easyfig, alignment of linear genome maps of SEP1 and its related phages revealed conserved functional modules (e.g., those involved in phage lysis and DNA packaging) and variable genomic regions (Fig. 3D). 3.4 Effects of Phage SEP1 on the Phenotype of Mice Infected with S. Paratyphi All mice in the challenge-only group succumbed within three days post-infection. In contrast, SEP1 treatment resulted in 100% survival, matching the PBS and phage-only control groups (Fig. 4A). Untreated infected mice exhibited significant weight loss (18.58 ± 0.56 g to 16.48 ± 0.62 g by day 2) and a rapid decline in BCS to 0 by day 3, accompanied by lethargy, hunched posture, and piloerection (Fig. 4B, C). Phage-treated mice maintained steady weight gain (18.88 ± 0.29 g to 20.40 ± 0.47 g over 7 days) and near-normal BCS, with only a transient decrease on day 2. The phage-treated group showed no significant difference from controls (p > 0.05) but differed from the challenge group by day 2 (p < 0.01). 3.5 Effect of phage SEP1 on bacterial load in liver and spleen of infected mice The bacterial challenge model established a rapidly lethal infection, with all mice in the challenge group succumbing by day 3 post-infection. This mortality was associated with uncontrolled bacterial proliferation in both the liver and spleen, as quantified in Fig. 5. In contrast, phage intervention proved effective in controlling the infection. By day 5, bacterial loads in the treated group had declined to approximately 10² CFU/g in both organs, and fell below the detection threshold by day 7. It is worth noting that throughout the study, neither the PBS nor the Phage-only group showed any detectable bacterial presence, ruling out nonspecific or phage-derived effects. 3.6 Effect of phage SEP1 on pathological damage of liver, spleen and gut in infected mice Histopathological examination revealed that phage SEP1 treatment reduced organ damage in mice challenged with S. Paratyphi (Fig. 6). In the infection-only group, severe pathological alterations were consistently observed in all tissues examined. Livers from these animals presented extensive hepatocellular necrosis accompanied by inflammatory foci and sinusoidal congestion, resulting in a score of 3. The spleen similarly showed substantial structural disruption, characterized by loss of lymphoid follicles and significant inflammatory infiltration (score: 3). In the gut, the damage manifested as villous atrophy, crypt necrosis, and diffuse inflammatory cell influx, also scoring 3. In contrast, the phage-treated group exhibited well-preserved tissue integrity across organs. Their livers displayed minimal inflammatory infiltrates while maintaining normal radial lobular organization, which corresponded to a score of 1. Splenic architecture remained largely intact, showing only mild red pulp congestion and minor follicular changes (score: 1). The gut tissue similarly demonstrated near-normal villous and crypt morphology, with just mild edema and limited inflammatory cell presence (score: 1). All control groups, whether receiving PBS or phage alone, showed normal histological features in all three organs, scoring 0 in each case. 3.7 Effects of phage SEP1 on peripheral blood cytokines in infected mice In the challenge group, cytokine levels increased by day 3 before universal mortality (Fig. 7). At day 1, IL-1β, IL-10, and IFN-γ levels in the phage group were comparable to the challenge group, while IL-6 was lower. By day 3, the phage group showed substantially reduced IL-1β, IL-6, and IFN-γ, and a sharp drop in IL-10. By day 5, IL-1β and IL-6 in the phage group were similar to controls, IL-10 returned to baseline, while IFN-γ remained slightly elevated. By day 7, all cytokines in the phage group normalized to control levels. 4. Discussion This study focused on the isolation and characterization of a novel lytic Salmonella phage, SEP1, and systematically evaluated its therapeutic potential in a mice model of S. Paratyphi infection. The results indicated that SEP1 possessed several good biological characteristics, including broad stability across a range of temperatures and pH levels, a relatively short incubation period, substantial burst production, and a distinct lytic cycle, with no detection of genes associated with lytic activity. These features aligned with its taxonomic classification within the Felixounavirus genus [ 22 , 23 ]. Members of this genus are generally reported to have a conserved genome structure and to lack virulence-associated genes, which further supported the taxonomic placement of SEP1 and its inherent biological advantages. These characteristics suggested that phage SEP1 held promising potential for biocontrol applications while circumventing the biosafety risks associated with temperate phages, such as the transfer of virulence factors and antibiotic resistance genes via lysogenic conversion [ 24 – 26 ]. However, it should be noted that although SEP1 was a virulent phage, this did not equate to absolute safety. Its genetic stability under prolonged environmental exposure and the potential for genetic material exchange with host bacterium required further investigation [ 27 , 28 ]. Furthermore, whole-genome sequencing analysis relied on comparisons with known databases; therefore, genes with NCBI functions or newly emerging potential risk genes might have been overlooked [ 29 ]. Additionally, while SEP1 exhibited a moderate host range, it encompassed several clinically relevant Salmonella serotypes, supporting its targeted application [ 30 , 31 ]. Nevertheless, we acknowledged that, compared to antibiotics, its narrower host range could limit broader application. This limitation might potentially be overcome in the future through well-designed phage cocktail therapies—for example, by combining phages with complementary host ranges capable of lysing multiple Salmonella serotypes (including Enteritidis , Typhimurium , and Infantis ) to enhance efficacy in complex infection models [ 32 , 33 ]. Thus, the application of SEP1 as a monotherapy appeared relatively limited, and its greater value might lie as a component of future cocktail therapies. Regarding in vivo therapeutic efficacy, SEP1 demonstrated the ability to rescue mice from lethal infection, indicating its potential for clinical application. However, it is important to emphasize that this result was obtained under a highly controlled laboratory infection model (using a single bacterial strain with specific infection dose and timing). The treatment regimen was designed based on the pathological process of salmonellosis. The initial intraperitoneal injection administered 30 minutes post-infection ensured rapid entry of the phage into the circulation to address early bacteremia. Subsequent injections over three consecutive days aimed to maintain effective phage concentrations in the blood and target organs for sustained bacterial clearance [ 34 , 35 ]. Accordingly, all treated animals survived, exhibiting very mild tissue damage and a rapid decrease in bacterial load in organs. These outcomes correlated with weight recovery, improved clinical scores, and normalization of inflammatory cytokine. These results were consistent with the effects reported for similar Salmonella phage therapies [ 36 , 37 ]. However, these positive indicators primarily reflected short-term outcomes; long-term survival, the possibility of infection recurrence, and potential delayed side effects were not assessed. Furthermore, SEP1 treatment suppressed the infection-induced cytokine storm, reducing levels of IL-1β, IL-6, and IFN-γ, which contributed to the restoration of immune balance [ 38 , 39 ]. This immunomodulatory effect should be interpreted cautiously, as it was likely an indirect consequence of reduced bacterial load rather than a direct immunomodulatory function of the phage. The specific mechanisms underlying this effect and its reproducibility under different immune contexts require further elucidation. Although probably indirect, the observation suggested that timely phage administration might help control excessive inflammation [ 40 , 41 ].However, directly linking it to critical determinants in the progression of sepsis or bacteremia is not sufficiently supported by current evidence, and more refined mechanistic studies are needed to establish causal relationships. A further consideration is that the single-strain and single-phage strategy used in this study did not fully reflect the genetic diversity of natural Salmonella populations or more complex infection [ 42 ]. Salmonella populations exhibit extensive genomic variation, including differences in surface receptors that may limit the host range of a single phage—a challenge that single-phage studies cannot fully address [ 43 , 44 ]. Moreover, the study did not evaluate SEP1's efficacy against a broader panel of clinical isolates (particularly multidrug-resistant strains), nor did it assess its effectiveness in co-infection settings or under microbial competition. Future work should assess the clinical therapeutic potential of SEP1 in more diverse contexts, such as polymicrobial infection or biofilm-associated models, and in other animal species. In summary, this study conducted experiments on the biological properties, genomics, and therapeutic efficacy of Salmonella phage SEP1. Based on its clear genetic background, good environmental tolerance, safety profile, and therapeutic effect, SEP1 merits further research and development as a component of a precision antibacterial strategy, especially amid the growing challenge of antibiotic resistance. However, it is crucial to recognize that translating phages from preclinical research to successful clinical application requires overcoming numerous hurdles. These include, but are not limited to: establishing scalable production and purification processes, studying formulation stability, conducting rigorous regulatory compliance assessments, and performing clinical trials to evaluate safety and efficacy in humans [ 45 – 47 ]. 5. Conclusions As a novel Felixounavirus , phage SEP1 exhibits potent lytic activity against S. Paratyphi , coupled with robust environmental stability. In a murine model, SEP1 administration ensured complete survival by effectively clearing bacterial loads, mitigating histopathological damage, and restoring immune balance. These findings position SEP1 as a promising therapeutic candidate for combating S. Paratyphi infections. Declarations Funding This work was supported by the Noncommunicable chronic Diseases National Science and Technology Major Project (2025ZD0549300), the National Natural Science Foundation of China (32460885) and the Key R&D Program of Henan Province (242102111032). Acknowledgments We thank Prof Xuerui Li and Dr.Jianhua Zhou for their technical support. Data availability The complete genome sequence of bacteriophage SEP1 reported in this study is publicly available in the GenBank database under the accession number MW311372.1. The full sequence can be accessed via the following link: https://www.ncbi.nlm.nih.gov/nuccore/MW311372.1. All other relevant data supporting the findings of this study are available from the corresponding authors upon reasonable request. Ethics approval and consent to participate This study was conducted in accordance with the guidelines approved by the Animal Ethics Committee of Tarim University (Approval No. 2023061). All animal experimental procedures strictly followed the Guidelines for the Ethical Review of Laboratory Animal Welfare (GB/T 39760 − 2021). This study did not involve human participants, so informed consent to participate was not applicable. Competing interests The authors declare no competing interests. Author details 1. College of Animal Science and Technology, Tarim University, Alar, Xinjiang 843300 China 2. Senior Department of Pulmonary and Critical Care Medicine, Chinese PLA General Hospital 3. Center for Animal Disease Control and Prevention, Yushu, Qinghai, 815000, China 4. Qinghai Academy of Animal Sciences and Veterinary Medicine, Qinghai University, Xining 810016, China 5. College of Veterinary Medicine, Henan Agricultural University, zhengzhou 450046, PR China 6. Engineering Laboratory of Tarim Animal Diseases Diagnosis and Control, Xinjiang Production & Construction Corps 843300 China *Correspondence authors: Chunyan Xu, PhD; College of Veterinary Medicine, Henan Agricultural University, zhengzhou, 450046, PR China E-mail: [email protected] Shengyi Han, PhD; College of Animal Science and Veterinary Science, Qinghai University, Xining, 810016, China E-mail: [email protected] References Andino A, Hanning I. 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Species-Scale Genomic Analysis of Staphylococcus aureus Genes Influencing Phage Host Range and Their Relationships to Virulence and Antibiotic Resistance Genes. mSystems. 2022;7(1):e0108321. 10.1128/msystems.01083-21 . Adler BA, Kazakov AE, Zhong C, Liu H, Kutter E, Lui LM, et al. The genetic basis of phage susceptibility, cross-resistance and host-range in Salmonella. Microbiol (Reading). 2021;167(12). 10.1099/mic.0.001126 . Gao D, Ji H, Wang L, Li X, Hu D, Zhao J, et al. Fitness Trade-Offs in Phage Cocktail-Resistant Salmonella enterica Serovar Enteritidis Results in Increased Antibiotic Susceptibility and Reduced Virulence. Microbiol Spectr. 2022;10(5):e0291422. 10.1128/spectrum.02914-22 . Petrovic Fabijan A, Iredell J, Danis-Wlodarczyk K, Kebriaei R, Abedon ST. Translating phage therapy into the clinic: Recent accomplishments but continuing challenges. PLoS Biol. 2023;21(5):e3002119. 10.1371/journal.pbio.3002119 . Faltus T. The Medicinal Phage-Regulatory Roadmap for Phage Therapy under EU Pharmaceutical Legislation. Viruses. 2024;16(3). 10.3390/v16030443 . Wdowiak M, Paczesny J, Raza S. Enhancing the Stability of Bacteriophages Using Physical, Chemical, and Nano-Based Approaches: A Review. Pharmaceutics. 2022;14(9). 10.3390/pharmaceutics14091936 . Tables Tables 1 to 4 are available in the Supplementary Files section. Additional Declarations No competing interests reported. Supplementary Files Table.zip Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-8201091","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":576168979,"identity":"d77f38e7-e41e-482a-a9a3-f429310059f8","order_by":0,"name":"Zhiyi 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13:24:17","extension":"html","order_by":6,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":128000,"visible":true,"origin":"","legend":"","description":"","filename":"earlyproof.html","url":"https://assets-eu.researchsquare.com/files/rs-8201091/v1/455cf9921c47ed81c5fa6717.html"},{"id":100687987,"identity":"b242eb66-9fd2-4c28-9802-3f16b6730f74","added_by":"auto","created_at":"2026-01-20 13:24:32","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":1900566,"visible":true,"origin":"","legend":"\u003cp\u003e(A) Plaque morphology of phage SEP1 on double-layer agar plates; (B) Transmission electron micrograph showing the phage structure of SEP1.\u003c/p\u003e","description":"","filename":"Figure1AandB.png","url":"https://assets-eu.researchsquare.com/files/rs-8201091/v1/949cdbf1a6974989beced493.png"},{"id":100688131,"identity":"ced686ec-f470-4050-a651-e7e09a7589f4","added_by":"auto","created_at":"2026-01-20 13:26:50","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":889046,"visible":true,"origin":"","legend":"\u003cp\u003eThe biological characteristics of phage SEP1.\u003c/p\u003e","description":"","filename":"Figure2ABCD.png","url":"https://assets-eu.researchsquare.com/files/rs-8201091/v1/064b4fb3a1996b42e85fdc1a.png"},{"id":100688047,"identity":"1e7d6522-144b-4c35-a347-2f99443d2e10","added_by":"auto","created_at":"2026-01-20 13:25:40","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":2079800,"visible":true,"origin":"","legend":"\u003cp\u003e(A) The genome annotation of phage SEP1;(B) Genome similarity comparison of phage SEP1 with other phages; (C) The phylogenetic tree base on the major capsid protein; (D) Genome-wide comparison of phage SEP1 and other phages.\u003c/p\u003e","description":"","filename":"Figure3ABCD.png","url":"https://assets-eu.researchsquare.com/files/rs-8201091/v1/4a6dae8c154116a25ba79bf0.png"},{"id":100688052,"identity":"e97a01e9-5f00-4856-a4fa-c5ff45f35497","added_by":"auto","created_at":"2026-01-20 13:25:59","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":1215357,"visible":true,"origin":"","legend":"\u003cp\u003e(A) Survival rate; (B) Body weight changes; (C) Body condition scoring.\u003c/p\u003e","description":"","filename":"Figure4ABC.png","url":"https://assets-eu.researchsquare.com/files/rs-8201091/v1/7db677740e26fac600d291bc.png"},{"id":100687978,"identity":"b2f4c0a1-495a-468b-89f5-5527dda674f5","added_by":"auto","created_at":"2026-01-20 13:24:14","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":530651,"visible":true,"origin":"","legend":"\u003cp\u003eBacterial loads in liver and spleen\u003c/p\u003e","description":"","filename":"Figure5.png","url":"https://assets-eu.researchsquare.com/files/rs-8201091/v1/1213a5b32cb38da7e0b6d763.png"},{"id":100688136,"identity":"c9375516-f622-4dd0-b75b-141e5e128ca4","added_by":"auto","created_at":"2026-01-20 13:26:56","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":7898610,"visible":true,"origin":"","legend":"\u003cp\u003ePathological changes in spleen, liver and gut\u003c/p\u003e","description":"","filename":"Figure6.png","url":"https://assets-eu.researchsquare.com/files/rs-8201091/v1/17c8c178a7a739c8508b8c6c.png"},{"id":100688151,"identity":"06246a3f-5619-45a3-a288-356f5bd9e1a0","added_by":"auto","created_at":"2026-01-20 13:27:09","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":902773,"visible":true,"origin":"","legend":"\u003cp\u003ePeripheral blood cytokines\u003c/p\u003e","description":"","filename":"Figure7.png","url":"https://assets-eu.researchsquare.com/files/rs-8201091/v1/6930d4211c361b7547917851.png"},{"id":102826646,"identity":"2727ec2e-412b-46d8-8a54-2c6998320bc2","added_by":"auto","created_at":"2026-02-17 08:57:29","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":16632725,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8201091/v1/37ad8799-9914-4a64-bcf5-120efcd74eda.pdf"},{"id":100688080,"identity":"99cccf68-0695-424d-b81a-880e504bd3eb","added_by":"auto","created_at":"2026-01-20 13:26:19","extension":"zip","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":44647,"visible":true,"origin":"","legend":"","description":"","filename":"Table.zip","url":"https://assets-eu.researchsquare.com/files/rs-8201091/v1/6208e62767fcf9e98c64e4f9.zip"}],"financialInterests":"No competing interests reported.","formattedTitle":"Isolation, Identification, and In Vivo Anti-Infective Efficacy of Lytic Bacteriophage SEP1 Against Salmonella Paratyphi","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003e \u003cem\u003eSalmonella\u003c/em\u003e, a facultative anaerobic, Gram-negative rod-shaped bacterium of the \u003cem\u003eEnterobacteriaceae\u003c/em\u003e family, represents a major global foodborne pathogen impacting both human and animal health [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. Its remarkable adaptability facilitates persistent colonization in diverse agricultural environments\u0026mdash;including soil, water, and animal feed\u0026mdash;with the fecal-oral route serving as the primary mode of transmission [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. While numerous serovars contribute to its global burden, \u003cem\u003eS. Paratyphi\u003c/em\u003e is particularly notable for causing enteric fever, a systemic illness whose severity contrasts sharply with the typically self-limiting gastroenteritis associated with other foodborne strains [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. Humans and domestic animals act as key reservoirs, and clinical outcomes range from asymptomatic carriage to life-threatening septicemia, largely influenced by host immunity and an array of sophisticated bacterial virulence mechanisms[\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. Although antibiotics remain the standard treatment for invasive salmonellosis, the increasing prevalence of multidrug-resistant strains and the emergence of persistent bacterial subpopulations have progressively compromised their efficacy [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e],This reality underscores the growing urgency for developing alternative therapeutic approaches.\u003c/p\u003e \u003cp\u003eIn this context, bacteriophages (phages)\u0026mdash;viruses that specifically infect and replicate within bacteria\u0026mdash;have regained prominence as promising antimicrobial agents. Their life cycle can proceed through a lytic pathway, directly lysing the host bacterium, or a lysogenic one, facilitating genetic exchange. Phages offer several unique advantages for clinical and biotechnological applications: high specificity, an inherent capacity for self-amplification at infection sites, and minimal toxicity to the host [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. Notably, \u003cem\u003eSalmonella\u003c/em\u003e-targeting phages have demonstrated potential in several areas, from enabling rapid pathogen detection and enhancing food safety through biocontrol to serving as effective therapeutics against active infections [\u003cspan additionalcitationids=\"CR11 CR12 CR13\" citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. Against this backdrop, the current study aims to contribute to the field by characterizing SEP1, a novel lytic phage targeting \u003cem\u003eS. Paratyphi\u003c/em\u003e. We present its complete genome sequence alongside detailed morphological and biological analyses. Furthermore, we critically evaluate the therapeutic potential of SEP1 using a murine infection model, highlighting its viability as an alternative to conventional antibiotics.\u003c/p\u003e"},{"header":"2. Materials and Methods","content":"\u003cp\u003e\u003cstrong\u003e2.1 Experimental Materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.1.1 Strains and Samples\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFor this study, 32 \u003cem\u003eSalmonella\u003c/em\u003e strains (including the one used for in vivo experiments) were obtained from the Engineering Laboratory of Tarim Animal Diseases Diagnosis and Control, Xinjiang Production \u0026amp; Construction Corps. Sewage samples used in the experiment were collected from various poultry farms in Lanzhou City (Sampling point coordinates: 102°36′-104°35′E, 35°34′-37°00′N).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.1.2 Experimental Animals\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFemale SPF BALB/c mice (6–8 weeks old, 16–22 g) were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. All animals were housed under standard laboratory conditions with ad libitum access to sterile water and a commercial rodent diet (Beijing Ke'ao Xieli Feed Co., Ltd.). After a two-day acclimatization period, experiments were conducted in accordance with guidelines approved by the Animal Ethics Committee of Tarim University (Approval No. 2023061), following the Guidelines for the Ethical Review of Laboratory Animal Welfare (GB/T 35892-2018).\u003c/p\u003e\n\u003cp\u003eNote: This study only involved animal experiments and did not include human clinical trials; thus, Clinical trial number: not applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.2 Isolation, Purification, Biological Characterization and Genomic Analysis of Phage SEP1\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.2.1 Phage isolation and purification\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA bacteriophage was isolated from poultry farm sewage using\u003cem\u003e\u0026nbsp;S. Paratyphi\u003c/em\u003e QH as the host. Approximately 20 g of sewage was suspended in 100 mL of SM buffer (0.01% gelatin (w/v), 100 mM NaCl, 10 mM MgSO₄, 50 mM Tris-HCl, pH 7.5) and centrifuged at 8,000 ×g for 10 min. The supernatant was filtered through a 0.22 μm membrane. Then, 10 mL of filtrate was mixed with 10 mL of a \u003cem\u003eS. Paratyphi\u003c/em\u003e QH culture (OD\u003csub\u003e600\u003c/sub\u003e ≈ 0.6) and incubated with shaking (180 r/min) at 37°C for 12 h. After centrifugation (8,000 ×g, 10 min), 100 µL of supernatant was adsorbed with 100 µL of fresh host culture (OD\u003csub\u003e600\u0026nbsp;\u003c/sub\u003e≈ 1.2) for 10 min at 37°C.\u003c/p\u003e\n\u003cp\u003ePlaque isolation was performed using the double-layer agar method: 200 µL of the adsorption mixture was mixed with 0.7% LB soft agar and overlaid onto solid LB agar plates. After incubation at 37°C for 12 h, visible plaques were subjected to five successive rounds of purification. Phage concentration and purification followed Gavrić and Knežević\u0026nbsp;[15], involving CsCl density gradient centrifugation (densities: 1.45, 1.50, 1.70 g/mL) and ultracentrifugation at 25,000 ×g for 4 h at 4°C.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.2.2\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eMorphological observation\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA purified phage suspension (10\u003csup\u003e9\u003c/sup\u003e–10\u003csup\u003e10\u003c/sup\u003ePFU/mL) was applied to a 400-mesh carbon-coated copper grid for 10 min. After blotting excess liquid, the grid was negatively stained with 1% phosphotungstic acid for 2 min. Samples were air-dried and imaged using an HT7700 TEM (Hitachi, Japan) at 50 kV.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.2.3 Host range\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe host range of SEP1 was determined against 31\u003cem\u003e\u0026nbsp;Salmonella\u003c/em\u003e strains using the standard double-layer agar plaque assay. Lytic activity was qualitatively assessed and quantified using a predefined infection efficiency scale (Table 1).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.2.4 One step growth curve\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eHost cultures (OD\u003csub\u003e600\u003c/sub\u003e = 0.6) were infected with phage at an MOI of 0.01 and incubated at 37°C with shaking (180 r/min). Samples (100 μL) were collected at 20 min intervals, centrifuged (8,000 ×g, 10 min, 4°C), and the supernatants were titrated using the double-layer agar method. Experiments were performed in triplicate.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.2.5 Optimal multiplicity of infection\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003ePhage serial dilutions were mixed with equal volumes of logarithmic-phase bacterial cultures to achieve MOIs of 0.0001 to 10. After adsorption (10 min, 37°C), mixtures were centrifuged (8,000 ×g, 10 min), pellets resuspended in 10 mL fresh LB broth, and cultured (180 r/min, 37°C) for 5 h. Cultures were centrifuged, supernatants filtered (0.22 μm), and phage titers determined. All experiments were conducted in triplicate.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.2.6 Temperature and pH stability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFor thermal stability, phage suspensions were incubated at 10–70°C (10°C increments) for 1 h, and viable particles were quantified. For pH stability, 100 µL phage suspension was mixed with 900 µL SM buffer adjusted to pH 2–13, incubated at 37°C for 1 h, and titrated. All assays were performed in triplicate.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.2.7\u003c/strong\u003e \u003cstrong\u003ePhage genome extraction, sequencing, and analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003ePhage genomic DNA was extracted using the Bacteriophage DNA Isolation Kit (Norgen Biotek, Canada). Sequencing libraries were prepared using the Illumina TruSeq Nano DNA LT Library Prep Kit. Raw reads were processed with Trimmomatic (v0.39) and assembled de novo using SPAdes (v3.12.0). ORFs were predicted and annotated via BLASTp. Antibiotic resistance and virulence genes were screened using ResFinder and VirulenceFinder databases. tRNA genes were predicted with tRNAscan-SE 2.0. A neighbor-joining phylogenetic tree was constructed in MEGA 7.0 [16]based on major capsid protein sequences. Intergenomic comparisons were performed with VIRIDIC [17], and genomic comparisons were visualized using Easyfig 2.2.5 [18]\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.3 In Vivo Experimental Design and Treatment\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.3.1 Preparation of challenge solution\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eS. Paratyphi\u003c/em\u003e QH was grown in LB medium at 37°C with shaking (180 r/min) to late logarithmic phase (OD\u003csub\u003e600\u003c/sub\u003e ≈ 5.0 × 10\u003csup\u003e8\u003c/sup\u003e CFU/mL). Cells were harvested by centrifugation (8,000 ×g, 10 min, 4°C), resuspended in sterile PBS, and adjusted to 2.0 × 10\u003csup\u003e8\u003c/sup\u003e CFU/mL.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.3.2 Grouping and processing\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eBALB/c mice were randomly divided into four groups (n=12 per group):\u003c/p\u003e\n\u003cp\u003eChallenge group: Intraperitoneal (i.p.) injection of 100 μL \u003cem\u003eS. Paratyphi\u003c/em\u003e QH suspension (2.0 × 10\u003csup\u003e8\u003c/sup\u003e CFU/mice). After 30 min, 100 μL sterile PBS was administered i.p., repeated daily for three days.\u003c/p\u003e\n\u003cp\u003ePhage treatment group: i.p. injection of bacterial suspension as above. After 30 min, 100 μL phage SEP1 suspension (5.0 × 10\u003csup\u003e10\u003c/sup\u003e PFU/mice, MOI=0.01) was given i.p., followed by an i.p. injection of the same dose daily for the next three days.\u003c/p\u003e\n\u003cp\u003ePBS control group: i.p. injection of 100 μL sterile PBS daily for three days.\u003c/p\u003e\n\u003cp\u003ePhage control group: i.p. injection of 100 μL phage SEP1 suspension (5.0 × 10\u003csup\u003e10\u003c/sup\u003ePFU/mL) daily for three days.\u003c/p\u003e\n\u003cp\u003eAfter treatments concluded on day 3, a seven-day observation period followed. General health status was monitored daily.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.4 Detection Indicators and Methods\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.4.1 Animal euthanasia method\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWhen it was necessary to collect tissue and blood samples (on days 1, 3, 5, and 7 post - infection), the mice were euthanized humanely following the ethical requirements of laboratory animal welfare. First, the mice were anesthetized with an intraperitoneal injection of pentobarbital sodium at a dosage of 50 mg/kg body weight. After confirming that the mice were completely unconscious (judged by the loss of corneal reflex and pain response), cervical dislocation was performed to ensure rapid and painless death. This euthanasia method was selected to minimize animal suffering and comply with the Guidelines for the Ethical Review of Laboratory Animal Welfare (GB/T 39760 − 2021).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.4.2 Mice phenotypic monitoring\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSurvival rates were recorded daily. Body weight was measured daily at 9:00 AM. Body condition scores (BCS) were assessed daily using a scale of 0 (dead) to 5 (healthy)(Table2) [19].\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.4.3 Detection of bacterial loads in liver and spleen\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eOn days 1, 3, 5, and 7 post-infection, liver and spleen samples were aseptically collected from three mice per group. Tissues (0.5 g) were homogenized in 4.5 mL PBS. Serial dilutions were plated on LB agar, incubated at 37°C for 12 h, and CFU/g calculated.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.4.4 Histopathological analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eLiver, spleen, and jejunum samples were fixed in 4% paraformaldehyde, dehydrated, embedded in paraffin, sectioned at 5 μm, and stained with H\u0026amp;E. Sections were examined under an Olympus BX53 microscope. Pathology scores were assigned as follows:[20]\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eLiver: 0 (normal), 1 (mild edema), 2 (moderate necrosis, localized inflammation), 3 (severe necrosis, inflammatory foci, congestion).\u003c/p\u003e\n\u003cp\u003eSpleen: 0 (normal), 1 (minimal apoptosis), 2 (moderate congestion, inflammation), 3 (severe necrosis, follicle loss, extensive inflammation).\u003c/p\u003e\n\u003cp\u003eGut: 0 (normal), 1 (mild villous edema), 2 (partial villous disruption, mild crypt damage), 3 (extensive villous loss, crypt necrosis, massive inflammation).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.4.4 Peripheral blood cytokine levels\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eBlood was collected on days 1, 3, 5, and 7. Plasma levels of IL-1β, IL-6, IL-10, and IFN-γ were quantified using ELISA kits according to the manufacturer’s instructions.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.5 Statistical analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eData are presented as mean ± standard deviation from three independent replicates. Statistical analyses were performed using GraphPad Prism 9.0: one-way ANOVA with LSD post-hoc test for multiple comparisons; Log-rank (Mantel-Cox) test for survival analysis; two-way ANOVA for phage properties under varying conditions. P \u0026lt; 0.05 was considered significant.\u003c/p\u003e"},{"header":"3. Results","content":"\u003cdiv id=\"Sec25\" class=\"Section2\"\u003e \u003ch2\u003e3.1 Isolation and microscopy\u003c/h2\u003e \u003cp\u003eUsing the double-layer agar method, we isolated a lytic bacteriophage from poultry farm sewage. After five purification rounds, SEP1 formed clear, circular plaques with halos (Fig.\u0026nbsp;1A). TEM revealed an icosahedral head (70\u0026thinsp;\u0026plusmn;\u0026thinsp;1 nm) and a contractile tail (145\u0026thinsp;\u0026plusmn;\u0026thinsp;2 nm) (Fig.\u0026nbsp;1B), morphologically typical of the \u003cem\u003eMyoviridae\u003c/em\u003e family.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec26\" class=\"Section2\"\u003e \u003ch2\u003e3.2 Biological Characteristics\u003c/h2\u003e \u003cp\u003eHost specificity profiling showed phage SEP1 exhibited lytic activity against a moderate range of \u003cem\u003eSalmonella\u003c/em\u003e isolates, infecting 10 out of 32 tested strains (31.25%; Table\u0026nbsp;3).\u003c/p\u003e \u003cp\u003eOne-step growth analysis was performed at an MOI of 0.01, showing that SEP1 had a latent period of 20 minutes, underwent exponential phage production until reaching a plateau at 120 minutes, with a mean burst size of 141 PFU/infected cell (Fig.\u0026nbsp;2A).\u003c/p\u003e \u003cp\u003eThe optimal MOI for SEP1 was determined to be 0.01, at which a maximal phage titer of 6.9\u0026times;10\u003csup\u003e7\u003c/sup\u003e PFU/mL was achieved after 4 hours of propagation in \u003cem\u003eS. Paratyphi\u003c/em\u003e QH (Fig.\u0026nbsp;2B).\u003c/p\u003e \u003cp\u003eSEP1 retained lytic activity across a certain temperature range (10\u0026ndash;50\u0026deg;C) but was irreversibly inactivated at temperatures\u0026thinsp;\u0026gt;\u0026thinsp;50\u0026deg;C (Fig.\u0026nbsp;2C) and showed a certain pH resilience, maintaining infectivity between pH 4 and 9 with complete inactivation only observed under extreme pH conditions (pH\u0026thinsp;\u0026lt;\u0026thinsp;3 or \u0026gt;\u0026thinsp;10; Fig.\u0026nbsp;2D).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec27\" class=\"Section2\"\u003e \u003ch2\u003e3.3 Genomic Characterization\u003c/h2\u003e \u003cp\u003eThe complete genome sequence of phage SEP1 (GenBank accession: MW311372.1) is an 85,703 bp linear double-stranded DNA molecule containing 123 predicted coding sequences, of which 67 ORFs were functionally annotated through homology analysis (the remaining 56 encode hypothetical proteins). Functional annotation grouped these ORFs into three distinct modules: structural components (major capsid, portal, tail sheath, tail fiber proteins), DNA replication/repair machinery (DNA helicase, primase, polymerase), and a host lysis system (holin, endolysin, spanin proteins). Notably, the genome lacks lysogeny-associated genes (e.g., integrases, excisionases, repressors) but encodes eight tRNA genes (for Asp, Trp, Arg, Leu, Pro, Met, Tyr, Thr) (Fig.\u0026nbsp;3A, Table\u0026nbsp;4).\u003c/p\u003e \u003cp\u003ePhylogenetic analysis showed SEP1 clusters most closely with Shigella phage Tf (GenBank: OR980947.1) and Escherichia phage garuso (GenBank: MN850566.1)\u0026mdash;both members of the \u003cem\u003eFelixounavirus\u003c/em\u003e genus. Escherichia phage garuso (isolated in Denmark in 2019) has an 85,798 bp genome encoding 126 protein-coding genes [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e], while Shigella phage Tf has a similarly sized genome (89,550 bp) with 132 predicted genes\u0026mdash;this shared phylogenetic lineage supports SEP1\u0026rsquo;s taxonomic placement within \u003cem\u003eFelixounavirus\u003c/em\u003e (Fig.\u0026nbsp;3B). Comparative analysis of the major capsid protein (MCP) further confirmed this placement, showing high amino acid identity to Salmonella phage MBP4696116 (GenBank: OP515798.1; Fig.\u0026nbsp;3C). Using Easyfig, alignment of linear genome maps of SEP1 and its related phages revealed conserved functional modules (e.g., those involved in phage lysis and DNA packaging) and variable genomic regions (Fig.\u0026nbsp;3D).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec28\" class=\"Section2\"\u003e \u003ch2\u003e3.4 Effects of Phage SEP1 on the Phenotype of Mice Infected with \u003cem\u003eS. Paratyphi\u003c/em\u003e\u003c/h2\u003e \u003cp\u003eAll mice in the challenge-only group succumbed within three days post-infection. In contrast, SEP1 treatment resulted in 100% survival, matching the PBS and phage-only control groups (Fig.\u0026nbsp;4A). Untreated infected mice exhibited significant weight loss (18.58\u0026thinsp;\u0026plusmn;\u0026thinsp;0.56 g to 16.48\u0026thinsp;\u0026plusmn;\u0026thinsp;0.62 g by day 2) and a rapid decline in BCS to 0 by day 3, accompanied by lethargy, hunched posture, and piloerection (Fig.\u0026nbsp;4B, C). Phage-treated mice maintained steady weight gain (18.88\u0026thinsp;\u0026plusmn;\u0026thinsp;0.29 g to 20.40\u0026thinsp;\u0026plusmn;\u0026thinsp;0.47 g over 7 days) and near-normal BCS, with only a transient decrease on day 2. The phage-treated group showed no significant difference from controls (p\u0026thinsp;\u0026gt;\u0026thinsp;0.05) but differed from the challenge group by day 2 (p\u0026thinsp;\u0026lt;\u0026thinsp;0.01).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec29\" class=\"Section2\"\u003e \u003ch2\u003e3.5 Effect of phage SEP1 on bacterial load in liver and spleen of infected mice\u003c/h2\u003e \u003cp\u003eThe bacterial challenge model established a rapidly lethal infection, with all mice in the challenge group succumbing by day 3 post-infection. This mortality was associated with uncontrolled bacterial proliferation in both the liver and spleen, as quantified in Fig.\u0026nbsp;5. In contrast, phage intervention proved effective in controlling the infection. By day 5, bacterial loads in the treated group had declined to approximately 10\u0026sup2; CFU/g in both organs, and fell below the detection threshold by day 7. It is worth noting that throughout the study, neither the PBS nor the Phage-only group showed any detectable bacterial presence, ruling out nonspecific or phage-derived effects.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec30\" class=\"Section2\"\u003e \u003ch2\u003e3.6 Effect of phage SEP1 on pathological damage of liver, spleen and gut in infected mice\u003c/h2\u003e \u003cp\u003eHistopathological examination revealed that phage SEP1 treatment reduced organ damage in mice challenged with \u003cem\u003eS. Paratyphi\u003c/em\u003e (Fig.\u0026nbsp;6). In the infection-only group, severe pathological alterations were consistently observed in all tissues examined. Livers from these animals presented extensive hepatocellular necrosis accompanied by inflammatory foci and sinusoidal congestion, resulting in a score of 3. The spleen similarly showed substantial structural disruption, characterized by loss of lymphoid follicles and significant inflammatory infiltration (score: 3). In the gut, the damage manifested as villous atrophy, crypt necrosis, and diffuse inflammatory cell influx, also scoring 3.\u003c/p\u003e \u003cp\u003eIn contrast, the phage-treated group exhibited well-preserved tissue integrity across organs. Their livers displayed minimal inflammatory infiltrates while maintaining normal radial lobular organization, which corresponded to a score of 1. Splenic architecture remained largely intact, showing only mild red pulp congestion and minor follicular changes (score: 1). The gut tissue similarly demonstrated near-normal villous and crypt morphology, with just mild edema and limited inflammatory cell presence (score: 1). All control groups, whether receiving PBS or phage alone, showed normal histological features in all three organs, scoring 0 in each case.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec31\" class=\"Section2\"\u003e \u003ch2\u003e3.7 Effects of phage SEP1 on peripheral blood cytokines in infected mice\u003c/h2\u003e \u003cp\u003eIn the challenge group, cytokine levels increased by day 3 before universal mortality (Fig.\u0026nbsp;7). At day 1, IL-1β, IL-10, and IFN-γ levels in the phage group were comparable to the challenge group, while IL-6 was lower. By day 3, the phage group showed substantially reduced IL-1β, IL-6, and IFN-γ, and a sharp drop in IL-10. By day 5, IL-1β and IL-6 in the phage group were similar to controls, IL-10 returned to baseline, while IFN-γ remained slightly elevated. By day 7, all cytokines in the phage group normalized to control levels.\u003c/p\u003e \u003c/div\u003e"},{"header":"4. Discussion","content":"\u003cp\u003eThis study focused on the isolation and characterization of a novel lytic \u003cem\u003eSalmonella\u003c/em\u003e phage, SEP1, and systematically evaluated its therapeutic potential in a mice model of \u003cem\u003eS. Paratyphi\u003c/em\u003e infection. The results indicated that SEP1 possessed several good biological characteristics, including broad stability across a range of temperatures and pH levels, a relatively short incubation period, substantial burst production, and a distinct lytic cycle, with no detection of genes associated with lytic activity. These features aligned with its taxonomic classification within the \u003cem\u003eFelixounavirus\u003c/em\u003e genus [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e, \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. Members of this genus are generally reported to have a conserved genome structure and to lack virulence-associated genes, which further supported the taxonomic placement of SEP1 and its inherent biological advantages. These characteristics suggested that phage SEP1 held promising potential for biocontrol applications while circumventing the biosafety risks associated with temperate phages, such as the transfer of virulence factors and antibiotic resistance genes via lysogenic conversion [\u003cspan additionalcitationids=\"CR25\" citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. However, it should be noted that although SEP1 was a virulent phage, this did not equate to absolute safety. Its genetic stability under prolonged environmental exposure and the potential for genetic material exchange with host bacterium required further investigation [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e, \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. Furthermore, whole-genome sequencing analysis relied on comparisons with known databases; therefore, genes with NCBI functions or newly emerging potential risk genes might have been overlooked [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. Additionally, while SEP1 exhibited a moderate host range, it encompassed several clinically relevant \u003cem\u003eSalmonella\u003c/em\u003e serotypes, supporting its targeted application [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e, \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. Nevertheless, we acknowledged that, compared to antibiotics, its narrower host range could limit broader application. This limitation might potentially be overcome in the future through well-designed phage cocktail therapies\u0026mdash;for example, by combining phages with complementary host ranges capable of lysing multiple \u003cem\u003eSalmonella serotypes\u003c/em\u003e (including \u003cem\u003eEnteritidis\u003c/em\u003e, \u003cem\u003eTyphimurium\u003c/em\u003e, and \u003cem\u003eInfantis\u003c/em\u003e) to enhance efficacy in complex infection models [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e, \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. Thus, the application of SEP1 as a monotherapy appeared relatively limited, and its greater value might lie as a component of future cocktail therapies.\u003c/p\u003e \u003cp\u003eRegarding in vivo therapeutic efficacy, SEP1 demonstrated the ability to rescue mice from lethal infection, indicating its potential for clinical application. However, it is important to emphasize that this result was obtained under a highly controlled laboratory infection model (using a single bacterial strain with specific infection dose and timing). The treatment regimen was designed based on the pathological process of salmonellosis. The initial intraperitoneal injection administered 30 minutes post-infection ensured rapid entry of the phage into the circulation to address early bacteremia. Subsequent injections over three consecutive days aimed to maintain effective phage concentrations in the blood and target organs for sustained bacterial clearance [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e, \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. Accordingly, all treated animals survived, exhibiting very mild tissue damage and a rapid decrease in bacterial load in organs. These outcomes correlated with weight recovery, improved clinical scores, and normalization of inflammatory cytokine. These results were consistent with the effects reported for similar \u003cem\u003eSalmonella\u003c/em\u003e phage therapies [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e, \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]. However, these positive indicators primarily reflected short-term outcomes; long-term survival, the possibility of infection recurrence, and potential delayed side effects were not assessed. Furthermore, SEP1 treatment suppressed the infection-induced cytokine storm, reducing levels of IL-1β, IL-6, and IFN-γ, which contributed to the restoration of immune balance [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e, \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e]. This immunomodulatory effect should be interpreted cautiously, as it was likely an indirect consequence of reduced bacterial load rather than a direct immunomodulatory function of the phage. The specific mechanisms underlying this effect and its reproducibility under different immune contexts require further elucidation. Although probably indirect, the observation suggested that timely phage administration might help control excessive inflammation [\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e, \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e].However, directly linking it to critical determinants in the progression of sepsis or bacteremia is not sufficiently supported by current evidence, and more refined mechanistic studies are needed to establish causal relationships.\u003c/p\u003e \u003cp\u003eA further consideration is that the single-strain and single-phage strategy used in this study did not fully reflect the genetic diversity of natural \u003cem\u003eSalmonella\u003c/em\u003e populations or more complex infection [\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e]. \u003cem\u003eSalmonella\u003c/em\u003e populations exhibit extensive genomic variation, including differences in surface receptors that may limit the host range of a single phage\u0026mdash;a challenge that single-phage studies cannot fully address [\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e, \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e]. Moreover, the study did not evaluate SEP1's efficacy against a broader panel of clinical isolates (particularly multidrug-resistant strains), nor did it assess its effectiveness in co-infection settings or under microbial competition. Future work should assess the clinical therapeutic potential of SEP1 in more diverse contexts, such as polymicrobial infection or biofilm-associated models, and in other animal species.\u003c/p\u003e \u003cp\u003eIn summary, this study conducted experiments on the biological properties, genomics, and therapeutic efficacy of \u003cem\u003eSalmonella\u003c/em\u003e phage SEP1. Based on its clear genetic background, good environmental tolerance, safety profile, and therapeutic effect, SEP1 merits further research and development as a component of a precision antibacterial strategy, especially amid the growing challenge of antibiotic resistance. However, it is crucial to recognize that translating phages from preclinical research to successful clinical application requires overcoming numerous hurdles. These include, but are not limited to: establishing scalable production and purification processes, studying formulation stability, conducting rigorous regulatory compliance assessments, and performing clinical trials to evaluate safety and efficacy in humans [\u003cspan additionalcitationids=\"CR46\" citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e].\u003c/p\u003e"},{"header":"5. Conclusions","content":"\u003cp\u003eAs a novel \u003cem\u003eFelixounavirus\u003c/em\u003e, phage SEP1 exhibits potent lytic activity against \u003cem\u003eS. Paratyphi\u003c/em\u003e, coupled with robust environmental stability. In a murine model, SEP1 administration ensured complete survival by effectively clearing bacterial loads, mitigating histopathological damage, and restoring immune balance. These findings position SEP1 as a promising therapeutic candidate for combating \u003cem\u003eS. Paratyphi\u003c/em\u003e infections.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by the Noncommunicable chronic Diseases National Science and Technology Major Project (2025ZD0549300), the National Natural Science Foundation of China (32460885) and the Key R\u0026amp;D Program of Henan Province (242102111032).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe thank Prof Xuerui Li and Dr.Jianhua Zhou for their technical support.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe complete genome sequence of bacteriophage SEP1 reported in this study is publicly available in the GenBank database under the accession number MW311372.1. The full sequence can be accessed via the following link: https://www.ncbi.nlm.nih.gov/nuccore/MW311372.1. All other relevant data supporting the findings of this study are available from the corresponding authors upon reasonable request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study was conducted in accordance with the guidelines approved by the Animal Ethics Committee of Tarim University (Approval No. 2023061). All animal experimental procedures strictly followed the Guidelines for the Ethical Review of Laboratory Animal Welfare (GB/T 39760\u0026thinsp;\u0026minus;\u0026thinsp;2021). This study did not involve human participants, so informed consent to participate was not applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor details\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e1. College of Animal Science and Technology, Tarim University, Alar, Xinjiang 843300 China\u003c/p\u003e\n\u003cp\u003e2. Senior Department of Pulmonary and Critical Care Medicine, Chinese PLA General Hospital\u003c/p\u003e\n\u003cp\u003e3. Center for Animal Disease Control and Prevention, Yushu, Qinghai, 815000, China\u003c/p\u003e\n\u003cp\u003e4. Qinghai Academy of Animal Sciences and Veterinary Medicine, Qinghai University, Xining 810016, China\u003c/p\u003e\n\u003cp\u003e5. College of Veterinary Medicine, Henan Agricultural University, zhengzhou 450046, PR China\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e6. Engineering Laboratory of Tarim Animal Diseases Diagnosis and Control, Xinjiang Production \u0026amp; Construction Corps 843300 China\u003c/p\u003e\n\u003cp\u003e*Correspondence authors:\u003c/p\u003e\n\u003cp\u003eChunyan Xu, PhD; College of Veterinary Medicine, Henan Agricultural University, zhengzhou, 450046, PR China\u003c/p\u003e\n\u003cp\u003eE-mail: [email protected]\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eShengyi Han, PhD; College of Animal Science and Veterinary Science, Qinghai University, Xining, 810016, China\u003c/p\u003e\n\u003cp\u003eE-mail: [email protected]\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eAndino A, Hanning I. Salmonella enterica: survival, colonization, and virulence differences among serovars. 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Pharmaceutics. 2022;14(9). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.3390/pharmaceutics14091936\u003c/span\u003e\u003cspan address=\"10.3390/pharmaceutics14091936\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"},{"header":"Tables","content":"\u003cp\u003eTables 1 to 4 are available in the Supplementary Files section.\u003c/p\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Salmonella Paratyphi, Bacteriophage, Biological Characteristics, Genomics, Phage therapy","lastPublishedDoi":"10.21203/rs.3.rs-8201091/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8201091/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e \u003cp\u003eThe emergence of multidrug-resistant \u003cem\u003eSalmonella Paratyphi\u003c/em\u003e has created a critical need for novel therapeutic options beyond conventional antibiotics. Bacteriophages, with their ability to selectively lyse bacterial hosts, offer a promising alternative. This study aimed to characterize the lytic phage SEP1 and evaluate its efficacy against \u003cem\u003eS. Paratyphi\u003c/em\u003e infection.\u003c/p\u003e\u003ch2\u003eMethods\u003c/h2\u003e \u003cp\u003eSEP1 was isolated from poultry farm sewage in Lanzhou, China, using \u003cem\u003eS. Paratyphi\u003c/em\u003e QH as the host. We performed transmission electron microscopy for morphological analysis, double-layer agar assays for host range, one-step growth curves for lifecycle characteristics, and stability tests under varying temperature and pH conditions. Genomic DNA was sequenced and analyzed. In vivo efficacy was assessed in a lethal murine model of \u003cem\u003eS. Paratyphi\u003c/em\u003e infection, with monitoring of survival, body weight, bacterial loads in liver and spleen, histopathology of liver/spleen/intestine, and serum cytokine levels (IL-1β, IL-6, IL-10, IFN-γ).\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003eSEP1 was classified as a member of the \u003cem\u003eFelixounavirus\u003c/em\u003e genus, featuring a double-stranded DNA genome of 85,703 bp with 39.00% GC content and 123 predicted open reading frames. No lysogenic or virulence genes were identified. The phage exhibited a host range infecting 31.25% of tested \u003cem\u003eSalmonella\u003c/em\u003e strains, a latent period of 20 minutes, and a burst size of approximately 141 PFU per cell. It remained stable at temperatures between 10\u0026ndash;50\u0026deg;C and pH values from 4 to 9. In the mice model, a single dose of SEP1 administered 30 minutes post-infection resulted in 100% survival, compared to 0% in the challenge control group (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001). Treatment prevented weight loss, reduced bacterial loads in organs (p\u0026thinsp;\u0026lt;\u0026thinsp;0.01), mitigated tissue damage, and restored cytokine levels to near baseline.\u003c/p\u003e","manuscriptTitle":"Isolation, Identification, and In Vivo Anti-Infective Efficacy of Lytic Bacteriophage SEP1 Against Salmonella Paratyphi","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-01-20 11:14:01","doi":"10.21203/rs.3.rs-8201091/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"3e1c6392-dc47-4169-b9bf-4a8c61a37196","owner":[],"postedDate":"January 20th, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2026-02-17T08:56:28+00:00","versionOfRecord":[],"versionCreatedAt":"2026-01-20 11:14:01","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8201091","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8201091","identity":"rs-8201091","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}