Phage PQ38 and application thereof as well as salmonella inhibiting medicine

Abstract

The invention belongs to the technical field of biological detection, and discloses a bacteriophage PQ38, application thereof and a salmonella inhibiting medicine, the bacteriophage PQ38 has the preservation number of GDMCC NO: 67169-B1, is preserved in Guangdong Microbial Culture Collection Center, the preservation time is October 27, 2025, and the bacteriophage PQ38 is named as Siphoviridae jerseyvirus in taxonomy; the bacteriophage has a good salmonella splitting capability in salmonella, and tests show that the bacteriophage has an excellent capability of splitting salmonella pullorum.

Description

Technical Field

[0001] This application relates to the field of biotechnology, and in particular to bacteriophage PQ38, its uses, and drugs for inhibiting Salmonella. Background Technology

[0002] Currently, researchers are exploring new alternatives to antibiotics, such as gene therapy, traditional Chinese medicine additives, and probiotics. In recent years, bacteriophages, as specific viruses capable of infecting bacteria, have been extensively studied. They are widely present in the natural environment and organisms, playing a crucial role in maintaining the dynamic balance of microorganisms in nature. Compared to antibiotics, bacteriophage preparations have attracted significant attention due to their advantages such as fewer adverse reactions, lower risk of developing drug resistance, high safety, no residue, and low cost. In recent years, scholars both domestically and internationally have published research findings on bacteriophage therapy technologies, demonstrating good therapeutic effects in agriculture, animal husbandry, aquaculture, and human diseases. Recently, the U.S. Food and Drug Administration (FDA) and the U.S. Department of Agriculture (USDA) approved Phageguard-E as a Generally Recognized As Safe (GRAS) product for food processing aids against E. coli O157, laying a solid foundation for future research on bacteriophage therapy for bacterial diseases.

[0003] Bacteriophages are a class of biological agents that lyse and kill bacteria. They exhibit strict host specificity and possess more efficient and specific bactericidal capabilities compared to traditional antibiotics. They do not disrupt the host's normal flora, can increase the proportion of beneficial bacteria in the gut, and are naturally excreted after killing bacteria, leaving no drug residue. Therefore, they are considered a potential ideal alternative to antibiotics.

[0004] The problem this solution aims to solve is: how to provide a bacteriophage suitable for Salmonella as a pathogen. Summary of the Invention

[0005] The purpose of this application is to provide a bacteriophage that has good lytic ability against Salmonella, and experiments have shown that the bacteriophage has excellent lytic ability against Salmonella pullorum.

[0006] To achieve the above objectives, this application discloses bacteriophage PQ38, with accession number GDMCC NO:67169-B1, deposited at Guangdong Provincial Center for Microbial Culture Collection on October 27, 2025, and taxonomically named Siphoviridae jerseyvirus.

[0007] In addition, this application also discloses the use of the bacteriophage preparation for the preparation of a drug that inhibits Salmonella as described above.

[0008] Preferably, the Salmonella is one of Salmonella pullorum, Salmonella enteritidis, Salmonella typhimurium, Salmonella Indiana, or Salmonella debilis.

[0009] Preferably, the Salmonella is Salmonella pullorum.

[0010] In addition, this application also discloses a drug for inhibiting Salmonella containing bacteriophage PQ38.

[0011] Preferably, the drug is a drug that inhibits Salmonella pullorum.

[0012] The beneficial effects of this application are:

[0013] This application provides a bacteriophage that exhibits good lytic ability against Salmonella, and experiments have shown that this bacteriophage has excellent lytic ability against Salmonella pullorum. Attached Figure Description

[0014] Figure 1 The inhibition curve of bacteriophage PQ38 against Salmonella Indiana;

[0015] Figure 2 The inhibition curve of phage PQ38 against Salmonella enteritidis;

[0016] Figure 3 The inhibition curve of bacteriophage PQ38 against Salmonella typhimurium;

[0017] Figure 4 The inhibition curve of bacteriophage PQ38 against Salmonella debilis;

[0018] Figure 5 The inhibition curve of bacteriophage PQ38 against Salmonella pullorum;

[0019] Figure 6 This is a one-step growth curve of phage PQ38;

[0020] Figure 7 A schematic diagram showing the optimal multiplicity of infection test results for phage PQ38;

[0021] Figure 8 A schematic diagram showing the temperature stability test results of bacteriophage PQ38;

[0022] Figure 9 This is a schematic diagram showing the pH stability test results of bacteriophage PQ38.

[0023] Figure 10 The graph shows the antibacterial effect of bacteriophage PQ38 at 4℃.

[0024] Figure 11This is a graph showing the antibacterial effect of bacteriophage PQ38 at 25℃.

[0025] Figure 12 The graph shows the antibacterial effect of bacteriophage PQ38 at 37℃.

[0026] Figure 13 A schematic diagram illustrating the inhibition of biofilm formation rate of Salmonella pullorum by bacteriophage PQ38;

[0027] Figure 14 A schematic diagram illustrating the disruption of the biofilm of Salmonella pullorum by bacteriophage PQ38;

[0028] Figure 15 This is a schematic diagram of Simpson index alpha diversity analysis of cecal microbiota.

[0029] Figure 16 This is a schematic diagram of the Shannon index alpha diversity analysis of the cecal microbiota.

[0030] Figure 17 This is a schematic diagram of the Observed_features index alpha diversity analysis of the cecal microbiota.

[0031] Figure 18 This is a schematic diagram of the Chao1 index alpha diversity analysis of the cecal microbiota.

[0032] Figure 19 A schematic diagram of the beta diversity analysis of PCoA in the cecal microbiota;

[0033] Figure 20 This is a schematic diagram of the beta diversity analysis of NMDS in the cecal microbiota. Detailed Implementation

[0034] The present application will be clearly and completely described below with reference to its embodiments. It should be noted that, unless specific conditions are specified in the embodiments, conventional conditions or conditions recommended by the manufacturer shall apply. Reagents or instruments whose manufacturers are not specified are all commercially available conventional products.

[0035] Example 1: Sequencing and physiological characteristics of bacteriophage PQ38

[0036] 1.1 Virus classification and whole genome sequencing

[0037] Phage genomes were extracted according to the instructions of the viral DNA extraction kit (Omega, D3892-01). 250 μL of the purified phage solution was taken, and 10 μL LOB Protease Solution, 4 μL L Inear Acrylamide, and 250 μL BL Buffer were added. The mixture was vortexed for 15 seconds and incubated in a 65°C water bath for 10 minutes. Then, 260 μL of anhydrous ethanol was added, vortexed, and transferred to a HiBind® DNAMini binding column. The column was centrifuged at 8000g for 1 minute at room temperature, and the filtrate and collection tube were discarded. 500 μL of HBC Buffer was added to the column, and the column was centrifuged at 8000g for 1 minute, and the filtrate was discarded. 700 μL of DNA Wash Buffer was added to the column, and the column was centrifuged at 8000g for 1 minute, and the filtrate was discarded. The column was then centrifuged at 12000g using HiBind®... After eluting the DNAMini column for 5 minutes to remove residual liquid (especially ethanol), insert the column into a new centrifuge tube, add 50 μL of preheated (65°C) Elution Buffer to the column, incubate at room temperature for 5 minutes, and then centrifuge at 8000g for 1 minute to recover the DNA. The above steps can be repeated for a second elution to improve the recovery rate. Subsequently, the nucleic acid concentration is determined using a microplate reader and detected by agarose gel electrophoresis. If the sample meets the delivery requirements, store it at -20°C for delivery.The extracted phage genome samples were sent to Shanghai Paiseno Biotechnology Co., Ltd. for sequencing. A whole-genome shotgun (WGS) strategy was used to construct libraries with different insert fragments. Next-generation sequencing (NGS) technology was employed on the Illumina NovaSeq platform to perform paired-end sequencing on these libraries. The sequencing data was saved in paired-end FASTQ format. The NGS data were statistically analyzed, and FASTP was used for quality control. Based on the quality control results, the data was filtered to obtain high-quality sequences. Subsequently, A5-MiSeq and SPAdes were used to de novo assemble the sequencing data (with adapter sequences removed) to construct contigs. MUMmer software was used for collinearity analysis to determine the positional relationships between contigs and to fill gaps. Pilon software was used to correct the results to obtain the final phage genome sequence. GeneMarkS software was then used to predict protein-coding genes in the phage genome, and the blastp function of Diamond software was used for alignment with NCBI. The NR database was used to perform functional annotation of coding genes. The Resfinder and VFDB databases were used to identify drug resistance genes and virulence genes. The phage genome map was drawn using the CGview online software. The MEGA 11 was used to perform genetic evolution analysis of the phages and draw a phylogenetic tree.

[0038] Results: Members of the genus Jerseyvirus in the family Siphoviridae showed the highest similarity to Salmonella phage vB_SalP_QS (GenBank accession number: PP537161.1), with coverage and homology of 93% and 96.45%, respectively.

[0039] 1.2 Morphological identification

[0040] Phages were concentrated using the PEG-8000 precipitation method. 2 mL of host bacteria cultured to the logarithmic growth phase and 2 mL of phage proliferation solution were simultaneously added to 200 mL of LB broth and incubated at 37°C and 200 rpm for 12 h. The mixture was then centrifuged at 4°C and 8000 rpm for 20 min, and the supernatant was collected. The sample was filtered through a 0.22 μm filter to remove bacterial debris. DNase I and RNase A were added to a final concentration of 1 μg / mL, and the mixture was incubated at 37°C for 1 h. NaCl was then added to the sample to a concentration of 1 mol / L, and the mixture was gently shaken until completely dissolved. The mixture was then incubated on ice for 2 h to promote the separation of phages from bacterial debris. The supernatant was collected after centrifugation at 4°C and 8000 rpm for 20 min, and PEG-8000 was added to a final concentration of 10%. The mixture was slowly stirred until completely dissolved and incubated overnight at 4°C to promote the precipitation of phage particles. The next day, the mixture was centrifuged at 4°C and 12000 rpm for 20 min, the supernatant was discarded, and 2 mL of the supernatant was used to concentrate the phage. The precipitate was collected with SM buffer, and an equal volume of chloroform was added to extract PEG-8000 and bacterial fragments. The mixture was inverted and mixed for 5 min, then centrifuged at 4000 rpm for 20 min at 4°C. The supernatant was collected as the concentrated phage solution, which was stored at 4°C for later use. Phages were purified using CsCl density gradient centrifugation (1.32, 1.45, 1.50, 1.70 g / mL). Following the order from high to low density, 9 mL of each gradient solution was added sequentially to a 38.5 mL open polypropylene thin-walled tube using a syringe. The solution was added slowly along the tube wall to ensure a clear interface between the gradient layers. The concentrated phage solution was then slowly added to the top layer of the density gradient centrifuge solution to a final volume of 38.5 mL. The mixture was balanced and transferred to a suitable SW32Ti rotor, then placed in an ultra-high-speed refrigerated centrifuge at 4°C. Centrifuge at 32,000 rpm for 4 hours. After centrifugation, slowly remove the sample and observe the position of the blue band. Use a syringe to slowly collect and remove the upper gradient liquid. Then, aspirate the liquid at the position of the blue band, which is the purified phage fluid. Dialyze the purified phage fluid to remove impurities such as cesium chloride. Use a dialysis bag with a pore size of 20 kDa and select SM buffer as the dialysis buffer. Dialyze overnight at 4°C, changing the dialysis buffer every 4 hours, for at least three times. After dialysis, the purified phage can be obtained and stored at 4°C for later use. Phage morphology was observed by transmission electron microscopy using phosphotungstic acid negative staining. 20 μL of the purified phage solution was dropped onto a copper grid with a support membrane and allowed to adsorb naturally for 5–10 min. Then, excess droplets were removed with filter paper, and 20 μL of 2% phosphotungstic acid solution was added for staining for 3–5 min. Excess staining solution was removed with filter paper, and the phage was dried under an incandescent lamp. Finally, the phage morphology was observed and photographed using an HT7700 (HITACHI, Japan) transmission electron microscope.

[0041] Results: Phages of the order *Longiformis* and family *Longiformis*

[0042] Example 2 Host analysis of bacteriophage PQ38

[0043] 2.1 Host spectrum analysis

[0044] Using clinical isolates of Salmonella with different serotypes (Enteritis, Pullorum disease, Typhus typhimurium, Kentucky, Indiana, Argonne, London, Derby, Corvallis, Wetford Raeden, Thompson, Xugochenglong, Duck, and Newport Salmonella) as host bacteria, the host spectrum of bacteriophages was identified by the drop method. 100 μL of different serotype Salmonella cultured to the logarithmic phase was taken, 10 mL of LB semi-solid medium was added and vortexed to mix, and then poured onto LB solid medium. After the medium solidified, 5 μL of bacteriophage proliferation solution was added. After the liquid was completely absorbed, the culture was incubated upside down in a 37°C incubator for 8 hours. The formation of phage plaques was observed, and the morphology, size, clarity, and presence of halos of the plaques were noted. The results were then recorded.

[0045] Results: It had a lytic effect on five serotypes of Salmonella: Pullorum disease, Enteritis disease, Typhoid fever, Indiana disease, and Derby disease.

[0046] 2.2 Antibacterial curves: Using five different serotypes of Salmonella (Enteritis, Pullorum disease, Typhus typhimurium, Indiana spp., and Derby spp.) as host bacteria, the antibacterial curves of bacteriophages were determined; Salmonella cultured to the logarithmic growth phase was serially diluted with LB broth to 1×10⁻⁶. 7 CFU / mL, according to different MOIs (MOI=10) 3 10 2 10 1 10 0 10 -1 10 -2 10 -3 Take 100 μL of the corresponding concentration of phage fluid and 100 μL of 1×10⁻⁶ solution. 7 The bacterial suspension was mixed with CFU / mL, and 800 μL of LB broth was added and vortexed. The control group was treated with the same volume of PBS buffer instead of phage solution. 200 μL of sample was added to each well of a 96-well plate and labeled. The plates were incubated at 37°C for 12 h, with the absorbance (OD) measured every 1 h. 600 The antibacterial curves of bacteriophages were plotted using the software Graphpad Prism.

[0047] Results: Reference Figure 1-5 ,in Figure 1 The inhibition curve of bacteriophage PQ38 against Salmonella Indiana;

[0048] Figure 2The inhibition curve of phage PQ38 against Salmonella enteritidis;

[0049] Figure 3 The inhibition curve of bacteriophage PQ38 against Salmonella typhimurium;

[0050] Figure 4 The inhibition curve of bacteriophage PQ38 against Salmonella debilis;

[0051] Figure 5 The inhibition curve of bacteriophage PQ38 against Salmonella pullorum;

[0052] As can be seen, the inhibition curves of bacteriophage PQ38 against the five Salmonella strains showed significant differences. Within the range of moi=0.001 to moi=1000, it exhibited a significant inhibitory effect on the host bacterium, Pullorum avianum P53, within 12 hours. However, in the inhibition curves of PQ38 against S202101-39 and 156, the bacteriophage showed a certain degree of inhibitory effect when the moi was 100 and 1000, but its inhibitory effect significantly decreased as the moi decreased.

[0053] Example 3

[0054] 3.1 Phage titer calculation

[0055] Phage titer refers to the number of phages contained in a unit volume (usually per milliliter) of sample, typically expressed as plaque forming units (PFU). The phage titer was determined using the double-layer agar plate method. Phage proliferation solution was serially diluted with PBS buffer. 100 μL of each dilution was mixed thoroughly with 100 μL of host bacterial culture (Salmonella pullorum) cultured to the logarithmic growth phase and transferred to 10 mL of LB semi-solid medium. After vortexing and mixing, the mixture was poured onto LB solid medium. The medium was then inverted and incubated at 37°C for 8 hours. Plaque counting was performed, and plaques with a count between 30 and 300 were considered valid. Phage titer = plaque count × dilution factor × 10 PFU / mL.

[0056] Result: 1.4 × 10 10 PFU / mL

[0057] 3.2 One-step growth curve

[0058] To determine the one-step growth curve of bacteriophages under optimal multiplicity of infection (MMI) conditions, 5 mL of host bacterial culture (Salmonella pullorum) cultured to the logarithmic phase was mixed with 5 mL of bacteriophage solution of the corresponding concentration. The mixture was incubated at 37°C for 15 min at 200 rpm to ensure that the bacteriophages fully adsorbed the host bacteria. Then, the mixture was centrifuged at 4°C for 10 min at 8000 rpm, the supernatant was discarded, and the precipitate was thoroughly washed with PBS buffer. The mixture was centrifuged again under the same conditions to remove the supernatant (to remove free bacteriophages). The precipitate was resuspended in 10 mL of LB broth (preheated in a 37°C water bath), and 100 μL of the sample was taken for phage titer determination using the double-layer agar plate method. The mixture was then quickly placed in a 37°C incubator at 200 rpm for 2.5 h. During this period, samples were taken every 10 min to determine the bacteriophage titer. This experiment was repeated three times.

[0059] Results: Reference Figure 6 Latency period 10 min, lysis period 60 min, lysis amount 23 PFU / cell;

[0060] The above results indicate that bacteriophages have characteristics such as a short latency period, a long lysis period, and a large lysis volume when infecting host bacteria, giving them significant advantages in terms of bactericidal efficiency and environmental adaptability.

[0061] 3.3 Optimal Multiplicity of Infection

[0062] The multiplicity of infection (MOI) refers to the average number of phages infecting each bacterium. The optimal MOI, under the same conditions, is the MOI that enables the phage to achieve optimal growth and replication efficiency in the host bacterium. The host bacterium (Salmonella pullorum) was cultured to the logarithmic growth phase (concentration approximately 1.0 × 10⁻⁶). 8 CFU / mL), phage solutions of different concentrations were obtained by serial dilution with SM buffer, according to an MOI of 10. 2 10 1 10 0 10 -1 10 -2 10 -3 10 -4 10 -5 10 -6 10 -7 10 -8100 μL of bacterial culture was mixed with 100 μL of phage culture and transferred to 1.8 mL of LB broth. The mixture was incubated at 37 °C and 200 rpm for 4 h, and then centrifuged at 8000 rpm for 20 min at 4 °C. The supernatant was collected and filtered through a 0.22 μm filter to remove bacteria. The phage titer was determined by serial dilution and double-layer plate method. The MOI with the highest titer was the optimal MOI. This experiment was repeated three times.

[0063] Results: Reference Figure 7 The optimal MOI is 1×10 -7 The valence is 6.50 × 10⁻⁶. 9 PFU / mL. Optimal multiplicity of infection assay results indicate strong lytic ability, achieving efficient lysis even under low MOI conditions.

[0064] 3.4 Temperature stability

[0065] Add 900 μL of PBS buffer solution to 2.0 mL sterile EP tubes, for a total of 8 tubes. Pre-cool one tube at 4°C for 1 h, and preheat the others in constant temperature water baths at 25°C, 37°C, 40°C, 50°C, 60°C, 70°C, 80°C, and 90°C for 1 h. After that, aseptically add 100 μL of phage fluid to the EP tubes at the above different temperature conditions, vortex mix, and quickly return to the corresponding temperature conditions for incubation. After 1 h, take 100 μL of phage fluid incubated at different temperature conditions, serially dilute it, and determine its phage titer using the double-layer plate method. This experiment is repeated three times.

[0066] Results: Reference Figure 8 After incubation at different temperatures for 1 hour, the phage titer remained essentially unchanged at temperatures ranging from 4 to 60°C, decreased slightly at 70°C and 80°C, and was completely inactivated at 90°C. Overall, the phage exhibits good temperature tolerance, maintaining a high titer even after incubation at temperatures between 4 and 60°C for 1 hour.

[0067] 3.5 pH stability

[0068] The pH of the PBS buffer solution was adjusted using HCl and NaOH to 2.0, 3.0, 4.0, 5.0, 6.0, 7.0, 8.0, 9.0, 10.0, 11.0, 12.0, and 13.0, respectively. 900 μL of PBS buffer solution at different pH values ​​was added to 2.0 mL sterile EP tubes and incubated in a 37°C water bath for 1 h. After incubation, 100 μL of phage fluid was aseptically added to the PBS buffer solution at different pH values, and the tubes were incubated again in a 37°C water bath for 1 h. 100 μL of phage fluid incubated at different pH conditions was then serially diluted and its phage titer was determined using the bilayer plate method. This experiment was repeated three times.

[0069] Results: Reference Figure 9 After incubation for 1 hour under different pH conditions, it was found that the bacteriophages remained relatively stable within the pH range of 3.0–11.0, with no significant decrease in titer; however, the titer decreased by 2.09 × 10⁻⁶ at pH 12.0. 6 PFU / mL; completely inactivated at pH ≥ 13.0 or pH ≤ 2.0.

[0070] 3.6 In vitro antibacterial effect

[0071] The antibacterial effect of bacteriophages on Salmonella planktonic bacteria (the planktonic form of Salmonella pullorum) at different temperature conditions (4℃, 25℃, 37℃) was evaluated by time-kill curves. Host bacterial cultures in the logarithmic growth phase were serially diluted to 1×10⁻⁶ with PBS buffer. 7 CFU / mL, at MOI=10 2 10 0 10 -2 Take 200 μL of phage fluid at different concentrations and 200 μL of phage solution with a concentration of 1×10 7CFU / mL bacterial suspension was mixed, and the control group was replaced with the same volume of PBS buffer solution instead of phage suspension. Then, 1.6 mL of LB broth was added and mixed thoroughly. The samples were incubated statically at different temperatures (4℃, 25℃, 37℃) for 24 h. At 0 h, 2 h, 4 h, 8 h, 12 h, and 24 h, 100 μL of sample solution was taken and serially diluted with PBS buffer solution. The bacterial content per unit volume of sample was determined using the pour method. 100 μL of each serially diluted solution was added dropwise to a sterile Petri dish, followed by 15–20 mL of dissolved and... Cool the XLT-4 agar medium to 45–55°C, immediately rotate the petri dish to thoroughly mix the diluent with the agar, and after it solidifies, invert the petri dish and incubate it at 37°C for 18–24 hours. Then, count the colonies and select those with colony counts in the range of 30–300 as valid counts. Bacterial concentration = colony count × dilution × 10 CFU / mL. This experiment was repeated three times. Finally, the time-kill curve was plotted using GraphpadPrism software, with time on the x-axis and the logarithm of bacterial concentration (logCFU / mL) on the y-axis.

[0072] Results: Reference Figure 10-12 It exhibits significant bactericidal effects within 12 hours at 4℃, 25℃, and 37℃. At 4℃, MOI=10. 2 MOI=10 0 MOI=10 -2 Both methods can reduce the host bacterial concentration by 3.38-3.88 logCFU / mL. The best results are observed at 25℃ and 37℃, with a reduction of approximately 5 logCFU / mL over 4 hours.

[0073] 3.7 Inhibition of biofilm formation

[0074] The host bacterium (Salmonella pullorum) was cultured at 37°C and 200 rpm for 6 hours until it reached the logarithmic growth phase. The bacterial culture was then serially diluted to 1×10⁻⁶ with LB broth. 6CFU / mL, take a sterile 96-well plate, add 100 μL of host bacterial dilution to each well, then add 100 μL of phage fluid of corresponding concentrations according to MOI=100, 1, 0.01 and mix well (the control group adds the same volume of PBS buffer solution). Place the 96-well plate in a constant temperature incubator at different temperatures (25℃, 37℃) and incubate for 48 h, with three replicates for each group. After incubation, the biofilm formation is determined by crystal violet staining. Remove the 96-well plate from the incubator, discard the bacterial suspension under sterile conditions, and thoroughly rinse with PBS buffer solution. Wash three times to remove airborne bacteria. After drying, add 200 μL of anhydrous methanol to each well for 15 min to fix. Then discard the anhydrous methanol and let it dry at room temperature. Next, add 200 μL of 1% crystal violet solution to stain for 15 min. Discard the excess crystal violet solution and wash away the unbound staining solution with PBS buffer. After completion, let it dry at room temperature for 15 min. Finally, add 200 μL of 33% acetic acid solution to each well to fully wash away the staining solution bound in the biofilm. Finally, use an ELISA reader to measure the OD595 value to evaluate the ability of bacteriophage to inhibit biofilm formation.

[0075] Results: Reference Figure 13 At 37℃, PQ38 with MOI=100, MOI=1, and MOI=0.01 reduced the biofilm formation rate of host bacteria (Salmonella pullorum) P53 anus by 63.89%, 54.79%, and 52.66%, respectively. All MOI phage treatment groups showed significant bactericidal effects on host bacteria P53 anus (reducing CFU / mL by 3–5 log CFU / mL) under different temperature conditions. However, at 25℃ and 37℃, the host bacteria showed relatively obvious tolerance after 12 hours of PQ38 treatment. In the time-kill curves at 4℃, when MOI=100, the concentration of the host bacteria decreased by 3.07, 3.88, 3.59, 3.62, and 3.38 log CFU / mL compared to the control group after 2 hours, 4 hours, 8 hours, 12 hours, and 24 hours of phage treatment, respectively. In the time-killing curves at 37℃, when MOI=100, the concentration of host bacteria decreased by 4.32, 5.33, 4.83, and 2.90 log CFU / mL compared to the control group after 2 h, 4 h, 8 h, and 12 h of phage treatment, respectively. Overall, although the bactericidal effects of bacteriophages varied due to differences in treatment temperature and concentration (MOI), they all exhibited significant bactericidal activity.

[0076] 3.8 Biofilm disruption

[0077] Take the host bacteria culture (Salmonella pullorum culture) cultured to the logarithmic growth phase and serially dilute it to 1×10⁻⁶ with LB broth. 6Add 200 μL of host bacterial dilution to each well of a sterile 96-well plate at CFU / mL. Incubate at 37°C for 48 hours to allow biofilm formation. Remove the 96-well plate and discard excess bacterial suspension under sterile conditions. Gently wash 3–5 times with sterile PBS buffer to remove airborne bacteria. Then, add 200 μL of different concentrations (1×10⁻⁶ CFU / mL) to each well. 4 PFU / mL, 1×10 6 PFU / mL, 1×10 8 Phage suspension (PFU / mL) was used (the control group was replaced with the same volume of PBS buffer solution). 96-well plates were incubated at different temperatures (4℃, 25℃, 37℃) for 12 h. The biofilm formation was then determined using crystal violet staining. The 96-well plates were removed, the bacterial suspension discarded, and the plates were gently washed 3–5 times with sterile PBS buffer solution. After drying, 200 μL of anhydrous methanol was added to each well for fixation for 15 min. After fixation, the anhydrous methanol was discarded, and the plates were dried at room temperature for 15 min. Then, 200 μL of 1% crystal violet solution was added to each well for staining for 15 min. After staining, excess dye was washed away with PBS buffer solution. After drying, 200 μL of 33% acetic acid solution was added to each well to thoroughly wash away the dye. Finally, the OD595 value was measured using a microplate reader to evaluate the phage's ability to disrupt biofilms.

[0078] Results: Reference Figure 14 At 37℃, different concentrations (10 8 PFU / mL, 10 6 PFU / mL, 10 4 Phage PQ38 (PFU / mL) reduced the biofilm of its host bacterium (Salmonella pullorum) P53 by 69.16%, 71.12%, and 53.06%, respectively; the inhibitory effect on biofilm was slightly better at 37℃ than at 25℃.

[0079] Example 4: Protective effect of bacteriophage PQ38 against pullorum disease in chicks.

[0080] 4.1 Evaluation of the protective effect of bacteriophage PQ38 against pullorum disease in chicks

[0081] Forty one-day-old SPF chicks were randomly divided into four groups of 10 chicks each: blank control group (NC), Salmonella challenge group (PC), bacteriophage gavage group (PA), and bacteriophage free drinking water group (PF). The chicks were housed in SPF chicken isolators, provided with ample clean drinking water and feed, with water changed every 8 hours. The experiment lasted 14 days. At two days of age, chicks in the bacteriophage gavage group (PA) were administered 0.5 mL of 1×10⁻⁶ protein via gavage. 8The PFU / mL phage preparation of PQ38 was administered to chicks continuously for 7 to 8 days. The other three groups were given the same volume of sterile PBS buffer solution by gavage under the same conditions. At 2 days old, the phage suspension was provided as drinking water to the chicks in the phage free drinking water group (PF), with a phage concentration of 1×10⁻⁶. 8 PFU / mL was administered continuously for 7 to 8 days, while the other three groups were provided with ample and clean drinking water under the same conditions. At 4 days of age, chicks in the Salmonella challenge group (PC), bacteriophage gavage group (PA), and bacteriophage free drinking water group (PF) were administered 1 mL of a 1×10⁻⁶ PFU / mL solution via gavage. 9 Chickens were given a CFU / mL Salmonella pullorum culture for two consecutive days, while a control group was given the same volume of PBS buffer solution under the same conditions. Chicks were fasted for 12 hours before challenge. During the experiment, chicks were observed daily for feed intake, water consumption, and mental state. Typical symptoms included lethargy, loss of appetite, and white, watery feces or pasty vent discharge. Disease incidence was recorded for each group. After the experiment, chicks were euthanized, and their internal organs were dissected and photographed. Cecal contents samples were collected, flash-frozen in liquid nitrogen, and stored at -80°C for later delivery.

[0082] (1) Daily weight gain: Starting from 4 days old, the chicks were weighed at the same time every day until the end of the experiment (14 days old). The average total weight gain of the chicks in each group from 4 days old to 14 days old was calculated. The average daily weight gain of the chicks in each group was calculated according to the formula: average daily weight gain = average total weight gain / number of days of the experiment.

[0083] Results: The average daily weight gain of the NC group and the PC group was 8.9±0.3g and 7.59±0.3g, respectively. The weight gain of the PA group chicks recovered to a level close to that of the NC group, with an average daily weight gain of 9.0±0.3g. The average daily weight gain of the PF group chicks was 8.1±0.5g.

[0084] (2) Uniformity: The uniformity of the weight of each group of chicks is reflected by the percentage of the number of chickens within ±10% of the weight and the coefficient of variation (CV). The coefficient of variation is the ratio of the standard deviation to the mean, usually expressed as a percentage. The lower the coefficient of variation, the higher the uniformity of the weight of the flock. The percentage uniformity and coefficient of variation of each group of chicks are calculated and recorded every day.

[0085] Results: The uniformity of chicks in each group was evaluated using two indicators: coefficient of variation (CV) and percentage evenness. The initial CVs of the chicks in each group (NC, PC, PA, PF) were 9.37%, 4.47%, 7.10%, and 6.67%, respectively, and the final CVs were 10.25%, 9.12%, 7.76%, and 13.02%, respectively. The initial percentage evennesses were 60%, 100%, 80%, and 80%, respectively, and the final percentage evennesses were 80%, 80%, 80%, and 30%, respectively. In summary, the PF group had the worst overall uniformity, with a CV of 13.02% and a percentage evenness of 30%, while the PA group had the best overall uniformity, with a CV of 7.76% and a percentage evenness of 80%, respectively.

[0086] 4.2 Alpha and Beta diversity analysis of cecal microbiota

[0087] Samples of cecal contents were collected from chicks, flash-frozen in liquid nitrogen, and stored at -80°C. The samples were then sent to Beijing Novogene Technology Co., Ltd. for microbial diversity sequencing and analysis. The specific methods are as follows:

[0088] (1) Sample collection: Collect cecal contents samples from chicks, flash freeze them with liquid nitrogen, and store them in a -80℃ freezer;

[0089] (2) DNA extraction: Four samples were randomly selected from each group, for a total of 16 samples, for microbial diversity sequencing and analysis. Total microbial DNA was extracted using a commercial DNA extraction kit.

[0090] (3) PCR amplification: This experiment used specific primers (338F-806R) to amplify the highly variable V3-V4 region of the 16S rRNA gene, with three replicates for each sample;

[0091] (4) Library construction and high-throughput sequencing: High-throughput sequencing was performed using the Illumina MiSeq / HiSeq platform;

[0092] (5) Bioinformatics analysis;

[0093] Results: Reference Figure 15-20The Chao1, Shannon, Simpson, and Observed_features indices in the PC group were significantly lower than those in the NC group, with the Shannon and Simpson indices showing the most significant decreases (p<0.05), indicating that Salmonella pullorum infection significantly reduced the cecal microbiota diversity in chicks. Furthermore, the microbial community diversity level in the PA group was higher than that in the PC and PF groups and closer to that in the NC group, indicating that phage intervention can effectively restore the species richness and evenness of the cecal microbiota in chicks. Further analysis using PCoA and NMDS to study Beta diversity revealed that the microbial community distribution in the PA group was closer to that in the NC group, indicating that phage intervention can effectively restore the cecal microbiota structure of infected chicks, bringing it closer to a normal state.

[0094] Note: NC: control group; PC: infection group; PA: phage gavage group; PF: phage free drinking water group.

[0095] The embodiments presented herein are merely selected implementations based on combinations of all possible embodiments. The appended claims should not be limited to the embodiments described herein. Some numerical ranges used in the claims include sub-ranges within them, and variations within these ranges should also be covered by the appended claims.

Claims

1. Bacteriophage PQ38, characterized in that, The accession number is GDMCC NO:67169-B1. It is deposited at the Guangdong Provincial Center for Microbial Culture Collection on October 27, 2025, and its taxonomic name is Siphoviridae jerseyvirus.

2. Use of the bacteriophage preparation for the preparation of a drug to inhibit Salmonella as described in claim 1.

3. The use according to claim 2, characterized in that, The Salmonella mentioned is one of Salmonella pullorum, Salmonella enteritidis, Salmonella typhimurium, Salmonella Indiana, or Salmonella debilis.

4. The use according to claim 2, characterized in that, The Salmonella mentioned is Salmonella pullorum.

5. A drug for inhibiting Salmonella, characterized in that, It contains bacteriophage PQ38.

6. The Salmonella drug according to claim 5, characterized in that, The drug is an inhibitor of Salmonella pullorum.

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