A bacteriophage preparation for controlling foodborne enterobacteriaceae and use thereof
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- THE SECOND HOSPITAL OF NANJING
- Filing Date
- 2026-04-17
- Publication Date
- 2026-07-14
Smart Images
Figure CN122382016A_ABST
Abstract
Description
Technical Field
[0001] This invention belongs to the field of microbial technology, specifically relating to a bacteriophage preparation for controlling foodborne Enterobacteriaceae and its application. Background Technology
[0002] Foodborne illnesses remain a significant public health challenge globally, causing numerous deaths each year, with over 200 diseases linked to the consumption of contaminated food. Among the various bacterial contaminants detected in food systems, Enterobacteriaceae (Enterobacteria spp.) are particularly prevalent. Enterobacter Enterobacter cloacae (ECC) has received increasing attention due to its pathogenic potential and growing antimicrobial resistance. In particular, Enterobacter cholerae (ECC), a major member of the Enterobacter cloacae complex (ECC), is a significant concern. Enterobacter hormaechei ), has become an important multidrug-resistant (MDR) pathogen in clinical and food-related environments.
[0003] Historically, *Enterobacter cholerae* has been considered a hospital-acquired opportunistic pathogen, associated with bloodstream infections, neonatal sepsis, respiratory infections, urinary tract infections, and wound complications. In recent years, it has also been identified as an emerging foodborne pathogen. Studies show its presence in raw meat, poultry products, street food, dairy products, vegetables, and animal feed, reflecting its strong environmental adaptability. For example, the contamination rate of *Enterobacteria* producing extended-spectrum β-lactamase (ESBL) in raw meat products is as high as 10%, including isolates of *Enterobacter cholerae* with multidrug resistance. Furthermore, reports indicate that *Enterobacter cholerae* can act as a pathogen for respiratory infections in animals and cause fatal diseases in fish, demonstrating zoonotic potential and raising concerns about cross-species transmission risks. Simultaneously, the issue of hormone residues in chicken feed highlights the risk of antimicrobial resistance spreading from the agricultural environment to the food chain.
[0004] Genome surveillance revealed that *Enterobacter holmieae* readily acquires resistance genes, including extended-spectrum β-lactamases and carbapenemases. This not only complicates clinical treatment but also exacerbates the global spread of carbapenem-resistant *Enterobacter* (CRE) strains. A ten-year epidemiological analysis showed a dramatic increase in carbapenem resistance rates among enteropathogenic *Escherichia coli* isolates, with *Enterobacter holmieae* subsp. *Hofmanni* (…) being particularly susceptible. E. hormaechei subsp. hoffmannii It has become the dominant bacterial species. Given the increasing difficulty in treating MDR or carbapenem-resistant Enterobacter cholerae infections, and their widespread detection in the food environment, there is an urgent need to develop alternative antimicrobial strategies to target foodborne contamination.
[0005] In recent years, bacteriophages have regained attention for their ability to specifically lyse host bacteria in controlling drug-resistant bacteria. While studies on bacteriophages targeting *Enterobacter holmieae* have been reported, most remain limited to single phages, and monotherapy is prone to failure due to host bacteria developing phage resistance. In contrast, phage combination therapy (cocktail therapy)—a strategy combining multiple lytic phages with non-overlapping receptor specificity—has become the preferred approach. Phage combination therapy offers synergistic advantages, including expanding the host range, reducing resistance development, improving eradication efficiency, and enhancing activity in complex environments such as biofilms or food matrices. However, current research largely focuses on the therapeutic application of clinical isolates, and the development of phage formulations for controlling foodborne *Enterobacter holmieae* contamination remains limited, especially systematic research on phage combination therapies applicable to different food matrices (such as fresh agricultural products and animal-derived foods) and complex environmental conditions. Therefore, developing a phage formulation that can effectively control foodborne *Enterobacter holmieae*, is applicable to multiple food matrices, and exhibits good stability is of significant practical importance for ensuring food safety and public health. Summary of the Invention
[0006] To address the aforementioned problems, this invention provides a phage preparation for controlling foodborne Enterobacteriaceae and its application. This phage preparation can effectively inhibit Enterobacter holmieae in various food matrices such as lettuce, pork, and milk, and can effectively prevent and treat bacteremia caused by it. It can serve as a highly efficient biological control agent for ensuring food safety and preventing infection.
[0007] To achieve the above objectives, the specific technical solution of the present invention is as follows: The first aspect of the present invention provides a phage preparation, the phage preparation comprising any one or more of phages ΦEAS1, ΦEHO11, ΦEHO14 and ΦECL22; The bacteriophage ΦEAS1 was classified as Enterobacter assbury bacteriophage. Enterobacter asburiae Phage is deposited at the China General Microbiological Culture Collection Center, with accession number CGMCC NO.47013; The bacteriophage ΦEHO11 was classified as Enterobacter holmie phage. Enterobacter wall Phage is deposited at the China General Microbiological Culture Collection Center, with accession number CGMCC NO.47014; The bacteriophage ΦEHO14 was classified as Enterobacter holmie phage. Enterobacter wallPhage is deposited at the China General Microbiological Culture Collection Center, with accession number CGMCC NO.47015; The bacteriophage ΦECL22 is classified as Enterobacter cloacae bacteriophage. Enterobacter cloaca bacteriophage is deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC NO.46281.
[0008] Phages ΦEAS1, ΦEHO11, ΦEHO14, and ΦECL22 exhibit typical T4-like myovirus morphology (icosahedral head, contractile tail, and tail fibers), with genome sizes of 173,107 bp, 178,437 bp, 173,643 bp, and 177,652 bp, respectively, and GC content ranging from 44.71% to 50.1%. All belong to the family Circular Double-Stranded DNA Climatoviridae. Physiologically, ΦEHO11 and ΦECL22 show similar UV resistance (40% infectivity retained after 10 minutes, inactivated after 20 minutes); ΦEHO14 has the lowest optimal MOI (0.0001), the highest infectivity, and moderate thermal stability (inactivated at 60°C); ΦEAS1 exhibits moderate thermal stability (inactivated at 50°C).
[0009] Furthermore, the total phage content in each liter of the phage preparation is ≥1×10⁻⁶. 10 PFU.
[0010] Furthermore, when the phage preparation comprises any two or more phages, the phages are mixed at a titer ratio of 1 to 10: 1 to 10.
[0011] A second aspect of the present invention provides the use of the above-described phage preparation in the preparation of products that inhibit or kill foodborne Enterobacteriaceae, wherein the foodborne Enterobacteriaceae is Enterobacter holmieae.
[0012] Furthermore, the product is a drug for preventing bacteremia, a drug for treating bacteremia, or an environmental bactericide.
[0013] Furthermore, the bacteremia is caused by Enterobacter holmieae, and the environmental bactericide is used to kill Enterobacter holmieae in the environment.
[0014] A third aspect of the present invention provides a medicament for the prevention or treatment of bacteremia, the medicament being composed of the phage preparation and excipients described above.
[0015] Furthermore, the bacteremia is caused by Enterobacter holmieae.
[0016] Furthermore, the dosage form of the drug is any dosage form that is veterinarily acceptable.
[0017] Furthermore, the drug is administered via intraperitoneal injection.
[0018] A fourth aspect of the present invention provides an environmental bactericide, which is composed of the bacteriophage preparation and an aqueous carrier described above.
[0019] Furthermore, the aqueous carrier comprises water, buffer solution, physiological saline, and culture medium.
[0020] Furthermore, the culture medium comprises LB medium; the buffer comprises SM buffer and phosphate buffer.
[0021] Furthermore, the environmental bactericide is used to inhibit or eliminate Enterobacter holmie in the food matrix.
[0022] Furthermore, the food matrix includes at least one of lettuce, pork, milk, and cherry tomatoes.
[0023] Compared with the prior art, the beneficial effects of the present invention are as follows: This invention provides a phage preparation for controlling foodborne Enterobacteriaceae and its application. The phage preparation includes any one or more of phages ΦEAS1, ΦEHO11, ΦEHO14, and ΦECL22. The accession numbers of phages ΦEAS1, ΦEHO11, ΦEHO14, and ΦECL22 are CGMCC NO.47013, CGMCC NO.47014, CGMCC NO.47015, and CGMCC NO.46281, respectively. The four phages provided by this invention can be used alone or combined into phage cocktails. The effects of phage preparations on Enterobacteriaceae in different ecological environments can be explored through phage combination therapy. The results showed that this phage preparation exhibited significant antibacterial activity against various food substrates contaminated with *Enterobacter cholerae*, including lettuce, pork, milk, and cherry tomatoes, and could be used for food preservation. Animal experiments showed that intraperitoneal injection of the phage preparation could effectively prevent bacteremia caused by *Enterobacter cholerae*. Therefore, the phage preparation can be considered as a potential biocontrol agent against *Enterobacter cholerae* contamination.
[0024] Information on the preservation of biological materials: The bacteriophage ΦEAS1 in this invention is classified and named as Asbury Enterobacter phage. Enterobacter asburiae Phage was deposited on January 8, 2026 at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC NO.47013. The bacteriophage ΦEHO11 in this invention is classified and named Enterobacter hopterii bacteriophage. Enterobacter wallPhage was deposited at the China General Microbiological Culture Collection Center on January 8, 2026, with accession number CGMCC NO.47014; The bacteriophage ΦEHO14 in this invention is classified and named Enterobacter hopterii bacteriophage. Enterobacter wall Phage was deposited on January 8, 2026, at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC NO.47015. The bacteriophage ΦECL22 in this invention is classified and named Enterobacter cloacae bacteriophage. Enterobacter cloaca bacteriophage was deposited on November 18, 2024, at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC NO.46281. Attached Figure Description
[0025] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.
[0026] Figure 1 A heatmap showing the host range distribution of isolated *Enterobacter holmie* phages. This heatmap illustrates the lytic activity of each phage against a group of *Enterobacter holmie* strains. Sensitivity scores are based on plaque formation intensity: strong lysis (red), moderate lysis (light red), or no detectable lysis (white). *Note: Strains EHO7622 (ST97) and EHO16040 (ST171) were isolated from Nanjing Drum Tower Hospital. **Note: Strains ECC193 (ST133) and ECC66 (ST418) were isolated from Shenzhen People's Hospital.
[0027] Figure 2 Morphages ФEAS1, ФEHO4B, ФEHO11, ФEHO14, and ФECL22 are shown. The top row of images shows phage plaques; the bottom row shows transmission electron microscopy (TEM) images. Scale bar: 200 nm.
[0028] Figure 3Biological characteristics of phages ФEAS1, ФEHO4B, ФEHO11, and ФEHO14. (A) The first row shows the thermal stability of the phages after incubation at different temperatures for 1 hour. Phage survival at 25°C is defined as 100%. (B) The second row shows the pH tolerance of the phages after incubation in buffer solutions with pH values ranging from 2 to 12 for 1 hour. Phage survival at pH 8.0 is defined as 100%. (C) The second row shows the sensitivity of the phages to ultraviolet (UV) irradiation; samples not exposed to UV irradiation serve as negative controls. (D) The second row shows the optimal multiple of infection (MOI) for each phage based on the highest progeny phage yield. (E) The third row shows the one-step growth curves of the phages, indicating the latency period and outbreak size.
[0029] Figure 4 This demonstrates the inhibitory effect of bacteriophages on the growth of *Enterobacter holmium*. The inhibition was achieved by using a microplate reader at a wavelength of 600 nm (OD). 600 ) Measure optical density and continuously monitor E.hormaechei 7-25 Growth in the presence of a single phage or different combinations of phages. (A) The figure shows the inhibitory effect of a single phage and a mixture containing all five phages (ФEAS1, ФEHO4B, ФEHO11, ФEHO14, and ФECL22) on growth. (B) The figure shows the inhibitory effect of a mixture of four phages ("-" indicates the absence of the corresponding single phage among the five phages) on growth. (C) The figure shows the growth inhibitory effect of phage ФEHO4B in combination with other phages. (D) The figure shows the effect of adding an additional phage to a mixture of ФEHO4B and ФECL22.
[0030] Figure 5 This is a heatmap showing the host range distribution of various phages and EHO-cocktail4 for clinically isolated ECC strains. Sensitivity was assessed based on plaque formation intensity: strong lysis (red), moderate lysis (light red), or no lysis (white).
[0031] Figure 6 To demonstrate the biofilm inhibition and clearance effects. (A) The figure shows the effect of using a single phage and EHO-cocktail4. E. wall Biofilm formation inhibition experiments were conducted on July 25, with uninfected bacteria serving as the control group. Figures (B) and (C) show the removal of bacteria by a single bacteriophage and EHO-cocktail4, respectively. E. hormaecheiMature biofilms were collected at 7-25°C. Biofilm quantity was quantified using CV (B) plot and CFU count (C) plot, with uninfected phage-infected LB medium serving as a negative control for biofilm removal. (D) shows a scanning electron microscope image of biofilm treated with EHO-cocktail 4, with an untreated sample as a control. In this study, 5-fold polymyxin B minimum inhibitory concentration (MIC) and 5 mM EDTA were used as positive anti-biofilm controls. One-way ANOVA combined with multiple comparison tests was used to analyze differences between groups. **** indicates... p <0.0001, ** indicates p <0.01, * indicates p <0.05. Scale bar: 10μm.
[0032] Figure 7 To assess the biocontrol efficacy of the EHO-cocktail4 phage mixture in different food matrices, the effects of the phage mixture on various food models were evaluated. E. hormaechei Antimicrobial activity was measured at 7-25°C. Bacterial reduction in lettuce (A, B), pork (C, D), and milk (E, F) was evaluated under storage conditions of 4°C or 25°C. The mixture also inhibited surface adhesion (G) and internalization (H) processes in cherry tomatoes. Samples inoculated with bacteria but not with bacteriophages served as negative controls. Polymyxin B (5×MIC) served as a positive antimicrobial control. Uninfected tomatoes served as a sterile control. Data represent the mean ± standard deviation of three independent experiments. Statistical significance between two groups was analyzed using the Student's t-test, and comparisons among multiple groups were performed using one-way ANOVA and multiple comparisons. **** indicates... p <0.0001, ** indicates p <0.01.
[0033] Figure 8 The therapeutic effect of bacteriophage mixtures in a mouse bacteremia model. (A) Figure shows... E. hormaechei Establishment of a bacteremia model. Mice were administered different doses of [a specific drug / method] via intraperitoneal injection (IP) or gavage (IG). E. hormaechei On July 25th, survival was monitored for 168 hours. Mice receiving PBS injection served as negative controls. (B) The figure shows the clinical scores of infected mice treated with EHO-cocktail4 (IP or IG). (C) The figure shows the weight changes of infected mice treated with EHO-cocktail4 (IP or IG). Polymyxin B (4 mg / kg) served as a positive antibacterial control. Statistical differences between groups were analyzed using the Student's t-test. * indicates... p<0.05. (D) The figure shows the Kaplan-Meier survival curves of mice treated with different regimens (including phage cocktail and control treatment). Survival differences were analyzed using the log-rank test. Detailed Implementation
[0034] The specific embodiments of the present invention are described in detail below, but it should be understood that the scope of protection of the present invention is not limited to the specific embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention. Unless otherwise specified, the experimental methods described in the embodiments of the present invention are conventional methods, and the materials and reagents used in the following embodiments are commercially available unless otherwise specified.
[0035] Tables 1-3 contain information on the strains used in this study.
[0036] Table 1. Strain Information Table 2 is a continuation of Table 1. Table 3 is a continuation of Table 2. Example 1: A bacteriophage preparation for controlling foodborne Enterobacteriaceae and its application. 1. Materials and Methods 1.1 Bacterial strains and growth conditions The bacterial strains used in this study are listed in Tables 1–3. For cloning, *E. coli* Stellar chemocompetent cells were purchased from Takara Bio. *Enterobacter* strains were obtained from the Clinical Sample Bank of Drug-Resistant Bacteria at Nanjing Second Hospital. All strains were cultured at 37°C in LB broth or LB agar on a shaker, with appropriate antibiotics added as needed. Plasmids were maintained using 50 μg / mL carbenicillin, 100 μg / mL apramycin, or 30 μg / mL sodium tellurate.
[0037] 1.2 Isolation, purification, and morphological characterization of bacteriophages Bacteriophages were isolated from untreated wastewater collected from Nanjing Public Health Medical Center using the standard double-layer agar plate method. Single plaques were picked and purified by three consecutive rounds of plaque separation in 1×SM buffer (Nanjing Sanger Biotechnology Co., Ltd., catalog number B458130). The purified plaques were then stored at -80°C in 20% glycerol solution for long-term preservation or at 4°C for routine use. Bacteriophage nomenclature followed the nomenclature guidelines for bacteria and archaea viruses.
[0038] Phage morphology analysis was performed using transmission electron microscopy (TEM) at the Electron Microscopy Center of Nanjing Agricultural University. The specific steps were as follows: phage lysate was filtered through a 0.22 μm filter membrane, coated onto a copper grid, negatively stained with 2% uranium acetate, blotted dry with filter paper, and then air-dried. Finally, the samples were observed using a Hitachi HT7700 TEM.
[0039] Host range was determined using spot testing to assess the susceptibility of bacterial isolates. Phage titers were quantified using the double agar plaque assay.
[0040] Four bacteriophages, ФEAS1, ФEHO11, ФEHO14, and ФECL22, are all deposited at the China General Microbiological Culture Collection Center (CGMCC), with accession numbers CGMCC NO.47013, CGMCC NO.47014, CGMCC NO.47015, and CGMCC NO.46281, respectively.
[0041] 1.3 Biological characteristics of bacteriophages To determine the optimal multiple of infection (MOI), E. hormaechei Strain 7-25 (sequencing results available in NCBI database accession number PRJNA1418209; the patent applicant guarantees to release this biological material to the public within twenty years from the application date) is Enterobacter hopterii ( E. hormaechei ) represents, will host E. hormaechei Cultured at 7-25 until mid-logarithmic growth phase (OD) 600 =0.5), collected by centrifugation, washed twice, and resuspended in LB broth. The bacterial suspension (1×10⁻⁵) 8 The phages (CFU / mL) were mixed with corresponding phages at different MOIs (0.00001, 0.0001, 0.001, 0.01, 0.1, 1, and 10). After incubation at 37°C for 4 hours, the mixture was centrifuged at 12000 rpm for 10 min, and the supernatant was filtered through a 0.22 μm syringe filter. The phage titer was determined using the double-layer agar method, and the MOI with the highest phage titer was defined as the optimal MOI. The experiment was designed with three independent replicates, each with two technical replicates.
[0042] To investigate the pH tolerance of bacteriophages, this invention prepared three buffer systems to generate conditions within the pH range of 2–12, including Na₂HPO₄-citrate buffer (pH 2–8), glycine-NaOH buffer (pH 9–10), and Na₂HPO₄-NaOH buffer (pH 11–12). For each pH value, 0.9 mL of buffer was aliquoted into sterile microtubes, followed by the addition of 0.1 mL of bacteriophage suspension. After incubation for 1 hour, the samples were diluted to appropriate concentrations, and the bacteriophage titer was determined using a double-layer plaque assay. To assess thermal stability, aliquots of the bacteriophage suspension were incubated at 4°C, 25°C, 40°C, 50°C, 60°C, and 70°C for 1 hour, and the bacteriophage titer was determined as described above. To assess the UV sensitivity of the bacteriophages, 5 mL of the bacteriophage suspension was placed in a sterile petri dish and exposed to UV light (20 W lamp, 50 cm away) for 1 hour. Collect 1 mL of sample every 10 minutes and titrate to assess the kinetics of UV quenching. The experiment was repeated three times.
[0043] To determine the single-step growth curve of bacteriophages, a single *Enterobacter holmieae* colony was inoculated into 6 mL of LB broth and cultured to mid-logarithmic growth phase. 1 mL of bacterial suspension (1 × 10⁻⁶) was then used. 8 100 µL of the corresponding phage (based on the optimal multiple of infection (MOI)) was used for adsorption at 37°C for 5 minutes, followed by centrifugation at 12000 rpm for 1 minute to remove unbound phage. The precipitate was washed once with preheated LB broth and resuspended in 1 mL LB. The suspension was then transferred to 100 mL of preheated LB broth, marked as time zero. At 0 min, 5 min, 10 min, 15 min, 20 min, 25 min, 30 min, 35 min, 40 min, and 45 min, 1 mL of sample was taken, cooled at 4°C, centrifuged at 12000 rpm for 2 minutes, and the supernatant was used for serial dilution. Phage titers were determined using the double-layer agar method, and the outbreak was calculated by dividing the average phage titer during the stationary phase by the number of infected bacteria. All experiments were performed in triplicate.
[0044] 1.4 Bioinformatics Analysis Genomic DNA was extracted using the SSNP-9600A automated nucleic acid extraction system and its accompanying kit, following the manufacturer's instructions. Phage genome sequencing was performed using an Illumina PE150 sequencer at Novogene. During phage genome processing and assembly, SAMtools (version 1.3.1) and Bowtie2 (version 2.5.4) were used to filter raw sequencing reads to remove host genome contamination and discard reads mapped to the host genome. The quality and integrity of the phage genome were assessed using CheckV. Pharokka (version 1.7.5) was used for functional annotation of the assembled phage genome; this tool is used to identify coding sequences and functional genomic elements. Linear alignment of the phage genome was performed using Easyfig 2(25).2.2 software (https: / / mjsull.github.io / Easyfig / ).
[0045] Interactive circular genome maps were generated using the Proksee online platform (https: / / proksee.ca / ) to visualize genomic features. In evolutionary analysis, researchers compared protein sequences with the NCBI database using BLASTp to identify closely related bacteriophages. Phylogenetic trees were constructed using MEGA11 software and visualized and annotated using iTOL (https: / / itol.embl.de / ).
[0046] The virulence factors in the genomes of five bacteriophages were analyzed using VFanalyzer, and antibiotic resistance genes were analyzed using the Global Pathogen Analysis Platform.
[0047] 1.5. Inhibition and clearance capabilities of phage preparations against biofilms To evaluate the ability of phage formulations to inhibit biofilm formation, E. hormaechei Cultured at 7-25°C until the exponential growth phase, LB broth containing 1% glucose was used to promote biofilm formation. The biofilm was collected by centrifugation and resuspended in fresh LB medium. OD 600 It is 0.01 (approximately 2.5 × 10⁻⁶). 6CFU / mL). Aliquots (100 μL) of bacterial suspension were transferred to 96-well polyvinyl chloride (PVC) microplates, followed by the addition of a single phage (ΦEAS1, ΦEHO4B, ΦEHO11, ΦEHO14, or ΦECL22) or a combination thereof (e.g., EHO-cocktail4), with a multiplicity of infection (MOI) of 10. Wells without phage inoculation and wells containing only LB medium served as positive and negative controls for biofilm formation, respectively. Polymyxin B (20 μg / mL, 5×MIC) and EDTA (5 mM) were used as reference anti-biofilm agents. After incubation at 37°C for 48 hours, biofilm formation was quantified by crystal violet (CV) staining. Each experiment was performed independently in triplicate, and results are expressed as mean ± standard deviation.
[0048] To determine the ability of bacteriophages to disrupt established biofilms, firstly... E. hormaechei On July 25th, biofilms were cultured in 96-well polyvinyl chloride plates for 48 hours. After removing planktonic cells, 100 μL of LB medium containing 1% glucose was added to each well, and the plates were cultured for another 48 hours to ensure full biofilm development. The resulting mature biofilms (4 days old) were gently rinsed once with fresh LB medium to remove non-adhesive bacteria. Individual bacteriophages (ΦEAS1, ΦEHO4B, ΦEHO11, ΦEHO14, and ΦECL22) and EHO-cocktail4 were diluted in LB medium to a final concentration of 1 × 10⁻⁶. 7 PFU / mL was added to the wells. Wells containing only LB medium served as untreated controls. Polymyxin B (20 μg / mL, 5×MIC) and EDTA (5 mM) were used as baseline biofilm removal treatments. After culturing at 37°C for 24 hours, biofilm biomass was assessed by crystal violet staining, and viable cells in selected groups were counted by colony forming units (CFU). All experiments were performed in biological triplicate, and data are presented as mean ± standard deviation.
[0049] To observe the structural visualization of biofilm disruption after phage preparation treatment, a single well was cut from a polyvinyl chloride microplate and processed using a scanning electron microscope (SEM; Clara).
[0050] 1.6 Evaluation of the antibacterial activity of bacteriophage preparations in food matrix models The method for evaluating the antibacterial activity of phage preparations against foodborne Enterobacter holmie is as follows.
[0051] Freshly purchased lettuce undergoes the following pretreatment process: first, it is continuously rinsed with sterile distilled water; then, it is surface-sterilized with 75% ethanol; and finally, it is rinsed with sterile PBS. Leaf tissue is then cut into 1×1 cm pieces. 2 Square sections were air-dried in a biosafety cabinet and then exposed to ultraviolet light for 20 minutes. Each section was then transferred to a sterile petri dish. E. hormaecheiThe logarithmic mid-growth culture from 7-25 was adjusted to 1×10⁻⁶. 5 CFU / mL concentration, take 0.1 mL of suspension and spread it evenly on each lettuce leaf (final load ≈ 1 × 10⁻⁶). 4 CFU / cm 2 After incubation at 25°C for 40 min, the samples were randomly divided into three groups: a phage group (containing 0.1 mL of phage preparation, MOI 100), a negative control group (containing 0.1 mL of sterile SM buffer), and a positive control group (containing 0.1 mL of polymyxin B, final concentration 20 µg / mL). The treated samples were incubated at 4°C (simulated cold chain) or 25°C (simulated treatment temperature) for 9 hours, respectively. At time points of 0 h, 3 h, 6 h, and 9 h, lettuce slices were transferred to 5 mL of SM buffer, vortexed for 5 min, serially diluted, and finally plated onto LB agar plates. Colony counts were determined using the standard plate count method, with three replicates for each experimental condition.
[0052] Dispense 12 mL of pasteurized whole milk into three sterile test tubes (4 mL per group). [The text then abruptly shifts to a seemingly unrelated topic:] ...logarithmic growth phase... E. wall Dilute the bacterial culture to 4×10⁻⁵ on July 25th. 5 CFU / mL, add 0.1 mL to each tube (final concentration ≈ 1×10⁻⁶). 4 CFU / mL). After static incubation at 25℃ for 40 min, phage preparation (MOI 100), SM buffer (equal volume negative control), and 20 µg / mL polymyxin B (positive control) were added to the samples. Milk samples were stored at 4℃ or 25℃ and collected at 0h, 12h, 24h, 3h6, and 48h. All samples were vortexed, serially diluted, and inoculated onto LB agar plates, and counted using the standard colony counting method. Three biological replicates were set up for the experiment.
[0053] Cut 500 grams of fresh pork tenderloin into 1 cm pieces using aseptic cutting. 3 The samples were then subjected to the following surface disinfection treatments: rinsing with sterile distilled water, immersing in 75% ethanol for 1 minute, and washing with sterile PBS. After drying in a biosafety cabinet, the samples were treated with ultraviolet light for 20 minutes. E. hormaechei Mid-log growth culture from 7-25°C was diluted to 1×10⁻⁶. 5 CFU / mL, take 0.1 mL and apply it to the surface of each square (final contamination concentration ≈ 1 × 10⁻⁶). 4 CFU / cm 2After adsorption at 25℃ for 40 min, pork pieces were randomly divided into three groups: the phage preparation group (MOI 100), the SM buffer control group, and the polymyxin B control group (final concentration 20 µg / mL). The treated pork pieces were stored at 4℃ or 25℃, respectively. At 0 h, 3 h, 6 h, and 9 h, one sample was transferred to 5 mL of sterile SM buffer, vortexed for 5 min to remove bacteria, serially diluted, and inoculated onto LB agar plates for CFU determination. Three replicates were set up for each treatment condition for analysis.
[0054] 1.7 Evaluation of the adhesion and internalization of Enterobacter hominis in cherry tomatoes by the phage preparation EHO-cocktail 4 To evaluate the activity of the phage preparation EHO-cocktail4 against the surface adhesion of *Enterobacter holmieae*, undamaged cherry tomatoes (approximately 7 g / tomato) were selected, thoroughly washed with sterile distilled water, disinfected with 75% ethanol, and rinsed with sterile saline to remove surface contaminants. Three tomatoes were immersed in 50 ml of (i) E. hormaechei 7-25 suspension (1×10 5 (i) Bacteria in EHO-cocktail 4 (MOI 100). Immersion was performed at 12°C for 30 minutes to simulate contamination under cold chain or refrigeration conditions. Polymyxin B (20 μg / mL, 5×MIC) was used as a positive control. After exposure, tomatoes were aseptically removed and rinsed with 50 mL of sterile saline. 100 μL of the rinse solution was aliquoted onto LB agar plates and incubated at 37°C for 24 hours. Bacterial load (CFU / mL) was quantified. Each condition was performed in triplicate.
[0055] To assess internalization control, cherry tomatoes were pre-incubated at 25°C to establish a temperature gradient favorable for bacterial penetration. As described above, three tomatoes were immersed in separate bacterial suspensions or bacterial-phage preparations (MOI 100) at 12°C for 30 minutes. Polymyxin B (20 μg / mL, 5×MIC) was again used as a positive control. After treatment, the tomatoes were rinsed once with 50 mL of sterile saline to remove externally attached bacteria, then immersed in 96% ethanol for 2 minutes and air-dried under sterile conditions for 30 minutes. These tomatoes were then transferred to sterile bags containing 50 mL of sterile saline for homogenization. The homogenate was sonicated at 40 kHz for 20 minutes using a YM-100 PLUS multi-functional sonicator. Solid residues were precipitated, resuspended in 1 mL of saline, inoculated onto LB agar, and incubated at 37°C for 24 hours. Bacterial counts (CFU / mL) were calculated. All conditions were tested in triplicate.
[0056] 1.8. Mouse model of infection induced by Enterobacter cholerae The experimental animals were 6-week-old, 30-35g female specific pathogen-free (SPF) Kunming (KM) mice. Mice were housed under standard laboratory conditions, with a regular light / dark cycle and room temperature, and were provided with food and water. All animals underwent a one-week acclimatization period before the experiment.
[0057] After the acclimatization period, mice were randomly assigned to experimental groups (n=5 per group). Before infection, all mice were fasted for 12 hours but allowed free access to water to minimize the potential impact of residual food on the experimental results. Infection models were established via intragastric gavage (IG) or intraperitoneal injection (IP): 4.0 × 10⁻⁶ mg / L, respectively. 9 CFU / each or 4.0 × 10 8 The dose of CFU / mouse was administered to mice by gavage in the form of 100 μL. E. hormaechei Bacterial suspension was prepared on July 25th. Control mice were administered 100 μL of sterile phosphate-buffered saline (PBS) via intraperitoneal injection (IP). After infection, mice were given normal feeding conditions and free access to sterile water and food. Clinical symptoms were monitored and scored every 4 hours for the first 48 hours; thereafter, they were assessed daily for a total observation period of 7 days. Clinical severity was assessed using a scoring system adapted from the Mouse Sepsis Score (MSS). Survival data were analyzed using Kaplan-Meier survival curves generated by GraphPad Prism software.
[0058] 1.9 Phage therapy model of bacteremia in mice Mice were randomly divided into five groups, with 10 mice in each group. Mice in the control group were intraperitoneally injected with 100 μL of sterile PBS. Mice in the other groups were intraperitoneally injected with 100 μL of sterile PBS. E. hormaechei 7-25 (4.0×10) 9 CFU / mouse). One hour post-infection (POI), mice in the control and infected groups were intraperitoneally injected with 100 μL of SM buffer. Mice in the phage preparation group were intraperitoneally injected with 100 μL of EHO-cocktail4 (1.0 × 10⁻⁶ CFU / mouse). 6 PFU / mouse), mice in the gavage group were orally administered 100 μL of EHO-cocktail 4 (1.0 × 10⁻⁶ PFU / mouse). 9 PFU / mouse). Mice in the polymyxin B group were intraperitoneally injected with polymyxin B (4 mg / kg). All phages used in this study were purified by cesium chloride density gradient ultracentrifugation, followed by endotoxin removal by dialysis.
[0059] After treatment, all mice were housed under standard feeding conditions with free access to food and water. Body weight and clinical scores were recorded daily during the experiment. On day 7 post-infection, surviving mice were anesthetized, and euthanized via retroorbital puncture followed by cervical dislocation. Blood bacterial counts were determined using the LB agar plate culture method. Complete blood count (CBC) analysis was performed to assess the peripheral blood immune cell profile. Survival data were analyzed using Kaplan-Meier survival curves generated by GraphPad Prism version 10.5.0 software.
[0060] II. Test Results 2.1 Isolation and Host Range Analysis of Bacteriophages To separate the target E.hormaechei Highly virulent bacteriophages were used in this study, and clinically isolated bacteriophages were employed. E.hormaechei Using the strain as a host, bacteriophages were isolated from hospital wastewater. Four bacteriophages were successfully isolated: ΦEHO1, ΦEHO4B, ΦEHO11, and ΦEHO14. For the bioinformatics and characteristics of ΦECL22, see: Optimizing Phage Therapy for Carbapenem-Resistant Enterobacter cloacae Bacteremia: Insights into Dose and Timing. Antimicrobial Agents and Chemotherapy, 2025, 69(4). The host strain of ΦEHO1 was identified as *Enterobacter assbury* by 16S rDNA sequence analysis. Enterobacter asburiae Therefore, it was named ΦEAS1. All host strains underwent whole-genome sequencing to determine their sequence types (STs). Among the 18 *Enterobacter holmie* strains analyzed... E.hormaechei Fourteen different STs were identified from the strains. The host range of each phage was preliminarily assessed using a dot assay. Although all isolated phages could lyse *Enterobacter holmieae* strains, they showed significant differences in host range. Figure 1 ).
[0061] To achieve broader lysis coverage, five phages (ΦEAS1, ΦEHO4B, ΦEHO11, ΦEHO14, and ΦECL22) were initially screened for the mixed formulation due to their broad host spectrum, complementarity, and ability to co-lyze all tested phages. E.hormaechei Strains, including epidemiologically important sequence types such as ST78, ST133, ST171, and ST418 ( Figure 1 It is worth noting that ST78 and ST171 are the main lineages of human infection in the United States and Europe, while ST133 and ST418 are among the most common sequence types in hospitals in southern China.
[0062] In this study, E. hormaechei 7-25 (ST171) exhibits resistance to both meropenem and polymyxin B (Table 4), and is classified as a carbapenem- and polymyxin-resistant strain. E. hormaechei The isolate offered limited clinical treatment options. Furthermore, this strain was susceptible to all five bacteriophages. Therefore, it was selected as a representative host strain for subsequent experiments.
[0063] Table 4. Conventional inhibitors against E. hormaechei Minimum inhibitory concentration of 7-25 2.2 Study on the biological characteristics of five bacteriophages The biological characteristics of these five bacteriophages were then characterized. ΦEAS1, ΦEHO11, ΦEHO14, and ΦECL22 formed small needle-like plaques, while ΦEHO4B produced relatively large plaques surrounded by distinct halos, suggesting the possible production of polysaccharide depolymerases. Transmission electron microscopy (TEM) showed that ΦEAS1, ΦEHO11, ΦEHO14, and ΦECL22 exhibited typical T4-like morphology, characterized by an icosahedral head, a contractile tail, and clearly visible tail fibers, and were classified as myoviruses. In contrast, ΦEHO4B had a long, non-contractile tail, consistent with the morphological characteristics of siphon viruses. Figure 2 ).
[0064] Thermal stability tests showed that ΦEAS1 and ΦEHO11 were moderately sensitive to temperature, almost completely inactivated after 1 hour at 50°C, while ΦEHO14 was completely inactivated only at 60°C. Notably, ΦEHO4B exhibited excellent heat resistance, retaining approximately 75% infectivity at 60°C and completely inactivated only at 70°C, indicating its strong thermal stability. Figure 3 Similarly, ΦEAS1, ΦEHO11, and ΦEHO14 exhibited typical pH tolerance characteristics, while ΦEHO4B showed significant alkali tolerance, with over 80% of phage particles still surviving at pH 12. Figure 3 After 10 minutes of UV irradiation, ΦEAS1, ΦEHO4B, and ΦEHO14 were almost completely inactivated. In contrast, ΦEHO11 showed stronger UV tolerance, retaining approximately 40% infectivity after 10 minutes of exposure and becoming completely inactivated only after 20 minutes. Figure 3 ).
[0065] The optimal multiples of infection (MOI) values for ΦEAS1, ΦEHO4B, ΦEHO11, and ΦEHO14 were 0.01, 0.01, 0.01, and 0.0001, respectively. Single-step growth curve analysis showed that ΦEAS1 had a latency period of 15 minutes and a lysis period of 15 minutes; ΦEHO4B had a shorter latency period of 5 minutes, followed by a 10-minute lysis phase; ΦEHO11 had a latency period of 15 minutes and a lysis period of 25 minutes; ΦEHO14 had a latency period of 15 minutes and a lysis period of 15 minutes. Figure 3 The phage bursts of ΦEAS1, ΦEHO4B, ΦEHO11, and ΦEHO14 in each infected bacterial cell were approximately 12, 22, 32, and 28 phage particles, respectively. ΦECL22 exhibited biological characteristics generally similar to ΦEHO11, including relatively high UV tolerance.
[0066] 2.3 Characterization and Comparative Genomics Study of Five Enterobacterial Phages The complete genome sizes of bacteriophages ΦEAS1, ΦEHO4B, ΦEHO11, ΦEHO14, and ΦECL22 were determined to be 173107 bp, 50416 bp, 178437 bp, 173643 bp, and 177652 bp, respectively, with GC contents of 49.4%, 50.2%, 50.1%, 49.6%, and 44.71%. All five bacteriophages possess circular double-stranded DNA genomes and belong to the class Caudaviviridae. ΦEAS1, ΦEHO11, ΦEHO14, and ΦECL22 are classified under the family Caudaviviridae, while ΦEHO4B belongs to the family Drellviridae (Table 5).
[0067] Table 5. Genomic characteristics of five Enterobacterial bacteriophages Whole-genome BLAST analysis showed that ΦEAS1 had 97% genome coverage and 97% nucleotide identity with Enterobacterial phage vB_EclM_Q7622 (NC_070778.1), originally isolated in Guangdong Province, China. ΦEHO4B had 76% coverage and 83% identity with Enterobacterial phage vB_EclS_AS5 (OR753409.1), indicating that ΦEHO4B is a novel phage. ΦEHO11 had 95% coverage and 98.49% sequence identity with Enterobacterial phage EC-F1. ΦEHO14 was highly similar to Enterobacterial phage vB_EclM_CIP9 (NC_048849), isolated in Canada, with 99% coverage and 97.32% sequence identity. ΦECL22 had 97% coverage and 99.72% sequence identity with Enterobacterial phage phi5. Linear genome alignment revealed that although these phages share sequence similarities with their closest Enterobacterial phage relatives, their genome structures differ significantly, suggesting that recombination events occurred during evolutionary differentiation.
[0068] A phylogenetic tree was constructed based on the amino acid sequences of the major capsid proteins of five bacteriophages. The resulting tree clearly divides into two independent branches: ΦEAS1, ΦEHO11, ΦEHO14, and ΦECL22 cluster in the same branch, while ΦEHO4B forms an independent branch, indicating that ΦEHO4B has a unique evolutionary trajectory. In the main branch, ΦEHO11 and ΦECL22 are adjacent, indicating a very high phylogenetic correlation, while ΦEAS1 and ΦEHO14 also cluster closely. In contrast, there is a large evolutionary distance between these two subgroups. Safety assessment of the genomes of these five bacteriophages was performed using virulence factor analysis tools and a global pathogen analysis platform. No virulence factors or antibiotic resistance genes were detected in any of the five bacteriophages.
[0069] 2.4 Antagonistic effect of bacteriophage ΦEHO4B in bacteriophage mixtures and preparation of bacteriophage preparation EHO-cocktail4 To prepare a high-coverage phage mixture, a phage mixture EHO-cocktail5 was first prepared, consisting of ΦEAS1, ΦEHO4B, ΦEHO11, ΦEHO14, and ΦECL22 (each phage was mixed at a 1:1 titer ratio). When used alone, ΦEAS1, ΦEHO4B, and ΦEHO14... E.hormaecheiThe inhibitory effect of 7-25 lasted for about 8 hours before bacterial regeneration began. In contrast, ΦEHO11 and ΦECL22 showed more significant inhibitory effects, suppressing bacterial growth to extremely low levels for at least 24 hours. However, the phage mixture EHO-cocktail5 only inhibited bacterial growth for 6 hours, after which bacterial proliferation rapidly returned to the level of the untreated control group, indicating that it completely lost its inhibitory efficacy. Figure 4 A).
[0070] To determine the source of the antagonistic effect, this invention combined multiple tetraphage combinations from five phages (each phage mixed in a 1:1 titer ratio). However, it was found that any mixture containing ΦEHO4B exhibited a weak inhibitory effect, while the only mixture without ΦEHO4B completely inhibited the antagonistic effect. E.hormaechei Growth was inhibited for 14 hours from 7-25 and remained strongly suppressed for 24 hours. Figure 4 B).
[0071] To further determine whether ΦEHO4B interferes with specific phages, this invention evaluated the interactions of pairwise phage combinations (each phage mixed at a 1:1 titer ratio). No direct antagonistic effect was observed between ΦEHO4B and the other four phages. Figure 4 C). Although ΦEHO4B and ΦECL22 exhibit moderate synergistic activity when used alone, the addition of a third phage (such as ΦEHO11 or ΦEHO14) reduces their synergistic activity, even if the additional phage (such as ΦEBU8) is effective against these conditions. E.hormaechei 7-25 lacks cytotoxic activity, and this synergistic effect is immediately disrupted. It is noteworthy that the antagonistic effect between ΦEHO4B and ΦECL22 is mitigated when heat-inactivated ΦEHO11 is added. Figure 4 D).
[0072] These results suggest that ΦEHO4B plays an "outlier" role among these phages and is not suitable for inclusion in multiphage mixtures.
[0073] Phages ΦEAS1, ΦEHO11, ΦEHO14 and ΦECL22 were mixed (each phage was mixed at a titer ratio of 1:1) to obtain a phage formulation, which was then labeled as EHO-cocktail4.
[0074] Spot test confirmed that EHO-cocktail4 was effective in 86.4% (19 / 22) of known ST cases. E.hormaechei The strain has an inhibitory effect ( Figure 1To further evaluate the coverage of EHO-cocktail4 against clinical Enterobacter isolates, 56 clinical isolates belonging to the Enterobacter cloacae complex were randomly selected, and their sensitivity was assessed using a dot blot assay. This phage preparation showed lysis activity against 78.6% (44 / 56) of the tested clinical isolates. Figure 5 ).
[0075] 2.5. Bacteriophage preparations can effectively inhibit and eliminate biofilms. To determine whether EHO-cocktail4 can effectively inhibit biofilm formation, its ability to prevent biofilm formation was first evaluated. E.hormaechei 7-25 Biofilm development capacity. Biofilms were cultured in LB broth in 96-well polyvinyl chloride plates containing different antimicrobial agents. Figure 6 The results from A showed that EHO-cocktail4 significantly reduced biofilm formation, exhibiting a stronger inhibitory effect than any single phage, and comparable to the activity of 5×MIC polymyxin B and 5mM EDTA.
[0076] Next, this invention evaluated the ability of EHO-cocktail4 to remove existing biofilms. E. wall After strain 7-25 was cultured in 96-well polyvinyl chloride plates for 4 days to form a mature biofilm, it was treated with a single bacteriophage (ΦEAS1, ΦEHO11, ΦEHO14 or ΦECL22) or EHO-cocktail4 preparation, and the elimination effect was compared.
[0077] The results showed that single phages exhibited limited biofilm removal activity, although ΦECL22 performed best in single phage treatment. In contrast, EHO-cocktail4 showed significantly higher biofilm removal efficiency than any single phage, even exceeding the activity of 5×MIC polymyxin B and 5mM EDTA. Figure 6 B). CFU quantification further confirmed that, compared with the untreated control group, the phage preparation EHO-cocktail4 reduced the viable bacterial load in mature biofilms by nearly 3 log units (B). Figure 6 C). Scanning electron microscopy (SEM) shows the untreated... E.hormaechei Strain 7-25 formed a dense, well-structured biofilm on the surface of polyvinyl chloride, while treatment with the phage preparation EHO-cocktail4 left only sparse and dispersed cells. Figure 6 D).
[0078] In summary, the above results indicate that the phage preparation EHO-cocktail4 can be used as a defense mechanism against... E. hormaechei Highly promising anti-biofilm agents.
[0079] 2.6. The bacteriophage preparation EHO-cocktail 4 has effects on food... E. hormaechei It has a significant bactericidal effect first, E.hormaechei 7-25 can rapidly proliferate on the surface of the above foods or in milk under both refrigerated (4℃) and room temperature (25℃) conditions.
[0080] When incubated at 25°C, the bacterial load in lettuce, pork, and milk increased by >2 log units, >2 log units, and >5 log units, respectively, within 9 hours. Figure 7 (AF). In contrast, the EHO-cocktail4 treatment group exhibited significant bactericidal activity: at 4°C, bacterial counts in all food matrices decreased by >1 log unit; at 25°C, bacterial counts in pork decreased significantly. E.hormaechei The 7-25 level decreased by approximately one log unit within 9 hours, and EHO-cocktail4 completely eliminated detectable bacterial contamination in lettuce within 6 hours (3 hours earlier than the clearance time of 5×MIC polymyxin B). In milk, EHO-cocktail4 treatment resulted in only a one log unit increase in bacterial proliferation within 48 hours, while the untreated control group showed an increase of 5 log units. Figure 7 AF).
[0081] Furthermore, using cherry tomatoes as a model, the EHO-cocktail4 pair was tested. E. hormaechei The control effect of surface adhesion and tissue internalization.
[0082] The results showed that while polymyxin B at 5 times the minimum inhibitory concentration (MIC) reduced surface-associated bacteria by more than 1 log unit, the phage preparation EHO-cocktail 4 achieved a reduction of more than 2 log units. Figure 7 G). For internalized bacteria, EHO-cocktail4 reduced the bacterial load from 8.2 × 10⁻⁶. 4 CFU decreased to 5.3×10 2 CFU, compared to the untreated group ( Figure 7 H), its efficacy is still significantly higher than that of the traditional antibiotic polymyxin B.
[0083] In summary, EHO-cocktail4 exhibited bactericidal activity superior to or comparable to 5×MIC polymyxin B in various food matrices, and can be used as a control agent. E. hormaechei Potential environmental disinfectants for food contamination.
[0084] 2.7 Intraperitoneal injection of the phage preparation EHO-cocktail4 can completely prevent Enterobacter hominis bacteremia. To establish a mouse model of *Enterobacter holmieae* infection, different routes of infection and bacterial doses were first evaluated. Mice were administered different concentrations of the bacteria via intragastric gavage or intraperitoneal injection. E.hormaechei Infection occurred on July 25th. Oral administration via gavage did not induce obvious disease symptoms; mice remained active and showed no significant difference compared to the uninfected control group. In contrast, intraperitoneal injection led to severe infection: when the *Enterobacter holmieae* infection dose was 4 × 10⁻⁵... 8 At CFU / mouse, 80% (4 / 5) of the mice died within 30 hours of infection, while the remaining mice gradually recovered; when the dose was increased to 4×10 9 At CFU / mouse, all mice died within 24 hours. Figure 8 A). These results demonstrate that *Enterobacter holmieae* can successfully establish a lethal bacteremia model in mice via the intraperitoneal route.
[0085] Based on this model, the therapeutic effects of phage formulations administered via intraperitoneal injection and oral gavage were further evaluated. Oral administration failed to produce a protective effect, and all infected mice died, even with a 1000-fold increase in phage dose. In contrast, intraperitoneal injection of the phage formulation EHO-cocktail4 provided significant protection. Within 24 hours of treatment, the mice's clinical symptoms significantly improved, and their weight gradually recovered. Figure 8 (B and C), ultimately achieving a 100% survival rate. Notably, this protective effect was superior to the polymyxin B treatment group (only 60% of mice survived). Figure 8 D). Seven days later, blood samples were collected from surviving mice for routine bacterial culture and hematological analysis. No live bacteria were detected in the blood of either the phage treatment group or the polymyxin B treatment group. Furthermore, the white blood cell (WBC) and neutrophil counts in the phage treatment group were comparable to those in the uninfected control group, indicating that the phage treatment effectively cleared the bacterial infection.
[0086] In summary, these results indicate that intraperitoneal injection of phage preparations can effectively protect mice from infection. E.hormaechei Induced bacteremia.
[0087] It should be noted that when numerical ranges are involved in this invention, it should be understood that both endpoints of each numerical range and any value between the two endpoints can be selected. Since the steps and methods used are the same as in the embodiments, preferred embodiments are described here to avoid redundancy. Although preferred embodiments of the invention have been described, those skilled in the art, once they understand the basic inventive concept, can make other changes and modifications to these embodiments. Therefore, the appended claims are intended to be interpreted as including the preferred embodiments as well as all changes and modifications falling within the scope of this invention.
[0088] Obviously, those skilled in the art can make various modifications and variations to this invention without departing from its spirit and scope. Therefore, if these modifications and variations fall within the scope of the claims of this invention and their equivalents, this invention also intends to include these modifications and variations.
Claims
1. A bacteriophage preparation, characterized in that, The phage preparation includes any one or more of phages ΦEAS1, ΦEHO11, ΦEHO14, and ΦECL22; the accession number of phage ΦEAS1 is CGMCC NO.47013; the accession number of phage ΦEHO11 is CGMCC NO.47014; the accession number of phage ΦEHO14 is CGMCC NO.47015; and the accession number of phage ΦECL22 is CGMCC NO.46281.
2. The phage preparation according to claim 1, characterized in that, The total phage content in each liter of the phage preparation is ≥1×10⁻⁶. 10 PFU.
3. The phage preparation according to claim 1, characterized in that, When the phage preparation comprises any two or more phages, the phages are mixed at a titer ratio of 1 to 10: 1 to 10.
4. The use of the phage preparation according to any one of claims 1 to 3 in the preparation of products that inhibit or kill foodborne Enterobacteriaceae, characterized in that, The foodborne enterobacter is Enterobacter holmieae.
5. The application according to claim 4, characterized in that, The product is a drug for preventing bacteremia, a drug for treating bacteremia, or an environmental bactericide.
6. A drug for the prevention or treatment of bacteremia, characterized in that, The drug is composed of the phage preparation and excipients as described in claim 1.
7. The drug according to claim 6, characterized in that, The bacteremia is caused by Enterobacter holmieae; the drug is in any veterinary-acceptable dosage form.
8. An environmental bactericide, characterized in that, The environmental bactericide is composed of the bacteriophage preparation described in claim 1 and an aqueous carrier; the aqueous carrier comprises water, buffer solution, physiological saline and culture medium.
9. The environmental bactericide according to claim 8, characterized in that, The environmental bactericide is used to inhibit or eliminate Enterobacter holmie in food substrates.
10. The environmental bactericide according to claim 9, characterized in that, The food matrix includes at least one of lettuce, pork, milk, and cherry tomatoes.