Broad-spectrum bacteriophage targeting mucoid escherichia coli, cronobacter and salmonella and applications thereof

Abstract

The application discloses a broad-spectrum bacteriophage targeting mucoid Escherichia coli, Cronobacter and Salmonella and application thereof Escherichia coli phage vB EcoP 3BB7B, the bacteriophage can specifically lyse colanic acid-producing mucoid Escherichia coli, and breaks through the technical bottleneck that a mucous layer limits adsorption of most bacteriophages; further has cross-species lysis capacity, not only has good lysis activity on mucoid Escherichia coli, but also can effectively lyse important foodborne pathogenic bacteria such as Salmonella of various serotypes and Cronobacter sakazakii, and significantly widens the host spectrum range. The bacteriophage can realize efficient amplification under the condition of a lower infection multiple, has good environmental stability, has strong lysis activity, does not carry drug resistance genes and virulence-related genes, has high safety, and has a good application prospect in clinical and food safety prevention and control.

Description

Technical Field

[0001] This invention relates to the field of microbial and pathogen control technology, and more specifically, to a broad-spectrum bacteriophage targeting mucoid Escherichia coli, Cronobacter, and Salmonella, and its applications. Background Technology

[0002] Antimicrobial resistance has become a major challenge in global public health in the 21st century, especially multidrug-resistant infections caused by Gram-negative bacteria, which often lead to limited clinical treatment options or even a situation where "no drugs are available." With the rate of antibiotic development lagging far behind the rate of emergence of drug-resistant bacteria, finding alternative or complementary antimicrobial strategies has become a hot topic in international research. Bacteriophages are a type of virus that specifically infects bacteria. They possess advantages such as strong host specificity, self-replication capability, non-disruption of normal flora, and continued killing effect on drug-resistant bacteria, and are considered an important alternative or complementary treatment to antibiotics.

[0003] The process of bacteriophage infection of bacteria depends on its recognition and adsorption to specific receptor structures on the bacterial surface, such as lipopolysaccharides, capsular polysaccharides, or outer membrane proteins. However, in recent years, Enterobacteriaceae with a mucoid phenotype have gradually emerged in clinical and animal sources, such as... Escherichia coli , Salmonella enterica , Klebsiella pneumoniae and Cronobacter sakazakii These strains form a thick mucus layer by excessively secreting extracellular polysaccharides, giving the colonies a viscous or stringy appearance. The mucus layer not only helps bacteria form biofilms, enhances environmental tolerance and immune evasion, but is also closely related to increased virulence, persistent infection, and improved drug resistance, posing a serious threat to clinical treatment and food safety. More importantly, the mucus layer can cover or mask common receptor structures on the bacterial surface, making it difficult for most bacteriophages to effectively adsorb and infect, thus leading to strong natural resistance of mucus-type strains to bacteriophages. Furthermore, when subjected to bacteriophage infection pressure, bacteria can rapidly form or enhance the mucus layer by regulating the synthesis and secretion of extracellular polysaccharides, serving as an important adaptive defense mechanism to prevent bacteriophages from contacting the cell surface and evading infection. This process can transform originally sensitive strains into mucus-resistant strains, often accompanied by enhanced biofilm formation, improved immune evasion, and increased virulence, further increasing the difficulty of infection control.

[0004] Currently reported bacteriophages mostly target non-mucous bacteria, exhibiting limited lytic ability against highly virulent and resistant mucous phenotypes or strains that can be induced to produce a mucous layer, making it difficult to effectively control the infection or spread of such pathogens. Therefore, isolating and screening bacteriophages capable of recognizing and infecting mucous structures not only helps to directly target naturally occurring mucous strains and overcome mucous-resistant strains resulting from phage selection pressure, but also holds promise for overcoming the bottleneck of reduced efficacy in traditional phage therapy due to the masking of surface receptors, thereby improving treatment stability and effectiveness.

[0005] However, existing technologies still have very limited effective phage resources targeting mucinous phenotype bacteria, especially lacking phages capable of specifically recognizing mucus structures and achieving efficient infection. Therefore, developing a phage capable of targeting mucinous phenotype bacteria and breaching the mucus barrier has significant clinical and industrial value for controlling infections caused by highly virulent and drug-resistant bacteria, as well as improving the effectiveness of phage therapy. This invention application is thus proposed. Summary of the Invention

[0006] The technical problem to be solved by this invention is to overcome the difficulties in the prevention and control of myxotropic Escherichia coli and the lack of effective phage resources for myxotropic bacteria. This invention provides a broad-spectrum phage targeting myxotropic Escherichia coli, Cronobacter, and Salmonella and its applications.

[0007] The first objective of this invention is to provide a strain of Escherichia coli bacteriophage. Escherichia coli phage vBEcoP 3BB7B.

[0008] A second objective of this invention is to provide Escherichia coli bacteriophages. Escherichia coli phage Applications of vB EcoP3BB7B.

[0009] A third objective of this invention is to provide a method for removing bacterial biofilms or for disinfection.

[0010] The fourth objective of this invention is to provide a product.

[0011] The above-mentioned objective of this invention is achieved through the following technical solution: This invention provides a strain of Escherichia coli bacteriophage Escherichia coli phage vB EcoP 3BB7B (abbreviated as P3BB7B), this bacteriophage was deposited on January 8, 2026, at the China General Microbiological Culture Collection Center (CGMCC), with accession number CGMCC No. 47012. The genome of bacteriophage P3BB7B identified in this invention is linear double-stranded DNA, belonging to... Autographiviridae Virology MelnykvirinaeThis subfamily, after comparison with similar phages in the NCBI database, showed a similarity of only about 70%, suggesting it may be a potential new genus of phage. The genome size is 42,677 bp, with a G+C content of 52%, and 48 open reading frames were successfully annotated. VirulenceFinder and ResFinder predictions indicate that the P3BB7B genome does not contain virulence factors or drug resistance genes.

[0012] The optimal multiplicity of infection (MOU) of bacteriophage P3BB7B against *Escherichia coli* was 0.000001:1, with a latency period of 10 min, a burst period of 70 min, and a burst yield of 54 ± 8 PFU / cell. It maintained good activity at 4–50 °C, indicating excellent temperature stability. Activity decreased at 60 °C and was completely inactivated at 70 °C, suggesting its potential application as a food preservative. It is inactivated after high-temperature cooking and poses no harm to consumers. When incubated for 1 h at pH 3–12, the phage titer did not change significantly and remained relatively stable, indicating good pH tolerance of phage P3BB7B.

[0013] Simultaneously, phage P3BB7B can target *Escherichia coli* myxomatosis, 13 different serotypes of *Salmonella*, and 4 ST-type *Cronobacter*. Identification revealed that the myxomatosis-specific *E. coli* targeted by this phage is *E. coli* that secretes colacin. If bacterial resistance to the phage is induced, the bacteria will not secrete colacin, and the relative adsorption efficiency of the phage will significantly decrease to about 10% of that of the host bacteria, indicating that the phage recognizes surface structures associated with colacin. It exhibits good bactericidal effects against myxomatosis *E. coli*; when MOI > 1000, phage P3BB7B can significantly inhibit bacterial growth without inducing phage resistance, demonstrating good application efficacy. Furthermore, bacteriophage P3BB7B has a good bactericidal effect on hospital-originating colacin-producing mucoid Escherichia coli, effectively removing biofilms formed by mucoid Escherichia coli, and also effectively removing bacteria on polyethylene surfaces. It has good disinfection effects and has the potential to be used in the clinical treatment of mucoid Escherichia coli that produce biofilms, as well as as a novel disinfectant. It can be used in the manufacture of biopharmaceuticals, chemical pharmaceutical raw materials, and formulations.

[0014] Therefore, the present invention provides Escherichia coli bacteriophages. Escherichia coli phage The following applications of vB EcoP 3BB7B: Application in the inhibition of mucoid Escherichia coli, Cronobacter and / or Salmonella for purposes other than disease diagnosis and treatment.

[0015] Application in the preparation of products that inhibit mucoid Escherichia coli, Cronobacter and / or Salmonella.

[0016] Preferably, the mucoid Escherichia coli is an Escherichia coli that can secrete Kolac.

[0017] Application in the preparation of disinfectant products.

[0018] Application in the preparation of therapeutic drugs for diseases caused by mucoid Escherichia coli.

[0019] Preferably, the product or drug can remove bacterial biofilm.

[0020] This invention provides a method for removing bacterial biofilms or disinfecting, using a product containing bacteriophage P3BB7B for treatment.

[0021] The present invention also provides a product containing bacteriophage. Escherichia coli phage vB EcoP 3BB7B .

[0022] Preferably, the product is a disinfectant, cleaning agent, external bactericide, medicine, food preservative, or feed additive.

[0023] The present invention has the following beneficial effects: This invention provides a bacteriophage capable of efficiently lysing mucinous Enterobacteriaceae. Escherichia coli phage vB EcoP 3BB7B, a bacteriophage, can specifically lyse collamyic, mucoid-type *Escherichia coli*, overcoming the technical bottleneck of limited adsorption of most bacteriophages by the mucus layer. Simultaneously, this bacteriophage exhibits cross-genera lysis capability, demonstrating good lysis activity not only against mucoid *E. coli* but also effectively lysing various serotypes of *Salmonella* and *Cronobacter sakazakii*, significantly broadening its host spectrum. This bacteriophage achieves efficient amplification under low multiple of infection (MOI) conditions, exhibits good environmental stability, and strong lysis activity. Genomic sequencing analysis shows that it does not carry drug resistance genes or virulence-related genes, indicating high safety and promising clinical and food safety control applications. Furthermore, its genome shows low homology with previously reported bacteriophages, and phylogenetic analysis suggests it may belong to a new taxonomic unit, possessing significant taxonomic innovation value. Attached Figure Description

[0024] Figure 1 This is a single-spot morphology of bacteriophage P3BB7B.

[0025] Figure 2 This is a transmission electron microscope image of bacteriophage P3BB7B.

[0026] Figure 3 This is the complete genome information of bacteriophage P3BB7B.

[0027] Figure 4 This is a phylogenetic tree of bacteriophage P3BB7B.

[0028] Figure 5 This is the one-step growth curve of bacteriophage P3BB7B.

[0029] Figure 6 This represents the optimal multiplicity of infection for phage P3BB7B.

[0030] Figure 7 The results of the stability evaluation of bacteriophage P3BB7B (A: temperature stability; B: pH stability).

[0031] Figure 8 The lysis effect of bacteriophage P3BB7B on different serotypes of Salmonella.

[0032] Figure 9 The in vitro bactericidal effect of bacteriophage P3BB7B.

[0033] Figure 10 It is a resistant bacterial morphology.

[0034] Figure 11 BW2513- yrfF Colony morphology of Y98N strain after rcsC mutation.

[0035] Figure 12 BW25113- yrfF Phage susceptibility after rcsC mutation in Y98N strain.

[0036] Figure 13 For the quantitative detection of colacid in bacteriophage-resistant bacteria.

[0037] Figure 14 This study aims to quantitatively detect colacid in mucoid Escherichia coli.

[0038] Figure 15 The relative adsorption efficiency of bacteriophage P3BB7B on bacteria.

[0039] Figure 16 The bactericidal curve of phage P3BB7B against hospital-derived mucoid Escherichia coli is shown.

[0040] Figure 17 Simulation of the bacteriophage disinfection effect on polyethylene surface.

[0041] Figure 18 To evaluate the ability of phage P3BB7B to inhibit and clear biofilms. Detailed Implementation

[0042] The present invention will be further described below with reference to the accompanying drawings and specific embodiments, but the embodiments do not limit the present invention in any way. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in this technical field.

[0043] Unless otherwise specified, all reagents and materials used in the following examples are commercially available.

[0044] The LB nutrient agar formula used in the examples is as follows: 10.0 g tryptone, 5.0 g yeast extract, 10.0 g sodium chloride, 15.0 g agar, and 1000 mL distilled water.

[0045] The recipe for LB broth is as follows: 10.0 g tryptone, 5.0 g yeast extract, 10.0 g sodium chloride, and 1000 mL deionized water.

[0046] The SM solution formula is as follows: 8.5g sodium chloride, 2g magnesium sulfate, 50 mL 1 mol / L Tris-HCl, 0.25 g gelatin, and 1000 mL deionized water.

[0047] The formula for LB semi-solid agar is: 7.2 g tryptone, 3.6 g yeast extract, 7.2 g sodium chloride, 7.0 g agar, and 1000 mL deionized water.

[0048] Aseptic double-concentrated broth with 2 mM CaCl2: 20 g LB broth powder, 88.78 mg CaCl2, 400 mL deionized water.

[0049] All pathogens used in the following examples were isolated and preserved in Professor Liu Jianhua's research group at the College of Veterinary Medicine, South China Agricultural University.

[0050] Example 1: Isolation, preparation, purification, and culture of bacteriophages 1. Selection of host bacteria The host bacterium selected was *Escherichia coli* 32M3BB (strain source: Tu J, Yang J, Song K, Wang J, Zhang N, He W, Liu J, Cai Z, Bai Y, Lv L, Zhu B, Tao P, Feng J, Liu JH. Colaninic acid-mediated phage resistance enhances virulence in high-risk globalclone *Escherichia coli* ST410. PLoS Pathog. 2025 Dec 22;21(12):e1013807. doi:10.1371 / journal.ppat.1013807. PMID: 41428763; PMCID: PMC12753057.). This strain was a phage-resistant strain screened during the team's phage resistance research. Compared with the original strain 32M, this strain showed an enhanced mucus phenotype, which was identified as increased colaninic acid secretion. Our research revealed that 32M3BB exhibits enhanced immune evasion and virulence, posing a potential risk during phage therapy. Furthermore, given the frequent detection of myxobacterial Escherichia coli in human and veterinary clinical settings, and the significant clinical harm it poses, this study selected myxobacterial Escherichia coli as the target for phage isolation.

[0051] 2. Sample pretreatment Pig manure was collected from a farm in Guangdong Province, preserved at low temperature, and then transported back to the laboratory. Fresh manure was added to SM buffer and thoroughly mixed by shaking for 4 hours. The sample was then centrifuged at 4000 g for 20 minutes to remove bacteria and other impurities. Bacterial sterilization was then performed using 0.45 μm and 0.22 μm microporous membranes, respectively.

[0052] 3. Isolation of bacteriophages Take 10 mL of sterile, double-concentrated broth containing 2 mmol / L CaCl2 and add 10 mL of clarified (or filtered) wastewater; inoculate with 0.1 mL of overnight cultured host bacteria and incubate gently (50 rpm / min) with shaking at a suitable growth temperature (usually 37 °C); after 24–48 h of incubation, centrifuge at 10000 g for 10 min. Pour the supernatant into a screw-cap vial or a stoppered test tube. Add 0.5 mL of chloroform to the clarified crude lysis buffer, shake gently, and store at 4 °C.

[0053] 4. Phage spot test Mix 100 μL of overnight cultured bacterial cells with 5 mL of thawed and incubated semi-solid culture medium at 50 °C, and pour the mixture onto a bottom agar plate to prepare bacterial growth for each strain. Add a few drops (5 μL) of the sample enrichment solution to the bacterial growth until the droplets are completely dry. Incubate the plate overnight at 37 °C. Check for the presence of clear, turbid lysis zones.

[0054] 5. Isolation of bacteriophages Add 0.9 mL of SM Buffer to sterile test tubes or centrifuge tubes, and number the centrifuge tubes according to the dilution. Add 0.1 mL of phage enrichment solution to the first tube, mix well, and then transfer 0.1 mL to the second tube. Perform 10-fold dilutions sequentially in this manner. Transfer 0.1 mL of each phage dilution to a warm semi-solid culture medium tube, immediately add 0.1 mL of host bacteria cultured to the logarithmic growth phase, mix well, and then pour the mixture onto the surface of the solid culture medium. After the semi-solid layer solidifies, incubate it upside down at the optimal culture temperature. Incubate for 8–10 h, and select plates with single plaques for phage purification.

[0055] 6. Purification of bacteriophages Using an autoclaved pipette tip, pick up a single phage plaque, suspend it in 1 mL of SM Buffer, and thoroughly vortex to dissolve the phage completely. Filter using a 0.22 μm pore size membrane to remove contaminants. Appropriately dilute the SM Buffer containing the phage plaque, and passage the phage continuously using a double-layer plate culture method. When the observed phage plaques are substantially consistent in size and morphology, purified phage individuals can be obtained. Figure 1 As shown, an Escherichia coli bacteriophage was obtained and named P3BB7B. Escherichia coli phage vB EcoP 3BB7B).

[0056] 7. Preservation of bacteriophages In a 2 mL preservation tube, mix 0.5 mL of phage stock solution with 0.5 mL of sterile glycerol to a final concentration of 50%. Store at -80 °C.

[0057] Example 2: Electron microscopic observation of bacteriophages Take the phage P3BB7B culture prepared in Example 1, and concentrate 50 mL of phage solution to 2 mL using a 100 kDa Amicon Ultra centrifuge ultrafiltration tube; take 15 μL of the concentrated phage solution and drop it onto a copper grid, and let it precipitate for 15 min; gently absorb the excess liquid on the copper grid with filter paper, and then add 2% phosphotungstic acid (PTA, pH=7.0) to the copper grid for staining for 10 min. After the copper grid dries, observe and photograph it under an electron microscope.

[0058] The results are as follows Figure 2 As shown, morphological observation of bacteriophage P3BB7B under a transmission microscope revealed that it is a short-tailed bacteriophage with a head structure diameter of approximately 51.37 nm.

[0059] Example 3: Extraction, sequencing, and preservation of bacteriophage genomes Take the phage culture P3BB7B prepared in Example 1 and concentrate 50 mL of phage solution to 2 mL using a 100 kDa Amicon Ultra centrifuge tube. Take 2 mL of phage sample that has passed through a 0.22 µm microporous membrane and place it in a 15 mL centrifuge tube that has been autoclaved and has a smooth surface. Add 2 µL of DNase I and RNase (1 mg / mL final concentration 1 µg / mL), gently invert and mix, and incubate at 37 °C for 1 h. In the 15 mL centrifuge tube, add 2 mL of formamide, 20 µL of EDTA, and 200 µL of 2 M Tris HCl / 0.2 M EDTA (TE), gently invert and mix, and let stand at room temperature for 30 min. Use the phage genome extraction kit (M13 Isolation Kit D6900) to complete the extraction of genomic DNA according to the instructions.

[0060] The extracted phage products were sent to Meiji Biotechnology Co., Ltd. for sequencing to obtain Escherichia coli phages. Escherichia coli phage The nucleotide sequence of vB EcoP 3BB7B. Genome annotation results are as follows: Figure 3 As shown, the genome of bacteriophage P3BB7B is linear double-stranded DNA, belonging to... Autographiviridae Virology Melnykvirinae The subfamily, after comparison with similar bacteriophages in the NCBI database, showed a similarity of only about 70%, and the phylogenetic tree showed it located on a separate branch, such as... Figure 4 As shown, these may be potential new genus bacteriophages, and their genomes have been submitted to the International Committee on Virology (ICTV) for verification. Among them, P3BB2B, P3BB5B, and PED39-1PT are bacteriophages previously isolated by the inventors, while the rest are bacteriophages from the NCBI database.

[0061] The P3BB7B genome is 42677 bp in size, with a G+C content of 52%, and has been successfully annotated to 48 open reading frames (ORFs). VirulenceFinder and ResFinder predictions indicate that the P3BB7B genome does not contain virulence factors or drug resistance genes.

[0062] The phage P3BB7B was then biopreserved, with the preservation information as follows: Escherichia coli phage. Escherichia coli phage vB EcoP 3BB7B was deposited on January 8, 2026, at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 47012.

[0063] Example 4: One-step growth curve determination (1) The host bacterium 32M3BB was cultured to the logarithmic growth phase and then plated for counting after being serially diluted tenfold; (2) The titer of phage P3BB7B was determined by a double-layer plate experiment; (3) Adjust the concentration of bacterial solution 32M3BB and the titer of phage solution P3BB7B to appropriate amounts, mix the phage solution and bacterial solution 1:1 with the optimal infection multiple, the final volume is 10 mL, and let stand for 10 min. (4) Centrifuge the mixture at 5000 rpm for 3 min to separate the supernatant and precipitate; (5) Filter the supernatant through 0.22 μm and perform a double-layer plate test to determine the phage titer; (6) The precipitate was resuspended in 10 mL of fresh LB broth and incubated with shaking at 37 °C and 180 rpm. The titer of phage P3BB7B was measured every 10 min. The entire process was recorded, and the experiment was repeated three times. The phage burst refers to the average number of phages released by each bacterium infected by a phage.

[0064] The calculation formula is: .

[0065] The results are as follows Figure 5 As shown, the incubation period of phage P3BB7B was 10 min, the outbreak period was 70 min, and the outbreak amount was 54±8 PFU / cell.

[0066] Example 5: Confirmation of the optimal multiplicity of infection for bacteriophages (1) The host bacterium 32M3BB was cultured to the logarithmic growth phase and then plated for counting by serial dilution of 10-fold; (2) The titer of phage P3BB7B was determined by a double-layer plate test. (3) Adjust the concentration of the bacterial solution and the titer of the phage solution to appropriate amounts. Mix the phage solution P3BB7B and the bacterial solution 32M3BB at a ratio of 1:1 with MOI = 10, 1, 0.1, 0.01, 0.001, 0.0001, 0.00001, and 0.000001 respectively, and let stand for 10 min. (4) Add fresh LB broth to bring the volume to 10 mL, and incubate at 37 °C and 180 rpm for 4 h with shaking. (5) Centrifuge the mixture at 10000 g for 2 min, take the supernatant and filter it through a 0.22 μm filter membrane; (6) The phage titer of each group of P3BB7B phage fluid was determined by a double-layer plate test. Each experimental group was repeated three times.

[0067] The results are as follows Figure 6 As shown, under the condition of MOI=0.000001, the titer of bacteriophage P3BB7B reached its highest level (4.07×10⁻⁶). 9 PFU / mL).

[0068] Example 6 Determination of phage stability 1. Temperature stability The host bacterium 32M3BB was cultured to the logarithmic growth phase. Metal baths were pre-set at 4 °C, 25 °C, 37 °C, 50 °C, 60 °C, and 70 °C. After the temperatures stabilized, the P3BB7B phage solution was incubated in each temperature for 1 h. After incubation, a double-layer plate assay was performed to determine the phage titer. Each experimental group was repeated in triplicate.

[0069] The results are as follows Figure 7 As shown, when bacteriophage P3BB7B was incubated at 4 ℃, 25 ℃, 37 ℃ and 50 ℃ for 1 h, the phage titer did not change significantly, and the phage maintained good activity, indicating that bacteriophage P3BB7B has good temperature stability. Its activity decreased at 60 ℃ and was completely inactivated at 70 ℃, showing potential for food preservation applications. It is inactivated after high-temperature cooking and is harmless to eat.

[0070] 2. pH stability The host bacterium 32M3BB was cultured to the logarithmic growth phase. The pH of LB broth was adjusted to 3–12 beforehand. P3BB7B bacteriophage was added to LB broth at different pH values ​​at a ratio of 1:10 and incubated at room temperature for 1 h. After incubation, a double-layer plate assay was performed to determine the bacteriophage titer. Each experimental group was performed in triplicate.

[0071] The results are as follows Figure 7As shown, when bacteriophage P3BB7B was incubated at pH 3 to 12 for 1 h, the phage titer did not change significantly and could maintain a relatively stable level, indicating that bacteriophage P3BB7B has good pH tolerance.

[0072] Example 7 Bacteriophage Escherichia coli phage Pyrolysis spectroscopy determination of vB EcoP 3BB7B This experiment selected 10 strains of mucoid Escherichia coli (plus one standard strain of Escherichia coli BW25113), 13 serotypes of Salmonella, 5 ST-type Kronoella, and different capsular types of Klebsiella pneumoniae (corresponding strain information is shown in the table below). All strains were cultured to the logarithmic growth phase. 200 μL of the bacterial culture was added to LB agar plates, along with 4.8 mL of semi-solid solution, and thoroughly mixed and cooled. 5 μL of a 1×10⁻⁶ titer was then taken. 8 A drop of PFU / mL P3BB7B bacteriophage solution was placed in the center of a semi-solid culture. After the liquid was completely absorbed, the culture was placed in a 37 °C incubator and incubated overnight. Bacteria that could form plaques were marked as "+" for a positive result, and "-" for a negative result.

[0073] Table 1 shows the lytic effect of phage P3BB7B on 10 strains of myxotrophic serovar Escherichia coli producing colacid. The results indicate that phage P3BB7B has a lytic effect on 10 strains of myxotrophic serovar Escherichia coli producing colacid, with a lysis rate of 100% (10 / 10).

[0074] Table 1. Lytic effect of phage P3BB7B on mucoid Escherichia coli

[0075] The lytic effect of bacteriophage P3BB7B on 13 serotypes of Salmonella is as follows: Figure 8 As shown, phage P3BB7B exhibits lytic activity against 13 serotypes of Salmonella, with a lysis rate of 57.02% (65 / 114), including Salmonella enteritidis, Salmonella vetavredensis, Salmonella typhimurium, Salmonella typhimurium monophasic variant, Salmonella delbrueckii, Salmonella Londonii, Salmonella Stanley, Salmonella St. Paulii, Salmonella Münsterii, Salmonella Brudenluppii, Salmonella Kif, Salmonella Argonne, and Salmonella Corvallis.

[0076] The lytic effects of phage P3BB7B on four serotypes of Kronoa are shown in Table 2. The results show that phage P3BB7B has a lytic effect on the four serotypes of Kronoa, with a lysis rate of 71.43% (5 / 7).

[0077] Table 2. Lysis effect of bacteriophage P3BB7B on different ST-type Kronobryozoans

[0078] Meanwhile, the results showed that phage P3BB7B had no lytic effect on Klebsiella pneumoniae, with a lysis rate of 0 (0 / 43).

[0079] Example 8: In vitro therapeutic effects of Escherichia coli bacteriophage The host bacterium 32M3BB was cultured to the logarithmic growth phase and then diluted to 10⁻⁶. 3 CFU / mL and 10 4 CFU / mL, phage P3BB7B cultured to 10 8 PFU / mL was used to mix 32M3BB phage suspension and P3BB7B bacterial suspension at a 1:1 ratio with MOIs of 1000, 10000, and 100000, and the mixtures were placed in 96-well plates. For the control group, LB broth was mixed with the bacterial suspension at a 1:1 ratio. The mixtures were incubated at 37°C with shaking at 800 rpm using a growth curve analyzer. OD was measured every 20 h using the growth curve analyzer. 600 Values. Each experimental group was repeated 3 times.

[0080] The results are as follows Figure 9 As shown, when the phage MOI > 1000, phage P3BB7B effectively kills bacteria and inhibits bacterial growth, indicating that phage P3BB7B has a good in vitro bactericidal effect and can effectively kill mucoid Escherichia coli.

[0081] Example 9 Receptor identification of bacteriophage P3BB7B 1. Screening of bacteriophage-resistant bacteria Phage resistant strains with different morphologies were selected from high MOI phage (P3BB7B)-bacteria (32M3BB) semi-solid plates and were designated as in vitro resistant strains, numbered 3BB7B-1, 3BB7B-2, 3BB7B-3, 3BB7B-4, and 3BB7B-5.

[0082] The colony morphology of the selected different phage-resistant strains is as follows: Figure 10 As shown, bacterial DNA was subsequently extracted using the Magen Bacterial Genome Kit, and a 350 bp microbial fragment library was constructed by Magen Biosciences. De novo sequencing (second-generation whole genome sequencing) was then performed using an Illumina NovaSeq 6000 (PE150). Long-read high-throughput sequencing (third-generation whole genome sequencing) was then performed using an Oxford Nanopore MinION sequencer.

[0083] After obtaining Illumina sequencing data, SPAde v3.8.7 was used to assemble the data, and Unicycler v0.4.7 was used to assemble and correct Nanopore sequencing data to obtain complete genome sequences. ABRicate v0.8 was used for analysis of drug resistance genes, plasmid replicons, and virulence genes. Bakta v1.9.3 was then used to annotate open reading frames of the genome sequences, and NCBI-blastp was used to compare and correct the annotation results. Finally, Snippy was used for single nucleotide polymorphism (SNP) analysis of the bacterial core genome.

[0084] Whole-genome sequencing analysis was performed on the five selected phage-resistant strains. The results are shown in Table 3. It was found that all five resistant strains... rcsC Mutations exist in the genes, with frameshift mutations occurring in the coding sequence of three strains. rcsC At amino acid 707 of the gene, the lysine residue became a stop codon, causing premature termination of the RcsC protein. One strain of bacteria had a deletion mutation at positions 543-547 of the RcsC protein, losing the five amino acids FSPRE. In addition, one strain of bacteria... rcsC Genes and their upstream atoS Large segments of the gene were deleted. The colony morphology of these five resistant strains changed significantly from that of the original strain, changing from a slime-like type to a non-slime type, as shown in Table 4.

[0085] Table 3. Mutation status of phage-resistant strains of 32M3BB

[0086] Table 4. Mutation status of 3BB7B-5

[0087] Since bacterial 32M3BB cannot perform CRISPR-Cas9 gene editing, we used the E. coli standard strain BW25113- yrfF The Y98N strain, as a CRISPR-Cas9 gene-editing strain, is due to BW25113- yrfF Y98N and 32M3BB yrfF The mutation sites are consistent, and they also exhibit the mucus phenotype associated with canalacid secretion. We used CRISPR-Cas9 in BW25113- yrfF Y98N introduced rcsC TAA mutations and rcsC Del mutation, visible in BW25113- yrfF Y98N -rcs TAA and BW25113 -yrfF The mucus phenotype of Y98N-rcsC Del disappeared, such as Figure 11 As shown, sensitivity to bacteriophages disappears, such as Figure 12 As shown.

[0088] 2. Transposon mutation library screening (Tn-seq) We constructed a 32M3BB-pSC189 transposon mutant library by conjugating E. coli 32M3BB with SM10 carrying a pSC189 transposon vector library. First, E. coli 32M3BB and the SM10-pSC189-containing strain were cultured in a shaker at 37 °C to the logarithmic growth phase. After concentration, equal volumes of 32M3BB were mixed with the donor strain SM10 and dropped onto a 0.45 μm nitrocellulose membrane (Millipore) on an LB agar plate, and incubated at 37 °C for 6 hours.

[0089] After conjugation, the bacterial cells were resuspended in LB liquid medium and spread onto LB agar plates containing 2 μg / mL triclosan to screen for conjugates. After incubation at 37 °C for 1 day, colonies were scraped into 30% LB glycerol broth and frozen at -80 °C. This is the initial Tn-seq library.

[0090] Add 20 μL of frozen bacterial culture to 2 mL of broth to thaw for 1 hour, then transfer the culture to 5 mL of broth and adjust the OD value to OD200. 600 =0.005 (approximately 2.5 × 10⁻⁵) 6 CFU / mL). Phage P3BB7B was added at an MOI of 1:1 and incubated at 37°C for 2 h / 4 h. The bacterial suspension was centrifuged at 5000 rpm for 5 min, resuspended in PBS, and centrifuged again, repeated three times to remove the phage. The bacterial suspension was serially diluted and plated onto LB agar plates. After 16 h, different single colonies were selected and cultured in LB broth at 37°C until the logarithmic growth phase. 100 μL of the bacterial suspension was added to 5 mL of melted and incubated at 50°C semi-solid medium, poured onto the bottom agar plate to prepare bacterial colonies for each strain. Several drops of 5 μL of P3BB7B phage solution were added to the bacterial colonies until the droplets were completely dry. The plates were incubated overnight at 37°C and the presence of clear phage plaques was checked. If no phage plaques were produced, it indicated that the strain had acquired phage resistance after transposon insertion into a gene. The whole genome of this strain was then sequenced.

[0091] The sequencing results are shown in Table 5. All Tn transposons were inserted into the RcsC or RcsD gene, consistent with the phage-resistant strains obtained under natural conditions, indicating that the changes in the rsc system are related to the resistance of phage P3BB7B and the phage receptor.

[0092] Table 5. Transposon insertion sites of phage-resistant bacteria screened by Tn-seq

[0093] 3. Quantitative detection of colacid The above-mentioned bacterial strains were streaked onto LB agar plates and incubated at 37 °C for 12 hours. Colonies were scraped into 1 mL of sterile distilled water, and the bacterial suspension was adjusted to OD0.05. 600 =4. The bacterial suspension was then heated at 100 °C for 20 minutes to inactivate the enzymes that degrade extracellular polysaccharides. After cooling, it was centrifuged at 16000 g for 20 minutes. The supernatant was collected and diluted 10-fold to bring its concentration within the range of the standard curve.

[0094] Take 111 μL of the diluted sample and add 500 μL of sulfuric acid / water mixture (6:1, volume ratio). Heat at 95 °C for 30 minutes and then cool to room temperature. Add each sample to two wells of a 96-well plate: (a) add 5 μL of 3% cysteine ​​hydrochloride (Cys·HCl) stock solution; (b) add 5 μL of ddH2O. Then add 200 μL of cooled acidified EPS mixture to each well. Measure the absorbance values ​​of (a) and (b) at wavelengths of 396 nm and 427 nm, respectively.

[0095] The absorbance values ​​without Cys·HCl were subtracted from the corresponding values ​​with Cys·HCl to obtain the corrected A396 and A427. The final absorbance value = A396 - A427. The results were converted to fucose concentration using a standard curve for L-fucose in the range of 5-100 μg / mL. The standard curve equation is: y = 0.0026x + 0.0197 (where y is A396 - A427, x is the L-fucose concentration (μg / mL), R...). 2 = 0.9947).

[0096] The results are as follows Figure 13 As shown, the rcsC mutant strain had a significantly reduced amount of L-fucose compared to the original strain, indicating a decrease in kolanitrate secretion. This suggests that phage resistance is mediated by a reduction in the receptors that bind to phages.

[0097] Furthermore, we also confirmed through quantitative detection of colacid that the colacid expression levels in hospital-derived mucoid Escherichia coli targeted by bacteriophages were significantly higher than those in the non-colacid-producing control Escherichia coli BW25113. Figure 14 As shown.

[0098] 4. Relative adsorption efficiency detection (1) The bacteria were cultured to the logarithmic growth phase and the P3BB7B phage fluid and bacterial suspension (3BB7B-1, 3BB7B-2, 3BB7B-3, 3BB7B-4, 3BB7B-5) were mixed with an MOI of 0.01.

[0099] (2) Place at room temperature for 10 min, take a sample into a 2 mL EP tube, and centrifuge at 8000 g for 1 min.

[0100] (3) Take the supernatant and filter it through a 0.22 µm filter membrane.

[0101] (4) Perform a double-layer plate test on the filtered liquid to determine the phage titer. Three replicates were performed for each group.

[0102] The formula for calculating the relative adsorption rate is: .

[0103] The results are as follows Figure 15 As shown, the results indicate that the adsorption efficiency of each strain was significantly lower than that of the original strain, suggesting that when the rcsC gene is mutated, the secretion of kolanitine decreases, the binding force between the phage and the strain decreases, thereby producing phage resistance.

[0104] The above experiments show that when bacteria develop resistance to bacteriophages, their morphology changes from mucoid to non-mucoid, the amount of kolanitin secreted decreases significantly, and the adsorption efficiency of bacteriophage P3BB7B decreases significantly, indicating that bacteriophage P3BB7B recognizes surface structures related to kolanitin.

[0105] Example 10: In vitro bactericidal effect of bacteriophage P3BB7B against hospital-derived colacin-producing mucoid Escherichia coli. Hospital-derived mucoid Escherichia coli GD25LH97952 and GD25LH73213, which produce colacid, were selected, and the bacterial culture was cultured to the logarithmic growth phase and then diluted to 10⁻⁶. 3 CFU / mL and 10 4 CFU / mL, phage P3BB7B cultured to 10 6 PFU / mL, phage fluid and bacterial culture were mixed 1:1 at an MOI of 1000 and placed in 96-well plates. For the control group, LB broth and bacterial culture were mixed 1:1. The plates were incubated at 37 °C with shaking at 800 rpm using a growth curve analyzer. OD was measured every 20 h using the growth curve analyzer. 600 Values. Each experimental group was repeated 3 times.

[0106] The results are as follows Figure 16 As shown, phage P3BB7B can effectively kill and inhibit hospital-derived mucinous Escherichia coli, indicating that phage P3BB7B has good potential for clinical application.

[0107] Example 11: Disinfection effect of bacteriophage P3BB7B on hospital-derived colacin-producing mucoid Escherichia coli. (1) Prepare a 25×25 mm polyethylene material, soak it in 75% alcohol for 10 minutes for disinfection, rinse it with pure water and dry it. Sterilize the cleaned and disinfected simulated surface at 121°C and dry it for later use.

[0108] (2) Place sterile culture dishes in the clean bench with the lids open, and use sterile forceps to transfer the autoclaved polyethylene material into the sterile culture dishes. Set up control group and phage treatment group respectively, and sterilize with ultraviolet light.

[0109] (3) Preparation of host bacteria and bacteriophages: Single bacterial clones were picked from the bacterial GD25LH97952 plate and placed in 3 mL of liquid culture medium. The culture was carried out overnight at 37°C with shaking. The overnight cultured bacteria were then transferred 1:100 and cultured for another 4 h at 37°C with shaking until the logarithmic growth phase. The bacteria were then serially diluted to 10⁻⁶. 5 CFU / mL. After enrichment with phage P3BB7B, the concentration was adjusted to 10. 9 PFU / mL.

[0110] (4) Experimental Procedure: 50 μL of resuspended bacterial suspension was dropped onto the surface of the polyethylene material and gently spread with a pipette tip. The container was then left uncovered and dried in a clean bench for 1 hour. Phage preparation was then drawn up with a pipette tip and dropped onto the dried polyethylene material surface, again gently spreading it to cover the entire area contaminated with bacteria. dd water was used as the control group. At 2h, 4h, 8h, and 24h, samples of the polyethylene material surface were placed in centrifuge tubes containing 1 mL of PBS buffer. The bacterial suspension was serially diluted 10-fold, and each dilution was dropped onto a solid plate. The plates were incubated overnight at 37°C. Bacterial growth was observed and the bacterial count was determined after overnight incubation.

[0111] The results are as follows Figure 17 As shown, bacteriophage P3BB7B can effectively reduce bacteria on the surface of polyethylene materials within 2-4 hours and can eliminate bacteria on the surface of polyethylene materials within 8 hours, indicating that bacteriophage P3BB7B has a good disinfection effect.

[0112] Example 12 Biofilm inhibition and clearance experiment of bacteriophage P3BB7B against hospital-derived colacid-producing mucoid Escherichia coli (1) Assessment of biofilm formation capacity: The overnight cultured bacterial suspension GD25LH96510 was transferred to fresh broth at a ratio of 1:100 and shaken for 4 hours until the logarithmic phase was reached. Then, it was diluted 100-fold using LB broth. 100 μL of the diluted bacterial suspension was inoculated into each well of a 96-well plate and incubated for 24 hours. To avoid edge effects and reduce water evaporation, the experimental wells were concentrated in the center of the plate, with 8 parallel samples per group. The remaining peripheral wells were filled with an equal volume of broth. After the culture was completed, the liquid in the wells was discarded and the wells were spun dry in a water bath. Then, the wells were rinsed three times with clean water and air-dried. Next, 120 μL of methanol was added to each well, and the wells were allowed to stand for 15 minutes to fix the biofilm. After discarding the fixative, the wells were air-dried again for about 10 minutes. Then, 130 μL of 0.1% crystal violet staining solution was added to each well to stain the biofilm, and the reaction was carried out in the dark for 15 minutes. After staining, repeat the washing step three times and air dry. Finally, add 150 μL of 33% acetic acid to dissolve the dye. After thorough shaking to ensure complete dye mixing, measure the OD of each well using a microplate reader. 550 The value is used to quantitatively assess the total amount of biofilm.

[0113] (2) For the inhibition experiment, the logarithmic phase bacterial solution was diluted 100 times with LB broth and co-inoculated with the phage solution with MOI=1000 in a 96-well plate, ensuring that the final volume of each well was 100 μL and that the phage solution and bacterial solution were mixed 1:1. Then, the plates were incubated at 37 °C for 24 hours to observe the blocking effect of the enzyme on membrane formation.

[0114] (3) For the removal experiment, 100 μL of diluted bacterial solution should be inoculated into the well plate and pre-cultured for 24 hours until the biofilm matures. The original solution should be discarded and the plate should be gently washed with sterile PBS to remove airborne bacteria. Then, 100 μL of phage solution with MOI=1000 should be added to each well and the plate should be treated at 37 °C for 4 hours to evaluate its degradation efficacy on the existing membrane structure.

[0115] Both sets of experiments required eight parallel wells, with the edge wells filled with broth to prevent evaporation. After treatment, the crystal violet staining method described above was followed.

[0116] The results are as follows Figure 18 As shown, phage P3BB7B has a weak ability to inhibit biofilm formation, but it can significantly remove existing biofilm structures, and has the potential for clinical application in the treatment of mucoid Escherichia coli that produce biofilms.

[0117] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. A strain of Escherichia coli bacteriophage Escherichia coli phage vB EcoP 3BB7B, characterized in that, The phage was deposited on January 8, 2026, at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 47012.

2. The bacteriophage according to claim 1 Escherichia coli phage Application of vB EcoP 3BB7B in the inhibition of mucoid Escherichia coli, Cronobacter and / or Salmonella for non-disease diagnosis and treatment purposes.

3. The bacteriophage according to claim 1 Escherichia coli phage vB Application of EcoP 3BB7B in the preparation of products that inhibit mucoid Escherichia coli, Cronobacter and / or Salmonella.

4. The application according to claim 2 or 3, characterized in that, The mucoid Escherichia coli is an Escherichia coli that can secrete colacid.

5. The bacteriophage according to claim 1 Escherichia coli phage Application of vB EcoP 3BB7B in the preparation of disinfectant products.

6. The bacteriophage according to claim 1 Escherichia coli phage Application of vB EcoP 3BB7B in the preparation of therapeutic drugs for diseases caused by mucoid Escherichia coli.

7. The application according to claim 5 or 6, characterized in that, The product or drug can remove bacterial biofilm.

8. A method for removing bacterial biofilm or disinfecting, characterized in that, Using the bacteriophage described in claim 1 Escherichia coli phage The product vB EcoP 3BB7B is being processed.

9. A product characterized in that, Contains the bacteriophage according to claim 1 Escherichia coli phage vB EcoP3BB7B.

10. The product according to claim 9, characterized in that, The products are disinfectants, cleaning agents, external bactericides, medicines, food preservatives, or feed additives.

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