Escherichia coli bacteriophage PE-2 with high blood entry efficiency and fermentation efficiency and application thereof

Abstract

The application belongs to the technical field of bioengineering, and particularly relates to a bacteriophage PE-2 of Escherichia coli with high blood entry efficiency and fermentation efficiency and application. The bacteriophage involved in the application has a preservation number of CCTCC NO: M20252973, and can specifically lyse pathogenic Escherichia coli O2 serotype and has high blood entry efficiency. The bacteriophage can enter blood circulation through oral administration, and can be widely distributed to the spleen, liver, heart and other tissues through blood circulation. The bacteriophage can be prepared in pilot production to obtain a very high titer, and the titer reaches 1.6x10 12 PFU / mL. Compared with the production of antibiotics, the production cost of the bacteriophage is low, and the bacteriophage provides a source and a reference example for industrial production of high-titer bacteriophage.

Description

Technical Field

[0001] This invention belongs to the field of bioengineering technology, specifically relating to a strain of Escherichia coli bacteriophage PE-2 with high blood entry efficiency and fermentation efficiency and its applications. Background Technology

[0002] Escherichia coli is one of the most common and economically damaging pathogens in livestock and poultry farming. It can exist as an intestinal symbiotic bacterium, but it can also cause disease under certain conditions, leading to various E. coli infections such as septicemia, peritonitis, diarrhea, and mastitis. There is an urgent need to develop green, safe, and efficient biological antibacterial agents such as targeted bacteriophages to fill the ecological niche.

[0003] Bacteriophages are the most abundant organisms on Earth, widely distributed in various ecological environments, especially densely distributed in water bodies and the intestines. Their ability to specifically recognize and lyse bacteria makes them an important candidate tool for combating drug-resistant bacterial infections. As a biological antimicrobial agent, bacteriophages have broad application prospects and have received widespread attention and application in medicine, aquaculture, livestock and poultry farming, and food preservation. Studies have shown that bacteriophages can effectively reduce biofilm formation in host bacteria, significantly reduce bacterial density, and also have a killing effect on drug-resistant strains. Oral administration of bacteriophages is one of the most common routes of administration in clinical practice, with advantages such as relative safety and ease of operation. Existing research shows that some bacteriophages can cross the intestinal barrier and enter the bloodstream via oral administration, achieving systemic antimicrobial effects. However, due to their inherent stability, weak tolerance to gastric acid and bile salts, and various factors related to the bacteriophage strain, most bacteriophages have low blood entry efficiency and unstable titers, limiting their clinical application and industrial development.

[0004] Therefore, screening out bacteriophages with high blood entry efficiency and high fermentation efficiency has important application value. Summary of the Invention

[0005] The purpose of this invention is to provide an Escherichia coli bacteriophage PE-2 with high blood entry efficiency and fermentation efficiency, wherein the bacteriophage has the CCTCC NO: M20252973.

[0006] Another object of the present invention is to provide the use of Escherichia coli phage PE-2 in the preparation of medicaments for the treatment or prevention of Escherichia coli O2 serotype infection.

[0007] Another objective of this invention is to provide a pilot-scale fermentation method for high-titer Escherichia coli phage PE-2. This method features high phage fermentation efficiency, simple operation, and yields a titer of 1.6 × 10⁻⁶ after only 5 hours of fermentation. 12 Phage fermentation broth with PFU / mL.

[0008] To achieve the above objectives, the present invention adopts the following technical measures:

[0009] The applicant isolated and screened a bacteriophage from domestic sewage in a chicken farm in Rizhao, Shandong Province. This bacteriophage was deposited at the China Center for Type Culture Collection on December 19, 2025, with the classification name: Escherichia coli bacteriophage PE-2, accession number CCTCC NO: M 20252973, address: Wuhan University, Wuhan, China.

[0010] The scope of protection of this invention also includes:

[0011] A composition comprising Escherichia coli bacteriophage PE-2.

[0012] A pilot-scale fermentation method for high-titer Escherichia coli phage PE-2 includes the following steps: inoculating Escherichia coli host bacteria into LB liquid medium at an inoculum concentration of 0.5–5%, and fermenting until OD200. 600 When the concentration is in the range of 0.15-0.6, inoculate with Escherichia coli phage PE-2 at an inoculation rate of 0.1%-1% and continue culturing until the fermentation broth becomes clear.

[0013] In the fermentation method described above, preferably, the inoculum size of *Escherichia coli* host bacteria is 1%, and the fermentation is cultured to OD0.05. 600 At 0.5-0.6, inoculate again with 1% E. coli phage PE-2;

[0014] The preferred fermentation conditions described above are: 36-38℃, agitator speed of 130-170 r / min, and aeration rate of 2-4 m³ / min. 3 / h, tank pressure 0.01-0.03mPa.

[0015] The preferred fermentation conditions described above are: 37℃, agitator speed of 150 r / min, and aeration rate of 2 m³ / min. 3 / h, tank pressure 0.02mPa.

[0016] The use of Escherichia coli phage PE-2 and / or the above-mentioned composition in the preparation of a medicament for the treatment or prevention of Escherichia coli O2 serotype infection.

[0017] The use of Escherichia coli bacteriophage PE-2 and / or the above-mentioned composition in the preparation of Escherichia coli O2 serotype antibacterial agents.

[0018] Compared with the prior art, the present invention has the following beneficial effects:

[0019] This invention screened a bacteriophage PE-2 that can rapidly cross the intestinal barrier after oral administration, be absorbed into the bloodstream, and be widely distributed to the spleen, liver, heart, and other tissues via the bloodstream.

[0020] Bacteriophage fermentation is highly efficient and easy to operate; a titer of 1.6 × 10⁻⁶ can be obtained with a co-fermentation time of 5 hours. 12 Phage fermentation broth at PFU / mL can extend product shelf life or reduce production costs to some extent. It has high clinical application and industrialization value.

[0021] The bacteriophage of this invention has high blood entry efficiency and high titer in pilot-scale fermentation, laying the foundation for the industrial development of bacteriophage PE-2 and its application in the prevention and control of pathogenic bacteria. Attached Figure Description

[0022] Figure 1 This is a transmission electron microscope image of Escherichia coli bacteriophage PE-2.

[0023] Figure 2 This is a graph showing the temperature stability of Escherichia coli phage PE-2.

[0024] Figure 3 This is a graph showing the acid-base stability of Escherichia coli bacteriophage PE-2.

[0025] Figure 4 This is a graph showing the blood entry performance of Escherichia coli bacteriophage PE-2.

[0026] Figure 5 This image shows the detection of Escherichia coli bacteriophage PE-2 in tissues and organs. Detailed Implementation

[0027] The present invention will be further explained and described below with reference to the accompanying drawings and specific embodiments.

[0028] Unless otherwise specified, the experimental methods used in the following examples are conventional methods; the raw materials and reagents used are commercially available products.

[0029] Example 1:

[0030] Obtaining and biological characteristics of Escherichia coli bacteriophage PE-2:

[0031] The applicant isolated and screened a bacteriophage from domestic sewage in a chicken farm in Rizhao, Shandong Province. The bacteriophage was deposited at the China Center for Type Culture Collection on [date missing] 2025, classified and named as Escherichia colibacteriophage PE-2, with accession number CCTCC NO: M 20252973, located at Wuhan University, Wuhan, China.

[0032] (1) Observation by transmission electron microscopy

[0033] The microscopic morphology of bacteriophages was observed using transmission electron microscopy with phosphotungstic acid negative staining. Figure 1 As shown, the head of bacteriophage PE-2 is an icosahedron, approximately 100 nm long and 80 nm wide, with a tail approximately 100 nm long. There is also a collar structure at the junction of the head and tail, and tail filaments are visible at the end of the tail. It belongs to the family Myoviridae.

[0034] (2) Optimal multiplicity of infection

[0035] Following standard procedures, 1% of the host bacteria and phage PE-2 were inoculated to proliferate, and the host bacteria concentration and phage titer were determined after proliferation. 5 × 10⁻⁶ 9 CFU / mL of E. coli host bacteria 24-29 (isolated by the applicant; other E. coli standard strains can be used as host bacteria for the propagation of this bacteriophage) and 5×10 8 Phages at PFU / mL were mixed at MOIs of 0.0001, 0.001, 0.01, and 0.1, and 1% was inoculated into Erlenmeyer flasks containing 50 mL of LB liquid medium. The mixtures were incubated at 37°C with shaking at 200 rpm until the fermentation broth changed from turbid to clear and bacterial fragments became visible. After incubation, the mixture was centrifuged at 10000g for 3 min, and the supernatant was appropriately diluted with PBS buffer. The phage titer in each Erlenmeyer flask was determined using the bilayer plate method.

[0036] The results are shown in Table 1. When the MOI was between 0.001 and 0.1, the titer of PE-2 phage was consistently above 10. 9 At PFU / mL or higher, the phage PE-2 titer reached a maximum of 1.1 × 10⁻⁶ when MOI = 0.01. 10 PFU / mL, therefore, the optimal multiplicity of infection for phage PE-2 is 0.01.

[0037] Table 1. Multivariable of infection of Escherichia coli phage PE-2

[0038] .

[0039] (3) Temperature stability

[0040] The potency is 2×10 9 400 μL of PE-2 phage proliferation medium with PFU / mL was incubated in water baths at 45℃, 50℃, 55℃, 60℃, and 65℃ for 60 min. After cooling to room temperature, the phage titer was measured after each treatment. A temperature stability curve of the phage was plotted with temperature on the x-axis and the logarithm of the phage titer on the y-axis.

[0041] Experimental results are as follows Figure 2 As shown, the titer of PE-2 bacteriophage treated at 45℃ and 50℃ for 60 min did not decrease significantly compared to the untreated result; however, the titer gradually decreased with increasing temperature. At 65℃ for 60 min, the bacteriophage was not completely inactivated, and the titer was 3 × 10⁻⁶. 4 PFU / mL.

[0042] (4) pH stability

[0043] The pH of LB liquid medium was adjusted to 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 and 12 with NaOH or hydrochloric acid solution. 100 μL of phage proliferation solution was mixed with 900 μL of medium at different pH values ​​and incubated at room temperature for 24 h before phage activity was detected.

[0044] The results are as follows Figure 3 As shown, bacteriophage PE-2 maintained its titer unchanged and stabilized at 10 after 24 h of treatment within a pH range of 5.0-10.0. 9 At PFU / mL and pH=12.0, the potency can be maintained at 10 after 24 hours of treatment. 7 PFU / mL; potency was 3.1 × 10⁻⁶ after 24 hours of treatment at pH 2.0. 4 PFU / mL.

[0045] Example 2:

[0046] The lysis of Escherichia coli phage PE-2 against Escherichia coli O2 serotype:

[0047] The lytic activity of purified bacteriophages against 21 strains of Escherichia coli O2 serotype was detected by the drop method: 100 μL of the test bacteria was added to soft agar containing culture medium, quickly mixed and poured onto a plate containing culture medium, and 5 μL of the bacteriophage proliferation solution after membrane filtration was dropped onto the surface of the solidified double-layer plate. The plates were incubated at 37℃ for 8 h, and the presence or absence of phage plaques was observed on each plate.

[0048] The results are shown in Table 2. Phage PE-2 could lyse 9 strains of Escherichia coli O2 serotype, with a lysis rate of 50%.

[0049] Table 2. Lysis spectrum of PE-2 phage against Escherichia coli

[0050] .

[0051] Note: + indicates plaque presence; – indicates plaque absence.

[0052] Example 3:

[0053] Evaluation of the application of Escherichia coli phage PE-2

[0054] (1) PE-2 security verification

[0055] Twelve healthy broiler chickens aged two days were randomly divided into two groups. PE-2 phage solution was added to the drinking water of the experimental group to achieve a final phage concentration of 1×10⁻⁶. 8 PFU / mL, for 7 consecutive days; the control group was given only regular drinking water. Both groups were isolated and reared for 7 days, with daily records of behavior, feed intake, and fecal condition. Dissection of the heart, liver, and spleen was performed. No lethargy, decreased feed intake, or visible tissue lesions were observed throughout the process, indicating that PE-2 has no adverse effects on chicks.

[0056] (2) Protective effect of PE-2 against Escherichia coli challenge

[0057] Thirty healthy 10-day-old chicks were divided into three groups: a blank control group, an untreated infection group (positive control), and an infection + PE-2 group (treatment group). The latter two groups were intraperitoneally injected with 5×10 8 CFU / mL avian pathogenic Escherichia coli O2 serotype 0.2 mL. In the treatment group, PE-2 was administered via drinking water starting from the day of infection. The final concentration of PE-2 in the drinking water was 10. 8 PFU / mL, for 5 consecutive days; the positive control group received an equal volume of physiological saline instead of phage solution; the blank group received no stimulation. Autopsy was performed 7 days later.

[0058] The positive control group developed significant diarrhea on day 2, and autopsy revealed pericardial fibrinous exudate, necrotic foci on the liver surface, and significant splenomegaly. The treatment and control groups showed formed stools with only mild splenomegaly and no fibrinous exudate or necrotic foci. These results indicate that PE-2 can rapidly inhibit E. coli infection and prevent the appearance of typical pathological changes.

[0059] Example 4:

[0060] Detection of the ability of Escherichia coli phage PE-2 to enter the bloodstream after oral administration

[0061] Nine healthy 10-day-old broiler chickens were divided into three groups of three. Each chicken was given 1×10 mg of the drug orally. 91 mL of PE-2 phage (PFU / mL) was administered orally. Blood was collected at 2 h, 4 h, 6 h, 8 h, 10 h, and 12 h using anticoagulant blood collection tubes. The supernatant was centrifuged at 10000 g for 5 min, filtered through a 0.22 μm filter, and serially diluted tenfold with PBS buffer. The phage content in the blood at different time points was detected using a double-layer plate method: 100 μL each of the host *E. coli* and the diluted supernatant were placed in 1.5 mL sterile centrifuge tubes and incubated at 37±1℃ for 5 min to allow phage adsorption onto the host cells. 200 μL of the incubation solution was transferred to a test tube containing 5 mL of LB soft agar, pre-warmed at 50℃ in a water bath, quickly mixed, and poured onto a nanoplate. The plate was allowed to solidify and then incubated at 37±1℃ for 6–8 h. Results were then statistically analyzed.

[0062] The results are as follows Figure 4 As shown, bacteriophages were detectable in the blood 2 hours after oral administration, with a titer of 1.2 × 10⁻⁶. 7 The PFU / mL phage titer in blood after 4 hours was 1.6 × 10⁻⁶. 8 The titer peaks at PFU / mL, then gradually decreases with increasing dosing time, remaining detectable in the blood for up to 12 hours, with a titer still reaching 10 at 12 hours. 5 PFU / mL.

[0063] Example 4:

[0064] Stepwise detection of tissues and organs after oral administration of PE-2 bacteriophage

[0065] Twelve healthy 10-day-old broiler chickens were divided into three groups of four each. Each chicken was given 1×10 mg of the drug orally. 9 One mL of PE-2 phage (PFU / mL) was administered orally. Heart, liver, and spleen were harvested via dissection at 2, 4, 8, and 12 hours post-oral administration. PBS buffer was added to each sample, and the tissue homogenates were homogenized using a homogenizer. The homogenates were then serially diluted tenfold with PBS buffer. After sterilization using a 0.22 μm filter, the phage titer in different tissues and organs at different sampling times was determined using a double-layer plate method.

[0066] The results are as follows Figure 5 As shown, the titers of bacteriophages in tissues and organs after oral administration showed a consistent trend with those in blood. Two hours after oral administration, only 13 PFU / mL of bacteriophages were detected in the heart, while the titers in the liver and spleen were 1.8 × 10⁻⁶. 6 PFU / mL and 2.0×10 6 PFU / mL. 4.2 × 10⁻⁶ PFU / mL was detected in the heart 4 hours after oral administration. 5The PFU / mL phage concentration was 5.8 × 10⁻⁶ in the liver and spleen, respectively. 6 PFU / mL and 1.1×10 7 PFU / mL, all reaching peak phage titer. 2.1 × 10⁻⁶ PFU / mL was detected in the heart 8 hours after oral administration. 4 PFU / mL phage, phage titers detected in the liver and spleen were 1.2 × 10⁻⁶. 6 PFU / mL and 1.1×10 5 PFU / mL. 1.6 × 10⁻⁶ PFU / mL was detected in the heart 12 hours after oral administration. 3 The PFU / mL phage concentration was 4.4 × 10⁻⁶ phage titers detected in the liver and spleen, respectively. 5 PFU / mL and 2.9×10 4 PFU / mL. The distribution of bacteriophages in the heart, liver, and spleen revealed that at the same time point, the phage concentration in the heart was significantly lower than that in the liver and spleen.

[0067] Example 5:

[0068] Simultaneous inoculation and fermentation of bacteriophage PE-2 and Escherichia coli host bacteria:

[0069] Preparation of secondary seed culture of host bacteria: Glycerol tubes containing the Escherichia coli host bacteria (ATCC 25922 used in this example) fermented by bacteriophage PE-2 were taken from a -80℃ freezer and streaked onto LB agar plates. The plates were incubated at 37℃ for 16 hours. Single colonies were picked and inoculated into LB liquid medium, and incubated at 37℃ and 200 rpm for 14 hours to obtain the primary seed culture. The primary seed culture was then transferred to 500 mL of LB liquid medium at a 1% (v / v) inoculation rate and incubated at 37℃ and 200 rpm for 3 hours. Oddi concentration (OD) was measured. 600 The value is in the range of 1.5-1.8.

[0070] Preparation of phage seed culture: 500 μL of phage and host bacteria primary seed culture from glycerol tubes were separately incubated in 50 mL LB liquid medium at 37 °C and 200 rpm for 3 h, then shaken to remove impurities. The titer of the phage seed culture was determined to be 8 × 10⁻⁶ using the double-layer plate method. 9 PFU / mL.

[0071] LB liquid culture medium was prepared in a 50L fermenter with a loading coefficient of 50%. The medium was sterilized by high-pressure saturated steam for 30 min using 1 kg of medium. The host bacteria secondary seed culture was inoculated at a rate of 1% using the flame inoculation method. At the same time, bacteriophage seed culture was inoculated at rates of 0.1%, 0.01%, and 0.001%, respectively.

[0072] The fermenter temperature was set to 37℃, the agitator speed to 150 r / min, and the aeration rate to 2 m³ / min. 3 / h, tank pressure 0.02mPa. After 3 hours of fermentation, the fermentation broth became clear and contained obvious cell debris. The results of phage titer detection using the double-layer plate method are shown in Table 3.

[0073] Table 3. Results of fermentation titer assays when host bacteria and bacteriophages at different inoculum sizes are simultaneously inoculated.

[0074] .

[0075] According to the data in Table 3, the maximum titer of fermentation when the host bacteria and bacteriophage are inoculated simultaneously can reach 1.0 × 10⁻⁶. 11 PFU / mL, with the lowest potency at 5×10⁻⁶ 9 PFU / mL.

[0076] Example 6:

[0077] Stepwise fermentation of bacteriophage PE-2 with Escherichia coli host bacteria:

[0078] A 50L pilot-scale fermentation was conducted using the secondary seed culture of the host bacteria and the bacteriophage seed culture from Example 5. The liquid culture medium was prepared in a 50L fermenter with a 50% loading coefficient, sterilized by autoclaving with 1 kg of saturated steam for 30 min, and inoculated with the secondary seed culture of the host bacteria at a 1% inoculum using the flame inoculation method. The fermenter temperature was set at 37°C, the agitator speed at 150 rpm, and the aeration rate at 2 m³ / min. 3 / h, tank pressure 0.02mPa. After fermentation for 1h and 2h respectively, the OD value of the fermentation broth was measured.

[0079] Fermentation was carried out by feeding 1% and 0.1% of phage seed liquid respectively, and fermentation continued until the fermentation broth changed from turbid to clear or the OD value dropped to a low level.

[0080] The titer determination results are shown in Table 4. The highest titer reached was 1.6 × 10⁻⁶ when 1% of the host bacteria was inoculated and fermented for 2 hours, followed by inoculation with 1% bacteriophage. 12 The PFU / mL concentration increased by 200 times compared to the seed culture. The lowest titer achieved by stepwise fermentation, where the host bacteria were cultured first and then inoculated with bacteriophages, was 1.2 × 10⁻⁶ PFU / mL. 11 PFU / mL. As shown in Table 3, during the PE-2 phage seed culture fermentation process, culturing the host bacteria for 2 hours before inoculating with the phage resulted in a 16-fold higher titer compared to simultaneously inoculating both the host and phage for final fermentation.

[0081] Table 4. Results of stepwise fermentation titers of host bacteria and bacteriophage PE-2

[0082] .

[0083] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit it. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the spirit and scope of the present invention, and all such modifications or substitutions should be covered within the scope of the claims of the present invention.

Claims

1. An isolated Escherichia coli bacteriophage ( Escherichia coli The phage (PE-2) has the accession number CCTCC NO: M 20252973.

2. A composition comprising the Escherichia coli bacteriophage PE-2 as described in claim 1.

3. The high-titer pilot-scale fermentation method for Escherichia coli bacteriophage PE-2 according to claim 1, comprising the steps of inoculating Escherichia coli host bacteria into LB liquid medium at an inoculum concentration of 0.5-5%, and fermenting until OD. 600 At a temperature of 0.15-0.6, inoculate with the Escherichia coli phage PE-2 described in claim 1 at an inoculation rate of 0.1%-1% and continue culturing until the fermentation broth becomes clear.

4. The method according to claim 3, wherein the inoculum amount of *Escherichia coli* host bacteria is 1%, and fermentation is carried out until OD... 600 At 0.5-0.6, inoculate with 1% E. coli phage PE-2.

5. The method according to claim 3, wherein the fermentation conditions are: 36-38℃, stirring speed of 130-170 r / min, and aeration rate of 2-4 m³ / min. 3 / h, tank pressure 0.01-0.03mPa.

6. The method according to claim 5, wherein the fermentation conditions are: 37°C, impeller speed of 150 r / min, and aeration rate of 2 m³ / min. 3 / h, tank pressure 0.02mPa.

7. The use of the Escherichia coli bacteriophage PE-2 of claim 1 and / or the composition of claim 2 in the preparation of a medicament for treating or preventing Escherichia coli O2 serotype infection.

8. The use of the Escherichia coli bacteriophage PE-2 of claim 1 and / or the composition of claim 2 in the preparation of an Escherichia coli O2 serotype antibacterial agent.

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