A bacteriophage XP2 for treating bacillus cereus endophthalmitis and application thereof

Abstract

This invention belongs to the field of biomedical engineering and ophthalmic anti-infective therapy technology, specifically relating to a bacteriophage XP2 for treating Bacillus cereus endophthalmitis and its applications. Bacteriophage XP2 was deposited on November 18, 2025, at the China General Microbiological Culture Collection Center (CGMCC), with accession number CGMCC No. 47001. The bacteriophage provided by this invention exhibits high specificity in killing host bacteria, acting only against the host bacteria without disrupting normal flora or inducing widespread drug resistance. Simultaneously, the bacteriophage itself is non-toxic to normal retinal function and demonstrates excellent preservation of retinal function, even superior to vancomycin treatment, with a shorter onset time than vancomycin, approximately 30 minutes. This invention confirms the effectiveness of bacteriophage XP2 in directly reducing the bacterial load in Bacillus cereus-induced endophthalmitis and indirectly controlling inflammatory damage. By rapidly killing bacteria, XP2 minimizes neutrophil-induced inflammatory responses while protecting retinal structure and function.

Description

Technical Field

[0001] This invention belongs to the field of biomedical engineering and ophthalmic anti-infective treatment technology, specifically relating to a bacteriophage XP2 that can be used to treat Bacillus cereus endophthalmitis and its application. Background Technology

[0002] Bacillus cereus is one of the leading pathogens causing traumatic endophthalmitis, second only to Staphylococcus epidermidis. Once infected, it can cause irreversible damage within 24 hours. Currently, treatment primarily involves intravitreal injection of vancomycin or combined with vitrectomy. Studies have shown that intravitreal injection of vancomycin within 4 hours of Bacillus cereus infection is crucial for saving vision. However, Bacillus cereus grows rapidly and is highly motile, progressing quickly and easily spreading to cause panophthalmitis. Vancomycin treatment has a slow onset of action, taking effect only after 2 hours. Furthermore, repeated use may lead to antibiotic resistance; Bacillus cereus can form biofilms, reducing sensitivity to vancomycin; and repeated intravitreal injections may trigger retinal toxicity, further damaging vision. Therefore, existing treatments have multiple shortcomings in terms of timeliness, targeting, and safety. Even with vancomycin treatment, 75%-91% of patients still fail to regain effective vision, and some may even become blind due to enucleation or evisceration.

[0003] Therefore, there is an urgent need for rapid and effective treatment methods to reduce the blindness rate. Summary of the Invention

[0004] The purpose of this invention is to overcome the shortcomings and deficiencies of the existing technology and to provide a bacteriophage XP2 that can be used to treat Bacillus cereus endophthalmitis and its application.

[0005] The technical solution adopted in this invention is as follows: a bacteriophage XP2 (Bacillus cereus phage), which can be used to treat Bacillus cereus endophthalmitis, belongs to the Hellerviridae family ( Herelleviridae ), Bastillevirus subfamily ( Bastillevirinae ), Blue-green virus genus ( Caeruleovirus It was deposited at the China General Microbiological Culture Collection Center (CGMCC) on November 18, 2025, with accession number CGMCC No. 47001.

[0006] The above-mentioned bacteriophage XP2 is used to prepare drugs for treating traumatic endophthalmitis caused by Bacillus cereus.

[0007] A drug for treating traumatic endophthalmitis caused by Bacillus cereus, the active ingredient of which is bacteriophage XP2 as described above.

[0008] Preferably, the dosage form is intravitreal injection solution.

[0009] The beneficial effects of this invention are as follows: The bacteriophage provided by this invention exhibits high specificity in killing host bacteria, acting solely against them without disrupting normal flora or inducing widespread drug resistance. Simultaneously, the bacteriophage itself is non-toxic to normal retinal function and demonstrates excellent preservation of retinal function, even surpassing vancomycin treatment, with a shorter onset time of approximately 30 minutes. This invention confirms the effectiveness of bacteriophage XP2 in directly reducing the bacterial load in Bacillus cereus-induced endophthalmitis and indirectly controlling inflammatory damage. By rapidly killing bacteria, XP2 minimizes neutrophil-induced inflammatory responses while protecting retinal structure and function. Attached Figure Description

[0010] 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, obtaining other drawings based on these drawings without creative effort still falls within the scope of the present invention.

[0011] Figure 1 The morphology, size, and plaque characteristics of bacteriophage XP2 are as follows: (A), (B) bacteriophage XP2 in non-shrink and shrink states under a transmission electron microscope; (C) bacteriophage on a double-layer plate; (D), (E), (F), and (G) are the length and width of the head and tail of bacteriophage XP2, respectively. Figure 2 shows the growth curves of Bacillus cereus cells by phage XP2 and VAN. Figure 3 shows the survival rate of Muller cells under different concentrations of XP2; Figure 4. Slit lamp images of the anterior segment of mice and clinical scoring diagrams: (A), (B), (C), (D) Normal mouse eyeballs, eyeballs 24 hours after infection with Bc2 bacteria, eyeballs treated with bacteriophage XP2 and eyeballs 18 hours after vancomycin treatment under slit lamp; (E) Clinical scoring diagrams of the eyeballs of the four groups of mice. Figure 5 shows the mouse retinal electrophysiology (A), a-wave amplitude (B), and b-wave amplitude (C). Detailed Implementation

[0012] To make the objectives, technical solutions, and advantages of the present invention clearer, the present invention will be further described in detail below with reference to the accompanying drawings.

[0013] Example 1: Phage isolation and identification: In this embodiment, bacteriophages were enriched from soil samples around Wenzhou Medical University. Using clinically isolated Bacillus cereus as the host bacterium, the phages were screened and purified to obtain the target phages. Phage XP2 was further isolated using a double-layer agar method, its morphology was observed by transmission electron microscopy, and its host spectrum was determined.

[0014] The specific separation steps are as follows: Melt the semi-solid LB medium by heating and incubate it in a 55°C water bath until ready for use. Remove the phage filtrate from the soil sample and 10 tubes of logarithmic-phase Bacillus cereus culture from a 4°C freezer and equilibrate to room temperature. Take 10 15 mL sterile centrifuge tubes and add 200 μL of logarithmic-phase Bacillus cereus culture and 200 μL of coarse phage filtrate to each, respectively, and mix thoroughly. After 10 min, add 5 mL of semi-solid LB medium to the mixture, invert to mix, and immediately pour onto 10 prepared solid LB plates to prepare double-layer agar plates. Place in a biosafety cabinet until the double-layer agar plates solidify, and record the host bacteria and other information on the plates. Then, incubate overnight in a 35°C bacterial incubator. The next day, check the double-layer agar plates for phage plaques. Prepare several 1.5 mL centrifuge tubes and add 0.5–1 mL of SM buffer. Using a sterile pipette tip, pick up the central region of a single phage plaque and repeatedly pipette-mix it into the SM buffer to ensure slow release of the phage. Label the centrifuge tubes with the host bacteria and other relevant information. Add 2 μL of host bacteria to each centrifuge tube, mix thoroughly, and incubate at 180 rpm and 37°C for 4 h to propagate the phage. Filter the phage propagation solution through a 0.22 μm filter membrane to remove bacteria and other impurities. Repeat the above steps three times to purify the phage until uniform phage plaques of consistent morphology and size are obtained on a double-layer agar plate. Finally, obtain purified stock solutions of different phage species and label them according to the host bacteria.

[0015] Phage characteristics: Phage XP2, belonging to the Myotail Phage family, was successfully isolated. Figure 1 As shown, electron microscopy revealed that the bacteriophage head exhibits an icosahedral symmetry structure, with a retractable tail, a structure beneficial for transmitting genetic material and infecting host bacteria. It has a broad host spectrum, with 105 out of 136 strains showing sensitivity. The optimal multiplicity of infection (MOF) is 0.1, with a latency period of 20 min, an outbreak period of 40 min, and an outbreak yield of approximately 227 PFU / cell. It demonstrates good stability under conditions of 4–45℃, pH 5–11, and sodium chloride concentrations below 500 mM, ensuring its effectiveness in complex physiological environments.

[0016] Genomic characteristics: The genome of bacteriophage XP2 is 157,896 bp in length, with a GC content of 39.78%. It encodes multiple functional proteins, including those related to structural assembly, cleavage, and DNA recombination and replication, as well as 13 tRNA genes, which help improve translation efficiency and enhance cleavage ability. Phylogenetic analysis determined its taxonomic position; bacteriophage XP2 is... Herellevirida e-science, Bastillevirinae This subfamily is a new member of the genus Caeruleovirus. Comparison with other bacteriophage genomes in the NCBI database revealed that the bacteriophages most similar to XP2 are Bacillus phages Z3, SP2, VP, PK16, and BCP01. Furthermore, the genome similarity heatmap further reveals their phylogenetic relationships, providing a basis for a deeper understanding of bacteriophage evolution and function.

[0017] Bacteriophage XP2 was deposited on November 18, 2025, at the China General Microbiological Culture Collection Center (CGMCC), accession number CGMCC No. 47001. Address: No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, 100101, China.

[0018] Example 2 In vitro activity experiment: The bactericidal effects of different concentrations of Bacillus cereus phage XP2 and vancomycin on Bacillus cereus were compared in vitro. Figure 2 As shown, under the action of Bacillus cereus phage XP2 group, the turbidity of Bacillus cereus showed a significant decreasing trend after 30 minutes, and the absorbance was close to 0 within 6 hours. In contrast, vancomycin took effect after 2 hours and only approached 0 after about 12 hours.

[0019] In vitro toxicity tests were conducted, and after Bacillus cereus phage XP2 acted on Müller cells, as... Figure 3 As shown, no toxic effects were found.

[0020] Example 3 In vivo experiment: Establishing a mouse endophthalmitis model: A single colony of Bacillus cereus Bc2 was picked and cultured in 5 mL of liquid LB medium at 180 rpm and 37°C until the logarithmic growth phase, then diluted to the desired concentration. The mouse endophthalmitis model was established according to previous methods. 0.5 μL of 2×10⁻⁶ Bacillus cereus solution was injected using a 36-gauge needle. 4 Intraocular inflammation was induced by injecting a 2×10⁻⁶ / μL solution of Bacillus cereus Bc2 into the vitreous humor. Six hours after bacterial injection, 0.5 μL of a 2×10⁻⁶ / μL solution was injected into the vitreous humor using a 36-gauge needle. 9PFU / mL phage XP2 or 2 μg / μL VAN solution. In addition, the negative control group received only 0.5 μL of physiological saline or phage XP2 injected into the eye.

[0021] The experimental group received intravitreal injections of 0.5 μL of 2×10⁻⁶ ions. 9 PFU / mL phage XP2, control group injected with 0.5μL of 2μg / μL vancomycin, such as Figure 4 As shown, intravitreal injection of bacteriophage XP2 to treat endophthalmitis in mice has a relieving effect on the eye disease and inhibits the progression of the disease.

[0022] An experiment was conducted in mice to investigate changes in retinal function following phage XP2 treatment. Figure 5 A showed that 24 hours after infection with Bacillus cereus Bc2 without any treatment, the ERG waveform indicated complete loss of retinal function. However, after treatment with phage XP2 or vancomycin, the ERG waveform showed a more normal morphology and decreased amplitude. Furthermore, the retinal waveforms of mice receiving only phage XP2 injection showed no significant difference from the normal control group, suggesting that phages themselves have no significant adverse effects on normal retinal function. Through observation... Figure 5 B and C show that the XP2 phage treatment group had a higher degree of amplitude preservation of both a and b waves than the vancomycin treatment group. The XP2 treatment alone had no significant effect on the amplitude of retinal ERG a and b waves, indicating that it itself has no toxic effect on normal retinal function and has a better effect on preserving retinal function than the vancomycin treatment group.

[0023] The above description discloses only preferred embodiments of the present invention and should not be construed as limiting the scope of the present invention. Therefore, equivalent variations made in accordance with the claims of the present invention are still within the scope of the present invention.

Claims

1. A bacteriophage XP2 that can be used to treat Bacillus cereus endophthalmitis, characterized in that, It belongs to Herelleviridae division, Bastillevirinae Subfamily, Caeruleovirus The genera were deposited on November 18, 2025, at the China General Microbiological Culture Collection Center (CGMCC), with accession number CGMCC No. 47001.

2. The use of the bacteriophage XP2 as described in claim 1 in the preparation of a drug for treating traumatic endophthalmitis caused by Bacillus cereus.

3. A drug for treating traumatic endophthalmitis caused by Bacillus cereus, characterized in that, Its active ingredient is the bacteriophage XP2 as described in claim 1.

4. The drug according to claim 3, characterized in that, Its dosage form is intravitreal injection.

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