A bacteriophage of bacillus cereus for treating high pathogenic bacillus cereus infection of chinese soft-shelled turtles

Abstract

The application discloses a bacillus cereus phage for treating high-pathogenicity bacillus cereus infection of Chinese soft-shelled turtles, which is named PSYJ-YH in Latin, is deposited in the Guangdong Microbial Culture Collection Center on October 25, 2023, and has a preservation number of GDMCC No.63912-B1. Bacillus cereus The application has a strong lysis effect on multiple strains of high-pathogenicity bacillus cereus and can be used for preventing and controlling high-pathogenicity bacillus cereus in a breeding environment or an animal body.

Description

Technical Field

[0001] This invention relates to the field of microbial technology, and in particular to a Bacillus cereus phage for treating highly pathogenic Bacillus cereus infection in Chinese soft-shelled turtles. Background Technology

[0002] Bacillus cereus ( Bacillus cereus Commonly found in soil, dust, sewage, feed, plants, and various raw and cooked foods, *Bacillus cereus* is classified as Group 18, Section II, Gram-positive, in Bergey's Manual of Bacteriology, Ninth Edition. It is a genus of *Bacillus* within the endospore-forming bacilli and cocci. *Bacillus cereus* is divided into non-toxic and toxic strains. Non-toxic strains are often used as probiotics, primarily for regulating the intestinal flora of humans and other animals and treating diarrhea. It is one of the microecological preparation strains approved for production in my country and has been widely used. Toxic strains of *Bacillus cereus* typically cause food poisoning and are pathogens causing gastrointestinal and non-intestinal infections, belonging to the category of opportunistic pathogens. *Bacillus cereus* infection can cause food poisoning in humans and can also lead to diarrhea, skin rot, ulceration, and even death in farmed animals. In recent years, a highly pathogenic *Bacillus cereus* strain has been found to cause parotitis in Chinese soft-shelled turtles, resulting in bleeding in multiple internal organs and, in severe cases, mass mortality. The pathogenic process of highly pathogenic Bacillus cereus generally involves five steps: adhesion, invasion, colonization, proliferation, and toxin production. Its pathogenicity is related to the type and quantity of virulence factors it carries. Currently, antibiotics are still the first-line treatment for the prevention and treatment of pathogenic Bacillus cereus infections. However, with the decreasing variety of available antibiotics and the emergence and spread of drug-resistant bacteria, there is an urgent need to develop antibiotic alternatives.

[0003] Bacteriophages are a class of viruses with extremely high specificity, capable of infecting microorganisms such as bacteria, fungi, actinomycetes, or spirochetes. They were first discovered by a British biologist in 1915. It is estimated that there are approximately 10 million bacteriophages in the Earth's biosphere. 31 Bacteriophages play a crucial role in regulating the environment and the gut microbiota of animals. They are classified into virulent phages and temperate phages. Virulent phages can proliferate and lyse susceptible host bacteria, and are also known as virulent phages, specifically killing pathogens. Virulent phages invade bacteria primarily by utilizing the host's synthetic mechanisms to exert pressure on the bacterial cell wall, disrupting bacterial metabolism, leading to bacterial lysis and rupture, and subsequently releasing the phage. Compared to other treatment methods, phage therapy is safe, reliable, efficient, rapid, economical, and practical, and holds promise as a superior antibacterial agent, especially in treating drug-resistant bacterial infections. However, currently, there are few phages targeting Bacillus cereus, especially virulent phages with strong lytic capabilities. Summary of the Invention

[0004] The purpose of this invention is to provide a Bacillus cereus phage for treating highly pathogenic Bacillus cereus infection in Chinese soft-shelled turtles. It has a strong lytic effect on multiple strains of highly pathogenic Bacillus cereus and a broad lytic spectrum against highly pathogenic Bacillus cereus. It can be used for the prevention and control of highly pathogenic Bacillus cereus in aquaculture environments or animals.

[0005] The technical solution adopted by this invention to solve its technical problem is: A Bacillus cereus phage for treating highly pathogenic Bacillus cereus infection in Chinese soft-shelled turtles, its Latin name is Bacillus cereus The phage, named PSYJ-YH, is deposited at the Guangdong Provincial Center for Microbial Culture Collection, located at 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, Guangdong Province, on October 25, 2023, with accession number GDMCC No. 63912-B1. Utilizing the unique properties of this phage, the drug resistance problem of highly pathogenic Bacillus cereus in clinical practice can be effectively overcome, providing an effective treatment option for the clinical treatment of highly pathogenic Bacillus cereus infection in Chinese soft-shelled turtles. Compared with existing Bacillus cereus phages, the therapeutic effect of the Bacillus cereus phage of this invention is significantly improved in treating highly pathogenic Bacillus cereus infection in Chinese soft-shelled turtles.

[0006] The application of the Bacillus cereus phage in the preparation of biological agents for preventing and treating highly pathogenic Bacillus infections in Chinese soft-shelled turtles.

[0007] A biological agent for the prevention and treatment of highly pathogenic Bacillus cereus infection, wherein the active ingredient of the biological agent is the Bacillus cereus bacteriophage.

[0008] The beneficial effects of this invention are: it has a strong lytic effect on multiple strains of highly pathogenic Bacillus cereus from Chinese soft-shelled turtles, and can be used for the prevention and control of highly pathogenic Bacillus cereus in the breeding environment or in animals.

[0009] This invention determined the inhibitory effect of bacteriophage PSYJ-YH on host bacteria, confirming that the host bacterial concentration remained at a low level within 9 hours. Using a double-layer plate method on four highly pathogenic Bacillus cereus strains, the PSYJ-YH phage was found to lyse all four strains with a 100% lysis rate. This result indicates that the phage has a broad lysis spectrum against highly pathogenic Bacillus cereus and shows promising potential for clinical application. Attached Figure Description

[0010] Figure 1 This is a diagram illustrating the phage plaque effect of the present invention; Figure 2 This is an electron microscope image of the bacteriophage of the present invention; Figure 3This is a sensitivity curve of the bacteriophage of the present invention to different temperatures; Figure 4 This is a sensitivity curve of the bacteriophage of the present invention to different pH values; Figure 5 This is a diagram showing the in vitro growth kinetics of the bacteriophage of the present invention; Figure 6 This is a diagram showing the effect of the bacteriophage of the present invention in vitro lysing pathogenic Bacillus cereus strains of different heights; Figure 7 This is the in vitro antibacterial OD600 curve of the bacteriophage of the present invention. Detailed Implementation

[0011] The technical solution of the present invention will be further described in detail below through specific embodiments.

[0012] In this invention, unless otherwise specified, all raw materials and equipment used are commercially available or commonly used in the field. The methods described in the following embodiments are conventional methods in the field, unless otherwise specified.

[0013] Example 1: Screening and purification of Bacillus cereus bacteriophage (1) Sample collection The Chinese soft-shelled turtle animal sample used in this invention was collected in October 2022 from a Chinese soft-shelled turtle farm in Hangzhou, Zhejiang Province.

[0014] (2) Specific amplification of Bacillus cereus phage in the sample Dissect the Chinese soft-shelled turtle, take the intestinal contents, add them to 15 mL of Trypticase Soy Broth (commercially available, TSB broth), then add 500 μL of highly pathogenic Bacillus cereus cultured overnight (SYJ-YH, provided by the Institute of Hydrobiology, Zhejiang Academy of Agricultural Sciences), mix well, and incubate overnight at 28°C with shaking at 200 rpm.

[0015] (3) Detection of the presence of bacteriophages using the double-layer plate method Prepare 1.5% TSB nutrient agar solid medium (Haibo Biotechnology). After autoclaving, allow it to stand at room temperature (approximately 40-60°C), then pour 10-15 mL into a petri dish, spreading it evenly at the bottom. Let it solidify at room temperature for 30 min, using this TSB nutrient agar as the bottom layer agar. Take the above-mentioned mixed culture of highly pathogenic Bacillus cereus and intestinal contents, centrifuge at 12000 rpm for 5 min, and filter the supernatant through a 0.22 μm microporous membrane to obtain the phage stock solution. Mix 100 μL of the phage stock solution with 100 μL of highly pathogenic Bacillus cereus and incubate at 28°C for 5-10 min. Add 5 mL of 0.7% TSB semi-solid nutrient agar medium (Haibo Biotechnology), which has been autoclaved and cooled to 55°C. After mixing by hand in the test tube, quickly pour the mixture onto the prepared bottom agar plate, and rotate the plate to distribute it evenly into the top agar. After the agar solidifies, incubate overnight at 28°C upside down, and observe for the presence of plaques. If plaques are present, proceed to step (4); all the above operations are performed under aseptic conditions.

[0016] The results of plaque observation were as follows: the plaques were round, translucent spots, approximately 3 mm in diameter. Figure 1 .

[0017] (4) Purification of phage samples The single phage plaque obtained in step (3) was removed and placed in 1 mL of TSB liquid medium (Haibo Biotechnology). Several sterile ceramic beads were added, and the mixture was shaken at 28°C for 1 h. Then, it was centrifuged at 12000 rpm for 5 min. The supernatant was filtered through a 0.22 μm microporous filter to obtain the phage stock solution. The phage stock solution was diluted 10-fold to a suitable dilution (to the point where single phage plaques were visible). The phage plaques were purified by the double-layer plate method. The purification was repeated 3 times to obtain phage plaques with basically the same size and morphology. The phage monoclonal monomer was obtained and named PSYJ-YH.

[0018] (5) Amplification of phage samples Extract the purified phage single spot from step (4), place it in 1 mL of TSB liquid medium, add several sterile ceramic beads, shake at 28℃ for 1 h, then centrifuge at 12000 rpm for 5 min. Filter the supernatant through a 0.22 μm microporous filter for sterilization and set aside. Take 100 μL of the supernatant filtrate and add it to 10 mL of highly pathogenic Bacillus cereus culture that has been cultured to the early logarithmic growth phase (OD600 = 0.3~0.6). Incubate at 28℃ for 5 h in a shaker at 200 rpm to obtain the phage proliferation solution.

[0019] (6) Preparation of phage preservation solution The phage propagation solution was prepared by mixing the autoclaved TSB nutrient broth (containing 50% glycerol) at a 1:1 volume ratio. The phage preservation solution was stored at -80°C.

[0020] Example 2: Morphological observation of Bacillus cereus bacteriophage Electron microscopy observation of bacteriophages was performed using the phosphotungstic acid negative staining method. 10 µL of concentrated bacteriophage solution was added to a copper grid for adsorption for 1 min. Excess liquid was blotted away with filter paper, followed by staining with 1 drop of 2% phosphotungstic acid for 1 min. Excess staining was then blotted away with filter paper, and the mixture was allowed to air dry before observation using a Hitachi H7650 transmission electron microscope at 80 kV. Measurements of the bacteriophages were performed using Nano Measure 1.2 software.

[0021] Electron microscopy revealed that phage PSYJ-YH is a long-tailed phage with an elliptical, symmetrical head approximately 93.1 nm long and 55.1 nm in diameter, and a tail approximately 139.5 nm long. Figure 2 This new Bacillus cereus phage is morphologically inconsistent with existing reports of Bacillus cereus phage DZ1 (Isolation and characterization of Bacillus cereus bacteriophage DZ1 and its application in foods), LY3W (Research and preliminary application of Bacillus cereus phage cocktail formulations), LY16F2 (Research and preliminary application of Bacillus cereus phage cocktail formulations), and BC-T25 (Isolation, identification and preliminary application of Bacillus cereus phage BC-T25).

[0022] Example 3: Detection of biological characteristics of bacteriophages (a) Phage counting methods The phage proliferation broth was serially diluted 10-fold using TSB nutrient broth, with 100 μL diluted to 10⁻⁶. -5 Up to 10 -7 The phage proliferation solution was mixed with 100 μL of highly pathogenic Bacillus cereus SYJ-YH bacterial solution and incubated at 28°C for 5-10 min. Double-layer plates were prepared using the method in step (3) of Example 1, with three replicates for each dilution. The plaques in the plates were observed, and plates with 30-300 plaques were used to count and determine the phage titer. Phage titer (PFU / mL) = average number of plaques × 10 × dilution factor.

[0023] Record the average number of phage plaques in the three parallel samples at this dilution and calculate the phage titer of the proliferation solution. The phage titer of this strain is 1.89 × 10⁻⁶. 10 PFU / mL.

[0024] (ii) Detection of bacteriophage thermal stability Take 1.0 mL of phage fluid with known titer into a 1.5 mL centrifuge tube and incubate it in a water bath at 28℃, 40℃, 50℃, 60℃ and 70℃ for 30 min. After the incubation time is over, remove the tube and immediately place it in an ice bath to cool. Determine the phage titer using the double-layer plate method.

[0025] When bacteriophages are exposed to temperatures between 28°C and 60°C for 30 minutes, their titer remains essentially unchanged, maintaining a level of 10. 10 After exposure at 70°C for 30 minutes, the phage titer decreased by 7 orders of magnitude, to approximately 10. 3 ( Figure 3 The above results indicate that the titer of the bacteriophage was not affected within a temperature range of 28℃ to 60℃, which is close to the water temperature in actual aquaculture production. Even in the hot summer, the titer of the bacteriophage will not be affected.

[0026] (III) Detection of pH stability of bacteriophages The pH of the liquid culture medium was adjusted with HCl and NaOH to 3.0, 4.0, 5.0, 6.0, 7.0, 8.0, 9.0, 10.0, 11.0, and 12.0, respectively. The culture was then filtered through a 0.22 µm microporous filter for sterilization before use. 100 µL of phage solution was placed in a 1.5 mL centrifuge tube, and 900 µL of liquid culture medium at different pH values ​​was added to each tube. After incubation at 28 °C for 2 h, the titer of the phage under each pH gradient was determined using the double-layer plate method.

[0027] The bacteriophages maintained high activity for 2 hours in an environment with a pH of 4–11. When the pH dropped below 3, the bacteriophage titer decreased by 7 orders of magnitude, to approximately 10. 3 Around 11; when the pH reached above 11, no bacteriophages were detected, indicating that the bacteriophages significantly lost their activity at pH below 3 or above 11. Figure 4 ).

[0028] (iv) Determination of the one-step growth curve of bacteriophage Add 1 mL of 6×10 7 PFU / mL phage solution and 1 mL 6×10 7A mixture of CFU / mL highly pathogenic Bacillus cereus SYJ-YH bacterial suspension was incubated at 28 ℃ for 5-10 min to allow adsorption, followed by centrifugation at 8000 g for 2 min. The supernatant was discarded, and the precipitate was diluted 10% with 1 mL of TSB liquid medium. -5 Incubate at 28℃ and 220 rpm with shaking for 10 mL of culture time. Measure the phage titer at appropriate time intervals using the double-layer plate method, repeating the process three times at each time point. Finally, plot the one-step growth curve of the phage with time on the x-axis and 1g PFU / mL on the y-axis to calculate the phage's latency period, outbreak period, stationary period, and lysis rate.

[0029] The results are as follows Figure 5 As shown, the incubation period of PSYJ-YH phage is approximately 20 min, the outbreak time is approximately 40 min (from the incubation period to the stationary phase), and the outbreak amount is 549 PFU / cell. Outbreak amount = phage titer at the end of the outbreak ÷ host bacterial concentration at the beginning of infection, i.e., 8.13 × 10⁻⁶. 9 (PFU / mL) / 1.48×10 7 (CFU / mL) = 549 PFU / cell.

[0030] Example 4: Determination of phage lysis profile Four highly pathogenic Bacillus cereus strains (provided by the Institute of Hydrobiology, Zhejiang Academy of Agricultural Sciences) were used as subjects. These four strains, SYJ-YH, SYJ-YXM, SYJ-NX, and SYJ-XJH, were all isolated from the Chinese soft-shelled turtle. The phage lysis profile was determined using the spot method. 6 mL of melted 0.7% agarose TSB semi-solid medium (Haibo Biotechnology) was mixed with 100 µL of fresh host bacteria. After thorough mixing, the mixture was poured onto 1.5% agarose TSB solid medium (Haibo Biotechnology). After the surface dried and solidified, 7 µL of phage solution was added, and the mixture was incubated overnight at 28°C. The formation of lysis zones was then observed.

[0031] The test results showed that bacteriophage PSYJ-YH could lyse four highly pathogenic Bacillus cereus strains, with a lysis rate of 100%. Figure 6 The highly pathogenic Bacillus cereus strains capable of lysis all originated from diseased Chinese soft-shelled turtles, indicating that this bacteriophage strain has high specificity for highly pathogenic Bacillus cereus from Chinese soft-shelled turtles.

[0032] Example 5: Determination of the in vitro antibacterial effect of bacteriophage Take two 50 mL bottles of liquid culture medium, add 500 µL of highly pathogenic Bacillus cereus SYJ-YH, and incubate at 28℃ with shaking at 220 rpm. When OD600 = 0.3, add bacteriophage with MO1 = 0.001 to one of the culture bottles. Measure OD600 every 30 min with a UV spectrophotometer for the first 3 h, and then every 1 h thereafter, for a total of 9 h.

[0033] The results showed that from 60 min to 540 min, the OD600 of the culture medium with added phage was significantly lower than that of the culture medium without added phage, with the lowest value being 0.308. Figure 7 The above results indicate that this bacteriophage has a strong lytic effect on the host bacteria.

[0034] Example 6: Determination of the efficacy of bacteriophage PSYJ-YH in preventing highly pathogenic Bacillus cereus Take the highly pathogenic Bacillus cereus SYJ-YH culture that has been cultured overnight, centrifuge at 8000 rpm for 5 min, discard the supernatant, reselect the bacterial pellet with 0.9% sterile physiological saline, and dilute the bacterial solution to a final concentration of 10. 4 CFU / mL was used as the bacterial suspension for subsequent challenge. A concentration of 10 CFU / mL was prepared using SM buffer (Haibo Biotechnology) as the solvent. 9 Different phage solutions (PSYJ-YH, DZ1, LY3W, LY16F2, BC-T25) at PFU / mL were used as phage biological agents. Experimental and control groups were set up, with three containers in each group, each containing 10 Chinese soft-shelled turtles. 100 µL of bacterial suspension was injected intraperitoneally into the experimental group, while 100 µL of the phage biological agent was injected into the control group simultaneously. The number of deaths was recorded daily, and the cumulative mortality rate was calculated. The immunoprotective rate of the phage biological agent was calculated after 7 days.

[0035] The results (Table 1) showed that the cumulative mortality rates over 7 days in the three tanks of the PSYJ-YH group were 60%, 60%, and 70%, respectively, with an average of 63.3%. All members of the DZ1, LY3W, LY16F2, BC-T25 groups, and the control group died, resulting in a cumulative mortality rate of 100%. The immunoprotective rate of the PSYJ-YH phage biological agent against highly pathogenic Bacillus cereus was 36.7%. In treating highly pathogenic Bacillus cereus infection in Chinese soft-shelled turtles, the therapeutic effect of the Bacillus cereus phage of this invention is significantly improved compared to existing technologies.

[0036] Table 1

[0037] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the present invention in any way. Other variations and modifications are possible without departing from the technical solutions described in the claims.

Claims

1. A Bacillus cereus bacteriophage for treating highly pathogenic Bacillus cereus infection in Chinese soft-shelled turtles, characterized in that: Its Latin name is Bacillus cereus Phage, named PSYJ-YH, is deposited at the Guangdong Provincial Center for Microbial Culture Collection on October 25, 2023, with accession number GDMCC No. 63912-B1.

2. The application of the Bacillus cereus phage as described in claim 1 in the preparation of a biological agent for preventing and treating highly pathogenic Bacillus cereus infection in Chinese soft-shelled turtles.

3. A biological agent for the prevention and treatment of highly pathogenic Bacillus cereus infection in Chinese soft-shelled turtles, characterized in that: The active ingredient of the biological agent is the Bacillus cereus phage as described in claim 1.

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