A thermoresistant Aeromonas hydrophila bacteriophage, its composition, and its applications

Abstract

This invention relates to the field of microbial technology, specifically to a thermoresistant Aeromonas guinea pig bacteriophage, its composition, and its applications. The bacteriophage was deposited on June 14, 2024, at the China General Microbiological Culture Collection Center (CGMCC), with accession number CGMCC No. 45946, and named RKP-AE24003. The composition comprises the aforementioned bacteriophage. The bacteriophage can be used to prepare products that kill or inhibit Aeromonas guinea pig; and products for the prevention and / or treatment of aquatic diseases caused by Aeromonas guinea pig. This bacteriophage RKP-AE24003 exhibits good thermoresistance and strong genetic stability, allowing for stable storage in high-temperature environments, significantly improving the stability of industrial production and its applicability to high-temperature scenarios.

Description

Technical Field

[0001] This invention relates to the field of microbial technology, specifically to a thermoresistant Aeromonas hydrophila bacteriophage, its composition, and its applications. Background Technology

[0002] Aeromonas spp. are a group of Gram-negative short bacilli belonging to the Vibrio family, widely found in freshwater, sewage, and soil. *Aeromonas guinea pig* is a species within the *Aeromonas* spp. that is pathogenic not only to aquatic animals but can also infect humans through seafood, causing intestinal diseases or food poisoning. Furthermore, *Aeromonas guinea pig* can infect mammals, leading to serious health problems.

[0003] Currently, treatment methods for Aeromonas vaginalis mainly include drug therapy and phage therapy. However, drug therapy suffers from drug resistance issues, and long-term use of antibiotics may lead to the spread of drug-resistant strains. Phage therapy, as an alternative therapy, has advantages such as high specificity, short development cycle, low cost, and significant therapeutic effect. However, traditional Aeromonas vaginalis phages are easily inactivated under high temperature environments, resulting in a significant reduction in their activity during production, storage, and transportation, which limits their practical application.

[0004] Therefore, providing a strain of Aeromonas guinea phage that is environmentally tolerant to high temperatures is of great practical significance for the production, storage and transportation of Aeromonas guinea phage.

[0005] The information disclosed in this background section is only intended to enhance the understanding of the background technology of this application and should not be construed as an admission or in any way implying that the information constitutes prior art known to those skilled in the art. Summary of the Invention

[0006] This invention addresses the shortcomings of existing technologies by providing a heat-resistant Aeromonas hydrophila phage through experiments. (Aeromonas caviae bacteriophage) RKP-AE24003, this bacteriophage strain RKP-AE24003 has good high temperature resistance and strong genetic stability. It can be stored stably in high temperature environments, which can significantly improve the stability of industrial production and its applicability in high temperature scenarios.

[0007] The technical solution of this invention is as follows:

[0008] On one hand, the present invention provides a thermoresistant Aeromonas guinea pig phage, wherein the phage is an Aeromonas guinea pig phage. (Aeromonas caviae bacteriophage)RKP-AE24003, this bacteriophage strain was isolated from a water sample in an aquaculture area in Shandong Province and was deposited on June 14, 2024, at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, with accession number CGMCCNo.45946.

[0009] On the other hand, the bacteriophage RKP-AE24003 survived for 7-8 weeks at 50°C.

[0010] On the one hand, the bacteriophage RKP-AE24003 exhibits the highest titer (1.36 × 10⁻¹⁰) when the multiplicity of infection is 1:1. 11 pfu / mL.

[0011] On the other hand, the incubation period of the bacteriophage RKP-AE24003 is 30 min, the logarithmic growth phase is 30-120 min, it tends to stabilize after 120 min, and the outbreak quantity is 10. 4 pfu / mL has the advantages of short latency and strong lysis.

[0012] On the other hand, when the pH is 2, the bacteriophage RKP-AE24003 is inactivated; when the pH is < 6, the bacteriophage titer decreases; when the pH is 6-10, the bacteriophage titer is relatively stable; when the pH is > 10, the bacteriophage titer decreases. This indicates that bacteriophage RKP-AE24003 has high tolerance to acids and alkalis, and is more resistant to acids than alkalis.

[0013] On the one hand, the titer of the bacteriophage RKP-AE24003 remained at 10 during 30 consecutive passages. 10 -10 11 With pfu / mL, stable phage titer, and good heritability, it is suitable for industrial production.

[0014] On the other hand, the present invention provides a composition comprising the above-described bacteriophage RKP-AE24003.

[0015] On the other hand, the present invention provides the application of the above-mentioned thermoresistant Aeromonas guinea pig phage or composition in the following (1) to (3):

[0016] (1) Prepare a product that kills Aeromonas hydrophila in guinea pigs;

[0017] (2) Prepare a product that inhibits Aeromonas hydrophila in guinea pigs;

[0018] (3) Prepare drugs for treating aquatic diseases caused by Aeromonas vaginalis.

[0019] On the other hand, in the application of the above-mentioned thermoresistant Aeromonas guinea pig phage or composition, the product or drug is an oral preparation, a wound debridement agent, or an injectable phage cocktail therapy.

[0020] On the other hand, the application is to prepare a drug for treating aeromonas disease in fish, and the drug or product can be added to the aquaculture water to reduce the amount of antibiotics used.

[0021] In another aspect, the application is for use in wastewater treatment systems to specifically remove Aeromonas hydrophila contamination from guinea pigs.

[0022] The beneficial effects achieved by this invention are as follows:

[0023] 1. The bacteriophage RKP-AE24003 of the present invention survives for 7-8 weeks at 50°C, exhibits high temperature tolerance, and can significantly improve the stability of industrial production and its applicability in high-temperature environments.

[0024] 2. The bacteriophage RKP-AE24003 of this invention can be used to prepare drugs or disinfectants for treating and preventing Aeromonas vaginalis infection in guinea pigs. Furthermore, this bacteriophage can also be used in aquaculture, food processing, and other fields as a biocontrol agent to reduce Aeromonas vaginalis contamination.

[0025] 3. The Aeromonas guinea pig phage RKP-AE24003 of this invention was used in experiments on 67 strains of Aeromonas guinea pig, and the phage lysis rate reached 91.04%, which is a good lysis effect.

[0026] 4. The Aeromonas guinea pig phage RKP-AE24003 in this invention can effectively kill Aeromonas guinea pig, improve the survival rate of Penaeus monodon after infection with Aeromonas guinea pig, and has a good prevention and control effect on Aeromonas guinea pig. Attached Figure Description

[0027] Figure 1 This is a plaque image of the bacteriophage RKP-AE24003 of this invention.

[0028] Figure 2 This is an electron microscope image of the bacteriophage RKP-AE24003 of this invention.

[0029] Figure 3 This is a phylogenetic tree analysis diagram of the bacteriophage RKP-AE24003 of this invention.

[0030] Figure 4 This is a diagram showing the infection complexity of the bacteriophage RKP-AE24003 of this invention.

[0031] Figure 5 This is a one-step growth curve of the bacteriophage RKP-AE24003 of this invention.

[0032] Figure 6 This is the pH diagram of the bacteriophage RKP-AE24003 of this invention.

[0033] Figure 7 This is a genetic stability diagram of the bacteriophage RKP-AE24003 of this invention.

[0034] Figure 8 This is a temperature stability diagram of the bacteriophage RKP-AE24003 of the present invention. Detailed Implementation

[0035] The technical solutions of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0036] In this invention, unless otherwise specified, the equipment and raw materials used are commercially available or commonly used in the field. The methods in the following embodiments, unless otherwise specified, are conventional methods in the field. Unless otherwise defined, all technical and scientific terms used in this specification have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. In this invention, Aeromonas guinea pig phage RKP-AE24003 may be simply referred to as "phage RKP-AE24003" or "RKP-AE24003".

[0037] Example 1: Isolation and purification of Aeromonas guinea pig bacteriophage

[0038] Water samples were collected from the aquaculture area, centrifuged at 12,000 rpm for 10 min, and sterilized using a 0.22 μm filter. 200 μL of the filtrate was mixed with 200 μL of Aeromonas guinea pig culture (concentration approximately 10). 8 The co-culture solution (CFU / mL) was placed in 5 mL of LB broth and incubated at 37°C and 160 rpm for 6 hours. The co-culture solution was then sterilized by passing it through a membrane for later use.

[0039] Dilute the co-culture medium 10 times, take 100 μL of the appropriate dilution and add it together with 100 μL of Aeromonas guinea pig culture to 5 mL of LB semi-solid medium, mix well, and quickly pour it onto the pre-prepared LB solid medium to make a double-layer plate. After solidification, invert the plate and incubate it overnight in a 37°C constant temperature incubator.

[0040] Select clear, bright phage plaques and place them in 5 mL of LB broth along with 100 μL of *Aeromonas guinea pig* bacterial suspension. Incubate at 37°C and 160 rpm for 6 hours on a shaker. Sterilize the proliferation solution through a membrane to obtain the phage filtrate. Observe the morphology of the plaques using the double-layer agar plate method. Repeat the process 3-5 times to obtain phage plaques of consistent shape and size, with a diameter of approximately 2 mm. (Image of phage plaques follows.) Figure 1 As shown.

[0041] Example 2 Electron Microscopy Experiment

[0042] Place the copper mesh on clean filter paper and use a micropipette to draw 20 μL of high-concentration phage suspension (titer 10). 10 A 2% phosphotungstic acid (PTA, pH 6.8) staining solution was added to a copper grid and allowed to stand at room temperature for 15 minutes. Excess liquid was then absorbed using filter paper via the edge siphon method, and the sample was allowed to air dry for 3 minutes. One drop of PFU / mL staining solution was then added to cover the sample area, and staining was performed for 10 minutes. After staining, excess staining solution was absorbed from the side using filter paper, and the sample was allowed to air dry for 3 minutes. After drying, the sample was observed under an electron microscope.

[0043] The results are as follows Figure 2 As shown, the morphology of Aeromonas guinea phage RKP-AE24003 observed under an electron microscope reveals that the phage has a regular polyhedral head and a non-contractile tail. The head is approximately 60-75 nm long, and the tail is approximately 95-110 nm long. According to the definition of the International Committee on Taxonomy of Viruses (ICTV), Aeromonas guinea phage RKP-AE24003 is classified as a phage of the Longtailidae family.

[0044] Example 3 Whole genome sequencing

[0045] After enrichment and propagation of single-strain bacteriophages, the bacteriophages were incubated overnight at 4°C in 10% PEG8000 and 0.5M NaCl. Bacteriophage nucleic acid was then extracted using the λ phage DNA extraction method. The whole genome was sent to BGI Genomics for whole-genome sequencing. The whole genome sequence is shown in SEQ ID NO: 1.

[0046] Example 4: Constructing an evolutionary tree

[0047] The gene sequence of bacteriophage RKP-AE24003 was compared with similar sequences using BLAST, and a phylogenetic tree was constructed between the gene sequence of bacteriophage RKP-AE24003 and similar gene sequences using MAGA software.

[0048] As shown in Figure 3, phage RKP-AE24003 has an independent branch in the phylogenetic tree, and can be identified as a new phage.

[0049] Example 5: Determination of the optimal multiplicity of infection for Aeromonas guinea pig bacteriophage

[0050] Aeromonas vaginalis and bacteriophage were proliferated separately, and the initial colony count and initial titer were determined. At different multiplicity of infection ratios (MOIs) of 100 μL of host bacterial culture and 100 μL of bacteriophage supernatant were added together to 5 mL LB broth tubes and incubated at 37°C with shaking for 6 h. Each MOI ratio was set up in triplicate. The bacteriophage titer at different MOIs was determined, and the average value was taken.

[0051] The results are as follows Figure 4 The results showed that the phage titer was highest at a multiplicity of infection of 1:1, reaching 1.36 × 10⁻⁶. 11 PFU / mL.

[0052] Example 6: Determination of one-step growth curve of Aeromonas guinea pig phage

[0053] Inoculate logarithmic-phase Aeromonas vaginalis and bacteriophages at the optimal multiplicity of infection ratio. Incubate in a water bath at 25°C for 15 min, then centrifuge at 8000 rpm for 2 min at room temperature. Discard the supernatant to remove free bacteriophages not adsorbed onto the host. Resuspend the precipitate in liquid culture medium and incubate at 37°C with timing. Samples are taken at 0, 5, 10, 15, 20, 30, 40, 60, 80, 100, 120, 140, 160, 180, 210, and 240 min to determine titer.

[0054] The results are as follows Figure 5 As shown, the phage titer showed no significant change within 0-30 minutes, representing the incubation period; 30-120 minutes was the logarithmic growth phase; and it tended to stabilize after 120 minutes. The phage burst size was approximately 10-1. 4 The PFU / mL indicates that the phage RKP-AE24003 has advantages such as short latency and strong lytic ability.

[0055] Example 7: pH test of Aeromonas vaginalis in guinea pigs

[0056] Add 0.9g NaCl to every 100mL of purified water to prepare a 0.9% NaCl solution. Adjust the pH of the solution to 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, and 13 using HCl and NaOH, respectively. Take 100μL of phage supernatant (titer 10). 10 PFU / mL was added to 900 μL of 0.9% NaCl at different pH values, mixed well, and allowed to stand at room temperature for 1 h. Immediately after mixing, samples were taken and diluted 10-fold. The phage titer after treatment at different pH values ​​was determined by the double plate method. The experiment was repeated 3 times.

[0057] Experimental results are as follows Figure 6 As shown, bacteriophages are inactivated at pH 2; their titer decreases at pH < 6; their titer remains relatively stable between pH 6 and 10; and decreases further at pH > 10. This indicates that bacteriophage RKP-AE24003 exhibits high tolerance to acids and alkalis, being more resistant to acid than alkali.

[0058] Example 8: Experiment on the lysis rate of Aeromonas guinea phage

[0059] Resuscitate 67 strains of Aeromonas guinea pig from the bacterial strain library. Take 100 μL of the bacterial suspension to be tested and add it to LB semi-solid medium. Pour the mixture into LB solid medium plates and allow it to solidify. Then, add 10 μL of bacteriophage suspension (titer 10). 9 PFU / mL was applied to the surface of a plate and incubated at 37°C for 18 hours.

[0060] The experimental results are shown in Table 1. The phage lysis rate of 67 strains of Aeromonas guinea pig was 91.04%, which showed good lysis effect.

[0061] Table 1. Experimental results of Aeromonas guinea pig phage lysis rate

[0062]

[0063] Note: "-" indicates no lysis, "+" indicates lysis and the plaque is relatively clear; "++" indicates lysis and the plaque is clear.

[0064] Example 9: Genetic stability experiment of Aeromonas guinea pig bacteriophage

[0065] Add 2% Aeromonas vaginalis host bacteria and bacteriophage to 5 mL LB tubes and incubate at 37°C in a shaker for 6 h. Repeat the process, subculturing the bacteriophage lysate with the host bacteria for a total of 30 subcultures. Determine the bacteriophage titer using the double-plate method.

[0066] The results are as follows Figure 7 As shown, the titer of bacteriophage RKP-AE24003 remained stable throughout continuous passages, indicating good genetic stability, making it suitable for industrial production.

[0067] Example 10 Temperature stability experiment of Aeromonas guinea pig bacteriophage

[0068] Take multiple 1 mL aliquots of phage supernatant (initial titer 10). 10The phage titer was determined by centrifuging at 1.5 mL in 1.5 mL EP tubes at constant temperatures of 4°C, 25°C, 30°C, 37°C, 40°C, 50°C, 60°C, and 70°C for 0 h, 1 h, 2 h, 6 h, 24 h, 48 h, 92 h, 168 h (1 w), 336 h (2 w), 672 h (4 w), 1344 h (8 w), 2016 h (12 w), and 2688 h (16 w) for 0 h, 1 h, 2 h, 6 h, 24 h (12 w), and 2688 h (16 w) for 24 h, 24 h, 48 h, 92 h, 168 h (1 w), 336 h (2 w), 672 h (4 w), 1344 h (8 w), 2016 h (12 w), and 2688 h (16 w) for 24 h, ... after incubation. After incubation, the centrifuge tubes were removed, cooled immediately, and appropriately diluted. The phage titer was then determined using the double-layer plate method.

[0069] The results are as follows Figure 8 As shown, Aeromonas guinea pig phages can survive for extended periods at temperatures of 50°C and below. At 4°C-30°C, phage titers showed no significant difference in quantity or magnitude over 16 weeks; at 37°C, phage titers showed no significant difference in quantity or magnitude over 8 weeks, after which the titer decreased by one order of magnitude; at 47°C, phage titers showed little change within 1 week, but decreased significantly after 1 week; at 50°C, phage titers were undetectable after 8 weeks; at 60°C, phage titers were undetectable after 48 hours; and at 70°C, phage titers were undetectable after 6 hours.

[0070] Example 11 Animal experiments with Aeromonas guinea pig bacteriophage

[0071] Aeromonas vulgaris phage (titer ≥10) 9 The phage preparation made by mixing PFU / mL with 0.5% sodium alginate carrier had a titer of 10. 7 PFU / mL.

[0072] Select healthy, pathogen-free (SPF) tiger prawns purchased from standardized hatcheries. Acclimate them for 7 days in tanks with a circulating water system, maintaining a water temperature of 28–30℃, salinity of 25‰, and pH of 7.8–8.2.

[0073] Place the tiger prawns in a solution containing 10 6 The shrimp were immersed in seawater containing CFU / mL Aeromonas vaginalis for 2 hours. 24 hours post-infection, three shrimp were randomly sampled, and hepatopancreatic tissue was homogenized and plated on TSA plates for counting. This confirmed successful infection.

[0074] Preparation of bacteriophage formulations: Aeromonas guinea phage (titer ≥10) 9 The phage preparation made by mixing PFU / mL with 0.5% sodium alginate carrier had a titer of 10. 7 PFU / mL.

[0075] Experimental Groups:

[0076] 1) Experimental group: Penaeus monodon infected with Aeromonas vaginalis was treated with a bacteriophage preparation. Phage RDS-1 disinfectant was added to the water immediately after infection, with a final concentration of 10. 8 PFU / mL.

[0077] 2) Positive control group: Penaeus monodon infected with Aeromonas vaginalis was treated with conventional antibiotics (such as enrofloxacin). Enrofloxacin was added (final concentration 5 mg / L).

[0078] 3) Negative control group: Healthy tiger prawns + sterile seawater treatment. An equal volume of sterile seawater was added.

[0079] 4) Control group: Penaeus monodon infected with Aeromonas vaginalis + sterile seawater treatment. An equal volume of sterile seawater was added.

[0080] Each experiment consisted of 30 shrimp of similar size, with a body length of 5–8 cm and a weight of 3–5 g, divided into 3 parallel aquariums (10 shrimp per aquarium).

[0081] Replace 50% of the water daily and refill with disinfectant / medication for 3 consecutive days. Observe mortality within 48 hours of infection and monitor continuously for 7 days to assess long-term effects. Record clinical symptoms and mortality numbers of the tiger prawns to determine survival rate.

[0082] Table 2. Results of animal experiments with Aeromonas guinea pig bacteriophage.

[0083]

[0084] The experimental results are shown in Table 2. The survival rate of the negative control group was 100%; the survival rate of the infected control group was 0%; the survival rate of the experimental group was 92%; and the survival rate of the positive control group was 65%. The results indicate that Aeromonas guinea pig phage RKP-AE24003 can effectively kill Aeromonas guinea pig, improve the survival rate of Penaeus monodon after infection with Aeromonas guinea pig, and has a good control effect against Aeromonas guinea pig.

[0085] The embodiments of the present invention described above do not constitute a limitation on the scope of protection of the present invention. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the scope of protection of the claims of the present invention.

Claims

1. A thermoresistant Aeromonas hydrophila phage (Aeromonas caviae bacteriophage) Its features are: It was deposited on June 14, 2024, at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 45946 and named RKP-AE24003.

2. A composition, characterized in that: Includes the thermoresistant Aeromonas guinea pig phage as described in claim 1.

3. The use of the thermoresistant Aeromonas guinea pig phage according to claim 1 or the composition according to claim 2 in the preparation of a product that kills Aeromonas guinea pig.

4. The application according to claim 3, characterized in that: The product is an oral preparation, a wound cleanser, or an injectable phage cocktail therapy.

5. The application according to claim 3, characterized in that: The product can be added to aquaculture water during use.

6. The application according to claim 3, characterized in that: The application is for use in wastewater treatment systems to specifically remove Aeromonas hydrophila contamination from guinea pigs.

7. The use of the thermoresistant Aeromonas guinea pig phage according to claim 1 or the composition according to claim 2 in the preparation of a medicament for treating aquatic diseases caused by Aeromonas guinea pig.

8. The application according to claim 7, characterized in that: The drug is an oral preparation, a wound debridement agent, or an injectable phage cocktail therapy.

9. The application according to claim 7, characterized in that: The application is for preparing a drug to treat aeromonas infection in fish, which can be added to the aquaculture water when used.

10. The application according to claim 7, characterized in that: The application is for use in wastewater treatment systems to specifically remove Aeromonas hydrophila contamination from guinea pigs.

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